RNAi Modulation of the RHO-A Gene and Uses Thereof

Abstract

compositions and methods for modulating the expression of the RhoA gene, and more particularly to the downregulation of RhoA by chemically modified oligonucleotides.

Description

RELATED APPLICATIONS

[0001]This application is a divisional application of U.S. application Ser. No. 11/491,367, filed on Jul. 21, 2006, which claims the benefit of U.S. Provisional Application No. 60/701,470, filed Jul. 21, 2005, U.S. Provisional Application No. 60/726,838, filed Oct. 14, 2005, and U.S. Provisional Application No. 60/748,316, filed Dec. 7, 2005. The contents of each of these priority applications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002]This application includes a Sequence Listing submitted electronically as a text file named "17189US_sequencelisting.txt," created on Jul. 30, 2010, with a size of 404,000 bytes. The sequence listing consists of 1165 sequences and is incorporated by reference.

TECHNICAL FIELD

[0003]The invention relates to compositions and methods for modulating the expression of RhoA, and more particularly to the downregulation of RhoA mRNA and RhoA protein levels by oligonucleotides via RNA interference, e.g., chemically modified oligonucleotides.

BACKGROUND

[0004]RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function.

[0005]Numerous myelin-derived axon growth inhibitors have been characterized (see, for review, David et al., WO995394547, 1999; Bandman et al. U.S. Pat. No. 5,858,708, 1999; Schwab, Neurochem. Res. 21:755-761, 1996). Several components of CNS white matter, NI35, NI250 (Nogo) and Myelin-associated glycoprotein (MAG), which have inhibitory activity for axonal extension, have been described as well (Schwab et al., WO9005191, 1990; Schwab et al., U.S. Pat. No. 5,684,133, 1997). In particular, RhoA is a member of the large family of Rho (Ras homologue) GTPases, itself belonging to the superfamily of Ras GTPases. All eukaryotes contain at least one Rho GTPase. During the process of evolution the number of Rho GTPases increased from 5 to 6 per organism (yeast) to over 20 (mammals) (Karnoub, A. E., et al., Breast Cancer Res. Treat. 2004, 84:61). Like other GTPases, RhoA has intrinsic GTPase activity and shuttles between an inactive GDP-bound state and an active GTP-bound state. In vitro, the exchange of GDP to GTP occurs very slowly, and is catalyzed by guanine nucleotide exchange factors (GEFs), which exchange GDP for GTP. GTPase activating proteins (GAPs) catalyze hydrolysis of the γ-phosphate of GTP. (Wheeler, A. P., Ridley, A. J., Exp. Cell Res. 2004, 301:43). A third set of regulatory proteins, the guanine nucleotide-dissociation inhibitors (GDIs), sequester GTPAses in the cytosol in the inactive, GDP-bound state.

[0006]The N-terminal half of Rho GTPases contains the majority of the amino acids involved in GTP binding and hydrolysis, together with the Switch 1 and 2 regions that change conformation between the GTP-bound and GDP-bound states (Bishop, A. L., Hall, A., Biochem. J. 2000, 348 (Pt. 2):241). The C-terminus of Rho family GTPases is essential for correct localization of the proteins. It is post-translationally modified by prenylation of a conserved C-terminal cysteine followed by methylation and proteolytic removal of the last three amino acids (Shao, F., Dixon, J. E., Adv. Exp. Med. Biol. 2003, 529:79). The prenyl group anchors the GTPases into membranes and this modification is essential for cell growth, transformation, and cytoskeleton organization (Allal, C., et al., J. Biol. Chem. 2000, 275:31001). Prenylation of Rho proteins appears to be important for their stability, inhibitors of enzymes that synthesize prenyl groups induce a decrease in Rho protein levels and their function (Stamatakis, K., et al., J. Biol. Chem. 2002, 277:49389). In the case of RhoA, prenylation adds a geranylgeranyl group. RhoA is mainly found in the cytoplasm or at the plasma membrane (Adamson, P., et al., J. Cell Biol. 1992, 119:617).

[0007]RhoA may bind to the intracellular portion of p75NTR and is activated by Nogo-R in a p75NTR-dependent manner (Wang, K. C., et al., Nature 2002, 420:74), which is how MAG, Nogo-66, and oligodendrocyte-myelin glycoprotein achieve RhoA activation. The central inhibitory domain of Nogo-A, NiG, distinct from Nogo-66, and Versican V2, a chondroitin-sulfate proteoglycan and another component of myelin, are able to activate RhoA in the absence of p75NTR, by an alternative pathway of RhoA activation remaining to be elucidated (Schweigreiter, R., et al., Mol. Cell. Neurosci. 2004, 27:163). Further pathways of activation may exist.

[0008]RhoA is part of the growth inhibitory machinery present in the central nervous system (CNS), but not in peripheral nerves, which prevents the regeneration of CNS tissue after injury. Both the expression and the activation of RhoA is induced in brain and spinal cord injury (Mueller, K., et al., Nature Reviews 2005, 4:387). Activation of RhoA leads to neuronal growth cone collapse, retraction bulb formation and neurite withdrawal. Inactivation of RhoA leads to neurite outgrowth in primary neurons on otherwise inhibitory substrates in vitro, and promotes axon regeneration and functional recovery after spinal cord injury in rats and mice in vivo (Lehmann, M. A., et al., J. Neurosci. 1999, 19:7537; Hara, M, et al., J. Neurosurg. 2000, 93:94; Dergham, P., et al., J. Neurosci. 2002, 22:6570). Furthermore, inactivation of Rho has been shown to protect endogenous cells of the spinal cord from apoptosis induced by spinal cord injury (Dubreuil, C. I., et al, J. Cell Biol. 2003, 162:233). These findings have clinical relevance because neuroprotective treatments after spinal cord injury lead to improved functional recovery (Liu, X. Z., et al., J. Neurosci. 1997, 17:5395).

[0009]Evidently, RhoA is a potential target for therapeutic intervention strategies aimed at diseases and conditions involving, e.g., the destruction and/or impaired regeneration of cells of the CNS. The present invention advances the art by providing methods and medicaments encompassing short dsRNAs leading to the downregulation of RhoA mRNA and protein levels in cells expressing the RhoA gene. These methods and medicaments may be used in the treatment of disorders or pathological processes mediated, at least in part, by RhoA, e.g., by preventing the RhoA inhibition of axonal elongation and regeneration, and consequently stimulating nerve growth and proliferation.

SUMMARY

[0010]The present invention is based, at least in part, on an investigation of the RhoA gene using iRNA agents and further testing of the iRNA agents that target the RhoA site. The present invention provides compositions and methods that are useful in reducing RhoA mRNA levels, RhoA protein levels and the treatment of pathological process mediated, at least in part, by RhoA, e.g. preventing RhoA inhibition of axonal elongation and regeneration, in a subject, e.g., a mammal, such as a human.

[0011]In one aspect, the invention provides iRNA agents comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836 as given in Table 1 below, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836.

[0012]In a further aspect, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and an antisense strand wherein the antisense strand comprises at least 15 contiguous nucleotides of the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and wherein the iRNA agent reduces the amount of RhoA mRNA present in cultured human cells after incubation with these agents by 40% or more compared to cells which have not been incubated with the agent.

[0013]In a further aspect, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell comprising a sense strand and an antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical to one of the sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit RhoA expression. Preferably, for such agents the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisense strand sequences of agent numbers 6523, 6524, 6530, 6614, 6650, 6656, 6657, 6661, 6662, 6703, 6712, 6713, 6732, 6751, 6756, 6767, 6769, 6787, 6789, 6790, 6832.

[0014]Evidently, in the above embodiments, the sense strands and/or antisense strands of the iRNA agents of the invention can also be identical to the sense strands and antisense strands of the agents, agent numbers 6477 to 6836.

[0015]The iRNA agents of the invention may comprise a modification, e.g a modification that causes the iRNA agent to have increased stability in a biological sample. For example, they may comprise a phosphorothioate, a 2'-modified nucleotide, a locked nucleotide, an abasic nucleotide, morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide. For purposes of the above embodiments, an iRNA agent is considered to comprise one of the sequences of the agents, agent numbers 6477 to 6836, irrespective of the potential presence of nucleotide modifications, i.e. a 2'-O-methyl guanosine would be considered a guanosine for such comparison. However, certain patterns of modifications are particularly preferred embodiments of the present invention. Consequently, in another embodiment, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell wherein the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisense strand sequences of agent numbers AL-DP-5972, AL-DP-5973, AL-DP-5974, AL-DP-5975, AL-DP-5976, AL-DP-5978, AL-DP-5979, AL-DP-5981, AL-DP-5982, AL-DP-5983, AL-DP-5984, AL-DP-5986, AL-DP-5987, AL-DP-5988, AL-DP-5989, AL-DP-5990, AL-DP-5991, AL-DP-5992, AL-DP-5993, AL-DP-5994, AL-DP-5995, AL-DP-6176, AL-DP-6177.

[0016]In the iRNA agents of the present invention, the antisense RNA strand may be 30 or fewer nucleotides in length, and the duplex region of the iRNA agent may be 15-30 nucleotide pairs in length.

[0017]A 2'-modified nucleotide according to the instant invention may comprise at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-guanine-3' (5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide.

[0018]The iRNA agents of the invention may be designed such that

[0019]every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is a 2'-modified in sense strand, and every 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in antisense strand, or

[0020]every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is 2'-modified in the sense and antisense strand, or

[0021]every pyrimidine nucleotide is 2'-modified in the sense strand, and every 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in the antisense strand, or

[0022]every pyrimidine nucleotide is 2'-modified in sense strand, and every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is 2'-modified in the antisense strand, or

[0023]every pyrimidine nucleotide in the sense strand is 2'-modified, and no nucleotide is 2'-modified in the antisense strand.

[0024]The 2'-modification in the iRNA agents of the invention may be selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O--N-methylacetamido (2'-O--NMA).

[0025]The iRNA agents of the invention may comprise a nucleotide overhang having 1 to 4 unpaired nucleotides, preferably 2 or 3 unpaired nucleotides. The nucleotide overhang may be at the 3'-end of the antisense strand of the iRNA agent. The iRNA agents may comprise a cholesterol moiety, which is preferably conjugated to the 3'-end of the sense strand of the iRNA agent. In a preferred embodiment, the iRNA agent is targeted for uptake by nerve cells or nerve sheath cells.

[0026]The present invention further provides methods for reducing the level of RhoA mRNA in a cell. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade RhoA mRNA in a cell and are comprised of the step of contacting a cell with one of the iRNA agents of the present invention. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the iRNA agents of the present invention. Reduction of RhoA mRNA in a cell results in a reduction in the amount of RhoA protein produced, and in an organism, may result in a decrease in RhoA specific pathological/disease effects, e.g. preventing RhoA inhibition of axonal elongation and regeneration.

[0027]In another aspect of the invention, a method of treating a human subject having a pathological process mediated in part by RhoA is provided, comprising administering an iRNA agent of the invention, e.g. wherein the iRNA agent comprises a sense strand wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences any one of the agents, agent numbers 6477 to 6836, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense strand sequences of any one of the agents, agent numbers 6477 to 6836.

[0028]In one embodiment of the above methods of the invention, the pathological process is the inhibition of nerve growth or elongation, preferably as a result of nerve injury or damage. In another preferred embodiment, the iRNA agent is administered in an amount sufficient to reduce the expression of RhoA in a cell or tissue of the subject. Preferably, the subject is a human.

[0029]In another aspect, the instant invention provides pharmaceutical compositions, comprising:

[0030]a.) an iRNA agent of the invention; and

[0031]b.) a pharmaceutically acceptable carrier

[0032]In another embodiment, the invention provides a cell comprising an iRNA agent of the invention.

[0033]In another embodiment, the invention provides a method for inhibiting the expression of a RhoA gene in a cell, the method comprising: [0034](a) introducing into the cell an iRNA agent of the invention; and [0035](b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the RhoA gene, thereby inhibiting expression of the RhoA gene in the cell.

[0036]In another embodiment, the invention provides a vector for inhibiting the expression of a RhoA gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an an iRNA agent of the invention.

[0037]In another embodiment, the invention provides a cell comprising the above vector.

[0038]The methods and compositions of the invention, e.g., the methods and iRNA compositions can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

[0039]The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from this description and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

[0040]FIG. 1 is a schematic illustrating the synthesis and structure of cholesterol conjugated RNA strands. The sphere represents the solid phase (controlled pore glass, CPG).

DETAILED DESCRIPTION

[0041]For ease of exposition the term "nucleotide" or "ribonucleotide" is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood that the usage of the term "ribonucleotide" or "nucleotide" herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety, as further described below, at one or more positions.

[0042]An "RNA agent" as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, each of which is described herein or is well known in the RNA synthetic art. While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those that have a 2' sugar modification, a modification in a single strand overhang, preferably a 3' single strand overhang, or, particularly if single stranded, a 5'-modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

[0043]An "iRNA agent" (abbreviation for "interfering RNA agent") as used herein, is an RNA agent, which can downregulate the expression of a target gene, e.g., RhoA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can be a double stranded iRNA agent.

[0044]A "ds iRNA agent" (abbreviation for "double stranded iRNA agent"), as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interstrand hybridization can form a region of duplex structure. A "strand" herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g., by a linker, e.g., a polyethyleneglycol linker, to form one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the "antisense strand." A second strand of the dsRNA agent, which comprises a region complementary to the antisense strand, is termed the "sense strand." However, a ds iRNA agent can also be formed from a single RNA molecule which is at least partly self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.

[0045]Although, in mammalian cells, long ds iRNA agents can induce the interferon response which is frequently deleterious, short ds iRNA agents do not trigger the interferon response, at least not to an extent that is deleterious to the cell and/or host (Manche et al., Mol. Cell. Biol. 12:5238, 1992; Lee et al., Virology 199:491, 1994; Castelli et al., J. Exp. Med. 186:967, 1997; Zheng et al., RNA 10:1934, 2004; Heidel et al., "Lack of interferon response in animals to naked siRNAs" Nature Biotechn. advance online publication doi:10.1038/nbt1038, Nov. 21, 2004). The iRNA agents of the present invention include molecules which are sufficiently short that they do not trigger a deleterious non-specific interferon response in normal mammalian cells. Thus, the administration of a composition including an iRNA agent (e.g., formulated as described herein) to a subject can be used to decreased expression of the RhoA genes in RhoA expressing cells in the subject, while circumventing an interferon response. Molecules that are short enough that they do not trigger a deleterious interferon response are termed siRNA agents or siRNAs herein. "siRNA agent" or "siRNA" as used herein, refers to an iRNA agent, e.g., a ds iRNA agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs.

[0046]The isolated iRNA agents described herein, including ds iRNA agents and siRNA agents, can mediate the decreased expression of a RhoA nucleic acid, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a nucleic acid is also referred to as a target gene. Preferably, the RNA to be silenced is a gene product of an endogenous RhoA gene.

[0047]As used herein, the phrase "mediates RNAi" refers to the ability of an agent to silence, in a sequence specific manner, a target gene. "Silencing a target gene" means the process whereby a cell containing and/or expressing a certain product of the target gene when not in contact with the agent, will contain and/or express at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.

[0048]As used herein, the term "complementary" is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule, e.g., a RhoA mRNA. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ from the target sequences by at least 4 nucleotides.

[0049]As used herein, an iRNA agent is "sufficiently complementary" to a target RNA, e.g., a target mRNA (e.g., a target RhoA mRNA) if the iRNA agent reduces the production of a protein encoded by the target RNA in a cell. The iRNA agent may also be "exactly complementary" to the target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A "sufficiently complementary" iRNA agent can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RhoA RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementarity is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference. Preferred iRNA agents will be based on or consist of or comprise the sense and antisense sequences provided in Table 1.

[0050]As used herein, "essentially identical" when used referring to a first nucleotide sequence in comparison to a second nucleotide sequence means that the first nucleotide sequence is identical to the second nucleotide sequence except for up to one, two or three nucleotide substitutions (e.g., adenosine replaced by uracil). "Essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells," as used herein referring to an iRNA agent not identical to but derived from one of the iRNA agents of Table 1 by deletion, addition or substitution of nucleotides, means that the derived iRNA agent possesses an inhibitory activity not more than 20% (in terms of remaining target mRNA) different from the inhibitory activity of the iRNA agent of Table 1 from which it was derived. For example, an iRNA agent derived from an iRNA agent of Table 1 which lowers the amount of RhoA mRNA present in cultured human Rho-A expressing cells by 70% may itself lower the amount of RhoA mRNA present in cultured human RhoA expressing cells by at least 50% in order to be considered as essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells. Optionally, an iRNA agent of the invention may lower the amount of RhoA mRNA present in cultured human RhoA expressing cells by at least 50%, or at least 40%.

[0051]As used herein, a "subject" refers to a mammalian organism undergoing treatment for a disorder mediated by RhoA protein expression. The subject can be any mammal, such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. In the preferred embodiment, the subject is a human.

[0052]Design and Selection of iRNA Agents

[0053]As used herein, "disorders associated with RhoA expression" refers to any biological or pathological state that (1) is mediated in part by the presence of RhoA mRNA and/or protein and (2) whose outcome can be affected by reducing the level of RhoA mRNA and/or protein present. Specific disorders associated with RhoA expression are noted below and are primarily based on the responsibility of RhoA action in inhibiting axonal elongation and regeneration.

[0054]The present invention is based on the design, synthesis and generation of iRNA agents that target RhoA and the demonstration of silencing of a RhoA gene in vitro in cultured cells after incubation with an iRNA agent, and the resulting RhoA-specific effect.

[0055]An iRNA agent can be rationally designed based on sequence information and desired characteristics. For example, an iRNA agent can be designed according to the relative melting temperature of the candidate duplex. Generally, the duplex should have a lower melting temperature at the 5' end of the antisense strand than at the 3' end of the antisense strand.

[0056]Candidate iRNA agents can also be designed by performing, for example, a gene walk analysis of the genes that will serve as the target gene. Overlapping, adjacent, or closely spaced candidate agents corresponding to all or some of the transcribed region can be generated and tested. Each of the iRNA agents can be tested and evaluated for the ability to down regulate the target gene expression (see below, "Evaluation of Candidate iRNA agents").

[0057]Herein, potential iRNA agents targeting RhoA were designed using the known sequences of RhoA for human, rat and mouse and other known RhoA sequences. The target sequences shown in Table 1 hereinabove were selected from those regions of the human RhoA mRNA sequences that show complete homology with the corresponding sequences in rat and mouse. Therefore, the siRNA agents, agent numbers 6477-6836 should show cross reactivity between these three species. Based on the results provided, the present invention provides iRNA agents that silence RhoA in cultured human RhoA expressing cells and in a subject.

[0058]Table 1 provides exemplary iRNA agents targeting RhoA

TABLE-US-00001 TABLE 1 Exemplary iRNA agents for targeting RhoA mRNA Start duplex sense strand antisense strand pos. SEQ design SEQ SEQ Agent in ID (over- ID ID number RNA^b NO. target sequence (5'-3') hang)^a NO. sequence (5'-3') NO. sequence (5'-3') 6477 288 1 ccggaagaaacuggugauuguug double 2 ggaagaaacuggugauuguTT 3 acaaucaccaguuucuuccTT 6478 289 4 cggaagaaacuggugauuguugg double 5 gaagaaacuggugauuguuTT 6 aacaaucaccaguuucuucTT 6479 290 7 ggaagaaacuggugauuguuggu double 8 aagaaacuggugauuguugTT 9 caacaaucaccaguuucuuTT 6480 291 10 gaagaaacuggugauuguuggug double 11 agaaacuggugauuguuggTT 12 ccaacaaucaccaguuucuTT 6481 292 13 aagaaacuggugauuguugguga double 14 gaaacuggugauuguugguTT 15 accaacaaucaccaguuucTT 6482 293 16 agaaacuggugauuguuggugau double 17 aaacuggugauuguuggugTT 18 caccaacaaucaccaguuuTT 6483 294 19 gaaacuggugauuguuggugaug double 20 aacuggugauuguuggugaTT 21 ucaccaacaaucaccaguuTT 6484 295 22 aaacuggugauuguuggugaugg double 23 acuggugauuguuggugauTT 24 aucaccaacaaucaccaguTT 6485 296 25 aacuggugauuguuggugaugga double 26 cuggugauuguuggugaugTT 27 caucaccaacaaucaccagTT 6486 297 28 acuggugauuguuggugauggag double 29 uggugauuguuggugauggTT 30 ccaucaccaacaaucaccaTT 6487 298 31 cuggugauuguuggugauggagc double 32 ggugauuguuggugauggaTT 33 uccaucaccaacaaucaccTT 6488 299 34 uggugauuguuggugauggagcc double 35 gugauuguuggugauggagTT 36 cuccaucaccaacaaucacTT 6489 300 37 ggugauuguuggugauggagccu double 38 ugauuguuggugauggagcTT 39 gcuccaucaccaacaaucaTT 6490 326 40 gaaagacaugcuugcugauaguc double 41 aagacaugcuugcucauagTT 42 cuaugagcaagcaugucuuTT 6491 327 43 aaagacaugcuugcugauagucu double 44 agacaugcuugcucauaguTT 45 acuaugagcaagcaugucuTT 6492 328 46 aagacaugcuugcucauagucuu double 47 gacaugcuugcucauagucTT 48 gacuaugagcaagcaugucTT 6493 329 49 agacaugcuugcugauagucuuc double 50 acaugcuugcucauagucuTT 51 agacuaugagcaagcauguTT 6494 330 52 gacaugcuugcugauagucuuca double 53 caugcuugcucauagucuuTT 54 aagacuaugagcaagcaugTT 6495 331 55 acaugcuugcucauagucuucag double 56 augcuugcucauagucuucTT 57 gaagacuaugagcaagcauTT 6496 332 58 caugcuugcucauagucuucagc double 59 ugcuugcucauagucuucaTT 60 ugaagacuaugagcaagcaTT 6497 333 61 augcuugcucauagucuucagca double 62 gcuugcucauagucuucagTT 63 cugaagacuaugagcaagcTT 6498 334 64 ugcuugcucauagucuucagcaa double 65 cuugcucauagucuucagcTT 66 gcugaagacuaugagcaagTT 6499 335 67 gcuugcucauagucuucagcaag double 68 uugcucauagucuucagcaTT 69 ugcugaagacuaugagcaaTT 6500 336 70 cuugcucauagucuucagcaagg double 71 ugcucauagucuucagcaaTT 72 uugcugaagacuaugagcaTT 6501 337 73 uugcucauagucuucagcaagga double 74 gcucauagucuucagcaagTT 75 cuugcugaagacuaugagcTT 6502 338 76 ugcucauagucuucagcaaggac double 77 cucauagucuucagcaaggTT 78 ccuugcugaagacuaugagTT 6503 339 79 gcucauagucuucagcaaggacc double 80 ucauagucuucagcaaggaTT 81 uccuugcugaagacuaugaTT 6504 340 82 cucauagucuucagcaaggacca double 83 cauagucuucagcaaggacTT 84 guccuugcugaagacuaugTT 6505 341 85 ucauagucuucagcaaggaccag double 86 auagucuucagcaaggaccTT 87 gguccuugcugaagacuauTT 6506 342 88 cauagucuucagcaaggaccagu double 89 uagucuucagcaaggaccaTT 90 ugguccuugcugaagacuaTT 6507 343 91 auagucuucagcaaggaccaguu double 92 agucuucagcaaggaccagTT 93 cugguccuugcugaagacuTT 6508 344 94 uagucuucagcaaggaccaguuc double 95 gucuucagcaaggaccaguTT 96 acugguccuugcugaagacTT 6509 345 97 agucuucagcaaggaccaguucc double 98 ucuucagcaaggaccaguuTT 99 aacugguccuugcugaagaTT 6510 346 100 gucuucagcaaggaccaguuccc double 101 cuucagcaaggaccaguucTT 102 gaacugguccuugcugaagTT 6511 347 103 ucuucagcaaggaccaguuccca double 104 uucagcaaggaccaguuccTT 105 ggaacugguccuugcugaaTT 6512 348 106 cuucagcaaggaccaguucccag double 107 ucagcaaggaccaguucccTT 108 gggaacugguccuugcugaTT 6513 349 109 uucagcaaggaccaguucccaga double 110 cagcaaggaccaguucccaTT 111 ugggaacugguccuugcugTT 6514 350 112 ucagcaaggaccaguucccagag double 113 agcaaggaccaguucccagTT 114 cugggaacugguccuugcuTT 6515 351 115 cagcaaggaccaguucccagagg double 116 gcaaggaccaguucccagaTT 117 ucugggaacugguccuugcTT 6516 352 118 agcaaggaccaguucccagaggu double 119 caaggaccaguucccagagTT 120 cucugggaacugguccuugTT 6517 353 121 gcaaggaccaguucccagaggug double 122 aaggaccaguucccagaggTT 123 ccucugggaacugguccuuTT 6518 354 124 caaggaccaguucccagaggugu double 125 aggaccaguucccagagguTT 126 accucugggaacugguccuTT 6519 425 127 gaaagcagguagaguuggcuuug double 128 aagcagguagaguuggcuuTT 129 aagccaacucuaccugcuuTT 6520 426 130 aaagcagguagaguuggcuuugu double 131 agcagguagaguuggcuuuTT 132 aaagccaacucuaccugcuTT 6521 535 133 gacagcccugauaguuuagaaaa double 134 cagcccugauaguuuagaaTT 135 uucuaaacuaucagggcugTT 6522 536 136 acagcccugauaguuuagaaaac double 137 agcccugauaguuuagaaaTT 138 uuucuaaacuaucagggcuTT 6523 537 139 cagcccugauaguuuagaaaaca double 140 gcccugauaguuuagaaaaTT 141 uuuucuaaacuaucagggcTT 6524 538 142 agcccugauaguuuagaaaacau double 143 cccugauaguuuagaaaacTT 144 guuuucuaaacuaucagggTT 6525 539 145 gcccugauaguuuagaaaacauc double 146 ccugauaguuuagaaaacaTT 147 uguuuucuaaacuaucaggTT 6526 540 148 cccugauaguuuagaaaacaucc double 149 cugauaguuuagaaaacauTT 150 auguuuucuaaacuaucagTT 6527 541 151 ccugauaguuuagaaaacauccc double 152 ugauaguuuagaaaacaucTT 153 gauguuuucuaaacuaucaTT 6529 542 154 cugauaguuuagaaaacauccca double 155 gauaguuuagaaaacauccTT 156 ggauguuuucuaaacuaucTT 6529 543 157 ugauaguuuagaaaacaucccag double 158 auaguuuagaaaacaucccTT 159 gggauguuuucuaaacuauTT 6530 544 160 gauaguuuagaaaacaucccaga double 161 auguuuagaaaacaucccaTT 162 ugggauguuuucuaaacuaTT 6531 545 163 auaguuuagaaaacaucccagaa double 164 aguuuagaaaacaucccagTT 165 cugggauguuuucuaaacuTT 6532 546 166 uaguuuagaaaacaucccagaaa double 167 guuuagaaaacaucccagaTT 168 ucugggauguuuucuaaacTT 6533 547 169 aguuuagaaaacaucccagaaaa double 170 uuuagaaaacaucccagaaTT 171 uucugggauguuuucuaaaTT 6534 548 172 guuuagaaaacaucccagaaaag double 173 uuagaaaacaucccagaaaTT 174 uuucugggauguuuucuaaTT 6535 549 175 uuuagaaaacaucccagaaaagu double 176 uagaaaacaucccagaaaaTT 177 uuuucugggauguuuucuaTT 6536 575 178 ccccagaagucaagcauuucugu double 179 ccagaagucaagcauuucuTT 180 agaaaugcuugacuucuggTT 6537 576 181 cccagaagucaagcauuucuguc double 182 cagaagucaagcauuucugTT 183 cagaaaugcuugacuucugTT 6538 577 184 ccagaagucaagcauuucugucc double 185 agaagucaagcauuucuguTT 186 acagaaaugcuugacuucuTT 6539 578 187 cagaagucaagcauuucuguccc double 188 gaagucaagcauuucugucTT 189 gacagaaaugcuugacuucTT 6540 579 190 agaagucaagcauuucuguccca double 191 aagucaagcauuucuguccTT 192 ggacagaaaugcuugacuuTT 6541 692 193 ugaaaccugaagaaggcagagau double 194 aaaccugaagaaggcagagTT 195 cucugccuucuucagguuuTT 6542 693 196 gaaaccugaagaaggcagagaua double 197 aaccugaagaaggcagagaTT 198 ucucugccuucuucagguuTT 6543 694 199 aaaccugaagaaggcagagauau double 200 accugaagaaggcagagauTT 201 aucucugccuucuucagguTT 6544 695 202 aaccugaagaaggcagagauaug double 203 ccugaagaaggcagagauaTT 204 uaucucugccuucuucaggTT 6545 696 205 accugaagaaggcagagauaugg double 206 cugaagaaggcagagauauTT 207 auaucucugccuucuucagTT 6546 697 208 ccugaagaaggcagagauauggc double 209 ugaagaaggcagagauaugTT 210 cauaucucugccuucuucaTT 6547 698 211 cugaagaaggcagagauauggca double 212 gaagaaggcagagauauggTT 213 ccauaucucugccuucuucTT 6548 699 214 ugaagaaggcagagauauggcaa double 215 aagaaggcagagauauggcTT 216 gccauaucucugccuucuuTT 6549 700 217 gaagaaggcagagauauggcaaa double 218 agaaggcagagauauggcaTT 219 ugccauaucucugccuucuTT 6550 701 220 aagaaggcagagauauggcaaac double 221 gaaggcagagauauggcaaTT 222 uugccauaucucugccuucTT 6551 702 223 agaaggcagagauauggcaaaca double 224 aaggcagagauauggcaaaTT 225 uuugccauaucucugccuuTT 6552 703 226 gaaggcagagauauggcaaacag double 227 aggcagagauauggcaaacTT 228 guuugccauaucucugccuTT 6553 704 229 aaggcagagauauggcaaacagg double 230 ggcagagauauggcaaacaTT 231 uguuugccauaucucugccTT 6554 705 232 aggcagagauauggcaaacagga double 233 gcagagauauggcaaacagTT 234 cuguuugccauaucucugcTT 6555 706 235 ggcagagauauggcaaacaggau double 236 cagagauauggcaaacaggTT 237 ccuguuugccauaucucugTT 6556 707 238 gcagagauauggcaaacaggauu double 239 agagauauggcaaacaggaTT 240 uccuguuugccauaucucuTT 6557 708 241 cagagauauggcaaacaggauug double 242 gagauauggcaaacaggauTT 243

