RNA Interference Mediated Inhibition of MAP Kinase Gene Expression or Expression of Genes Involved in MAP Kinase Pathway Using Short Interfering Nucleic Acid (SiNA)

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR § 1.52(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “SequenceListing45USCNT”, created on Aug. 27, 2008, which is 468,031 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases that respond to the modulation of mitogen activated protein kinase (MAP kinase) gene expression and/or activity. The present invention also concerns compounds, compositions, methods relating to the modulation of expression or activity of genes involved in the MAP kinase pathway. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against genes involved in the Jun amino-terminal kinase (JNK), p38, and/or ERK pathway, such as c-JUN. More specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Jun amino-terminal kinase (JNK), p38, ERK, and/or c-JUN genes.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in siRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describe specific chemically-modified siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using RNAi. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (greater than 25 nucleotide) constructs that mediate RNAi.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods useful for modulating the expression of genes associated with mitogen activated protein kinase (MAP kinase) gene expression pathways (see for example FIG. 12) by RNA interference (RNAi) using short interfering nucleic acid (siNA) molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of MAP kinase genes, including c-JUN, JNK genes such as JNK1 and JNK2, ERK genes such as ERK1 and ERK2, and p38 genes. A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating telomerase gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding MAP kinase proteins, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as MAP kinases. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary MAP kinases such as JNK1 (also referred to as MAPK8, for example Genbank Accession No. NM_—002750), p38 (also referred to as MAPK14, for example Genbank Accession No. NM_—139012), ERK2 (also referred to as MAPK1, for example Genbank Accession No. NM_—002745), and ERK1 (also referred to as MAPK3, for example Genbank Accession XM_—055766) genes. However, the various aspects and embodiments are also directed to other MAP kinases referred to by Accession number in Table 1 and other genes involved in MAP kinase pathways such those genes encoding c-JUN (for example Genbank Accession No. NM_—002228), TNF-alpha (for example Genbank Accession No. M10988), interleukins such as IL-8 (for example Genbank Accession No. M68932), and activating proteins such as AP-1 (for example Genbank Accession No. NM_—013277). The various aspects and embodiments are also directed to other genes that are involved in the MAP kinase pathways of gene expression. Those additional genes can be analyzed for target sites using the methods described for MAP kinase genes herein. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

In one embodiment, the invention features a siNA molecule that down-regulates expression of a MAP kinase gene, for example, wherein the MAP kinase gene comprises MAP kinase encoding sequence (e.g., c-JUN, JNK1, JNK2, p38, ERK1, or ERK2).

In one embodiment, the invention features a siNA molecule having RNAi activity against MAP kinase RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having MAP kinase encoding sequence, such as those sequences having MAP kinase GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against MAP kinase RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having other MAP kinase encoding sequence, such as mutant MAP kinase genes, splice variants of MAP kinase genes, and other MAP kinase ligands and receptors. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention.

In another embodiment, the invention features a siNA molecule having RNAi activity against a MAP kinase gene, wherein the siNA molecule comprises nucleotide sequence complementary to nucleotide sequence of a MAP kinase gene, such as those MAP kinase sequences having GenBank Accession Nos. shown in Table I or other MAP kinase encoding sequence, such as mutant MAP kinase genes, splice variants of MAP kinase genes, and other MAP kinase ligands and receptors. In another embodiment, a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a MAP kinase gene and thereby mediate silencing of MAP kinase gene expression, for example, wherein the siNA mediates regulation of MAP kinase gene expression by cellular processes that modulate the chromatin structure of the MAP kinase gene and prevent transcription of the MAP kinase gene.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule, that is complementary to a nucleotide sequence or portion of sequence of a MAP kinase gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a MAP kinase gene sequence or a portion thereof.

In one embodiment, the antisense region of ERK2 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1-163, or 1113-1116. The antisense region can also comprise sequence having any of SEQ ID NOs. 164-326, 1133-1136, 1141-1144, or 1149-1152. In another embodiment, the sense region of ERK2 siNA constructs can comprise sequence having any of SEQ ID NOs. 1-163, 1113-1116, 1129-1132, 1137-1140, or 1145-1148.