auccuguuugccauaucucTT 6558 709 244 agagauauggcaaacaggauugg double 245 agauauggcaaacaggauuTT 246 aauccuguuugccauaucuTT 6559 710 247 gagauauggcaaacaggauuggc double 248 gauauggcaaacaggauugTT 249 caauccuguuugccauaucTT 6560 711 250 agauauggcaaacaggauuggcg double 251 auauggcaaacaggauuggTT 252 ccaauccuguuugccauauTT 6561 712 253 gauauggcaaacaggauuggcgc double 254 uauggcaaacaggauuggcTT 255 gccaauccuguuugccauaTT 6562 713 256 auauggcaaacaggauuggcgcu double 257 auggcaaacaggauuggcgTT 258 cuccaauccuguuugccauTT 6563 714 259 uauggcaaacaggauuggcgcuu double 260 uggcaaacaggauuggcgcTT 261 gcgccaauccuguuugccaTT 6564 715 262 auggcaaacaggauuggcgcuuu double 263 ggcaaacaggauuggcgcuTT 264 agcgccaauccuguuugccTT 6565 716 265 uggcaaacaggauuggcgcuuuu double 266 gcaaacaggauuggcgcuuTT 267 aagcgccaauccuguuugcTT 6566 717 268 ggcaaacaggauuggcgcuuuug double 269 caaacaggauuggcgcuuuTT 270 aaagcgccaauccuguuugTT 6567 718 271 gcaaacaggauuggcgcuuuugg double 272 aaacaggauuggcgcuuuuTT 273 aaaagcgccaauccuguuuTT 6568 719 274 caaacaggauuggcgcuuuuggg double 275 aacaggauuggcgcuuuugTT 276 caaaagcgccaauccuguuTT 6569 720 277 aaacaggauuggcgcuuuugggu double 278 acaggauuggcgcuuuuggTT 279 ccaaaagcgccaauccuguTT 6570 721 280 aacaggauuggcgcuuuugggua double 281 caggauuggcgcuuuugggTT 282 cccaaaagcgccaauccugTT 6571 722 283 acaggauuggcgcuuuuggguac double 284 aggauuggcgcuuuuggguTT 285 acccaaaagcgccaauccuTT 6572 723 286 caggauuggcgcuuuuggguaca double 287 ggauuggcgcuuuuggguaTT 288 uacccaaaagcgccaauccTT 6573 724 289 aggauuggcgcuuuuggguacau double 290 gauuggcgcuuuuggguacTT 291 guacccaaaagcgccaaucTT 6574 725 292 ggauuggcgcuuuuggguacaug double 293 auuggcgcuuuuggguacaTT 294 uguacccaaaagcgccaauTT 6575 726 295 gauuggcgcuuuuggguacaugg double 296 uuggcgcuuuuggguacauTT 297 auguacccaaaagcgccaaTT 6576 727 298 auuggcgcuuuuggguacaugga double 299 uggcgcuuuuggguacaugTT 300 cauguacccaaaagcgccaTT 6577 728 301 uuggcgcuuuuggguacauggag double 302 ggcgcuuuuggguacauggTT 303 ccauguacccaaaagcgccTT 6578 729 304 uggcgcuuuuggguacauggagu double 305 gcgcuuuuggguacauggaTT 306 uccauguacccaaaagcgcTT 6579 730 307 ggcgcuuuuggguacauggagug double 308 cgcuuuuggguacauggagTT 309 cuccauguacccaaaagcgTT 6580 731 310 gcgcuuuuggguacauggagugu double 311 gcuuuuggguacauggaguTT 312 acuccauguacccaaaagcTT 6581 732 313 cgcuuuuggguacauggaguguu double 314 cuuuuggguacauggagugTT 315 cacuccauguacccaaaagTT 6582 733 316 gcuuuuggguacauggaguguuc double 317 uuuuggguacauggaguguTT 318 acacuccauguacccaaaaTT 6583 734 319 cuuuuggguacauggaguguuca double 320 uuuggguacauggaguguuTT 321 aacacuccauguacccaaaTT 6584 735 322 uuuuggguacauggaguguucag double 323 uuggguacauggaguguucTT 324 gaacacuccauguacccaaTT 6585 736 325 uuuggguacauggaguguucagc double 326 uggguacauggaguguucaTT 327 ugaacacuccauguacccaTT 6586 737 328 uuggguacauggaguguucagca double 329 ggguacauggaguguucagTT 330 cugaacacuccauguacccTT 6587 738 331 uggguacauggaguguucagcaa double 332 gguacauggaguguucagcTT 333 gcugaacacuccauguaccTT 6588 739 334 ggguacauggaguguucagcaaa double 335 guacauggaguguucagcaTT 336 ugcugaacacuccauguacTT 6589 740 337 gguacauggaguguucagcaaag double 338 uacauggaguguucagcaaTT 339 uugcugaacacuccauguaTT 6590 741 340 guacauggaguguucagcaaaga double 341 acauggaguguucagcaaaTT 342 uugcugaacacuccauguaTT 6591 742 343 uacauggaguguucagcaaagac double 344 cauggaguguucagcaaagTT 345 cuuugcugaacacuccaugTT 6592 743 346 acauggaguguucagcaaagacc double 347 auggaguguucagcaaagaTT 348 ucuuugcugaacacuccauTT 6593 744 349 cauggaguguucagcaaagacca double 350 uggaguguucagcaaagacTT 351 gucuuugcugaacacuccaTT 6594 745 352 auggaguguucagcaaagaccaa double 353 ggaguguucagcaaagaccTT 354 ggucuuugcugaacacuccTT 6595 746 355 uggaguguucagcaaagaccaaa double 356 gaguguucagcaaagaccaTT 357 uggucuuugcugaacacucTT 6596 747 358 ggaguguucagcaaagaccaaag double 359 aguguucagcaaagaccaaTT 360 uuggucuuugcugaacacuTT 6597 748 361 gaguguucagcaaagaccaaaga double 362 guguucagcaaagaccaaaTT 363 uuuggucuuugcugaacacTT 6598 749 364 aguguucagcaaagaccaaagau double 365 uguucagcaaagaccaaagTT 366 cuuuggucuuugcugaacaTT 6599 750 367 guguucagcaaagaccaaagaug double 368 guucagcaaagaccaaagaTT 369 ucuuuggucuuugcugaacTT 6600 770 370 auggagugagagagguuuuugaa double 371 ggagugagagagguuuuugTT 372 caaaaaccucucucacuccTT 6601 771 373 uggagugagagagguuuuugaaa double 374 gagugagagagguuuuugaTT 375 ucaaaaaccucucucacucTT 6602 797 376 cuacgagagcugcucugcaagcu double 377 acgagagcugcucugcaagTT 378 cuugcagagcagcucucguTT 6603 798 379 uacgagagcugcucugcaagcua double 380 cgagagcugcucugcaagcTT 381 gcuugcagagcagcucucgTT 6604 799 382 acgagagcugcucugcaagcuag double 383 gagagcugcucugcaagcuTT 384 agcuugcagagcagcucucTT 6605 800 385 cgagagcugcucugcaagcuaga double 386 agagcugcucugcaagcuaTT 387 uagcuugcagagcagcucuTT 6606 801 388 gagagcugcucugcaagcuagac double 389 gagcugcucugcaagcuagTT 390 cuagcuugcagagcagcucTT 6607 802 391 agagcugcucugcaagcuagacg double 392 agcugcucugcaagcuagaTT 393 ucuagcuugcagagcagcuTT 6608 803 394 gagcugcucugcaagcuagacgu double 395 gcugcucugcaagcuagacTT 396 gucuagcuugcagagcagcTT 6609 804 397 agcugcucugcaagcuagacgug double 398 cugcucugcaagcuagacgTT 399 cgucuagcuugcagagcagTT 6610 895 400 uugaagugcuguuuauuaaucuu double 401 gaagugcuguuuauuaaucTT 402 gauuaauaaacagcacuucTT 6611 896 403 ugaagugcuguuuauuaaucuua double 404 aagugcuguuuauuaaucuTT 405 agauuaauaaacagcacuuTT 6612 897 406 gaagugcuguuuauuaaucuuag double 407 agugcuguuuauuaaucuuTT 408 aagauuaauaaacagcacuTT 6613 898 409 aagugcuguuuauuaaucuuagu double 410 gugcuguuuauuaaucuuaTT 411 uaagauuaauaaacagcacTT 6614 899 412 agugcuguuuauuaaucuuagug double 413 ugcuguuuauuaaucuuagTT 414 cuaagauuaauaaacagcaTT 6615 900 415 gugcuguuuauuaaucuuagugu double 416 gcuguuuauuaaucuuaguTT 417 acuaagauuaauaaacagcTT 6616 901 418 ugcuguuuauuaaucuuagugua double 419 cuguuuauuaaucuuagugTT 420 cacuaagauuaauaaacagTT 6617 902 421 gcuguuuauuaaucuuaguguau double 422 uguuuauuaaucuuaguguTT 423 acacuaagauuaauaaacaTT 6618 903 424 cuguuuauuaaucuuaguguaug double 425 guuuauuaaucuuaguguaTT 426 uacacuaagauuaauaaacTT 6619 904 427 uguuuauuaaucuuaguguauga double 428 uuuauuaaucuuaguguauTT 429 auacacuaagauuaauaaaTT 6620 905 430 guuuauuaaucuuaguguaugau double 431 uuauuaaucuuaguguaugTT 432 cauacacuaagauuaauaaTT 6621 906 433 uuuauuaaucuuaguguaugauu double 434 uauuaaucuuaguguaugaTT 435 ucauacacuaagauuaauaTT 6622 907 436 uuauuaaucuuaguguaugauua double 437 auuaaucuuaguguaugauTT 438 aucauacacuaagauuaauTT 6623 908 439 uauuaaucuuaguguaugauuac double 440 uuaaucuuaguguaugauuTT 441 aaucauacacuaagauuaaTT 6624 909 442 auuaaucuuaguguaugauuacu double 443 uaaucuuaguguaugauuaTT 444 uaaucauacacuaagauuaTT 6625 910 445 uuaaucuuaguguaugauuacug double 446 aaucuuaguguaugauuacTT 447 guaaucauacacuaagauuTT 6626 911 448 uaaucuuaguguaugauuacugg double 449 aucuuaguguaugauuacuTT 450 aguaaucauacacuaagauTT 6627 912 451 aaucuuaguguaugauuacuggc double 452 ucuuaguguaugauuacugTT 453 caguaaucauacacuaagaTT 6628 913 454 aucuuaguguaugauuacuggcc double 455 cuuaguguaugauuacuggTT 456 ccaguaaucauacacuaagTT 6629 914 457 ucuuaguguaugauuacuggccu double 458 uuaguguaugauuacuggcTT 459 gccaguaaucauacacuaaTT 6630 915 460 cuuaguguaugauuacuggccuu double 461 uaguguaugauuacuggccTT 462 ggccaguaaucauacacuaTT 6631 916 463 uuaguguaugauuacuggccuuu double 464 aguguaugauuacuggccuTT 465 aggccaguaaucauacacuTT 6632 917 466 uaguguaugauuacuggccuuuu double 467 guguaugauuacuggccuuTT 468 aaggccaguaaucauacacTT 6633 918 469 aguguaugauuacuggccuuuuu double 470 uguaugauuacuggccuuuTT 471 aaaggccaguaaucauacaTT 6634 919 472 guguaugauuacuggccuuuuuc double 473 guaugauuacuggccuuuuTT 474 aaaaggccaguaaucauacTT 6635 939 475 uucauuuaucuauaauuuaccua double 476 cauuuaucuauaauuuaccTT 477 gguaaauuauagauaaaugTT 6636 940 478 ucauuuaucuauaauuuaccuaa double 479 auuuaucuauaauuuaccuTT 480 agguaaauuauagauaaauTT 6637 941 481 cauuuaucuauaauuuaccuaag double 482 uuuaucuauaauuuaccuaTT 483 uagguaaauuauagauaaaTT 6638 942 484 auuuaucuauaauuuaccuaaga double 485 uuaucuauaauuuaccuaaTT 486 uuagguaaauuauagauaaTT 6639 943 487 uuuaucuauaauuuaccuaagau double 488 uaucuauaauuuaccuaagTT 489 cuuagguaaauuauagauaTT 6640 944 490 uuaucuauaauuuaccuaagauu double 491 aucuauaauuuaccuaagaTT 492 ucuuagguaaauuauagauTT

6641 945 493 uaucuauaauuuaccuaagauua double 494 ucuauaauuuaccuaagauTT 495 aucuuagguaaauuauagaTT 6642 946 496 aucuauaauuuaccuaagauuac double 497 cuauaauuuaccuaagauuTT 498 aaucuuagguaaauuauagTT 6643 947 499 ucuauaauuuaccuaagauuaca double 500 uauaauuuaccuaagauuaTT 501 uaaucuuagguaaauuauaTT 6644 948 502 cuauaauuuaccuaagauuacaa double 503 auaauuuaccuaagauuacTT 504 guaaucuuagguaaauuauTT 6645 949 505 uauaauuuaccuaagauuacaaa double 506 uaauuuaccuaagauuacaTT 507 uguaaucuuagguaaauuaTT 6646 950 508 auaauuuaccuaagauuacaaau double 509 aauuuaccuaagauuacaaTT 510 uuguaaucuuagguaaauuTT 6647 951 511 uaauuuaccuaagauuacaaauc double 512 auuuaccuaagauuacaaaTT 513 uuuguaaucuuagguaaauTT 6648 952 514 aauuuaccuaagauuacaaauca double 515 uuuaccuaagauuacaaauTT 516 auuuguaaucuuagguaaaTT 6649 953 517 auuuaccuaagauuacaaaucag double 518 uuaccuaagauuacaaaucTT 519 gauuuguaaucuuagguaaTT 6650 954 520 uuuaccuaagauuacaaaucaga double 521 uaccuaagauuacaaaucaTT 522 ugauuuguaaucuuagguaTT 6651 975 523 agaagucaucuugcuaccaguau double 524 aagucaucuugcuaccaguTT 525 acugguagcaagaugacuuTT 6652 975 526 gaagucaucuugcuaccaguauu double 527 agucaucuugcuaccaguaTT 528 uacugguagcaagaugacuTT 6653 976 529 aagucaucuugcuaccaguauuu double 530 gucaucuugcuaccaguauTT 531 auacugguagcaagaugacTT 6654 977 532 agucaucuugcuaccaguauuua double 533 ucaucuugcuaccaguauuTT 534 aauacugguagcaagaugaTT 6655 978 535 gucaucuugcuaccaguauuuag double 536 caucuugcuaccaguauuuTT 537 aaauacugguagcaagaugTT 6656 979 538 ucaucuugcuaccaguauuuaga double 539 aucuugcuaccaguauuuaTT 540 uaaauacugguagcaagauTT 6657 980 541 caucuugcuaccaguauuuagaa double 542 ucuugcuaccaguauuuagTT 543 cuaaauacugguagcaagaTT 6658 981 544 aucuugcuaccaguauuuagaag double 545 cuugcuaccaguauuuagaTT 546 ucuaaauacugguagcaagTT 6659 982 547 ucuugcuaccaguauuuagaagc double 548 uugcuaccaguauuuagaaTT 549 uucuaaauacugguagcaaTT 6660 983 550 cuugcuaccaguauuuagaagcc double 551 ugcuaccaguauuuagaagTT 552 cuucuaaauacugguagcaTT 6661 984 553 uugcuaccaguauuuagaagcca double 554 gcuaccaguauuuagaagcTT 555 gcuucuaaauacugguagcTT 6662 985 556 ugcuaccaguauuuagaagccaa double 557 cuaccaguauuuagaagccTT 558 ggcuucuaaauacugguagTT 6663 986 559 gcuaccaguauuuagaagccaac double 560 uaccaguauuuagaagccaTT 561 uggcuucuaaauacugguaTT 6664 987 562 cuaccaguauuuagaagccaacu double 563 accaguauuuagaagccaaTT 564 uuggcuucuaaauacugguTT 6665 988 565 uaccaguauuuagaagccaacua double 566 ccaguauuuagaagccaacTT 567 guuggcuucuaaauacuggTT 6666 1131 568 cuugcuucuuucuagaaagagaa double 569 ugcuucuuucuagaaagagTT 570 cucuuucuagaaagaagcaTT 6667 1132 571 uugcuucuuucuagaaagagaaa double 572 gcuucuuucuagaaagagaTT 573 ucucuuucuagaaagaagcTT 6668 1133 574 ugcuucuuucuagaaagagaaac double 575 cuucuuucuagaaagagaaTT 576 uucucuuucuagaaagaagTT 6669 1134 577 gcuucuuucuagaaagagaaaca double 578 uucuuucuagaaagagaaaTT 579 uuucucuuucuagaaagaaTT 6670 1135 580 cuucuuucuagaaagagaaacag double 581 ucuuucuagaaagagaaacTT 582 guuucucuuucuagaaagaTT 6671 1136 583 uucuuucuagaaagagaaacagu double 584 cuuucuagaaagagaaacaTT 585 uguuucucuuucuagaaagTT 6672 1137 586 ucuuucuagaaagagaaacaguu double 587 uuucuagaaagagaaacagTT 588 cuguuucucuuucuagaaaTT 6673 1138 589 cuuucuagaaagagaaacaguug double 590 uucuagaaagagaaacaguTT 591 acuguuucucuuucuagaaTT 6674 1139 592 uuucuagaaagagaaacaguugg double 593 ucuagaaagagaaacaguuTT 594 aacuguuucucuuucuagaTT 6675 1140 595 uucuagaaagagaaacaguuggu double 596 cuagaaagagaaacaguugTT 597 caacuguuucucuuucuagTT 6676 1141 598 ucuagaaagagaaacaguuggua double 599 uagaaagagaaacaguuggTT 600 ccaacuguuucucuuucuaTT 6677 1142 601 cuagaaagagaaacaguugguaa double 602 agaaagagaaacaguugguTT 603 accaacuguuucucuuucuTT 6678 1143 604 uagaaagagaaacaguugguaac double 605 gaaagagaaacaguugguaTT 606 uaccaacuguuucucuuucTT 6679 1144 607 agaaagagaaacaguugguaacu double 608 aaagagaaacaguugguaaTT 609 uuaccaacuguuucucuuuTT 6680 1145 610 gaaagagaaacaguugguaacuu double 611 aagagaaacaguugguaacTT 612 guuaccaacuguuucucuuTT 6681 1146 613 aaagagaaacaguugguaacuuu double 614 agagaaacaguugguaacuTT 615 aguuaccaacuguuucucuTT 6682 1147 616 aagagaaacaguugguaacuuuu double 617 gagaaacaguugguaacuuTT 618 aaguuaccaacuguuucucTT 6683 1148 619 agagaaacaguugguaacuuuug double 620 agaaacaguugguaacuuuTT 621 aaaguuaccaacuguuucuTT 6684 1149 622 gagaaacaguugguaacuuuugu double 623 gaaacaguugguaacuuuuTT 624 aaaaguuaccaacuguuucTT 6685 1150 625 agaaacaguugguaacuuuugug double 626 aaacaguugguaacuuuugTT 627 caaaaguuaccaacuguuuTT 6686 1151 628 gaaacaguugguaacuuuuguga double 629 aacaguugguaacuuuuguTT 630 acaaaaguuaccaacuguuTT 6687 1152 631 aaacaguugguaacuuuugugaa double 632 acaguugguaacuuuugugTT 633 cacaaaaguuaccaacuguTT 6688 1153 634 aacaguugguaacuuuugugaau double 635 caguugguaacuuuugugaTT 636 ucacaaaaguuaccaacugTT 6689 1154 637 acaguugguaacuuuugugaauu double 638 aguugguaacuuuugugaaTT 639 uucacaaaaguuaccaacuTT 6690 1155 640 caguugguaacuuuugugaauua double 641 guugguaacuuuugugaauTT 642 auucacaaaaguuaccaacTT 6691 1156 643 aguugguaacuuuugugaauuag double 644 uugguaacuuuugugaauuTT 645 aauucacaaaaguuaccaaTT 6692 1157 646 guugguaacuuuugugaauuagg double 647 ugguaacuuuugugaauuaTT 648 uaauucacaaaaguuaccaTT 6693 1158 649 uugguaacuuuugugaauuaggc double 650 gguaacuuuugugaauuagTT 651 cuaauucacaaaaguuaccTT 6694 1159 652 ugguaacuuuugugaauuaggcu double 653 guaacuuuugugaauuaggTT 654 ccuaauucacaaaaguuacTT 6695 1160 655 gguaacuuuugugaauuaggcug double 656 uaacuuuugugaauuaggcTT 657 gccuaauucacaaaaguuaTT 6696 1161 658 guaacuuuugugaauuaggcugu double 659 aacuuuugugaauuaggcuTT 660 agccuaauucacaaaaguuTT 6697 1162 661 uaacuuuugugaauuaggcugua double 662 acuuuugugaauuaggcugTT 663 cagccuaauucacaaaaguTT 6698 1163 664 aacuuuugugaauuaggcuguaa double 665 cuuuugugaauuaggcuguTT 666 acagccuaauucacaaaagTT 6699 1164 667 acuuuugugaauuaggcuguaac double 668 uuuugugaauuaggcuguaTT 669 uacagccuaauucacaaaaTT 6700 1165 670 cuuuugugaauuaggcuguaacu double 671 uuugugaauuaggcuguaaTT 672 uuacagccuaauucacaaaTT 6701 1166 673 uuuugugaauuaggcuguaacua double 674 uugugaauuaggcuguaacTT 675 guuacagccuaauucacaaTT 6702 1167 676 uuugugaauuaggcuguaacuac double 677 ugugaauuaggcuguaacuTT 678 aguuacagccuaauucacaTT 6703 1168 679 uugugaauuaggcuguaacuacu double 680 gugaauuaggcuguaacuaTT 681 uaguuacagccuaauucacTT 6704 1169 682 ugugaauuaggcuguaacuacuu double 683 ugaauuaggcuguaacuacTT 684 guaguuacagccuaauucaTT 6705 1170 685 gugaauuaggcuguaacuacuuu double 686 gaauuaggcuguaacuacuTT 687 aguaguuacagccuaauucTT 6706 1171 688 ugaauuaggcuguaacuacuuua double 689 aauuaggcuguaacuacuuTT 690 aaguaguuacagccuaauuTT 6707 1172 691 gaauuaggcuguaacuacuuuau double 692 auuaggcuguaacuacuuuTT 693 aaaguaguuacagccuaauTT 6708 1173 694 aauuaggcuguaacuacuuuaua double 695 uuaggcuguaacuacuuuaTT 696 uaaaguaguuacagccuaaTT 6709 1174 697 auuaggcuguaacuacuuuauaa double 698 uaggcuguaacuacuuuauTT 699 auaaaguaguuacagccuaTT 6710 1175 700 uuaggcuguaacuacuuuauaac double 701 aggcuguaacuacuuuauaTT 702 uauaaaguaguuacagccuTT 6711 1176 703 uaggcuguaacuacuuuauaacu double 704 ggcuguaacuacuuuauaaTT 705 uuauaaaguaguuacagccTT 6712 1177 706 aggcuguaacuacuuuauaacua double 707 gcuguaacuacuuuauaacTT 708 guuauaaaguaguuacagcTT 6713 1178 709 ggcuguaacuacuuuauaacuaa double 710 cuguaacuacuuuauaacuTT 711 aguuauaaaguaguuacagTT 6714 1179 712 gcuguaacuacuuuauaacuaac double 713 uguaacuacuuuauaacuaTT 714 uaguuauaaaguaguuacaTT 6715 1180 715 cuguaacuacuuuauaacuaaca double 716 guaacuacuuuauaacuaaTT 717 uuaguuauaaaguaguuacTT 6716 1181 718 uguaacuacuuuauaacuaacau double 719 uaacuacuuuauaacuaacTT 720 guuaguuauaaaguaguuaTT 6717 1182 721 guaacuacuuuauaacuaacaug double 722 aacuacuuuauaacuaacaTT 723 uguuaguuauaaaguaguuTT 6718 1183 724 uaacuacuuuauaacuaacaugu double 725 acuacuuuauaacuaacauTT 726 auguuaguuauaaaguaguTT 6719 1184 727 aacuacuuuauaacuaacauguc double 728 cuacuuuauaacuaacaugTT 729 cauguuaguuauaaaguagTT 6720 1185 730 acuacuuuauaacuaacaugucc double 731 uacuuuauaacuaacauguTT 732 acauguuaguuauaaaguaTT 6721 1186 733 cuacuuuauaacuaacauguccu double 734 acuuuauaacuaacaugucTT 735 gacauguuaguuauaaaguTT 6722 1187 736 uacuuuauaacuaacauguccug double 737 cuuuauaacuaacauguccTT 738 gacauguuaguuauaaaguTT 6723 1188 739 acuuuauaacuaacauguccugc double 740 uuuauaacuaacauguccuTT 741 aggacauguuaguuauaaaTT 6724 1189 742 cuuuauaacuaacauguccugcc double 743 uuauaacuaacauguccugTT 744 caggacauguuaguuauaaTT

6725 1190 745 uuuauaacuaacauguccugccu double 746 uauaacuaacauguccugcTT 747 gcaggacauguuaguuauaTT 6726 1191 748 uuauaacuaacauguccugccua double 749 auaacuaacauguccugccTT 750 ggcaggacauguuaguuauTT 6727 1192 751 uauaacuaacauguccugccuau double 752 uaacuaacauguccugccuTT 753 aggcaggacauguuaguuaTT 6728 1193 754 auaacuaacauguccugccuauu double 755 aacuaacauguccugccuaTT 756 uaggcaggacauguuaguuTT 6729 1352 757 uggcagaguuacaguucuguggu double 758 gcagaguuacaguucugugTT 759 cacagaacuguaacucugcTT 6730 1353 760 ggcagaguuacaguucugugguu double 761 cagaguuacaguucuguggTT 762 ccacagaacuguaacucugTT 6731 1354 763 gcagaguuacaguucugugguuu double 764 agaguuacaguucugugguTT 765 accacagaacuguaacucuTT 6732 1374 766 uuucauguuaguuaccuuauagu double 767 ucauguuaguuaccuuauaTT 768 uauaagguaacuaacaugaTT 6733 1375 769 uucauguuaguuaccuuauaguu double 770 cauguuaguuaccuuauagTT 771 cuauaagguaacuaacaugTT 6734 1376 772 ucauguuaguuaccuuauaguua double 773 auguuaguuaccuuauaguTT 774 acuauaagguaacuaacauTT 6735 1377 775 cauguuaguuaccuuauaguuac double 776 uguuaguuaccuuauaguuTT 777 aacuauaagguaacuaacaTT 6736 1378 778 auguuaguuaccuuauaguuacu double 779 guuaguuaccuuauaguuaTT 780 uaacuauaagguaacuaacTT 6737 1379 781 uguuaguuaccuuauaguuacug double 782 uuaguuaccuuauaguuacTT 783 guaacuauaagguaacuaaTT 6738 1380 784 guuaguuaccuuauaguuacugu double 785 uaguuaccuuauaguuacuTT 786 aguaacuauaagguaacuaTT 6739 1381 787 uuaguuaccuuauaguuacugug double 788 aguuaccuuauaguuacugTT 789 caguaacuauaagguaacuTT 6740 1382 790 uaguuaccuuauaguuacugugu double 791 guuaccuuauaguuacuguTT 792 acaguaacuauaagguaacTT 6741 1383 793 aguuaccuuauaguuacugugua double 794 uuaccuuauaguuacugugTT 795 cacaguaacuauaagguaaTT 6742 1384 796 guuaccuuauaguuacuguguaa double 797 uaccuuauaguuacuguguTT 798 acacaguaacuauaagguaTT 6743 1385 799 uuaccuuauaguuacuguguaau double 800 accuuauaguuacuguguaTT 801 uacacaguaacuauaagguTT 6744 1386 802 uaccuuauaguuacuguguaauu double 803 ccuuauaguuacuguguaaTT 804 uuacacaguaacuauaaggTT 6745 1387 805 accuuauaguuacuguguaauua double 806 cuuauaguuacuguguaauTT 807 auuacacaguaacuauaagTT 6746 1388 808 ccuuauaguuacuguguaauuag double 809 uuauaguuacuguguaauuTT 810 aauuacacaguaacuauaaTT 6747 1389 811 cuuauaguuacuguguaauuagu double 812 uauaguuacuguguaauuaTT 813 uaauuacacaguaacuauaTT 6748 1390 814 uuauaguuacuguguaauuagug double 815 auaguuacuguguaauuagTT 816 cuaauuacacaguaacuauTT 6749 1391 817 uauaguuacuguguaauuagugc double 818 uaguuacuguguaauuaguTT 819 acuaauuacacaguaacuaTT 6750 1392 820 auaguuacuguguaauuagugcc double 821 aguuacuguguaauuagugTT 822 cacuaauuacacaguaacuTT 6751 1393 823 uaguuacuguguaauuagugcca double 824 guuacuguguaauuagugcTT 825 gcacuaauuacacaguaacTT 6752 1394 826 aguuacuguguaauuagugccac double 827 uuacuguguaauuagugccTT 828 ggcacuaauuacacaguaaTT 6753 1395 829 guuacuguguaauuagugccacu double 830 uacuguguaauuagugccaTT 831 uggcacuaauuacacaguaTT 6754 1396 832 uuacuguguaauuagugccacuu double 833 acuguguaauuagugccacTT 834 guggcacuaauuacacaguTT 6755 1397 835 uacuguguaauuagugccacuua double 836 cuguguaauuagugccacuTT 837 aguggcacuaauuacacagTT 6756 1398 838 acuguguaauuagugccacuuaa double 839 uguguaauuagugccacuuTT 840 aaguggcacuaauuacacaTT 6756 1399 841 cuguguaauuagugccacuuaau double 842 guguaauuagugccacuuaTT 843 uaaguggcacuaauuacacTT 6758 1400 844 uguguaauuagugccacuuaaug double 845 uguaauuagugccacuuaaTT 846 uuaaguggcacuaauuacaTT 6759 1401 847 guguaauuagugccacuuaaugu double 848 guaauuagugccacuuaauTT 849 auuaaguggcacuaauuacTT 6760 1402 850 uguaauuagugccacuuaaugua double 851 uaauuagugccacuuaaugTT 852 cauuaaguggcacuaauuaTT 6761 1403 853 guaauuagugccacuuaauguau double 854 aauuagugccacuuaauguTT 855 acauuaaguggcacuaauuTT 6762 1404 856 uaauuagugccacuuaauguaug double 857 auuagugccacuuaauguaTT 858 uacauuaaguggcacuaauTT 6763 1405 859 aauuagugccacuuaauguaugu double 860 uuagugccacuuaauguauTT 861 auacauuaaguggcacuaaTT 6764 1406 862 auuagugccacuuaauguauguu double 863 uagugccacuuaauguaugTT 864 cauacauuaaguggcacuaTT 6765 1407 865 uuagugccacuuaauguauguua double 866 agugccacuuaauguauguTT 867 acauacauuaaguggcacuTT 6766 1408 868 uagugccacuuaauguauguuac double 869 gugccacuuaauguauguuTT 870 aacauacauuaaguggcacTT 6767 1409 871 agugccacuuaauguauguuacc double 872 ugccacuuaauguauguuaTT 873 uaacauacauuaaguggcaTT 6768 1410 874 gugccacuuaauguauguuacca double 875 gccacuuaauguauguuacTT 876 guaacauacauuaaguggcTT 6769 1411 877 ugccacuuaauguauguuaccaa double 878 ccacuuaauguauguuaccTT 879 gguaacauacauuaaguggTT 6770 1412 880 gccacuuaauguauguuaccaaa double 881 cacuuaauguauguuaccaTT 882 ugguaacauacauuaagugTT 6771 1413 883 ccacuuaauguauguuaccaaaa double 884 acuuaauguauguuaccaaTT 885 uugguaacauacauuaaguTT 6772 1414 886 cacuuaauguauguuaccaaaaa double 887 cuuaauguauguuaccaaaTT 888 uuugguaacauacauuaagTT 6773 1415 889 acuuaauguauguuaccaaaaau double 890 uuaauguauguuaccaaaaTT 891 uuuugguaacauacauuaaTT 6774 1416 892 cuuaauguauguuaccaaaaaua double 893 uaauguauguuaccaaaaaTT 894 uuuuugguaacauacauuaTT 6775 1435 895 aauaaauauaucuaccccagacu double 896 uaaauauaucuaccccagaTT 897 ucugggguagauauauuuaTT 6776 1436 898 auaaauauaucuaccccagacua double 899 aaauauaucuaccccagacTT 900 gucugggguagauauauuuTT 6777 1437 901 uaaauauaucuaccccagacuag double 902 aauauaucuaccccagacuTT 903 agucugggguagauauauuTT 6778 1438 904 aaauauaucuaccccagacuaga double 905 auauaucuaccccagacuaTT 906 uagucugggguagauauauTT 6779 1439 907 aauauaucuaccccagacuagau double 908 uauaucuaccccagacuagTT 909 cuagucugggguagauauaTT 6780 1440 910 auauaucuaccccagacuagaug double 911 auaucuaccccagacuagaTT 912 ucuagucugggguagauauTT 6781 1441 913 uauaucuaccccagacuagaugu double 914 uaucuaccccagacuagauTT 915 aucuagucugggguagauaTT 6782 1442 916 auaucuaccccagacuagaugua double 917 aucuaccccagacuagaugTT 918 caucuagucugggguagauTT 6783 1443 919 uaucuaccccagacuagauguag double 920 ucuaccccagacuagauguTT 921 acaucuagucugggguagaTT 6784 1444 922 aucuaccccagacuagauguagu double 923 cuaccccagacuagauguaTT 924 uacaucuagucugggguagTT 6785 1445 925 ucuaccccagacuagauguagua double 926 uaccccagacuagauguagTT 927 cuacaucuagucugggguaTT 6786 1446 928 cuaccccagacuagauguaguau double 929 accccagacuagauguaguTT 930 acuacaucuagucugggguTT 6787 1447 931 uaccccagacuagauguaguauu double 932 ccccagacuagauguaguaTT 933 uacuacaucuagucuggggTT 6788 1448 934 accccagacuagauguaguauuu double 935 cccagacuagauguaguauTT 936 auacuacaucuagucugggTT 6789 1449 937 ccccagacuagauguaguauuuu double 938 ccagacuagauguaguauuTT 939 aauacuacaucuagucuggTT 6790 1450 940 cccagacuagauguaguauuuuu double 941 cagacuagauguaguauuuTT 942 aaauacuacaucuagucugTT 6791 1451 943 ccagacuagauguaguauuuuuu double 944 agacuagauguaguauuuuTT 945 aaaauacuacaucuagucuTT 6792 1452 946 cagacuagauguaguauuuuuug double 947 gacuagauguaguauuuuuTT 948 aaaaauacuacaucuagucTT 6793 1453 949 agacuagauguaguauuuuuugu double 950 acuagauguaguauuuuuuTT 951 aaaaaauacuacaucuaguTT 6794 1454 952 gacuagauguaguauuuuuugua double 953 cuagauguaguauuuuuugTT 954 caaaaaauacuacaucuagTT 6795 1455 955 acuagauguaguauuuuuuguau double 956 uagauguaguauuuuuuguTT 957 acaaaaaauacuacaucuaTT 6796 1456 958 cuagauguaguauuuuuuguaua double 959 agauguaguauuuuuuguaTT 960 uacaaaaaauacuacaucuTT 6797 1457 961 uagauguaguauuuuuuguauaa double 962 gauguaguauuuuuuguauTT 963 auacaaaaaauacuacaucTT 6798 1458 964 agauguaguauuuuuuguauaau double 965 auguaguauuuuuuguauaTT 966 uauacaaaaaauacuacauTT 6799 1459 967 gauguaguauuuuuuguauaauu double 968 uguaguauuuuuuguauaaTT 969 uuauacaaaaaauacuacaTT 6800 1460 970 auguaguauuuuuuguauaauug double 971 guaguauuuuuuguauaauTT 972 auuauacaaaaaauacuacTT 6801 1461 973 uguaguauuuuuuguauaauugg double 974 uaguauuuuuuguauaauuTT 975 aauuauacaaaaaauacuaTT 6802 1462 976 guaguauuuuuuguauaauugga double 977 aguauuuuuuguauaauugTT 978 caauuauacaaaaaauacuTT 6803 1463 979 uaguauuuuuuguauaauuggau double 980 guauuuuuuguauaauuggTT 981 ccaauuauacaaaaaauacTT 6804 1464 982 aguauuuuuuguauaauuggauu double 983 uauuuuuuguauaauuggaTT 984 uccaauuauacaaaaaauaTT 6805 1465 985 guauuuuuuguauaauuggauuu double 986 auuuuuuguauaauuggauTT 987 auccaauuauacaaaaaauTT 6806 1466 988 uauuuuuuguauaauuggauuuc double 989 uuuuuuguauaauuggauuTT 990 aauccaauuauacaaaaaaTT 6807 1467 991 auuuuuuguauaauuggauuucc double 992 uuuuuguauaauuggauuuTT 993 aaauccaauuauacaaaaaTT 6808 1468 994 uuuuuuguauaauuggauuuccu double 995 uuuuguauaauuggauuucTT 996

gaaauccaauuauacaaaaTT 6809 1469 997 uuuuuguauaauuggauuuccua double 998 uuuguauaauuggauuuccTT 999 ggaaauccaauuauacaaaTT 6810 1470 1000 uuuuguauaauuggauuuccuaa double 1001 uuguauaauuggauuuccuTT 1002 aggaaauccaauuauacaaTT 6811 1471 1003 uuuguauaauuggauuuccuaau double 1004 uguauaauuggauuuccuaTT 1005 uaggaaauccaauuauacaTT 6812 1472 1006 uuguauaauuggauuuccuaaua double 1007 guauaauuggauuuccuaaTT 1008 uuaggaaauccaauuauacTT 6813 1473 1009 uguauaauuggauuuccuaauac double 1010 uauaauuggauuuccuaauTT 1011 auuaggaaauccaauuauaTT 6814 1540 1012 guauuuggaaauaaagucagaug double 1013 auuuggaaauaaagucagaTT 1014 ucugacuuuauuuccaaauTT 6815 1541 1015 uauuuggaaauaaagucagaugg double 1016 uuuggaaauaaagucagauTT 1017 aucugacuuuauuuccaaaTT 6816 1542 1018 auuuggaaauaaagucagaugga double 1019 uuggaaauaaagucagaugTT 1020 caucugacuuuauuuccaaTT 6817 1543 1021 uuuggaaauaaagucagauggaa double 1022 uggaaauaaagucagauggTT 1023 ccaucugacuuuauuuccaTT 6818 1544 1024 uuggaaauaaagucagauggaaa double 1025 ggaaauaaagucagauggaTT 1026 uccaucugacuuuauuuccTT 6819 1545 1027 uggaaauaaagucagauggaaaa double 1028 gaaauaaagucagauggaaTT 1029 uuccaucugacuuuauuucTT 6820 1796 1030 ucccucccagaggagccaccagu double 1031 ccucccagaggagccaccaTT 1032 ugguggcuccucugggaggTT 6821 1797 1033 cccucccagaggagccaccaguu double 1034 cucccagaggagccaccagTT 1035 cugguggcuccucugggagTT 6822 1798 1036 ccucccagaggagccaccaguuc double 1037 ucccagaggagccaccaguTT 1038 acugguggcuccucugggaTT 6823 1799 1039 cucccagaggagccaccaguucu double 1040 cccagaggagccaccaguuTT 1041 aacugguggcuccucugggTT 6823 1800 1042 ucccagaggagccaccaguucuc double 1043 ccagaggagccaccaguucTT 1044 gaacugguggcuccucuggTT 6825 1801 1045 cccagaggagccaccaguucuca double 1046 cagaggagccaccaguucuTT 1047 agaacugguggcuccucugTT 6826 1843 1048 cuucucuccagcugacuaaacuu double 1049 ucucuccagcugacuaaacTT 1050 guuuagucagcuggagagaTT 6827 1869 1051 uucuguaccaguuaauuuuucca double 1052 cuguaccaguuaauuuuucTT 1053 gaaaaauuaacugguacagTT 6828 1870 1054 ucuguaccaguuaauuuuuccaa double 1055 uguaccaguuaauuuuuccTT 1056 ggaaaaauuaacugguacaTT 6829 1871 1057 cuguaccaguuaauuuuuccaac double 1058 guaccaguuaauuuuuccaTT 1059 uggaaaaauuaacugguacTT 6830 1872 1060 uguaccaguuaauuuuuccaacu double 1061 uaccaguuaauuuuuccaaTT 1062 uuggaaaaauuaacugguaTT 6831 1873 1063 guaccaguuaauuuuuccaacua double 1064 accaguuaauuuuuccaacTT 1065 guuggaaaaauuaacugguTT 6832 1874 1066 uaccaguuaauuuuuccaacuac double 1067 ccaguuaauuuuuccaacuTT 1068 aguuggaaaaauuaacuggTT 6833 1875 1069 accaguuaauuuuuccaacuacu double 1070 caguuaauuuuuccaacuaTT 1071 uaguuggaaaaauuaacugTT 6834 1897 1072 uaauagaauaaaggcaguuuucu double 1073 auagaauaaaggcaguuuUTT 1074 aaaacugccuuuauucuauTT 6835 1898 1075 aauagaauaaaggcaguuuucua double 1076 uagaauaaaggcaguuuucTT 1077 gaaaacugccuuuauucuaTT 6836 1899 1078 auagaauaaaggcaguuuucuaa double 1079 agaauaaaggcaguuuucuTT 1080 agaaaacugccuuuauucuTT ^aSingle: Single overhang design 21 mer sense, corresponding 23 mer antisense with 2 nucleotides overhang at 3' end; Double: double overhang design corresponding 19 mer sense and antisense strand each with 2 dT nucleotide overhang at 3' end ^b"Start position" corresponds to the position within the sequence of human RhoA (Genbank accession no. NM_001664) mRNA to which the 5'-most nucleotide of the sense strand corresponds for single overhang designs; for double overhang designs, the 5'-most ribonucleotide of the sense strand corresponds to (Start position +2)

[0059]Based on these results, the invention specifically provides an iRNA agent that includes a sense strand having at least 15 contiguous nucleotides of the sense strand sequences of the agents provided in Table 1 under agent numbers 6477-6836, and an antisense strand having at least 15 contiguous nucleotides of the antisense sequences of the agents provided in Table 1 under agent numbers 6477 to 6836.

[0060]The iRNA agents shown in Table 1 are composed of two strands of 19 nucleotides in length which are complementary or identical to the target sequence, plus a 3'-TT overhang. The present invention provides agents that comprise 15 contiguous nucleotides from these agents. However, while these lengths may potentially be optimal, the iRNA agents are not meant to be limited to these lengths. The skilled person is well aware that shorter or longer iRNA agents may be similarly effective, since, within certain length ranges, the efficacy is rather a function of the nucleotide sequence than strand length. For example, Yang, et al., PNAS 99:9942-9947 (2002), demonstrated similar efficacies for iRNA agents of lengths between 21 and 30 base pairs. Others have shown effective silencing of genes by iRNA agents down to a length of approx. 15 base pairs (Byrom, et al., "Inducing RNAi with siRNA Cocktails Generated by RNase III" Tech Notes 10(1), Ambion, Inc., Austin, Tex.).

[0061]Therefore, it is possible and contemplated by the instant invention to select from the sequences provided in Table 1 under agent numbers 6477 to 6836 a partial sequence of between 15 to 22 nucleotides for the generation of an iRNA agent derived from one of the sequences provided in Table 1 under agent numbers 6477 to 6836. Alternatively, one may add one or several nucleotides to one of the sequences provided in Table 1 under agent numbers 6477 to 6836, or an agent comprising 15 contiguous nucleotides from one of these agents, preferably, but not necessarily, in such a fashion that the added nucleotides are complementary to the respective sequence of the target gene, e.g., RhoA. For example, the first 15 nucleotides from one of the agents can be combined with the 8 nucleotides found 5' to these sequence in the RhoA mRNA to obtain an agent with 23 nucleotides in the sense and antisense strands. All such derived iRNA agents are included in the iRNA agents of the present invention, provided they essentially retain the ability to inhibit RhoA expression in cultured human RhoA expressing cells.

[0062]The antisense strand of an iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30, nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

[0063]The sense strand of an iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

[0064]The double stranded portion of an iRNA agent should be equal to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

[0065]Generally, the iRNA agents of the instant invention include a region of sufficient complementarity to the respective RhoA gene, and are of sufficient length in terms of nucleotides, that the iRNA agent, or a fragment thereof, can mediate down regulation of the RhoA gene. The ribonucleotide portions of the antisense strands of the iRNA agents of Table 1 under agent numbers 6477 to 6836 are fully complementary to the mRNA sequences of the RhoA gene, respectively, and ribonucleotide portion of their sense strands are fully complementary to the ribonucleotide portions of the respective antisense strands, except for the two 3'-terminal nucleotides on the antisense strand in single overhang design iRNA agents. However, it is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of a RhoA mRNA.

[0066]Therefore, the iRNA agents of the instant invention include agents comprising a sense strand and antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical, as defined below, to one of the sequences of Table 1 under agent numbers 6477 to 6836, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells, respectively. These agents will therefore possess at least 15 nucleotides identical to one of the sequences of Table 1 under agent numbers 6477 to 6836, but 1, 2 or 3 base mismatches with respect to either the target RhoA mRNA sequence or between the sense and antisense strand are introduced. Mismatches to the target RhoA mRNA sequence, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5' and/or 3' terminus, most preferably within 6, 5, 4, or 3 nucleotides of the 5'-terminus of the sense strand or the 3'-terminus of the antisense strand. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character of the molecule.

[0067]It is preferred that the sense and antisense strands be chosen such that the iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, an iRNA agent contains sense and antisense strands, preferably paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred siRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 to 4, or preferably 2 or 3 nucleotides, in length, at one or both ends of the iRNA agent. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The unpaired nucleotides forming the overhang can be ribonucleotides, or they can be deoxyribonucleotides, preferably thymidine. 5'-ends are preferably phosphorylated.

[0068]Preferred lengths for the duplexed region are between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA agent range discussed above. siRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the siRNA agent are linked, e.g., covalently linked, are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3' overhang are also within the invention.

Evaluation of Candidate iRNA Agents

[0069]A candidate iRNA agent can be evaluated for its ability to downregulate target gene expression. For example, a candidate iRNA agent can be provided, and contacted with a cell, that expresses the target gene, e.g., the RhoA gene, either endogenously or because it has been transfected with a construct from which a RhoA protein can be expressed. The level of target gene expression prior to and following contact with the candidate iRNA agent can be compared, e.g., on an mRNA or protein level. If it is determined that the amount of RNA or protein expressed from the target gene is lower following contact with the iRNA agent, then it can be concluded that the iRNA agent downregulates target gene expression. The level of target RhoA RNA or RhoA protein in the cell can be determined by any method desired. For example, the level of target RNA can be determined by Northern blot analysis, reverse transcription coupled with polymerase chain reaction (RT-PCR), or RNAse protection assay. The level of protein can be determined, for example, by Western blot analysis or immuno-fluorescence. Preferably, the assay also tests the ability of the iRNA agent to inhibit RhoA expression on a functional level, e.g. by assessing the ability of the iRNA agent to facilitate neuronal growth, e.g. the restoration of neurite outgrowth on an otherwise inhibitory substrate, e.g a substrate comprising myelin.

Stability Testing, Modification, and Retesting of iRNA Agents

[0070]A candidate iRNA agent can be evaluated with respect to stability, e.g., its susceptibility to cleavage by an endonuclease or exonuclease, such as when the iRNA agent is introduced into the body of a subject. Methods can be employed to identify sites that are susceptible to modification, particularly cleavage, e.g., cleavage by a component found in the body of a subject.

[0071]When sites susceptible to cleavage are identified, a further iRNA agent can be designed and/or synthesized wherein the potential cleavage site is made resistant to cleavage, e.g. by introduction of a 2'-modification on the site of cleavage, e.g. a 2'-O-methyl group. This further iRNA agent can be retested for stability, and this process may be iterated until an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

[0072]An iRNA agent identified as being capable of inhibiting RhoA gene expression can be tested for functionality in vivo in an animal model (e.g., in a mammal, such as in mouse or rat). For example, the iRNA agent can be administered to an animal, and the iRNA agent evaluated with respect to its biodistribution, stability, and its ability to inhibit RhoA gene expression or reduce a biological or pathological process mediated at least in part by RhoA.

[0073]The iRNA agent can be administered directly to the target tissue, e.g. the spinal cord, and, in the case of a spinal cord injury model, to the site of spinal cord injury, such as by injection. Preferably, the iRNA agent is administered to the animal model in the same manner that it would be administered to a human.

[0074]The iRNA agent can also be evaluated for its intracellular distribution. The evaluation can include determining whether the iRNA agent was taken up into the cell. The evaluation can also include determining the stability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNA agent in vivo can be facilitated by use of an iRNA agent conjugated to a traceable marker (e.g., a fluorescent marker such as fluorescein; a radioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; or antigen particles for immunohistochemistry).

[0075]The iRNA agent can be evaluated with respect to its ability to down regulate RhoA gene expression. Levels of RhoA gene expression in vivo can be measured, for example, by in situ hybridization, or by the isolation of RNA from tissue prior to and following exposure to the iRNA agent. Where the animal needs to be sacrificed in order to harvest the tissue, an untreated control animal will serve for comparison. RhoA mRNA can be detected by any desired method, including but not limited to RT-PCR, Northern blot, branched-DNA assay, or RNAase protection assay. Alternatively, or additionally, RhoA gene expression can be monitored by performing Western blot analysis on tissue extracts treated with the iRNA agent.

[0076]Animal models may be used to establish the concentration necessary to achieve a certain desired effect (e.g., EC₅₀ or ED₅₀). Such animal models may include transgenic animals that express a human gene, e.g., a gene that produces a target human RhoA RNA. In another embodiment, the composition for testing includes an iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RhoA RNA in the animal model and the target RhoA RNA in a human.

[0077]iRNA Chemistry

[0078]Described herein are isolated iRNA agents, e.g., ds RNA agents that mediate RNAi to inhibit expression of a RhoA gene.

[0079]RNA agents discussed herein include otherwise unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. Nucleic Acids Res. 22: 2183-2196, 1994. Such rare or unusual RNAs, often termed modified RNAs (apparently because they are typically the result of a post-transcriptional modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of the above are discussed herein.

[0080]Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. It may be desirable to modify one or both of the antisense and sense strands of an iRNA agent. As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most, cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. Similarly, a modification may occur on the sense strand, antisense strand, or both. In some cases, the sense and antisense strand will have the same modifications or the same class of modifications, but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it may be desirable to modify only one strand, e.g. the sense strand.

[0081]Two prime objectives for the introduction of modifications into iRNA agents is their stabilization towards degradation in biological environments and the improvement of pharmacological properties, e.g. pharmacodynamic properties, which are further discussed below. Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described in co-owned PCT Application No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can include a non-naturally occurring base, such as the bases described in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule. Exemplary features of non-naturally occurring sugars for use in iRNA agents are described in co-owned PCT Application No. PCT/US2004/11829, filed Apr. 16, 2003.

[0082]An iRNA agent can include an internucleotide linkage (e.g., the chiral phosphorothioate linkage) useful for increasing nuclease resistance. In addition, or in the alternative, an iRNA agent can include a ribose mimic for increased nuclease resistance. Exemplary internucleotide linkages and ribose mimics for increased nuclease resistance are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

[0083]An iRNA agent can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10, 2004.

[0084]An iRNA agent can have a ZXY structure, such as is described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

[0085]An iRNA agent can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with iRNA agents are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

[0086]In another embodiment, the iRNA agent can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type. iRNA agents complexed to a delivery agent are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

[0087]An iRNA agent can have non-canonical pairings, such as between the sense and antisense sequences of the iRNA duplex. Exemplary features of non-canonical iRNA agents are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

[0088]An iRNA agent, e.g., an iRNA agent that targets RhoA, can have enhanced resistance to nucleases. Naked RNA is often an easy prey for nucleolytic enzymes, such as exonucleases and endonucleases, which are omnipresent in biological media, such as the cellular cytoplasm, blood, or cerebrospinal fluid (CSF). Quick degradation can severly hamper the ability of an siRNA to inhibit the expression of a target gene. The vulnerability towards nucleolytic degradation can be greatly reduced by chemically modifying certain nucleotides of an siRNA. However, adding modifications in order to stabilize an siRNA sometimes represents a trade-off with its activity, and stabilizing modifications may even introduce toxic effects. It is therefore desirable to introduce the minimum number of modifications that still imparts the desired level of stability. Modifications in the sense strand usually have less impact on the activity of an siRNA.

[0089]In order to increase the stability of an siRNA towards nucleolytic degradation by endonucleases, it is therefore advantageous to modify only a limited number of nucleotides in particularly degradation prone positions, as described in co-owned U.S. Application No. 60/559,917, filed on May 4, 2004, co-owned U.S. Application No. 60/574,744, filed on May 27, 2004, and co-owned international application PCT/US2005/018931, filed May 27, 2005. We have determined that pyrimidine nucleotides, and specifically the 5' nucleotide in a 5'-ua-3' sequence context, a 5'-ug-3' sequence context, a 5'-ca-3' sequence context, a 5'-uu-3' sequence context, or a 5'-cc-3' sequence context are particularly prone to degradative attack, in that approximate order. Sufficiently stable and highly active siRNAs have been obtained by our laboratory when the 5'-most pyrimidines in all occurrences of the sequence contexts 5'-ua-3' and 5'-cc-3', or in all occurrences of 5'-ua-3',5'-ca-3', and 5'-uu-3', or in all occurrences of 5'-ua-3',5'-ca-3',5'-uu-3', and 5'-ug-3' were replaced by 2'-modified nucleotides, such as 2'-O-methyl nucleotides, in both strands. Alternatively, 2'-modifying all pyrimidine nucleotides in the sense strand and the 5'-most pyrimidines in all occurrences of the sequence contexts 5'-ua-3' and 5'-ca-3' in the antisense strand has given good results in terms of activity and stability. Sometimes, it has been necessary to 2'-modify all pyrimidine nucleotides in the sense strand and the 5'-most pyrimidines in all occurrences of the sequence contexts 5'-ua-3',5'-ca-3',5'-uu-3', and 5'-ug-3' in the antisense strand. The iRNA agent can include at least 2, at least 3, at least 4 or at least 5 of such dinucleotides.

[0090]Preferably, the 2'-modified nucleotides include, for example, a 2'-modified ribose unit, e.g., the 2'-hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0091]Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE and aminoalkoxy, O(CH₂)_nAMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

[0092]"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.

[0093]Preferred substitutents are 2'-methoxyethyl, 2'-OCH₃, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0094]The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An iRNA agent can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3'-3' linkage. In another alternative, the 3'-terminus can be blocked with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3'-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0095]Nucleolytic cleavage can also be inhibited by the introduction of phosphate linker modifications, e.g., phosphorothioate linkages. Thus, preferred iRNA agents include nucleotide dimers enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at a nonbridging position normally occupied by oxygen. The heteroatom can be S, Se, Nr₂, or Br₃. When the heteroatom is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Modified phosphate linkages are particularly efficient in inhibiting exonucleolytic cleavage when introduced near the 5'- or 3'-terminal positions, and preferably the 5'-terminal positions, of an iRNA agent.

[0096]5' conjugates can also inhibit 5'-3' exonucleolytic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5'-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0097]An iRNA agent can have increased resistance to nucleases when a duplexed iRNA agent includes a single-stranded nucleotide overhang on at least one end. In preferred embodiments, the nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment, the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide pair contains a purine base, and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs. In further embodiments, the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in an exemplary embodiment the nucleotide overhang is 5'-GC-3'. In preferred embodiments, the nucleotide overhang is on the 3'-end of the antisense strand. In one embodiment, the iRNA agent includes the motif 5'-CGC-3' on the 3'-end of the antisense strand, such that a 2-nt overhang 5'-GC-3' is formed.

[0098]Thus, an iRNA agent can include modifications so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or Nuclease Resistance promoting Monomers, the corresponding modifications as NRM modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC, or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

[0099]One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent.

[0100]NRM modifications include some which can be placed only at the terminus and others which can go at any position. Some NRM modifications can inhibit hybridization so it is preferable to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of a sequence which targets a subject sequence or gene, particularly on the antisense strand. They can be used anywhere in a sense strand, provided that sufficient hybridization between the two strands of the ds iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sense strand, as it can minimize off-target silencing.

[0101]In most cases, NRM modifications will be distributed differently depending on whether they are comprised on a sense or antisense strand. If on an antisense strand, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference). Cleavage of the target occurs about in the middle of a 20 or 21 nt antisense strand, or about 10 or 11 nucleotides upstream of the first nucleotide on the target mRNA which is complementary to the antisense strand. As used herein cleavage site refers to the nucleotides on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means the nucleotides within 1, 2, or 3 nucleotides of the cleavagee site, in either direction.

[0102]Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sense or antisense strand.

Tethered Ligands

[0103]The properties of an iRNA agent, including its pharmacological properties, can be influenced and tailored, for example, by the introduction of ligands, e.g. tethered ligands.

[0104]A wide variety of entities, e.g., ligands, can be tethered to an iRNA agent, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but that is only preferred, entities can be coupled at other points to an iRNA agent.

[0105]Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. The ligand or tethered ligand may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a "precursor" ligand-conjugated monomer subunit after a "precursor" ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., TAP--(CH₂)_nNH₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.

[0106]In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

[0107]Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

[0108]Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophilic molecules, lipids, lectins, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.

[0109]The ligand may be a naturally occurring or recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0110]Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a thyrotropin, melanotropin, surfactant protein A, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGD peptide mimetic.

[0111]Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. low density lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA), or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0112]The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0113]In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0114]A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[0115]In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

[0116]In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include the B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.

[0117]In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

5'-Phosphate Modifications

[0118]In preferred embodiments, iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus 5'-phosphate modifications of the antisense strand include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate ((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'), 5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)--O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-).

[0119]The sense strand can be modified in order to inactivate the sense strand and prevent formation of an active RISC, thereby potentially reducing off-target effects. This can be accomplished by a modification which prevents 5'-phosphorylation of the sense strand, e.g., by modification with a 5'-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5'-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5'-phosphate turning it into a phosphodiester linkage.

Transport of iRNA Agents into Cells

[0120]Not wishing to be bound by any theory, the chemical similarity between cholesterol-conjugated iRNA agents and certain constituents of lipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) may lead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL) in blood and/or the interaction of the iRNA agent with cellular components having an affinity for cholesterol, e.g. components of the cholesterol transport pathway. Lipoproteins as well as their constituents are taken up and processed by cells by various active and passive transport mechanisms, for example, without limitation, endocytosis of LDL-receptor bound LDL, endocytosis of oxidized or otherwise modified LDLs through interaction with Scavenger receptor A, Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver, pinocytosis, or transport of cholesterol across membranes by ABC (ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 or ABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoy facilitated uptake by cells possessing such transport mechanisms, e.g. cells of the liver. As such, the present invention provides evidence and general methods for targeting iRNA agents to cells expressing certain cell surface components, e.g. receptors, by conjugating a natural ligand for such component (e.g. cholesterol) to the iRNA agent, or by conjugating a chemical moiety (e.g. cholesterol) to the iRNA agent which associates with or binds to a natural ligand for the component (e.g. LDL, HDL).

Other Embodiments

[0121]An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.

[0122]Formulation

[0123]The iRNA agents described herein can be formulated for administration to a subject.

[0124]For ease of exposition, the formulations, compositions, and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions, and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

[0125]A formulated iRNA agent composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA agent is in an aqueous phase, e.g., in a solution that includes water.

[0126]The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA agent composition is formulated in a manner that is compatible with the intended method of administration.

[0127]An iRNA agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an iRNA agent, e.g., a protein that complexes with the iRNA agent to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0128]In one embodiment, the iRNA agent preparation includes two or more iRNA agent(s), e.g., two or more iRNA agents that can mediate RNAi with respect to the same gene, or different alleles of the gene, or with respect to different genes. Such preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different iRNA agent species. Such iRNA agents can mediate RNAi with respect to a similar number of different genes.

[0129]Where the two or more iRNA agents in such preparation target the same gene, they can have target sequences that are non-overlapping and non-adjacent, or the target sequences may be overlapping or adjacent.

[0130]Disorders Associated with RhoA Expression

[0131]An iRNA agent that targets RhoA, e.g., an iRNA agent described herein, can be used to treat a subject, e.g., a human having or at risk for developing a disease or disorder associated with RhoA gene expression or treating a subject where a biological process mediated by RhoA is unwanted. Since Nogo-L, RhoA, and Nogo-R participate in inhibiting axonal growth and elongations, the iRNA agents of the present invention are used to reverse this inhibition leading to nerve/axonal growth and elongation. Such a treatment is useful in treating injuries to the nervous system such as spinal cord injury or peripheral nerve death (caused by, e.g., Metastatic cancers of the CNS, e.g., gliomas (such as glioblastomas, astrocytomas, oligodendrogliomas, ependymomas), meningiomas, medulloblastomas, neuroblastomas, choroid plexus papillomas, sarcomas can also be treated by the iRNA agents described herein. Other indications include diseases of the central nervous system, including but not limited to encephalomyelitis, ischemic stroke, Alzheimer's Disease, spongiform encephalopathy, Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), multiple sclerosis, transverse myelitis, motor neuron disease, Guillan Bane, Anterior Spinal Artery Syndrome, and schizophrenia.

[0132]For example, an iRNA agent that targets RhoA mRNA can be used to treat a subject with a spinal cord injury or a subject having another pathological state which can be ameliorated, at least in part, by nerve growth and elongation. In such a use, an iRNA agent of the present invention is administered preferably locally at the site of nerve damage or the site at which the inhibitory effects of RhoA is desired to be reversed. Administration of the iRNA agent leads to decrease in RhoA protein resulting in reversing Nogo mediated inhibition of axonal elongation and growth.

[0133]Treatment Methods and Routes of Delivery

[0134]A composition that includes an iRNA agent, e.g., an iRNA agent that targets RhoA, can be delivered to a subject by a variety of routes to achieve either local delivery to the site of action of systemic delivery to the subject. Exemplary routes include direct injection to the site of treatment, intrathecal, parenchymal, intravenous, nasal, oral, and ocular delivery. The preferred means of administering the iRNA agents of the present invention is through direct injection or infusion to the site of treatment.

[0135]An iRNA agent can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more species of an iRNA agent and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0136]The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0137]The route of delivery can be dependent on the disorder of the patient. In general, the delivery of the iRNA agents of the present invention is done to achieve systemic delivery into the subject. One preferred means of achieving this is through parenteral administration. In a particularly preferred embodiment, the application is achieved by direct application of the pharmaceutical composition to the site of nerve injury, such as the site of spinal cord injury. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

[0138]Using the small interfering RNA vectors previously described, the invention also provides devices, systems, and methods for delivery of small interfering RNA to target locations in the nervous system and or/the brain. The envisioned route of delivery is through the use of implanted, indwelling, intrathecal or intraparenchymal catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into local nerves or local brain tissue. The proximal end of these catheters may be connected to an implanted, intrathecal or intracerebral access port surgically affixed to the patient's body or cranium, or to an implanted drug pump located in the patient's torso.

[0139]Alternatively, implantable delivery devices, such as an implantable pump may be employed. Examples of the delivery devices within the scope of the invention include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously in the body or on the cranium, and provides an access port through which therapeutic agents may be delivered to the nerves or brain. Delivery occurs through a stereotactically implanted polyurethane catheter. Two models of catheters that can function with the Model 8506 access port include the Model 8770 ventricular catheter by Medtronic, Inc., for delivery to the intracerebral ventricles, which is disclosed in U.S. Pat. No. 6,093,180, incorporated herein by reference, and the IPA1 catheter by Medtronic, Inc., for delivery to the brain tissue itself (i.e., intraparenchymal delivery), disclosed in U.S. Ser. Nos. 09/540,444 and 09/625,751, which are incorporated herein by reference. The latter catheter has multiple outlets on its distal end to deliver the therapeutic agent to multiple sites along the catheter path. In addition to the aforementioned device, the delivery of the small interfering RNA vectors in accordance with the invention can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference. Using the teachings of the invention and those of skill in the art will recognize that these and other devices and systems may be suitable for delivery of small interfering RNA vectors for the treatment of pain in accordance with the invention.

[0140]In one such embodiment, the method further comprises the steps of implanting a pump outside the body or brain, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of the at least one small interfering RNA or small interfering RNA vector through the discharge portion of the catheter. A further embodiment comprises the further step of periodically refreshing a supply of the at least one small interfering RNA or small interfering RNA vector to the pump outside said body or brain.

[0141]Thus, the invention includes the delivery of small interfering RNA vectors using an implantable pump and catheter, like that taught in U.S. Pat. Nos. 5,735,814 and 6,042,579, and further using a sensor as part of the infusion system to regulate the amount of small interfering RNA vectors delivered to the nerves or brain, like that taught in U.S. Pat. No. 5,814,014. Other devices and systems can be used in accordance with the method of the invention, for example, the devices and systems disclosed in U.S. Ser. Nos. 09/872,698 (filed Jun. 1, 2001) and 09/864,646 (filed May 23, 2001), which are incorporated herein by reference.

[0142]Preferably, the outlet of the pump or catheter is placed in close proximity of the desired site of action of the pharmaceutical composition, such as near the site of spinal cord, or other nerve, injury.

[0143]Administration can be provided by the subject or by another person, e.g., a caregiver. A caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.

[0144]The term "therapeutically effective amount" is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

[0145]The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect.

[0146]The term "pharmaceutically acceptable carrier" means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

[0147]The term "co-administration" refers to administering to a subject two or more agents, and in particular two or more iRNA agents. The agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulation and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be co-administered. In one embodiment, both Nogo-L, RhoA, and Nogo-R iRNA agents are co-administered.

[0148]The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

[0149]Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame Amino acids include alanine and glycine, with glycine being preferred.

[0150]Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

[0151]Dosage. An iRNA agent can be administered at a unit dose less than about 75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of bodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of iRNA agent per kg of bodyweight. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular, intrathecally, or directly into an organ), an inhaled dose, or a topical application.

[0152]Delivery of an iRNA agent directly to an organ (e.g., directly to the liver) can be at a dosage on the order of about 0.00001 mg to about 3 mg per organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

[0153]The dosage can be an amount effective to treat or prevent a disease or disorder.

[0154]In one embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. Because iRNA agent mediated silencing can persist for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen.

[0155]In one embodiment, a subject is administered an initial dose, and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into an siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 to 75 mg/kg of body weight per day, e.g., 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of body weight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

[0156]The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

[0157]Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.001 g to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

[0158]The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary, or topical, such as intrathecal or at the site of nerve injury. For example, topical formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning.

[0159]Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of an iRNA agent such as an siRNA used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays. For example, the subject can be monitored after administering an iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

[0160]Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient, or of drug accumulation at the site of application when delivering locally, e.g. at the site of nerve injusry, e.g. at the site of spinal cord injury. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models as described above.

[0161]The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES

[0162]Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 2.

TABLE-US-00002 TABLE 2 _Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds. Abbreviation^a Nucleotide(s) A, a 2'-deoxy-adenosine-5'-phosphate, adenosine-5'-phosphate C, c 2'-deoxy-cytidine-5'-phosphate, cytidine-5'-phosphate G, g 2'-deoxy-guanosine-5'-phosphate, guanosine-5'-phosphate T, t 2'-deoxy-thymidine-5'-phosphate, thymidine-5'-phosphate U, u 2'-deoxy-uridine-5'-phosphate, uridine-5'-phosphate N, n any 2'-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) am 2'-O-methyladenosine-5'-phosphate cm 2'-O-methylcytidine-5'-phosphate gm 2'-O-methylguanosine-5'-phosphate tm 2'-O-methyl-thymidine-5'-phosphate um 2'-O-methyluridine-5'-phosphate A, C, G, T, U, a, underlined: nucleoside-5'-phosphorothioate c, g, t, u -Chol 1-{6-[cholester-3-yloxycarbonylamino]-hexanoyl}-4-hydroxy- pyrrolidin-3-phosphorothioate diester ^acapital letters represent 2'-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)

Source of Reagents

[0163]Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Example 1

Selection of Sequences

[0164]Sequence alignment was performed to identify regions within the sequence of human RhoA mRNA with full homology to the respective sequences in both mouse and rat RhoA mRNA (human RhoA mRNA: Genbank accession no. NM_--001664; mouse RhoA mRNA: Genbank accession no. NM_--016802; rat RhoA mRNA: Genbank accession no. NM_--057132). Within the regions of homology thus identified, all possible contiguous sequences of 19 nucleotides were examined by further BLAST comparison for potential cross-reactivity of an siRNA comprising such sequence to other mRNA sequences present in humans. Only sequences with 3 or more mismatches to any other human mRNA or genomic sequence were chosen. The resulting set of 19 nt sequences is represented in the sense strand ribonucleotide sequences of the double-overhang iRNA agents given in Table 1.

[0165]In order to maximise the stability of the siRNAs for testing in biological media, particularly towards nucleolytic attack by endo- and exonucleases, the siRNAs were synthesized such that in the sense strands, all cytidine and uridine nucleotides comprise a 2'-O-methyl group, and in the antisense strand, all cytidines and uridines appearing in a sequence context of 5'-ca-3' or 5'-ua-3' comprise a 2'-O-methyl group.

[0166]To the same end, phosphorothioate linkages were introduced between 3'-terminal 5'-TT-3'-group thymidines. It has been our experience that the most active exonucleases in serum and other biological media relevant for the in vivo activity of siRNAs act by degrading siRNA strands 3'-5'. It has proven advantageous, and often sufficient, to replace the 2 penultimate nucleotides in the antisense strand by 2'-O-methyl-5'-phosphorothioate-modified nucleotides (e.g. the nucleotides in positions 21 and 22, counting 5' to 3', of a 23-nucleotide antisense strand); sometimes it is sufficient to modify only the penultimate nucleotide, or to use only 5'-phosphorothioate-modified nucleotides, or both. The sense strand may be protected in a similar fashion, and/or it may be 3'-conjugated to a tethered ligand via a phosphodiester or a phosphorothioate diester.

[0167]In addition to the sequences selected as described above, four siRNAs were synthesized which corresponded to four of those utilized by the authors of Ahmed, Z., et al, Mol Cell Neurosci. 2005, 28:509-23. AL-DP-5850 corresponds to RHO-A1 of Ahmed et al., supra, AL-DP-5851 to RHO-A2, AL-DP-5852 to RHO-A5 and AL-DP-5853 to RHO-A4 of Ahmed et al., supra.

Example 2

siRNA Synthesis

[0168]Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Glen Research, Sterling Va.) as solid support. RNA and RNA containing 2'-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2'-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

[0169]Deprotection and purification by anion exchange HPLC of the crude oligoribonucleotides were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The purified RNA solution was stored at -20° C. until use.

[0170]As a result of the synthesis strategy described above, all oligonucleotides synthesized as described above do not comprise a phosphate group on their 5'-most nucleotide.

[0171]Cholesterol was 3'-conjugated to siRNA as illustrated in FIG. 1. For the synthesis of these 3'-cholesterol-conjugated siRNAs, an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

##STR00001##

[0173]A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until the completion of reaction was ascertained by TLC (19 h). After 19 h which it was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl- ]-amino}-propionic acid ethyl ester AB

##STR00002##

[0175]Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. the completion of the reaction was ascertained by TLC. The reaction mixture was concentrated in vacuum and to the ethylacetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC

##STR00003##

[0177]3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hex- anoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated in vacuum and the residue water was added and the product was extracted with ethyl acetate. The crude product was purified by converting into hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1- 5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h- exanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

##STR00004##

[0179]The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15- ,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-he- xanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

##STR00005##

[0181]Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stiffing within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to a residue. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1- 7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF

##STR00006##

[0183]Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated in vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1- -yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1- 7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG

##STR00007##

[0185]Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated in vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimet- hyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH

##STR00008##

[0187]Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

##STR00009##

[0189]Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mm/g) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement (37 mM/g).

[0190]The synthesis and structure of cholesterol conjugated RNA strands is illustrated in FIG. 1.

[0191]The siRNAs listed Table 3 were synthesized for activity screening.

TABLE-US-00003 TABLE 3 siRNAs specific for RhoA Agent SEQ SEQ C. a. number Sense strand ID NO. Antisense strand ID NO. #¹ AL-DP-5850 gauuaugaccgucugaggcTT 1081 gccucagucggucauaaucTT 1082 n.a. AL-DP-5851 ggaucuucggaaugaugagTT 1083 cucaucauuccgaagauccTT 1084 n.a. AL-DP-5852 agaccaaagacggagugagTT 1085 cucacuccgucuuuggucuTT 1086 n.a. AL-DP-5853 ugaagcaggagccgguaaaTT 1087 uuuaccggcuccugcuucaTT 1088 n.a. AL-DP-5972 gcmumacmcmagumaumumumagaagcmTT 1089 gcuucumaaaumacuggumagcTT 1090 6661 AL-DP-5973 cmumacmcmagumaumumumagaagcmcmTT 1091 ggcuucumaaaumacuggumagTT 1092 6662 AL-DP-5974 gcmumgumaacmumacmumumumaumaacmTT 1093 guumaumaaagumaguumacmagcTT 0194 6712 AL-DP-5975 gumumacmumgumgumaaumumagumgcmTT 1095 gcmacumaauumacmacmagumaacTT 1096 6751 AL-DP-5976 cmcmacmumumaaumgumaumgumumacmcmTT 1097 ggumaacmaumacmauumaaguggTT 1098 6769 AL-DP-5977 cmagcmcmcmumgaumagumumumagaaTT 1099 uucumaaacumaucmagggcugTT 1100 6521 AL-DP-5978 gcmcmcmumgaumagumumumagaaaaTT 1101 uuuucumaaacumaucmagggcTT 1102 6523 AL-DP-5979 cmcmcmumgaumagumumumagaaaacmTT 1103 guuuucumaaacumaucmagggTT 1104 6524 AL-DP-5980 gaumagumumumagaaaacmaumcmcmTT 1105 ggauguuuucumaaacumaucTT 1106 6528 AL-DP-5981 umagumumumagaaaacmaumcmcmcmaTT 1107 ugggauguuuucumaaacumaTT 1108 6530 AL-DP-5982 cmagacmumagaumgumagumaumumumTT 1109 aaaumacumacmaucumagucugTT 1110 6790 AL-DP-5983 cmcmcmcmagacm8maga8mg8mag8maTT 1111 umacumacmaucumagucuggggTT 1112 6787 AL-DP-5984 cmcmagacmumagaumgumagumaumumTT 1113 aaumacumacmaucumagucuggTT 1114 6789 AL-DP-5985 cmcmcmagacmumagaumgumagumaumTT 1115 aumacumacmaucumagucugggTT 1116 6788 AL-DP-5986 umgcmcmacmumumaaumgumaumgumumaTT 1117 umaacmaumacmauumaaguggcmaTT 1118 6767 AL-DP-5987 umgcmumgumumumaumumaaumcmumumagTT 1119 cumaagauumaaumaaacmagcmaTT 1120 6614 AL-DP-5988 umcmaumgumumagumumacmcmumumaumaTT 1121 umaumaaggumaacumaacmaugaTT 1122 6732 AL-DP-5989 cmcmagumumaaumumumumumcmcmaacmumTT 1123 aguuggaaaauumaacuggTT 1124 6832 AL-DP-5990 umacmcmumaagaumumacmaaaumcmaTT 1125 ugauuugumaaucuugammumaTT 1126 6650 AL-DP-5991 umcmumumgcmumacmcmagumaumumumagTT 1127 cumaaaumacuggumagcmaagaTT 1128 6657 AL-DP-5992 umgumgumaaumumagumgcmcmacmumumTT 1129 aaguggcmacumaauumacmacmaTT 1130 6756 AL-DP-5993 aumcmumumgcmumacmcmagumaumumumaTT 1131 umaaaumacuggumagcmaagauTT 1132 6656 AL-DP-5994 cmumgumaacmumacmumumumaumaacmumTT 1133 aguumaumaaagumaguumacmagTT 1134 6713 AL-DP-5995 gumgaaumumaggcmumgumaacmumaTT 1135 umaguumacmagccumaauucmacTT 1136 6703 AL-DP-6176 cmumacmcmagumaumumumagaagcmcmTT-Chol 1137 ggcuucumaaaumacuggumagTT 1138 6662 AL-DP-6177 umgcmumgumumumaumumaaumcmumumagTT-Chol 1139 cumaagauumaaumaaacmagcmaTT 1140 6614 ¹C. a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 3 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2'-O-methyl modifications and phosphorothioate linkages, are disregarded

Example 3

siRNA Activity Testing

[0192]The ability of the iRNA agents represented in Table 3 to inhibit the expression of human RhoA was tested in human cell lines expressing the respective gene product from an expression construct, or in cell lines constitutively expressing the respective gene product. The iRNA agent is transfected into the cells, e.g., by transfection or electroporation, allowed to act on the cells for a certain time, e.g., 24 hours, and levels of RhoA expression were determined by measurement of RhoA mRNA concentrations in cell lysates. These expression levels were then compared to RhoA expression levels in cells treated equivalently but without addition of the iRNA agent, or to expression levels of housekeeping genes (e.g. GAPDH), and the ability of the iRNA agents represented in Table 3 to inhibit the expression of human RhoA thereby assessed.

Screening for Inhibition of RhoA Expression

[0193]One day before transfection, Neuroscreen-1 cells (Cellomics Inc., Pittsburgh, USA) were seeded at 1.5×10⁴ cells/well on 96-well collagen-coated plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl of growth medium (RPMI 1640, 10% horse serum, 5% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, 2 mM L-glutamine, Biochrom AG, Berlin, Germany). Transfections were performed in triplicates. For each well 0.5 μl Lipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. 2 μl of a 5 μM solution of siRNA in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride) were mixed with 10.5 μl Opti-MEM per well, combined with the Lipofectamine-2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. During this incubation, growth medium was removed from cells and replaced by 75 μl/well of fresh medium. The 25 μl solution of siRNA-Lipofectamine-2000-complex were added, resulting in an overall 100 nM siRNA concentration in the 100 μl incubation volume, and the cells were incubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau).

[0194]mRNA levels in cell lysates were quantitated by a commercially available branched DNA hybridization assay (QuantiGene bDNA-kit, Genospectra, Fremont, USA). Cells were harvested by applying 50 μl additional growth medium and 75 μl of Lysis Mixture (from QuantiGene bDNA-kit) to each well and were lysed at 53° C. for 30 min 50 μl of the lysates were incubated with probes specific to rat RhoA and rGAPDH (sequence of probes given in Table 4 and Table 5) according to the manufacturer's protocol for the QuantiGene bDNA kit assay. Finally, chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with RhoA probes were normalized to the respective GAPDH values for each well. Mock transfected cells (following the same protocol except that no siRNA was added) served as controls and forcomparison of mRNA levels.

[0195]Effective siRNAs from the screen were further characterized by establishment of dose response curves and calculation of IC₅₀ concentrations (the concentration at which 50% inhibition of gene expression would be observed). For dose response assessment, transfections were performed at the following concentrations: 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, 137 pM, 46 pM, 15 pM, 5 pM and mock (no siRNA) by serially diluting the 5 μM siRNA stock solution with annealing buffer and using 2 μl of the diluted stock according to the above protocol. The IC₅₀ was determined by curve fitting using the computer software X1fit using the following parameters: Dose Response One Site, 4 Parameter Logistic Model, fit=(A+((B-A)/(1+4(10 C)/x) D)))), inv=((10 C)/(((((B-A)/(y-A))-1) (1/D))), res=(y-fit).

TABLE-US-00004 TABLE 4 Rat RhoA probes Probe SEQ type¹ Nucleotide sequence ID NO. CE CCATTTTTCTGGGATGTTTTCTAAATTTTT 1141 CTCTTGGAAAGAAAGT CE ACAGAAATGCTTGACTTCTGGAGTTTTTTC 1142 TCTTGGAAAGAAAGT CE CTTCAGGTTTTACCGGCTCCTTTTTCTCTT 1143 GGAAAGAAAGT CE CTGTTTGCCATATCTCTGCCTTTTTTTCTC 1144 TTGGAAAGAAAGT CE TTGGTCTTTGCTGAACACTCCATTTTTCTC 1145 TTGGAAAGAAAGT CE CCCGCGTCTAGCTTGCAGATTTTTCTCTTG 1146 GAAAGAAAGT LE AGGATGATGGGCACATTTGGTTTTTAGGCA 1147 TAGGACCCGTGTCT LE GCCTTGTGTGCTCATCATTCCTTTTTAGGC 1148 ATAGGACCCGTGTCT LE TGCTTCATTTTGGCTAACTCCCTTTTTAGG 1149 CATAGGACCCGTGTCT LE TGTACCCAAAAGCGCCAATCTTTTTAGGCA 1150 TAGGACCCGTGTCT LE GCAGCTCTCGTGGCCATCTTTTTTAGGCAT 1151 AGGACCCGTGTCT LE AGGCACCCCGACTTTTTCTTTTTTTAGGCA 1152 TAGGACCCGTGTCT BL CTATCAGGGCTGTCGATGGAA 1153 BL GAAGATCCTTCTTGTTCCCAACT 1154 BL CAAAAACCTCTCTCACTCCGTCT 1155 ¹CE = Capture Extender probe; LE = Label Extender probe; BL = blocking probe

TABLE-US-00005 TABLE 5 Rat GAPDH probes Probe SEQ type¹ Nucleotide sequence ID NO. CE CCAGCTTCCCATTCTCAGCCTTTTTCTCTTG 1156 GAAAGAAAGT CE TCTCGCTCCTGGAAGATGGTTTTTTCTCTTG 1157 GAAAGAAAGT CE CCCATTTGATGTTAGCGGGATTTTTCTCTTG 1158 GAAAGAAAGT CE CGGAGATGATGACCCTTTTGGTTTTTCTCTT 1159 GGAAAGAAAGT LE GATGGGTTTCCCGTTGATGATTTTTAGGCAT 1160 AGGACCCGTGTCT LE GACATACTCAGCACCAGCATCACTTTTTAGG 1161 CATAGGACCCGTGTCT LE CCCAGCCTTCTCCATGGTGGTTTTTAGGCAT 1162 AGGACCCGTGTCT BL TTGACTGTGCCGTTGAACTTG 1163 BL CCCCACCCTTCAGGTGAGC 1164 BL GGCATCAGCGGAAGGGG 1165 ¹CE = Capture Extender probe; LE = Label Extender probe; BL = blocking probe

[0196]Table 6 lists the agent number, the position of the nucleotide within the human RhoA mRNA sequence (Genbank accession number NM_--001664) corresponding to the 5'-most nucleotide of the sense strand of the agent, the amount of total RhoA mRNA remaining in cells treated with the agent at 100 nM concentration in % of controls, and the IC₅₀ value for selected agents.

TABLE-US-00006 TABLE 6 Ability of siRNAs specific for RhoA to reduce RhoA mRNA levels in cultured cells Rem. RhoA Rem. RhoA mRNA at 100 nM mRNA at 100 nM IC₅₀ Agent Pos. in agent, first agent, second RhoA number mRNA¹ screen screen [nM] AL-DP-5850 73 ± 8% 142 AL-DP-5852 11 ± 4% 3.1 AL-DP-5853 18 ± 3% 2.8 AL-DP-5854 17 ± 1% 4.2 AL-DP-5972 986 30 ± 9% 17 ± 2% AL-DP-5973 987 21 ± 2% 15 ± 1% 0.003 AL-DP-5974 1179 44 ± 12% 48 ± 2% AL-DP-5975 1395 33 ± 4% 27 ± 10% AL-DP-5976 1413 26 ± 3% 17 ± 2% AL-DP-5977 537 n.d. 30 ± 1% AL-DP-5978 539 58 ± 4% 51 ± 1% AL-DP-5979 540 12 ± 2% 15 ± 2% 0.06 AL-DP-5980 544 75 ± 3% 95 ± 3% AL-DP-5981 546 17 ± 2% 16 ± 1% 0.13 AL-DP-5982 1452 18 ± 2% 22 ± 2% 0.13 AL-DP-5983 1449 37 ± 4% 29 ± 3% AL-DP-5984 1451 26 ± 1% 33 ± 4% AL-DP-5985 1450 n.d. 33 ± 1% 0.37 AL-DP-5986 1411 18 ± 1% 22 ± 1% 0.4 AL-DP-5987 901 22 ± 5% 10 ± 0% 0.01 AL-DP-5988 1376 17 ± 1% 16 ± 1% 0.34 AL-DP-5989 1876 20 ± 1% 25 ± 3% 3.1 AL-DP-5990 956 16 ± 2% 17 ± 1% 0.36 AL-DP-5991 982 55 ± 5% 33 ± 5% AL-DP-5992 1400 55 ± 6% 55 ± 6% AL-DP-5993 981 32 ± 2% 33 ± 3% AL-DP-5994 1180 23 ± 2% 20 ± 1% 0.24 AL-DP-5995 1170 25 ± 2% 26 ± 2% 6.0 AL-DP-6176 987 14 ± 2% 1.17 AL-DP-6177 901 19 ± 5% 0.005 ¹Position of nucleotide within human Nogo-R mRNA corresponding to the 5'-most nucleotide of the sense strand of the agent

[0197]In summary, agents AL-DP-5979, AL-DP-5990, AL-DP-5988, AL-DP-5981, AL-DP-5982, AL-DP-5986, AL-DP-5989 AL-DP-6176, and AL-DP-6177 were able to reduce the expression of RhoA mRNA by 80% or more, AL-DP-5973, AL-DP-5987, AL-DP-5994, AL-DP-5995, AL-DP-5976, AL-DP-5984, and AL-DP-5972 were able to reduce the expression of RhoA mRNA by 70% or more, AL-DP-5993, AL-DP-5975, and AL-DP-5983 were able to reduce the expression of RhoA mRNA by 60% or more, AL-DP-5974 was able to reduce the expression of RhoA mRNA by 50% or more, and AL-DP-5991, AL-DP-5992, and AL-DP-5978 were able to reduce the expression of RhoA mRNA by 40% or more. The high activity of AL-DP-6176 and AL-DP-6177 shwos that a cholesteryl moiety may be conjugated to the 3'-end of the sense strand of an siRNA without significant loss of activity. AL-DP-6176 and AL-DP-6177 are identical to AL-DP-5973 and AL-DP-5987, respectively, except for the 3'-conjugated cholesteryl moiety on the sense strand.

Example 4

Stability Testing

[0198]In order to verify the stability of siRNAs in the biological matrix most relevant to their intended physiological application, cerebrospinal fluid (CSF), we established a method for determining the degradation half life of siRNAs in this medium. This method comprises the incubation of siRNAs with CSF followed by Proteinase K treatment of the CSF sample and the separation of CSF sample constituents on an HPLC.

[0199]The example below shows the analyses of CSF samples which were contacted with siRNAs in vitro. However, this method can equally be applied to biological samples ex vivo, i.e. obtained from a subject which was contacted with an siRNA in vivo.

[0200]Bovine CSF was obtained from a calf (Bos bovis), age 6 months (Prof. Dr. J. Rehage, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany). Porcine CSF was pooled from 3 healthy weaner pigs (Sus scrofa domesticus), age 3-4 months (Prof. Dr. M. Wendt, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany). Rat CSF was pooled from 20 male Sprague Dawley rats (Rattus norvegicus), 175-200 g in weight (Charles River Laboratories, L'Arbresle Cedex, France). Proteinase K (20 mg/ml) was obtained from peQLab (Erlangen, Germany; Cat.-No. 04-1075) and diluted 1:1 with deionized water (18.2 mΩ) to a final concentration of 10 mg/ml Proteinase K. Proteinase K Buffer (4.0 ml TRIS-HCl 1M pH 7.5, 1.0 ml EDTA 0.5M, 1.2 ml NaC15M, 4.0 ml SDS 10%) was prepared fresh and kept at 50° C. until use to avoid precipitation.

[0201]A 40 mer of poly(L-dT), (L-dT)₄₀ was obtained from Noxxon Pharma AG (Berlin, Germany) and used as an internal standard. Polymers of the L-enantiomers of nucleic acids show an extraordinary stability towards nucleolytic degradation (Klussman S, et al., Nature Biotechn. 1996, 14:1112) but otherwise very similar properties when compared to naturally occurring nucleic acids consisting of R-enantiomers.

Proteinase K Treatment of siRNA Incubation Samples 6 μl of a 50 μM solution of the respective siRNA in phosphate buffered saline (PBS, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was incubated with 54 μl CSF at 37° C. for 30 min, 1, 2, 4, 8, 16, 24 or 48 hours. To terminate the siRNA-degradation, 25 μl of Proteinase K buffer were added to incubation samples immediately after expiry of the respective incubation period, the mixture vortexed at highest speed for 5 s (Vortex Genie 2, Scientific Industries, Inc., Bohemia, N.Y., USA, cat. no. SI 0256), 8 μl Proteinase K (10 mg/ml) were added followed by vortexing for 5 s, and finally the mixture was incubated for 20 min in a thermomixer at 42° C. and 1050 rpm.

[0202]5 μl of a 50 μM solution (250 pmole) of (L-dT)₄₀ were added as an internal standard to each well, the solution was vortexed for 5 s, and the tube centrifuged for 1 min in a tabletop centrifuge to collect all droplets clinging to the inner surfaces of the wells at the bottom. The solution was transferred to a 96 well Captiva 0.2 μm filter plate (Varian, Germany, Cat. No. A5960002) and filtered by centrifugation at 21900 rcf for 45 min.

[0203]The incubation wells were washed with 47.5 μl deionized water (18.2 mΩ), the wash filtered through the Captiva Filter Unit at 21900 rcf for 15 min, and the wash step repeated. Approximately 180 μl of the theoretical total volume of 200 μl are on average recovered after the second washing step.

Ion exchange chromatographic separation of siRNA single strands from each other and from degradation products:

[0204]A Dionex BioLC HPLC-system equipped with inline-degasser, autosampler, column oven and fixed wavelength UV-detector (Dionex GmbH, Idstein, Germany) was used under denaturing conditions. Standard run parameters were:

TABLE-US-00007 Column: Dionex DNA-Pac100; 4 × 250 mm Temperature: 75° C. Eluent A: 10 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile, pH = 8.0 Eluent B: 800 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile, pH = 8.0 Detection: @ 260 nm Gradient: 0-1 min: 10% B 1-11 min: 10% -> 35% B 11-12 min: 35% B -> 100% B 12-14 min: 100% B -> 10% B 14-16 min: 10% B for column reequilibration Injection volume: 20 μl

[0205]Where separation between the two strands of an siRNA was not satisfactory or a degradation fragment co-eluted with one strand, the chromatographic parameters were adjusted by changing temperature, pH, replacement of NaClO₄ by NaBr, the concentration of acetonitrile, and/or adjusting the slope of the eluent gradient until separation was achieved which allowed separate quantitation of the peaks from sense and antisense strand.

[0206]Peak areas for full length strands were obtained by integration of the UV detector signal using software supplied by the manufacturer of the instrument (Chromeleon 6.6; Dionex GmbH, Idstein, Germany).

Data Analysis:

[0207]Integrated sense strand, antisense strand, and internal standard peak areas were obtained for all samples and the normalization control.

[0208]A correction factor CF, accounting for liquid losses in the filtration and washing steps, was determined for every sample by calculating the ratio of experimental to theoretical internal standard peak area. The theoretical internal standard peak area is obtained, e.g. from a calibration curve of the internal standard obtained by injecting 50 μl each of a serial dilution of the 50 μM solution of (L-dT)₄₀ onto the HPLC column, and calculation of the theoretical peak area corresponding to 25 pmole (L-dT)₄₀ with the equation obtained by linear least square fit to the peak areas from the dilution series. The correction factor CF to be applied to the peak areas of the sense and antisense strand is the obtained as:

CF=PeakArea.sub.intStd(theoretical)/PeakArea.sub.intStd(Sample)

[0209]This treatment assumes that, by virtue of washing the filter twice, virtually complete recovery is achieved in the combined filtrates, and corrects for the variable volume of wash water retained in the filter, such that peak areas from different samples can be compared.

[0210]The peak areas obtained for the sense and antisense strand peaks for each time point are then multiplied with the correction factor CF to obtain Normalized Peak Areas (NPA_sense,t, NPA.sub.antisense,t):

NPA_sense or antisense,t=(Peak Area_sense or antisense,t)×CF

[0211]To obtain the relative amount of remaining Full Length Product (% FLP) for the sense and antisense strands at time t, the Normalized Peak Area for each strand at time t=0 min (NPA_sense,t=0, NPA.sub.antisense,t=0) is set as 100%, and the NPAs from other time points are divided by these values.

% FLP_t=1,2,3 . . . n=(NPA_t=1,2,3 . . . n/NPA_t=0)*100

[0212]The value obtained from the control sample, where the siRNA was incubated with annealing buffer only, may serve as a control of the accuracy of the method. The % FLP for both strands should lie near 100%, within error margins, regardless of time of incubation.

[0213]The degradation half life t₁/2 may then be calculated for each strand, assuming first order kinetics, from the slope of a linear least square fit to a plot of ln(% FLP) versus time as:

t₁/2=ln(0,5)/slope

Stability of siRNAs specific for NogoL and RhoA in rat, bovine and porcine CSF

[0214]Table 7 shows the results for select siRNAs of the determination of the relative amount of full length dsRNA present in porcine, rat, and bovine CSF, and PBS, after 48 h of incubation in the respective medium. In addition, the degradation half life was determined for the sense and antisense strands separately for some siRNAs.

TABLE-US-00008 TABLE 7 Stability of various siRNAs specific for NogoL and RhoA in rat, bovine and porcine CSF % full length duplex present after 48 h in Agent Porcine Rat Bovine Specific Modifi- C.a. number CSF CSF CSF PBS for cation¹ #² AL-DP-5973 95 3 95 100 RhoA 3/TTs 6662 AL-DP-5979 99 108 RhoA 3/TTs 6524 AL-DP-5981 96 103 RhoA 3/TTs 6530 AL-DP-5982 56 98 RhoA 3/TTs 6790 AL-DP-5986 100 105 RhoA 3/TTs 6767 AL-DP-5987 87 97 RhoA 3/TTs 6614 AL-DP-5988 41 99 RhoA 3/TTs 6732 AL-DP-5989 87 101 RhoA 3/TTs 6832 AL-DP-5990 76 92 RhoA 3/TTs 6650 ¹⁰ = no 2'-modifications; 1 = 5'-nucleotide in 5'-ua-3', 5'-uu-3', 5'-ca-3', and 5'-ug-3' motifs is 2'-modified in sense strand, 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in antisense strand; 2 = 5'-nucleotide in 5'-ua-3', 5'-uu-3', 5'-ca-3', and 5'-ug-3' motifs is 2'-modified in sense and antisense strand, 3 = all pyrmidine nucleotides are 2'-modified in sense strand, 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in antisense strand; 4 = all pyrimidine nucleotides are 2'-modified in sense strand, 5'-nucleotide in 5'-ua-3', 5'-uu-3', 5'-ca-3', and 5'-ug-3' motifs is 2'-modified in antisense strand; 5 = all pyrimidine nucleotides are 2'-modified in sense strand, no 2'-modifications in antisense strand; TT = 21 nucleotides and 3'-terminal TT single strand overhangs in sense and antisense strands; TTs = 21 nucleotides and 3'-terminal TT single strand overhangs in sense and antisense strands; 23 = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3'-end of antisense strand; 23s = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3'-end of antisense strand, nucleotides comprise 5'-phosphorothioate groups in positions 21 and 22 of antisense strand ²C.a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 2 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2'-O-methyl modifications and phosphorothioate linkages, are disregarded

[0215]As is evident from Table 7, the modification of siRNAs in select sites vulnerable to degradation can lead to agents with excellent properties in terms of activity and stability. For example, AL-DP-5871, AL-DP-5938, AL-DP-5963, AL-DP-5973, AL-DP-5979, AL-DP-5981, AL-DP-5986, AL-DP-5987, AL-DP-5989, and AL-DP-5990 all inhibit their respective target gene by more than 70% in the in vitro assays described above, and more than 70% full length duplex remain after incubation with porcine CSF for 48 h. However, there is some indication that rat CSF is more aggressive towards siRNAs than porcine or bovine CSF.

Example 5

Inhibition of RhoA Expression in Rat Primary Dorsal Root Ganglia (DRG) Cells in Culture

[0216]The inhibition of RhoA expression was assessed in in rat primary dorsal root ganglia (DRG) cells in culture in order to validate results obtained using Neuroscreen 1 cells as described above.

[0217]DRG cells were isolated from Sprague-Dawley rats at postnatal day 3 to 6. Rats were dissected and cells dissociated into single cells by addition of 1.3 ml (0.28 Wunsch units/ml) Liberase Blendzyme (Roche) in S-MEM (Invitrogen Gibco, Carlsbad Calif., USA) and incubated for 35 min at 37° C. The cell suspension was pre-plated on tissue-culture plates to remove non-neuronal cells. Neurons were then plated onto tissue-culture Biocoat® PDL Poly-D-Lysine/Laminin 96 well plates (BD Biosciences, Bedford Mass., USA) in F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5% fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heat inactivated) (both Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp., Madison Wis., USA) and kept at 37° C., 5% CO₂ in a humidified incubator until transfection.

[0218]A rhoA-specific siRNA, agent number AL-DP-5987, was tested in DRG cultures at 200 nM concentration using TransMessenger® Transfection reagent (Qiagen GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid formulation, specific RNA-condensing reagent (Enhancer R®) and an RNA-condensing buffer (Buffer EC-R®) keeping siRNA:Enhancer R® ratio (μg:μl) constant at 1:2, and siRNA:TransMessenger® ratio (μg:μl) constant at 1:12.

[0219]DRG neurons were transfected 24 h post-plating. For each well 0.52 μl Enhancer R® were first mixed with 13.68 μl Buffer EC-R®. 0.8 μl of a 25 μM solution of AL-DP-5987 (0.26 μg) in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), or 0.8 μl of annealing buffer (siRNA-free control) were added and the mixture incubated for 5 min at RT. 3.12 μl TransMesssenger® Transfection Reagent were diluted with 6.88 μl Buffer EC-R®, added to the mixture, and the mixture incubated for another 10 min at room temperature to allow transfection-complex formation. 75 μl serum free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad Calif., USA) were added to the transfection complexes and complete mixing achieved by gently pipetting up and down. The growth medium was removed from the DRG cells, and 90 μl of the above transfection complex mixture were added onto the cells. After 8 h of incubation at 37° C., 5% CO₂ in a humidified incubator supernatant was removed from the cells, fresh F12-HAM's medium containing glutamine supplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA) was added, the cells were incubated for another 16 h at 37° C., 5% CO₂ in a humidified incubator, and rhoA mRNA was quantified.

[0220]RhoA mRNA levels were measured using the QuantiGene® bDNA kit (Genospectra, Fremont, USA) according to manufacturer's protocol. Briefly, the supernatant was removed from the DRG cells, and the cells were lysed by addition of 150 μl of Lysis Working Reagent (1 volume of Lysis Mixture plus 2 volumes of medium) and incubation at 52° C. for 30 min 40 μl of the lysates were incubated at 52° C. for 40 min with the probe sets specific to rat RhoA and rat GAPDH given above in Table 4 and Table 5. Chemoluminescence was read on a Victor²-Light® (PerkinElmer Life And Analytical Sciences, Inc., Boston Mass., USA) as Relative Light Units (RLU). RLU for RhoA were normalized to GAPDH RLU for each well. Normalized RhoA/GAPDH ratios were then compared to the siRNA-free control, which was set as 100%.

[0221]In several independent experiments, rhoA mRNA was reduced in primary DRG cells treated with AL-DP-5987 in culture consistently to 20-25% of rhoA mRNA levels found in the siRNA free controls.

Sequence CWU 1

1165123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1ccggaagaaa cuggugauug uug 23221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 2ggaagaaacu ggugauugut t 21321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 3acaaucacca guuucuucct t 21423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 4cggaagaaac uggugauugu ugg 23521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 5gaagaaacug gugauuguut t 21621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 6aacaaucacc aguuucuuct t 21723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 7ggaagaaacu ggugauuguu ggu 23821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 8aagaaacugg ugauuguugt t 21921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 9caacaaucac caguuucuut t 211023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10gaagaaacug gugauuguug gug 231121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 11agaaacuggu gauuguuggt t 211221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 12ccaacaauca ccaguuucut t 211323RNAArtificial SequenceDescription of Artificial Sequence Syntheticoligonucleotide 13aagaaacugg ugauuguugg uga 231421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14gaaacuggug auuguuggut t 211521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15accaacaauc accaguuuct t 211623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 16agaaacuggu gauuguuggu gau 231721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17aaacugguga uuguuggugt t 211821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18caccaacaau caccaguuut t 211923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19gaaacuggug auuguuggug aug 232021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20aacuggugau uguuggugat t 212121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21ucaccaacaa ucaccaguut t 212223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 22aaacugguga uuguugguga ugg 232321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23acuggugauu guuggugaut t 212421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24aucaccaaca aucaccagut t 212523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25aacuggugau uguuggugau gga 232621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26cuggugauug uuggugaugt t 212721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27caucaccaac aaucaccagt t 212823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 28acuggugauu guuggugaug gag 232921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 29uggugauugu uggugauggt t 213021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 30ccaucaccaa caaucaccat t 213123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 31cuggugauug uuggugaugg agc 233221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 32ggugauuguu ggugauggat t 213321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 33uccaucacca acaaucacct t 213423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 34uggugauugu uggugaugga gcc 233521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 35gugauuguug gugauggagt t 213621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36cuccaucacc aacaaucact t 213723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37ggugauuguu ggugauggag ccu 233821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38ugauuguugg ugauggagct t 213921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39gcuccaucac caacaaucat t 214023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40gaaagacaug cuugcucaua guc 234121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41aagacaugcu ugcucauagt t 214221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42cuaugagcaa gcaugucuut t 214323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43aaagacaugc uugcucauag ucu 234421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 44agacaugcuu gcucauagut t 214521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 45acuaugagca agcaugucut t 214623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 46aagacaugcu ugcucauagu cuu 234721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 47gacaugcuug cucauaguct t 214821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 48gacuaugagc aagcauguct t 214923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 49agacaugcuu gcucauaguc uuc 235021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 50acaugcuugc ucauagucut t 215121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 51agacuaugag caagcaugut t 215223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 52gacaugcuug cucauagucu uca 235321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 53caugcuugcu cauagucuut t 215421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 54aagacuauga gcaagcaugt t 215523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 55acaugcuugc ucauagucuu cag 235621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 56augcuugcuc auagucuuct t 215721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 57gaagacuaug agcaagcaut t 215823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 58caugcuugcu cauagucuuc agc 235921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 59ugcuugcuca uagucuucat t 216021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 60ugaagacuau gagcaagcat t 216123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 61augcuugcuc auagucuuca gca 236221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 62gcuugcucau agucuucagt t 216321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 63cugaagacua ugagcaagct t 216423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64ugcuugcuca uagucuucag caa 236521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65cuugcucaua gucuucagct t 216621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66gcugaagacu augagcaagt t 216723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67gcuugcucau agucuucagc aag 236821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68uugcucauag ucuucagcat t 216921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69ugcugaagac uaugagcaat t 217023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70cuugcucaua gucuucagca agg 237121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71ugcucauagu cuucagcaat t 217221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72uugcugaaga cuaugagcat t 217323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73uugcucauag ucuucagcaa gga 237421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 74gcucauaguc uucagcaagt t 217521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 75cuugcugaag acuaugagct t 217623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 76ugcucauagu cuucagcaag gac 237721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 77cucauagucu ucagcaaggt t 217821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 78ccuugcugaa gacuaugagt t 217923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 79gcucauaguc uucagcaagg acc 238021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 80ucauagucuu cagcaaggat t 218121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81uccuugcuga agacuaugat t 218223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 82cucauagucu ucagcaagga cca 238321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83cauagucuuc agcaaggact t 218421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 84guccuugcug aagacuaugt t 218523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 85ucauagucuu cagcaaggac cag 238621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 86auagucuuca gcaaggacct t 218721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 87gguccuugcu gaagacuaut t 218823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 88cauagucuuc agcaaggacc agu 238921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 89uagucuucag caaggaccat t 219021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 90ugguccuugc ugaagacuat t 219123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 91auagucuuca gcaaggacca guu 239221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 92agucuucagc aaggaccagt t 219321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 93cugguccuug cugaagacut t 219423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 94uagucuucag caaggaccag uuc 239521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 95gucuucagca aggaccagut t 219621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 96acugguccuu gcugaagact t 219723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 97agucuucagc aaggaccagu ucc 239821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 98ucuucagcaa ggaccaguut t 219921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 99aacugguccu ugcugaagat t 2110023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 100gucuucagca aggaccaguu ccc 2310121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 101cuucagcaag gaccaguuct t 2110221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 102gaacuggucc uugcugaagt t 2110323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 103ucuucagcaa ggaccaguuc cca 2310421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 104uucagcaagg accaguucct t 2110521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 105ggaacugguc cuugcugaat t 2110623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 106cuucagcaag gaccaguucc cag 2310721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 107ucagcaagga ccaguuccct t

2110821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 108gggaacuggu ccuugcugat t 2110923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 109uucagcaagg accaguuccc aga 2311021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 110cagcaaggac caguucccat t 2111121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 111ugggaacugg uccuugcugt t 2111223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 112ucagcaagga ccaguuccca gag 2311321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 113agcaaggacc aguucccagt t 2111421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 114cugggaacug guccuugcut t 2111523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 115cagcaaggac caguucccag agg 2311621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 116gcaaggacca guucccagat t 2111721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 117ucugggaacu gguccuugct t 2111823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 118agcaaggacc aguucccaga ggu 2311921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 119caaggaccag uucccagagt t 2112021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 120cucugggaac ugguccuugt t 2112123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 121gcaaggacca guucccagag gug 2312221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 122aaggaccagu ucccagaggt t 2112321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 123ccucugggaa cugguccuut t 2112423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 124caaggaccag uucccagagg ugu 2312521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 125aggaccaguu cccagaggut t 2112621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 126accucuggga acugguccut t 2112723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 127gaaagcaggu agaguuggcu uug 2312821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 128aagcagguag aguuggcuut t 2112921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 129aagccaacuc uaccugcuut t 2113023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 130aaagcaggua gaguuggcuu ugu 2313121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 131agcagguaga guuggcuuut t 2113221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 132aaagccaacu cuaccugcut t 2113323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 133gacagcccug auaguuuaga aaa 2313421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 134cagcccugau aguuuagaat t 2113521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 135uucuaaacua ucagggcugt t 2113623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 136acagcccuga uaguuuagaa aac 2313721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 137agcccugaua guuuagaaat t 2113821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 138uuucuaaacu aucagggcut t 2113923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 139cagcccugau aguuuagaaa aca 2314021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 140gcccugauag uuuagaaaat t 2114121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 141uuuucuaaac uaucagggct t 2114223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 142agcccugaua guuuagaaaa cau 2314321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 143cccugauagu uuagaaaact t 2114421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 144guuuucuaaa cuaucagggt t 2114523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 145gcccugauag uuuagaaaac auc 2314621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 146ccugauaguu uagaaaacat t 2114721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 147uguuuucuaa acuaucaggt t 2114823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 148cccugauagu uuagaaaaca ucc 2314921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 149cugauaguuu agaaaacaut t 2115021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 150auguuuucua aacuaucagt t 2115123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 151ccugauaguu uagaaaacau ccc 2315221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 152ugauaguuua gaaaacauct t 2115321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 153gauguuuucu aaacuaucat t 2115423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 154cugauaguuu agaaaacauc cca 2315521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 155gauaguuuag aaaacaucct t 2115621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 156ggauguuuuc uaaacuauct t 2115723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 157ugauaguuua gaaaacaucc cag 2315821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 158auaguuuaga aaacauccct t 2115921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 159gggauguuuu cuaaacuaut t 2116023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 160gauaguuuag aaaacauccc aga 2316121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 161uaguuuagaa aacaucccat t 2116221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 162ugggauguuu ucuaaacuat t 2116323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 163auaguuuaga aaacauccca gaa 2316421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 164aguuuagaaa acaucccagt t 2116521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 165cugggauguu uucuaaacut t 2116623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 166uaguuuagaa aacaucccag aaa 2316721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 167guuuagaaaa caucccagat t 2116821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 168ucugggaugu uuucuaaact t 2116923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 169aguuuagaaa acaucccaga aaa 2317021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 170uuuagaaaac aucccagaat t 2117121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 171uucugggaug uuuucuaaat t 2117223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 172guuuagaaaa caucccagaa aag 2317321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 173uuagaaaaca ucccagaaat t 2117421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 174uuucugggau guuuucuaat t 2117523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 175uuuagaaaac aucccagaaa agu 2317621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 176uagaaaacau cccagaaaat t 2117721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 177uuuucuggga uguuuucuat t 2117823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 178ccccagaagu caagcauuuc ugu 2317921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 179ccagaaguca agcauuucut t 2118021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 180agaaaugcuu gacuucuggt t 2118123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 181cccagaaguc aagcauuucu guc 2318221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 182cagaagucaa gcauuucugt t 2118321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 183cagaaaugcu ugacuucugt t 2118423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 184ccagaaguca agcauuucug ucc 2318521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 185agaagucaag cauuucugut t 2118621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 186acagaaaugc uugacuucut t 2118723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 187cagaagucaa gcauuucugu ccc 2318821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 188gaagucaagc auuucuguct t 2118921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 189gacagaaaug cuugacuuct t 2119023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 190agaagucaag cauuucuguc cca 2319121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 191aagucaagca uuucugucct t 2119221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 192ggacagaaau gcuugacuut t 2119323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 193ugaaaccuga agaaggcaga gau 2319421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 194aaaccugaag aaggcagagt t 2119521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 195cucugccuuc uucagguuut t 2119623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 196gaaaccugaa gaaggcagag aua 2319721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 197aaccugaaga aggcagagat t 2119821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 198ucucugccuu cuucagguut t 2119923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 199aaaccugaag aaggcagaga uau 2320021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 200accugaagaa ggcagagaut t 2120121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 201aucucugccu ucuucaggut t 2120223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 202aaccugaaga aggcagagau aug 2320321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 203ccugaagaag gcagagauat t 2120421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 204uaucucugcc uucuucaggt t 2120523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 205accugaagaa ggcagagaua ugg 2320621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 206cugaagaagg cagagauaut t 2120721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 207auaucucugc cuucuucagt t 2120823RNAArtificial SequenceDescription of Artificial Sequence Synthetic

oligonucleotide 208ccugaagaag gcagagauau ggc 2320921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 209ugaagaaggc agagauaugt t 2121021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 210cauaucucug ccuucuucat t 2121123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 211cugaagaagg cagagauaug gca 2321221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 212gaagaaggca gagauauggt t 2121321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 213ccauaucucu gccuucuuct t 2121423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 214ugaagaaggc agagauaugg caa 2321521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 215aagaaggcag agauauggct t 2121621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 216gccauaucuc ugccuucuut t 2121723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 217gaagaaggca gagauauggc aaa 2321821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 218agaaggcaga gauauggcat t 2121921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 219ugccauaucu cugccuucut t 2122023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 220aagaaggcag agauauggca aac 2322121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 221gaaggcagag auauggcaat t 2122221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 222uugccauauc ucugccuuct t 2122323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 223agaaggcaga gauauggcaa aca 2322421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 224aaggcagaga uauggcaaat t 2122521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 225uuugccauau cucugccuut t 2122623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 226gaaggcagag auauggcaaa cag 2322721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 227aggcagagau auggcaaact t 2122821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 228guuugccaua ucucugccut t 2122923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 229aaggcagaga uauggcaaac agg 2323021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 230ggcagagaua uggcaaacat t 2123121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 231uguuugccau aucucugcct t 2123223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 232aggcagagau auggcaaaca gga 2323321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 233gcagagauau ggcaaacagt t 2123421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 234cuguuugcca uaucucugct t 2123523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 235ggcagagaua uggcaaacag gau 2323621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 236cagagauaug gcaaacaggt t 2123721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 237ccuguuugcc auaucucugt t 2123823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 238gcagagauau ggcaaacagg auu 2323921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 239agagauaugg caaacaggat t 2124021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 240uccuguuugc cauaucucut t 2124123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 241cagagauaug gcaaacagga uug 2324221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 242gagauauggc aaacaggaut t 2124321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 243auccuguuug ccauaucuct t 2124423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 244agagauaugg caaacaggau ugg 2324521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 245agauauggca aacaggauut t 2124621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 246aauccuguuu gccauaucut t 2124723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 247gagauauggc aaacaggauu ggc 2324821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 248gauauggcaa acaggauugt t 2124921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 249caauccuguu ugccauauct t 2125023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 250agauauggca aacaggauug gcg 2325121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 251auauggcaaa caggauuggt t 2125221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 252ccaauccugu uugccauaut t 2125323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 253gauauggcaa acaggauugg cgc 2325421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 254uauggcaaac aggauuggct t 2125521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 255gccaauccug uuugccauat t 2125623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 256auauggcaaa caggauuggc gcu 2325721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 257auggcaaaca ggauuggcgt t 2125821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 258cgccaauccu guuugccaut t 2125923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 259uauggcaaac aggauuggcg cuu 2326021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 260uggcaaacag gauuggcgct t 2126121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 261gcgccaaucc uguuugccat t 2126223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 262auggcaaaca ggauuggcgc uuu 2326321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 263ggcaaacagg auuggcgcut t 2126421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 264agcgccaauc cuguuugcct t 2126523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 265uggcaaacag gauuggcgcu uuu 2326621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 266gcaaacagga uuggcgcuut t 2126721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 267aagcgccaau ccuguuugct t 2126823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 268ggcaaacagg auuggcgcuu uug 2326921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 269caaacaggau uggcgcuuut t 2127021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 270aaagcgccaa uccuguuugt t 2127123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 271gcaaacagga uuggcgcuuu ugg 2327221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 272aaacaggauu ggcgcuuuut t 2127321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 273aaaagcgcca auccuguuut t 2127423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 274caaacaggau uggcgcuuuu ggg 2327521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 275aacaggauug gcgcuuuugt t 2127621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 276caaaagcgcc aauccuguut t 2127723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 277aaacaggauu ggcgcuuuug ggu 2327821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 278acaggauugg cgcuuuuggt t 2127921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 279ccaaaagcgc caauccugut t 2128023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 280aacaggauug gcgcuuuugg gua 2328121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 281caggauuggc gcuuuugggt t 2128221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 282cccaaaagcg ccaauccugt t 2128323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 283acaggauugg cgcuuuuggg uac 2328421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 284aggauuggcg cuuuugggut t 2128521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 285acccaaaagc gccaauccut t 2128623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 286caggauuggc gcuuuugggu aca 2328721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 287ggauuggcgc uuuuggguat t 2128821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 288uacccaaaag cgccaaucct t 2128923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 289aggauuggcg cuuuugggua cau 2329021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 290gauuggcgcu uuuggguact t 2129121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 291guacccaaaa gcgccaauct t 2129223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 292ggauuggcgc uuuuggguac aug 2329321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 293auuggcgcuu uuggguacat t 2129421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 294uguacccaaa agcgccaaut t 2129523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 295gauuggcgcu uuuggguaca ugg 2329621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 296uuggcgcuuu uggguacaut t 2129721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 297auguacccaa aagcgccaat t 2129823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 298auuggcgcuu uuggguacau gga 2329921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 299uggcgcuuuu ggguacaugt t 2130021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 300cauguaccca aaagcgccat t 2130123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 301uuggcgcuuu uggguacaug gag 2330221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 302ggcgcuuuug gguacauggt t 2130321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 303ccauguaccc aaaagcgcct t 2130423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 304uggcgcuuuu ggguacaugg agu 2330521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 305gcgcuuuugg guacauggat t 2130621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 306uccauguacc caaaagcgct t 2130723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 307ggcgcuuuug gguacaugga gug 2330821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 308cgcuuuuggg uacauggagt t

2130921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 309cuccauguac ccaaaagcgt t 2131023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 310gcgcuuuugg guacauggag ugu 2331121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 311gcuuuugggu acauggagut t 2131221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 312acuccaugua cccaaaagct t 2131323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 313cgcuuuuggg uacauggagu guu 2331421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 314cuuuugggua cauggagugt t 2131521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 315cacuccaugu acccaaaagt t 2131623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 316gcuuuugggu acauggagug uuc 2331721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 317uuuuggguac auggagugut t 2131821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 318acacuccaug uacccaaaat t 2131923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 319cuuuugggua cauggagugu uca 2332021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 320uuuggguaca uggaguguut t 2132121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 321aacacuccau guacccaaat t 2132223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 322uuuuggguac auggaguguu cag 2332321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 323uuggguacau ggaguguuct t 2132421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 324gaacacucca uguacccaat t 2132523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 325uuuggguaca uggaguguuc agc 2332621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 326uggguacaug gaguguucat t 2132721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 327ugaacacucc auguacccat t 2132823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 328uuggguacau ggaguguuca gca 2332921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 329ggguacaugg aguguucagt t 2133021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 330cugaacacuc cauguaccct t 2133123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 331uggguacaug gaguguucag caa 2333221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 332gguacaugga guguucagct t 2133321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 333gcugaacacu ccauguacct t 2133423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 334ggguacaugg aguguucagc aaa 2333521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 335guacauggag uguucagcat t 2133621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 336ugcugaacac uccauguact t 2133723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 337gguacaugga guguucagca aag 2333821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 338uacauggagu guucagcaat t 2133921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 339uugcugaaca cuccauguat t 2134023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 340guacauggag uguucagcaa aga 2334121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 341acauggagug uucagcaaat t 2134221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 342uuugcugaac acuccaugut t 2134323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 343uacauggagu guucagcaaa gac 2334421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 344cauggagugu ucagcaaagt t 2134521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 345cuuugcugaa cacuccaugt t 2134623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 346acauggagug uucagcaaag acc 2334721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 347auggaguguu cagcaaagat t 2134821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 348ucuuugcuga acacuccaut t 2134923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 349cauggagugu ucagcaaaga cca 2335021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 350uggaguguuc agcaaagact t 2135121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 351gucuuugcug aacacuccat t 2135223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 352auggaguguu cagcaaagac caa 2335321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 353ggaguguuca gcaaagacct t 2135421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 354ggucuuugcu gaacacucct t 2135523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 355uggaguguuc agcaaagacc aaa 2335621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 356gaguguucag caaagaccat t 2135721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 357uggucuuugc ugaacacuct t 2135823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 358ggaguguuca gcaaagacca aag 2335921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 359aguguucagc aaagaccaat t 2136021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 360uuggucuuug cugaacacut t 2136123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 361gaguguucag caaagaccaa aga 2336221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 362guguucagca aagaccaaat t 2136321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 363uuuggucuuu gcugaacact t 2136423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 364aguguucagc aaagaccaaa gau 2336521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 365uguucagcaa agaccaaagt t 2136621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 366cuuuggucuu ugcugaacat t 2136723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 367guguucagca aagaccaaag aug 2336821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 368guucagcaaa gaccaaagat t 2136921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 369ucuuuggucu uugcugaact t 2137023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 370auggagugag agagguuuuu gaa 2337121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 371ggagugagag agguuuuugt t 2137221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 372caaaaaccuc ucucacucct t 2137323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 373uggagugaga gagguuuuug aaa 2337421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 374gagugagaga gguuuuugat t 2137521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 375ucaaaaaccu cucucacuct t 2137623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 376cuacgagagc ugcucugcaa gcu 2337721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 377acgagagcug cucugcaagt t 2137821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 378cuugcagagc agcucucgut t 2137923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 379uacgagagcu gcucugcaag cua 2338021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 380cgagagcugc ucugcaagct t 2138121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 381gcuugcagag cagcucucgt t 2138223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 382acgagagcug cucugcaagc uag 2338321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 383gagagcugcu cugcaagcut t 2138421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 384agcuugcaga gcagcucuct t 2138523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 385cgagagcugc ucugcaagcu aga 2338621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 386agagcugcuc ugcaagcuat t 2138721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 387uagcuugcag agcagcucut t 2138823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 388gagagcugcu cugcaagcua gac 2338921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 389gagcugcucu gcaagcuagt t 2139021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 390cuagcuugca gagcagcuct t 2139123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 391agagcugcuc ugcaagcuag acg 2339221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 392agcugcucug caagcuagat t 2139321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 393ucuagcuugc agagcagcut t 2139423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 394gagcugcucu gcaagcuaga cgu 2339521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 395gcugcucugc aagcuagact t 2139621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 396gucuagcuug cagagcagct t 2139723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 397agcugcucug caagcuagac gug 2339821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 398cugcucugca agcuagacgt t 2139921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 399cgucuagcuu gcagagcagt t 2140023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 400uugaagugcu guuuauuaau cuu 2340121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 401gaagugcugu uuauuaauct t 2140221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 402gauuaauaaa cagcacuuct t 2140323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 403ugaagugcug uuuauuaauc uua 2340421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 404aagugcuguu uauuaaucut t 2140521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 405agauuaauaa acagcacuut t 2140623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 406gaagugcugu uuauuaaucu uag 2340721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 407agugcuguuu auuaaucuut t 2140821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 408aagauuaaua aacagcacut t 2140923RNAArtificial SequenceDescription of

Artificial Sequence Synthetic oligonucleotide 409aagugcuguu uauuaaucuu agu 2341021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 410gugcuguuua uuaaucuuat t 2141121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 411uaagauuaau aaacagcact t 2141223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 412agugcuguuu auuaaucuua gug 2341321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 413ugcuguuuau uaaucuuagt t 2141421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 414cuaagauuaa uaaacagcat t 2141523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 415gugcuguuua uuaaucuuag ugu 2341621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 416gcuguuuauu aaucuuagut t 2141721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 417acuaagauua auaaacagct t 2141823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 418ugcuguuuau uaaucuuagu gua 2341921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 419cuguuuauua aucuuagugt t 2142021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 420cacuaagauu aauaaacagt t 2142123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 421gcuguuuauu aaucuuagug uau 2342221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 422uguuuauuaa ucuuagugut t 2142321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 423acacuaagau uaauaaacat t 2142423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 424cuguuuauua aucuuagugu aug 2342521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 425guuuauuaau cuuaguguat t 2142621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 426uacacuaaga uuaauaaact t 2142723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 427uguuuauuaa ucuuagugua uga 2342821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 428uuuauuaauc uuaguguaut t 2142921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 429auacacuaag auuaauaaat t 2143023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 430guuuauuaau cuuaguguau gau 2343121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 431uuauuaaucu uaguguaugt t 2143221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 432cauacacuaa gauuaauaat t 2143323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 433uuuauuaauc uuaguguaug auu 2343421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 434uauuaaucuu aguguaugat t 2143521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 435ucauacacua agauuaauat t 2143623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 436uuauuaaucu uaguguauga uua 2343721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 437auuaaucuua guguaugaut t 2143821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 438aucauacacu aagauuaaut t 2143923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 439uauuaaucuu aguguaugau uac 2344021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 440uuaaucuuag uguaugauut t 2144121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 441aaucauacac uaagauuaat t 2144223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 442auuaaucuua guguaugauu acu 2344321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 443uaaucuuagu guaugauuat t 2144421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 444uaaucauaca cuaagauuat t 2144523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 445uuaaucuuag uguaugauua cug 2344621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 446aaucuuagug uaugauuact t 2144721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 447guaaucauac acuaagauut t 2144823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 448uaaucuuagu guaugauuac ugg 2344921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 449aucuuagugu augauuacut t 2145021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 450aguaaucaua cacuaagaut t 2145123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 451aaucuuagug uaugauuacu ggc 2345221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 452ucuuagugua ugauuacugt t 2145321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 453caguaaucau acacuaagat t 2145423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 454aucuuagugu augauuacug gcc 2345521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 455cuuaguguau gauuacuggt t 2145621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 456ccaguaauca uacacuaagt t 2145723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 457ucuuagugua ugauuacugg ccu 2345821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 458uuaguguaug auuacuggct t 2145921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 459gccaguaauc auacacuaat t 2146023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 460cuuaguguau gauuacuggc cuu 2346121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 461uaguguauga uuacuggcct t 2146221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 462ggccaguaau cauacacuat t 2146323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 463uuaguguaug auuacuggcc uuu 2346421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 464aguguaugau uacuggccut t 2146521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 465aggccaguaa ucauacacut t 2146623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 466uaguguauga uuacuggccu uuu 2346721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 467guguaugauu acuggccuut t 2146821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 468aaggccagua aucauacact t 2146923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 469aguguaugau uacuggccuu uuu 2347021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 470uguaugauua cuggccuuut t 2147121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 471aaaggccagu aaucauacat t 2147223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 472guguaugauu acuggccuuu uuc 2347321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 473guaugauuac uggccuuuut t 2147421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 474aaaaggccag uaaucauact t 2147523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 475uucauuuauc uauaauuuac cua 2347621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 476cauuuaucua uaauuuacct t 2147721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 477gguaaauuau agauaaaugt t 2147823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 478ucauuuaucu auaauuuacc uaa 2347921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 479auuuaucuau aauuuaccut t 2148021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 480agguaaauua uagauaaaut t 2148123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 481cauuuaucua uaauuuaccu aag 2348221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 482uuuaucuaua auuuaccuat t 2148321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 483uagguaaauu auagauaaat t 2148423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 484auuuaucuau aauuuaccua aga 2348521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 485uuaucuauaa uuuaccuaat t 2148621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 486uuagguaaau uauagauaat t 2148723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 487uuuaucuaua auuuaccuaa gau 2348821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 488uaucuauaau uuaccuaagt t 2148921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 489cuuagguaaa uuauagauat t 2149023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 490uuaucuauaa uuuaccuaag auu 2349121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 491aucuauaauu uaccuaagat t 2149221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 492ucuuagguaa auuauagaut t 2149323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 493uaucuauaau uuaccuaaga uua 2349421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 494ucuauaauuu accuaagaut t 2149521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 495aucuuaggua aauuauagat t 2149623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 496aucuauaauu uaccuaagau uac 2349721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 497cuauaauuua ccuaagauut t 2149821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 498aaucuuaggu aaauuauagt t 2149923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 499ucuauaauuu accuaagauu aca 2350021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 500uauaauuuac cuaagauuat t 2150121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 501uaaucuuagg uaaauuauat t 2150223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 502cuauaauuua ccuaagauua caa 2350321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 503auaauuuacc uaagauuact t 2150421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 504guaaucuuag guaaauuaut t 2150523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 505uauaauuuac cuaagauuac aaa 2350621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 506uaauuuaccu aagauuacat t 2150721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 507uguaaucuua gguaaauuat t 2150823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 508auaauuuacc uaagauuaca aau 2350921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 509aauuuaccua

agauuacaat t 2151021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 510uuguaaucuu agguaaauut t 2151123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 511uaauuuaccu aagauuacaa auc 2351221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 512auuuaccuaa gauuacaaat t 2151321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 513uuuguaaucu uagguaaaut t 2151423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 514aauuuaccua agauuacaaa uca 2351521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 515uuuaccuaag auuacaaaut t 2151621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 516auuuguaauc uuagguaaat t 2151723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 517auuuaccuaa gauuacaaau cag 2351821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 518uuaccuaaga uuacaaauct t 2151921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 519gauuuguaau cuuagguaat t 2152023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 520uuuaccuaag auuacaaauc aga 2352121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 521uaccuaagau uacaaaucat t 2152221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 522ugauuuguaa ucuuagguat t 2152323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 523agaagucauc uugcuaccag uau 2352421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 524aagucaucuu gcuaccagut t 2152521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 525acugguagca agaugacuut t 2152623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 526gaagucaucu ugcuaccagu auu 2352721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 527agucaucuug cuaccaguat t 2152821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 528uacugguagc aagaugacut t 2152923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 529aagucaucuu gcuaccagua uuu 2353021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 530gucaucuugc uaccaguaut t 2153121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 531auacugguag caagaugact t 2153223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 532agucaucuug cuaccaguau uua 2353321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 533ucaucuugcu accaguauut t 2153421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 534aauacuggua gcaagaugat t 2153523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 535gucaucuugc uaccaguauu uag 2353621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 536caucuugcua ccaguauuut t 2153721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 537aaauacuggu agcaagaugt t 2153823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 538ucaucuugcu accaguauuu aga 2353921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 539aucuugcuac caguauuuat t 2154021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 540uaaauacugg uagcaagaut t 2154123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 541caucuugcua ccaguauuua gaa 2354221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 542ucuugcuacc aguauuuagt t 2154321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 543cuaaauacug guagcaagat t 2154423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 544aucuugcuac caguauuuag aag 2354521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 545cuugcuacca guauuuagat t 2154621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 546ucuaaauacu gguagcaagt t 2154723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 547ucuugcuacc aguauuuaga agc 2354821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 548uugcuaccag uauuuagaat t 2154921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 549uucuaaauac ugguagcaat t 2155023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 550cuugcuacca guauuuagaa gcc 2355121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 551ugcuaccagu auuuagaagt t 2155221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 552cuucuaaaua cugguagcat t 2155323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 553uugcuaccag uauuuagaag cca 2355421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 554gcuaccagua uuuagaagct t 2155521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 555gcuucuaaau acugguagct t 2155623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 556ugcuaccagu auuuagaagc caa 2355721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 557cuaccaguau uuagaagcct t 2155821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 558ggcuucuaaa uacugguagt t 2155923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 559gcuaccagua uuuagaagcc aac 2356021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 560uaccaguauu uagaagccat t 2156121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 561uggcuucuaa auacugguat t 2156223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 562cuaccaguau uuagaagcca acu 2356321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 563accaguauuu agaagccaat t 2156421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 564uuggcuucua aauacuggut t 2156523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 565uaccaguauu uagaagccaa cua 2356621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 566ccaguauuua gaagccaact t 2156721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 567guuggcuucu aaauacuggt t 2156823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 568cuugcuucuu ucuagaaaga gaa 2356921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 569ugcuucuuuc uagaaagagt t 2157021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 570cucuuucuag aaagaagcat t 2157123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 571uugcuucuuu cuagaaagag aaa 2357221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 572gcuucuuucu agaaagagat t 2157321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 573ucucuuucua gaaagaagct t 2157423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 574ugcuucuuuc uagaaagaga aac 2357521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 575cuucuuucua gaaagagaat t 2157621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 576uucucuuucu agaaagaagt t 2157723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 577gcuucuuucu agaaagagaa aca 2357821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 578uucuuucuag aaagagaaat t 2157921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 579uuucucuuuc uagaaagaat t 2158023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 580cuucuuucua gaaagagaaa cag 2358121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 581ucuuucuaga aagagaaact t 2158221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 582guuucucuuu cuagaaagat t 2158323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 583uucuuucuag aaagagaaac agu 2358421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 584cuuucuagaa agagaaacat t 2158521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 585uguuucucuu ucuagaaagt t 2158623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 586ucuuucuaga aagagaaaca guu 2358721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 587uuucuagaaa gagaaacagt t 2158821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 588cuguuucucu uucuagaaat t 2158923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 589cuuucuagaa agagaaacag uug 2359021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 590uucuagaaag agaaacagut t 2159121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 591acuguuucuc uuucuagaat t 2159223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 592uuucuagaaa gagaaacagu ugg 2359321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 593ucuagaaaga gaaacaguut t 2159421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 594aacuguuucu cuuucuagat t 2159523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 595uucuagaaag agaaacaguu ggu 2359621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 596cuagaaagag aaacaguugt t 2159721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 597caacuguuuc ucuuucuagt t 2159823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 598ucuagaaaga gaaacaguug gua 2359921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 599uagaaagaga aacaguuggt t 2160021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 600ccaacuguuu cucuuucuat t 2160123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 601cuagaaagag aaacaguugg uaa 2360221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 602agaaagagaa acaguuggut t 2160321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 603accaacuguu ucucuuucut t 2160423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 604uagaaagaga aacaguuggu aac 2360521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 605gaaagagaaa caguugguat t 2160621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 606uaccaacugu uucucuuuct t 2160723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 607agaaagagaa acaguuggua acu 2360821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 608aaagagaaac aguugguaat t 2160921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 609uuaccaacug uuucucuuut t

2161023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 610gaaagagaaa caguugguaa cuu 2361121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 611aagagaaaca guugguaact t 2161221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 612guuaccaacu guuucucuut t 2161323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 613aaagagaaac aguugguaac uuu 2361421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 614agagaaacag uugguaacut t 2161521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 615aguuaccaac uguuucucut t 2161623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 616aagagaaaca guugguaacu uuu 2361721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 617gagaaacagu ugguaacuut t 2161821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 618aaguuaccaa cuguuucuct t 2161923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 619agagaaacag uugguaacuu uug 2362021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 620agaaacaguu gguaacuuut t 2162121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 621aaaguuacca acuguuucut t 2162223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 622gagaaacagu ugguaacuuu ugu 2362321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 623gaaacaguug guaacuuuut t 2162421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 624aaaaguuacc aacuguuuct t 2162523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 625agaaacaguu gguaacuuuu gug 2362621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 626aaacaguugg uaacuuuugt t 2162721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 627caaaaguuac caacuguuut t 2162823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 628gaaacaguug guaacuuuug uga 2362921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 629aacaguuggu aacuuuugut t 2163021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 630acaaaaguua ccaacuguut t 2163123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 631aaacaguugg uaacuuuugu gaa 2363221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 632acaguuggua acuuuugugt t 2163321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 633cacaaaaguu accaacugut t 2163423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 634aacaguuggu aacuuuugug aau 2363521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 635caguugguaa cuuuugugat t 2163621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 636ucacaaaagu uaccaacugt t 2163723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 637acaguuggua acuuuuguga auu 2363821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 638aguugguaac uuuugugaat t 2163921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 639uucacaaaag uuaccaacut t 2164023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 640caguugguaa cuuuugugaa uua 2364121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 641guugguaacu uuugugaaut t 2164221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 642auucacaaaa guuaccaact t 2164323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 643aguugguaac uuuugugaau uag 2364421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 644uugguaacuu uugugaauut t 2164521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 645aauucacaaa aguuaccaat t 2164623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 646guugguaacu uuugugaauu agg 2364721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 647ugguaacuuu ugugaauuat t 2164821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 648uaauucacaa aaguuaccat t 2164923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 649uugguaacuu uugugaauua ggc 2365021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 650gguaacuuuu gugaauuagt t 2165121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 651cuaauucaca aaaguuacct t 2165223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 652ugguaacuuu ugugaauuag gcu 2365321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 653guaacuuuug ugaauuaggt t 2165421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 654ccuaauucac aaaaguuact t 2165523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 655gguaacuuuu gugaauuagg cug 2365621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 656uaacuuuugu gaauuaggct t 2165721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 657gccuaauuca caaaaguuat t 2165823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 658guaacuuuug ugaauuaggc ugu 2365921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 659aacuuuugug aauuaggcut t 2166021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 660agccuaauuc acaaaaguut t 2166123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 661uaacuuuugu gaauuaggcu gua 2366221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 662acuuuuguga auuaggcugt t 2166321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 663cagccuaauu cacaaaagut t 2166423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 664aacuuuugug aauuaggcug uaa 2366521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 665cuuuugugaa uuaggcugut t 2166621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 666acagccuaau ucacaaaagt t 2166723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 667acuuuuguga auuaggcugu aac 2366821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 668uuuugugaau uaggcuguat t 2166921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 669uacagccuaa uucacaaaat t 2167023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 670cuuuugugaa uuaggcugua acu 2367121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 671uuugugaauu aggcuguaat t 2167221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 672uuacagccua auucacaaat t 2167323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 673uuuugugaau uaggcuguaa cua 2367421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 674uugugaauua ggcuguaact t 2167521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 675guuacagccu aauucacaat t 2167623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 676uuugugaauu aggcuguaac uac 2367721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 677ugugaauuag gcuguaacut t 2167821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 678aguuacagcc uaauucacat t 2167923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 679uugugaauua ggcuguaacu acu 2368021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 680gugaauuagg cuguaacuat t 2168121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 681uaguuacagc cuaauucact t 2168223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 682ugugaauuag gcuguaacua cuu 2368321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 683ugaauuaggc uguaacuact t 2168421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 684guaguuacag ccuaauucat t 2168523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 685gugaauuagg cuguaacuac uuu 2368621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 686gaauuaggcu guaacuacut t 2168721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 687aguaguuaca gccuaauuct t 2168823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 688ugaauuaggc uguaacuacu uua 2368921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 689aauuaggcug uaacuacuut t 2169021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 690aaguaguuac agccuaauut t 2169123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 691gaauuaggcu guaacuacuu uau 2369221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 692auuaggcugu aacuacuuut t 2169321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 693aaaguaguua cagccuaaut t 2169423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 694aauuaggcug uaacuacuuu aua 2369521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 695uuaggcugua acuacuuuat t 2169621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 696uaaaguaguu acagccuaat t 2169723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 697auuaggcugu aacuacuuua uaa 2369821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 698uaggcuguaa cuacuuuaut t 2169921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 699auaaaguagu uacagccuat t 2170023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 700uuaggcugua acuacuuuau aac 2370121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 701aggcuguaac uacuuuauat t 2170221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 702uauaaaguag uuacagccut t 2170323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 703uaggcuguaa cuacuuuaua acu 2370421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 704ggcuguaacu acuuuauaat t 2170521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 705uuauaaagua guuacagcct t 2170623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 706aggcuguaac uacuuuauaa cua 2370721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 707gcuguaacua cuuuauaact t 2170821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 708guuauaaagu aguuacagct t 2170923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 709ggcuguaacu acuuuauaac uaa 2371021DNAArtificial SequenceDescription of Artificial Sequence Synthetic

oligonucleotide 710cuguaacuac uuuauaacut t 2171121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 711aguuauaaag uaguuacagt t 2171223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 712gcuguaacua cuuuauaacu aac 2371321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 713uguaacuacu uuauaacuat t 2171421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 714uaguuauaaa guaguuacat t 2171523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 715cuguaacuac uuuauaacua aca 2371621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 716guaacuacuu uauaacuaat t 2171721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 717uuaguuauaa aguaguuact t 2171823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 718uguaacuacu uuauaacuaa cau 2371921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 719uaacuacuuu auaacuaact t 2172021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 720guuaguuaua aaguaguuat t 2172123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 721guaacuacuu uauaacuaac aug 2372221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 722aacuacuuua uaacuaacat t 2172321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 723uguuaguuau aaaguaguut t 2172423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 724uaacuacuuu auaacuaaca ugu 2372521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 725acuacuuuau aacuaacaut t 2172621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 726auguuaguua uaaaguagut t 2172723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 727aacuacuuua uaacuaacau guc 2372821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 728cuacuuuaua acuaacaugt t 2172921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 729cauguuaguu auaaaguagt t 2173023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 730acuacuuuau aacuaacaug ucc 2373121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 731uacuuuauaa cuaacaugut t 2173221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 732acauguuagu uauaaaguat t 2173323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 733cuacuuuaua acuaacaugu ccu 2373421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 734acuuuauaac uaacauguct t 2173521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 735gacauguuag uuauaaagut t 2173623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 736uacuuuauaa cuaacauguc cug 2373721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 737cuuuauaacu aacaugucct t 2173821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 738ggacauguua guuauaaagt t 2173923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 739acuuuauaac uaacaugucc ugc 2374021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 740uuuauaacua acauguccut t 2174121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 741aggacauguu aguuauaaat t 2174223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 742cuuuauaacu aacauguccu gcc 2374321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 743uuauaacuaa cauguccugt t 2174421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 744caggacaugu uaguuauaat t 2174523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 745uuuauaacua acauguccug ccu 2374621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 746uauaacuaac auguccugct t 2174721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 747gcaggacaug uuaguuauat t 2174823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 748uuauaacuaa cauguccugc cua 2374921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 749auaacuaaca uguccugcct t 2175021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 750ggcaggacau guuaguuaut t 2175123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 751uauaacuaac auguccugcc uau 2375221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 752uaacuaacau guccugccut t 2175321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 753aggcaggaca uguuaguuat t 2175423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 754auaacuaaca uguccugccu auu 2375521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 755aacuaacaug uccugccuat t 2175621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 756uaggcaggac auguuaguut t 2175723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 757uggcagaguu acaguucugu ggu 2375821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 758gcagaguuac aguucugugt t 2175921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 759cacagaacug uaacucugct t 2176023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 760ggcagaguua caguucugug guu 2376121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 761cagaguuaca guucuguggt t 2176221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 762ccacagaacu guaacucugt t 2176323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 763gcagaguuac aguucugugg uuu 2376421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 764agaguuacag uucuguggut t 2176521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 765accacagaac uguaacucut t 2176623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 766uuucauguua guuaccuuau agu 2376721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 767ucauguuagu uaccuuauat t 2176821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 768uauaagguaa cuaacaugat t 2176923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 769uucauguuag uuaccuuaua guu 2377021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 770cauguuaguu accuuauagt t 2177121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 771cuauaaggua acuaacaugt t 2177223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 772ucauguuagu uaccuuauag uua 2377321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 773auguuaguua ccuuauagut t 2177421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 774acuauaaggu aacuaacaut t 2177523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 775cauguuaguu accuuauagu uac 2377621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 776uguuaguuac cuuauaguut t 2177721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 777aacuauaagg uaacuaacat t 2177823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 778auguuaguua ccuuauaguu acu 2377921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 779guuaguuacc uuauaguuat t 2178021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 780uaacuauaag guaacuaact t 2178123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 781uguuaguuac cuuauaguua cug 2378221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 782uuaguuaccu uauaguuact t 2178321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 783guaacuauaa gguaacuaat t 2178423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 784guuaguuacc uuauaguuac ugu 2378521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 785uaguuaccuu auaguuacut t 2178621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 786aguaacuaua agguaacuat t 2178723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 787uuaguuaccu uauaguuacu gug 2378821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 788aguuaccuua uaguuacugt t 2178921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 789caguaacuau aagguaacut t 2179023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 790uaguuaccuu auaguuacug ugu 2379121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 791guuaccuuau aguuacugut t 2179221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 792acaguaacua uaagguaact t 2179323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 793aguuaccuua uaguuacugu gua 2379421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 794uuaccuuaua guuacugugt t 2179521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 795cacaguaacu auaagguaat t 2179623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 796guuaccuuau aguuacugug uaa 2379721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 797uaccuuauag uuacugugut t 2179821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 798acacaguaac uauaagguat t 2179923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 799uuaccuuaua guuacugugu aau 2380021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 800accuuauagu uacuguguat t 2180121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 801uacacaguaa cuauaaggut t 2180223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 802uaccuuauag uuacugugua auu 2380321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 803ccuuauaguu acuguguaat t 2180421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 804uuacacagua acuauaaggt t 2180523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 805accuuauagu uacuguguaa uua 2380621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 806cuuauaguua cuguguaaut t 2180721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 807auuacacagu aacuauaagt t 2180823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 808ccuuauaguu acuguguaau uag 2380921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 809uuauaguuac uguguaauut t 2181021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 810aauuacacag uaacuauaat t

2181123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 811cuuauaguua cuguguaauu agu 2381221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 812uauaguuacu guguaauuat t 2181321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 813uaauuacaca guaacuauat t 2181423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 814uuauaguuac uguguaauua gug 2381521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 815auaguuacug uguaauuagt t 2181621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 816cuaauuacac aguaacuaut t 2181723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 817uauaguuacu guguaauuag ugc 2381821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 818uaguuacugu guaauuagut t 2181921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 819acuaauuaca caguaacuat t 2182023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 820auaguuacug uguaauuagu gcc 2382121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 821aguuacugug uaauuagugt t 2182221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 822cacuaauuac acaguaacut t 2182323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 823uaguuacugu guaauuagug cca 2382421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 824guuacugugu aauuagugct t 2182521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 825gcacuaauua cacaguaact t 2182623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 826aguuacugug uaauuagugc cac 2382721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 827uuacugugua auuagugcct t 2182821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 828ggcacuaauu acacaguaat t 2182923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 829guuacugugu aauuagugcc acu 2383021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 830uacuguguaa uuagugccat t 2183121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 831uggcacuaau uacacaguat t 2183223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 832uuacugugua auuagugcca cuu 2383321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 833acuguguaau uagugccact t 2183421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 834guggcacuaa uuacacagut t 2183523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 835uacuguguaa uuagugccac uua 2383621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 836cuguguaauu agugccacut t 2183721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 837aguggcacua auuacacagt t 2183823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 838acuguguaau uagugccacu uaa 2383921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 839uguguaauua gugccacuut t 2184021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 840aaguggcacu aauuacacat t 2184123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 841cuguguaauu agugccacuu aau 2384221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 842guguaauuag ugccacuuat t 2184321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 843uaaguggcac uaauuacact t 2184423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 844uguguaauua gugccacuua aug 2384521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 845uguaauuagu gccacuuaat t 2184621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 846uuaaguggca cuaauuacat t 2184723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 847guguaauuag ugccacuuaa ugu 2384821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 848guaauuagug ccacuuaaut t 2184921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 849auuaaguggc acuaauuact t 2185023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 850uguaauuagu gccacuuaau gua 2385121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 851uaauuagugc cacuuaaugt t 2185221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 852cauuaagugg cacuaauuat t 2185323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 853guaauuagug ccacuuaaug uau 2385421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 854aauuagugcc acuuaaugut t 2185521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 855acauuaagug gcacuaauut t 2185623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 856uaauuagugc cacuuaaugu aug 2385721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 857auuagugcca cuuaauguat t 2185821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 858uacauuaagu ggcacuaaut t 2185923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 859aauuagugcc acuuaaugua ugu 2386021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 860uuagugccac uuaauguaut t 2186121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 861auacauuaag uggcacuaat t 2186223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 862auuagugcca cuuaauguau guu 2386321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 863uagugccacu uaauguaugt t 2186421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 864cauacauuaa guggcacuat t 2186523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 865uuagugccac uuaauguaug uua 2386621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 866agugccacuu aauguaugut t 2186721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 867acauacauua aguggcacut t 2186823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 868uagugccacu uaauguaugu uac 2386921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 869gugccacuua auguauguut t 2187021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 870aacauacauu aaguggcact t 2187123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 871agugccacuu aauguauguu acc 2387221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 872ugccacuuaa uguauguuat t 2187321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 873uaacauacau uaaguggcat t 2187423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 874gugccacuua auguauguua cca 2387521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 875gccacuuaau guauguuact t 2187621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 876guaacauaca uuaaguggct t 2187723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 877ugccacuuaa uguauguuac caa 2387821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 878ccacuuaaug uauguuacct t 2187921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 879gguaacauac auuaaguggt t 2188023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 880gccacuuaau guauguuacc aaa 2388121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 881cacuuaaugu auguuaccat t 2188221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 882ugguaacaua cauuaagugt t 2188323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 883ccacuuaaug uauguuacca aaa 2388421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 884acuuaaugua uguuaccaat t 2188521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 885uugguaacau acauuaagut t 2188623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 886cacuuaaugu auguuaccaa aaa 2388721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 887cuuaauguau guuaccaaat t 2188821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 888uuugguaaca uacauuaagt t 2188923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 889acuuaaugua uguuaccaaa aau 2389021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 890uuaauguaug uuaccaaaat t 2189121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 891uuuugguaac auacauuaat t 2189223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 892cuuaauguau guuaccaaaa aua 2389321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 893uaauguaugu uaccaaaaat t 2189421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 894uuuuugguaa cauacauuat t 2189523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 895aauaaauaua ucuaccccag acu 2389621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 896uaaauauauc uaccccagat t 2189721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 897ucugggguag auauauuuat t 2189823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 898auaaauauau cuaccccaga cua 2389921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 899aaauauaucu accccagact t 2190021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 900gucuggggua gauauauuut t 2190123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 901uaaauauauc uaccccagac uag 2390221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 902aauauaucua ccccagacut t 2190321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 903agucuggggu agauauauut t 2190423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 904aaauauaucu accccagacu aga 2390521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 905auauaucuac cccagacuat t 2190621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 906uagucugggg uagauauaut t 2190723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 907aauauaucua ccccagacua gau 2390821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 908uauaucuacc ccagacuagt t 2190921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 909cuagucuggg guagauauat t 2191023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 910auauaucuac cccagacuag aug 2391121DNAArtificial SequenceDescription of

Artificial Sequence Synthetic oligonucleotide 911auaucuaccc cagacuagat t 2191221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 912ucuagucugg gguagauaut t 2191323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 913uauaucuacc ccagacuaga ugu 2391421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 914uaucuacccc agacuagaut t 2191521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 915aucuagucug ggguagauat t 2191623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 916auaucuaccc cagacuagau gua 2391721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 917aucuacccca gacuagaugt t 2191821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 918caucuagucu gggguagaut t 2191923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 919uaucuacccc agacuagaug uag 2392021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 920ucuaccccag acuagaugut t 2192121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 921acaucuaguc ugggguagat t 2192223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 922aucuacccca gacuagaugu agu 2392321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 923cuaccccaga cuagauguat t 2192421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 924uacaucuagu cugggguagt t 2192523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 925ucuaccccag acuagaugua gua 2392621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 926uaccccagac uagauguagt t 2192721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 927cuacaucuag ucugggguat t 2192823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 928cuaccccaga cuagauguag uau 2392921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 929accccagacu agauguagut t 2193021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 930acuacaucua gucuggggut t 2193123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 931uaccccagac uagauguagu auu 2393221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 932ccccagacua gauguaguat t 2193321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 933uacuacaucu agucuggggt t 2193423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 934accccagacu agauguagua uuu 2393521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 935cccagacuag auguaguaut t 2193621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 936auacuacauc uagucugggt t 2193723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 937ccccagacua gauguaguau uuu 2393821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 938ccagacuaga uguaguauut t 2193921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 939aauacuacau cuagucuggt t 2194023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 940cccagacuag auguaguauu uuu 2394121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 941cagacuagau guaguauuut t 2194221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 942aaauacuaca ucuagucugt t 2194323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 943ccagacuaga uguaguauuu uuu 2394421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 944agacuagaug uaguauuuut t 2194521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 945aaaauacuac aucuagucut t 2194623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 946cagacuagau guaguauuuu uug 2394721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 947gacuagaugu aguauuuuut t 2194821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 948aaaaauacua caucuaguct t 2194923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 949agacuagaug uaguauuuuu ugu 2395021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 950acuagaugua guauuuuuut t 2195121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 951aaaaaauacu acaucuagut t 2195223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 952gacuagaugu aguauuuuuu gua 2395321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 953cuagauguag uauuuuuugt t 2195421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 954caaaaaauac uacaucuagt t 2195523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 955acuagaugua guauuuuuug uau 2395621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 956uagauguagu auuuuuugut t 2195721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 957acaaaaaaua cuacaucuat t 2195823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 958cuagauguag uauuuuuugu aua 2395921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 959agauguagua uuuuuuguat t 2196021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 960uacaaaaaau acuacaucut t 2196123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 961uagauguagu auuuuuugua uaa 2396221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 962gauguaguau uuuuuguaut t 2196321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 963auacaaaaaa uacuacauct t 2196423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 964agauguagua uuuuuuguau aau 2396521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 965auguaguauu uuuuguauat t 2196621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 966uauacaaaaa auacuacaut t 2196723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 967gauguaguau uuuuuguaua auu 2396821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 968uguaguauuu uuuguauaat t 2196921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 969uuauacaaaa aauacuacat t 2197023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 970auguaguauu uuuuguauaa uug 2397121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 971guaguauuuu uuguauaaut t 2197221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 972auuauacaaa aaauacuact t 2197323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 973uguaguauuu uuuguauaau ugg 2397421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 974uaguauuuuu uguauaauut t 2197521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 975aauuauacaa aaaauacuat t 2197623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 976guaguauuuu uuguauaauu gga 2397721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 977aguauuuuuu guauaauugt t 2197821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 978caauuauaca aaaaauacut t 2197923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 979uaguauuuuu uguauaauug gau 2398021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 980guauuuuuug uauaauuggt t 2198121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 981ccaauuauac aaaaaauact t 2198223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 982aguauuuuuu guauaauugg auu 2398321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 983uauuuuuugu auaauuggat t 2198421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 984uccaauuaua caaaaaauat t 2198523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 985guauuuuuug uauaauugga uuu 2398621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 986auuuuuugua uaauuggaut t 2198721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 987auccaauuau acaaaaaaut t 2198823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 988uauuuuuugu auaauuggau uuc 2398921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 989uuuuuuguau aauuggauut t 2199021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 990aauccaauua uacaaaaaat t 2199123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 991auuuuuugua uaauuggauu ucc 2399221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 992uuuuuguaua auuggauuut t 2199321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 993aaauccaauu auacaaaaat t 2199423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 994uuuuuuguau aauuggauuu ccu 2399521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 995uuuuguauaa uuggauuuct t 2199621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 996gaaauccaau uauacaaaat t 2199723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 997uuuuuguaua auuggauuuc cua 2399821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 998uuuguauaau uggauuucct t 2199921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 999ggaaauccaa uuauacaaat t 21100023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1000uuuuguauaa uuggauuucc uaa 23100121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1001uuguauaauu ggauuuccut t 21100221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1002aggaaaucca auuauacaat t 21100323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1003uuuguauaau uggauuuccu aau 23100421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1004uguauaauug gauuuccuat t 21100521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1005uaggaaaucc aauuauacat t 21100623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1006uuguauaauu ggauuuccua aua 23100721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1007guauaauugg auuuccuaat t 21100821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1008uuaggaaauc caauuauact t 21100923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1009uguauaauug gauuuccuaa uac 23101021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1010uauaauugga uuuccuaaut t 21101121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1011auuaggaaau

ccaauuauat t 21101223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1012guauuuggaa auaaagucag aug 23101321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1013auuuggaaau aaagucagat t 21101421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1014ucugacuuua uuuccaaaut t 21101523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1015uauuuggaaa uaaagucaga ugg 23101621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1016uuuggaaaua aagucagaut t 21101721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1017aucugacuuu auuuccaaat t 21101823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1018auuuggaaau aaagucagau gga 23101921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1019uuggaaauaa agucagaugt t 21102021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1020caucugacuu uauuuccaat t 21102123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1021uuuggaaaua aagucagaug gaa 23102221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1022uggaaauaaa gucagauggt t 21102321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1023ccaucugacu uuauuuccat t 21102423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1024uuggaaauaa agucagaugg aaa 23102521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1025ggaaauaaag ucagauggat t 21102621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1026uccaucugac uuuauuucct t 21102723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1027uggaaauaaa gucagaugga aaa 23102821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1028gaaauaaagu cagauggaat t 21102921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1029uuccaucuga cuuuauuuct t 21103023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1030ucccucccag aggagccacc agu 23103121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1031ccucccagag gagccaccat t 21103221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1032ugguggcucc ucugggaggt t 21103323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1033cccucccaga ggagccacca guu 23103421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1034cucccagagg agccaccagt t 21103521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1035cugguggcuc cucugggagt t 21103623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1036ccucccagag gagccaccag uuc 23103721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1037ucccagagga gccaccagut t 21103821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1038acugguggcu ccucugggat t 21103923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1039cucccagagg agccaccagu ucu 23104021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1040cccagaggag ccaccaguut t 21104121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1041aacugguggc uccucugggt t 21104223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1042ucccagagga gccaccaguu cuc 23104321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1043ccagaggagc caccaguuct t 21104421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1044gaacuggugg cuccucuggt t 21104523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1045cccagaggag ccaccaguuc uca 23104621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1046cagaggagcc accaguucut t 21104721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1047agaacuggug gcuccucugt t 21104823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1048cuucucucca gcugacuaaa cuu 23104921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1049ucucuccagc ugacuaaact t 21105021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1050guuuagucag cuggagagat t 21105123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1051uucuguacca guuaauuuuu cca 23105221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1052cuguaccagu uaauuuuuct t 21105321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1053gaaaaauuaa cugguacagt t 21105423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1054ucuguaccag uuaauuuuuc caa 23105521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1055uguaccaguu aauuuuucct t 21105621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1056ggaaaaauua acugguacat t 21105723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1057cuguaccagu uaauuuuucc aac 23105821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1058guaccaguua auuuuuccat t 21105921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1059uggaaaaauu aacugguact t 21106023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1060uguaccaguu aauuuuucca acu 23106121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1061uaccaguuaa uuuuuccaat t 21106221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1062uuggaaaaau uaacugguat t 21106323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1063guaccaguua auuuuuccaa cua 23106421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1064accaguuaau uuuuccaact t 21106521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1065guuggaaaaa uuaacuggut t 21106623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1066uaccaguuaa uuuuuccaac uac 23106721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1067ccaguuaauu uuuccaacut t 21106821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1068aguuggaaaa auuaacuggt t 21106923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1069accaguuaau uuuuccaacu acu 23107021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1070caguuaauuu uuccaacuat t 21107121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1071uaguuggaaa aauuaacugt t 21107223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1072uaauagaaua aaggcaguuu ucu 23107321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1073auagaauaaa ggcaguuuut t 21107421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1074aaaacugccu uuauucuaut t 21107523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1075aauagaauaa aggcaguuuu cua 23107621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1076uagaauaaag gcaguuuuct t 21107721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1077gaaaacugcc uuuauucuat t 21107823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1078auagaauaaa ggcaguuuuc uaa 23107921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1079agaauaaagg caguuuucut t 21108021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1080agaaaacugc cuuuauucut t 21108121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1081gauuaugacc gucugaggct t 21108221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1082gccucagucg gucauaauct t 21108321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1083ggaucuucgg aaugaugagt t 21108421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1084cucaucauuc cgaagaucct t 21108521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1085agaccaaaga cggagugagt t 21108621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1086cucacuccgu cuuuggucut t 21108721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1087ugaagcagga gccgguaaat t 21108821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1088uuuaccggcu ccugcuucat t 21108921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1089gcuaccagua uuuagaagct t 21109021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1090gcuucuaaau acugguagct t 21109121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1091cuaccaguau uuagaagcct t 21109221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1092ggcuucuaaa uacugguagt t 21109321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1093gcuguaacua cuuuauaact t 21109421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1094guuauaaagu aguuacagct t 21109521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1095guuacugugu aauuagugct t 21109621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1096gcacuaauua cacaguaact t 21109721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1097ccacuuaaug uauguuacct t 21109821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1098gguaacauac auuaaguggt t 21109921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1099cagcccugau aguuuagaat t 21110021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1100uucuaaacua ucagggcugt t 21110121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1101gcccugauag uuuagaaaat t 21110221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1102uuuucuaaac uaucagggct t 21110321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1103cccugauagu uuagaaaact t 21110421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1104guuuucuaaa cuaucagggt t 21110521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1105gauaguuuag aaaacaucct t 21110621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1106ggauguuuuc uaaacuauct t 21110721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1107uaguuuagaa aacaucccat t 21110821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1108ugggauguuu ucuaaacuat t 21110921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1109cagacuagau guaguauuut t 21111021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1110aaauacuaca ucuagucugt t 21111121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1111ccccagacua gauguaguat t

21111221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1112uacuacaucu agucuggggt t 21111321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1113ccagacuaga uguaguauut t 21111421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1114aauacuacau cuagucuggt t 21111521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1115cccagacuag auguaguaut t 21111621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1116auacuacauc uagucugggt t 21111721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1117ugccacuuaa uguauguuat t 21111821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1118uaacauacau uaaguggcat t 21111921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1119ugcuguuuau uaaucuuagt t 21112021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1120cuaagauuaa uaaacagcat t 21112121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1121ucauguuagu uaccuuauat t 21112221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1122uauaagguaa cuaacaugat t 21112321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1123ccaguuaauu uuuccaacut t 21112421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1124aguuggaaaa auuaacuggt t 21112521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1125uaccuaagau uacaaaucat t 21112621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1126ugauuuguaa ucuuagguat t 21112721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1127ucuugcuacc aguauuuagt t 21112821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1128cuaaauacug guagcaagat t 21112921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1129uguguaauua gugccacuut t 21113021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1130aaguggcacu aauuacacat t 21113121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1131aucuugcuac caguauuuat t 21113221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1132uaaauacugg uagcaagaut t 21113321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1133cuguaacuac uuuauaacut t 21113421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1134aguuauaaag uaguuacagt t 21113521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1135gugaauuagg cuguaacuat t 21113621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1136uaguuacagc cuaauucact t 21113721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1137cuaccaguau uuagaagcct t 21113821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1138ggcuucuaaa uacugguagt t 21113921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1139ugcuguuuau uaaucuuagt t 21114021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1140cuaagauuaa uaaacagcat t 21114146DNARattus norvegicus 1141ccatttttct gggatgtttt ctaaattttt ctcttggaaa gaaagt 46114245DNARattus norvegicus 1142acagaaatgc ttgacttctg gagttttttc tcttggaaag aaagt 45114341DNARattus norvegicus 1143cttcaggttt taccggctcc tttttctctt ggaaagaaag t 41114443DNARattus norvegicus 1144ctgtttgcca tatctctgcc tttttttctc ttggaaagaa agt 43114543DNARattus norvegicus 1145ttggtctttg ctgaacactc catttttctc ttggaaagaa agt 43114640DNARattus norvegicus 1146cccgcgtcta gcttgcagat ttttctcttg gaaagaaagt 40114744DNARattus norvegicus 1147aggatgatgg gcacatttgg tttttaggca taggacccgt gtct 44114845DNARattus norvegicus 1148gccttgtgtg ctcatcattc ctttttaggc ataggacccg tgtct 45114946DNARattus norvegicus 1149tgcttcattt tggctaactc cctttttagg cataggaccc gtgtct 46115044DNARattus norvegicus 1150tgtacccaaa agcgccaatc tttttaggca taggacccgt gtct 44115143DNARattus norvegicus 1151gcagctctcg tggccatctt ttttaggcat aggacccgtg tct 43115244DNARattus norvegicus 1152aggcaccccg actttttctt tttttaggca taggacccgt gtct 44115321DNARattus norvegicus 1153ctatcagggc tgtcgatgga a 21115423DNARattus norvegicus 1154gaagatcctt cttgttccca act 23115523DNARattus norvegicus 1155caaaaacctc tctcactccg tct 23115641DNARattus norvegicus 1156ccagcttccc attctcagcc tttttctctt ggaaagaaag t 41115741DNARattus norvegicus 1157tctcgctcct ggaagatggt tttttctctt ggaaagaaag t 41115841DNARattus norvegicus 1158cccatttgat gttagcggga tttttctctt ggaaagaaag t 41115942DNARattus norvegicus 1159cggagatgat gacccttttg gtttttctct tggaaagaaa gt 42116044DNARattus norvegicus 1160gatgggtttc ccgttgatga tttttaggca taggacccgt gtct 44116147DNARattus norvegicus 1161gacatactca gcaccagcat cactttttag gcataggacc cgtgtct 47116244DNARattus norvegicus 1162cccagccttc tccatggtgg tttttaggca taggacccgt gtct 44116321DNARattus norvegicus 1163ttgactgtgc cgttgaactt g 21116419DNARattus norvegicus 1164ccccaccctt caggtgagc 19116517DNARattus norvegicus 1165ggcatcagcg gaagggg 17

Claims

1. An iRNA agent for inhibiting the expression of a RhoA gene in a cell comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836.

2. An iRNA agent for inhibiting the expression of a RhoA gene in a cell comprising a sense strand wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and an antisense strand wherein the antisense strand comprises at least 15 contiguous nucleotides of the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and wherein the iRNA agent reduces the amount of RhoA mRNA present in cultured human cells after incubation with these agents by 40% or more compared to cells which have not been incubated with the agent.

3. An iRNA agent for inhibiting the expression of a RhoA gene in a cell comprising a sense strand and an antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical to one of the sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit RhoA expression.

4. The iRNA agent of claim 1, wherein the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisense strand sequences of agent numbers 6523, 6524, 6530, 6614, 6650, 6656, 6657, 6661, 6662, 6703, 6712, 6713, 6732, 6751, 6756, 6767, 6769, 6787, 6789, 6790, 6832

5. The iRNA agent of claim 1, wherein the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisens strand sequences of agent numbers AL-DP-5972, AL-DP-5973, AL-DP-5974, AL-DP-5975, AL-DP-5976, AL-DP-5978, AL-DP-5979, AL-DP-5981, AL-DP-5982, AL-DP-5983, AL-DP-5984, AL-DP-5986, AL-DP-5987, AL-DP-5988, AL-DP-5989, AL-DP-5990, AL-DP-5991, AL-DP-5992, AL-DP-5993, AL-DP-5994, AL-DP-5995, AL-DP-6176, AL-DP-6177.

6. The iRNA agent of claim 1, wherein the antisense RNA strand is 30 or fewer nucleotides in length, and the duplex region of the iRNA agent is 15-30 nucleotide pairs in length.

7. The iRNA agent of claim 1, comprising a modification that causes the iRNA agent to have increased stability in a biological sample.

8. The iRNA agent of claim 7, wherein said modification is a phosphorothioate, a 2'-modified nucleotide, a locked nucleotide, an abasic nucleotide, morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.

9. The iRNA agent of claim 1, comprising at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-guanine-3' (5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide.

10. The iRNA agent of claim 1,wherein every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is 2'-modified in the sense strand, and every 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in the antisense strand, orwherein every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is 2'-modified in the sense and antisense strand, orwherein every pyrimidine nucleotide is 2'-modified in the sense strand, and every 5'-nucleotide in 5'-ua-3' and 5'-ca-3' motifs is 2'-modified in the antisense strand, orwherein every pyrimidine nucleotide is 2'-modified in sense strand, and every 5'-nucleotide in 5'-ua-3',5'-uu-3',5'-ca-3', and 5'-ug-3' motifs is 2'-modified in the antisense strand, orwherein every pyrimidine nucleotide in the sense strand is 2'-modified, and no nucleotide is 2'-modified in the antisense strand.

11. The iRNA agent of any one of claim 8, wherein the 2'-modification is selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O--N-methylacetamido(2'-O-NMA).

12. The iRNA agent of claim 1, comprising a nucleotide overhang having 1 to 4 unpaired nucleotides.

13. The iRNA agent of claim 1, comprising a cholesterol moiety.

14. The iRNA agent of claim 13, wherein the cholesterol moiety is conjugated to the 3'-end of the sense strand of the iRNA agent.

15. The iRNA agent of claim 1, wherein the iRNA agent is targeted for uptake by nerve cells or nerve sheath cells.

16. A method of treating a subject having a pathological process mediated in part by RhoA comprising administering to the subject an iRNA agent of claim 1.

17. The method of claim 16, wherein the pathological process is the inhibition of nerve growth or elongation.

18. The method of claim 17, wherein the pathological process is the inhibition of nerve growth or elongation as a result of nerve injury or damage.

19. The method of any one of claim 16, wherein the iRNA agent is administered in an amount sufficient to reduce the expression of RhoA in a cell or tissue of the subject.

20. The method of any one of claim 16, wherein the subject is a human.

21. A pharmaceutical composition, comprising:a.) an iRNA agent of any one of claim 1; andb.) a pharmaceutically acceptable carrier.

22. A cell comprising an iRNA agent of any one of claim 1.

23. A method for inhibiting the expression of a RhoA gene in a cell, the method comprising:(a) introducing into the cell an iRNA agent of any one of claim 1; and(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the RhoA gene, thereby inhibiting expression of the RhoA gene in the cell.

Images