In one embodiment, the antisense region of ERK1 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 327-431, or 1117-1120. The antisense region can also comprise sequence having any of SEQ ID NOs. 432-536, 1157-1160, 1165-1168, or 1173-1176. In another embodiment, the sense region of ERK1 siNA constructs can comprise sequence having any of SEQ ID NOs. 327-431, 1117-1120, 1153-1156, 1161-1164, or 1169-1172.

In one embodiment, the antisense region of JNK1 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 537-615 or 1121-1124. The antisense region can also comprise sequence having any of SEQ ID NOs. 616-694, 1181-1184, 1189-1192, 1197-1200, 1237, 1239, 1241, 1243, 1245, or 1246. In another embodiment, the sense region of JNK1 constructs can comprise sequence having any of SEQ ID NOs. 537-615, 1121-1124, 1177-1180, 1185-1188, 1193-1196, 1236, 1238, 1240, 1242, or 1244. The sense region can comprise a sequence of SEQ ID NO. 1225 and the antisense region can comprise a sequence of SEQ ID NO. 1226. The sense region can comprise a sequence of SEQ ID NO. 1227 and the antisense region can comprise a sequence of SEQ ID NO. 1228. The sense region can comprise a sequence of SEQ ID NO. 1229 and the antisense region can comprise a sequence of SEQ ID NO. 1230. The sense region can comprise a sequence of SEQ ID NO. 1231 and the antisense region can comprise a sequence of SEQ ID NO. 1232. The sense region can comprise a sequence of SEQ ID NO. 1233 and the antisense region can comprise a sequence of SEQ ID NO. 1234. The sense region can comprise a sequence of SEQ ID NO. 1231 and the antisense region can comprise a sequence of SEQ ID NO. 1235.

In one embodiment, the antisense region of p38 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 695-903 or 1125-1128. The antisense region can also comprise sequence having any of SEQ ID NOs. 904-1112, 1205-1208, 1213-1216, or 1221-1224. In another embodiment, the sense region of p38 siNA constructs can comprise sequence having any of SEQ ID NOs. 695-903, 1125-1128, 1201-1204, 1209-1212, or 1217-1220.

In one embodiment, the antisense region of c-JUN siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1247-1427 or 1609-1616. In one embodiment, the antisense region of c-JUN siNA constructs can comprise sequence having any of SEQ ID NOs. 1428-1608, 1625-1632, 1641-1648, 1657-1664, 1673-1680, 1698, 1700, 1702, 1705, 1707, 1709, 1711, or 1714. In another embodiment, the sense region of c-JUN siNA constructs can comprise sequence having any of SEQ ID NOs. 1247-1427, 1609-1616, 1617-1624, 1633-1640, 1649-1656, 1665-1672, 1697, 1699, 1701, 1703, 1704, 1706, 1708, 1710, 1712, or 1713.

In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-1714. The sequences shown in SEQ ID NOs: 1-1714 are not limiting. A siNA molecule of the invention can comprise any contiguous MAP kinase sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23, 24 or 25 contiguous MAP kinase nucleotides).

In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables III and IV and described herein can be applied to any siRNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a MAP kinase protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a MAP kinase protein, and wherein said siNA further comprises a sense region having about 19 to about 29 nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a MAP kinase protein or a portion thereof. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a MAP kinase gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a MAP kinase protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a MAP kinase gene or a portion thereof.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a MAP kinase gene. Because MAP kinase genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of MAP kinase genes (and associated receptor or ligand genes) or alternately specific MAP kinase genes by selecting sequences that are either shared amongst different MAP kinase targets or alternatively that are unique for a specific MAP kinase target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of MAP kinase RNA sequence having homology between several MAP kinase genes so as to target several MAP kinase genes (e.g., different MAP kinase isoforms, splice variants, mutant genes etc.) with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific MAP kinase RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplexes with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for MAP kinase expressing nucleic acid molecules, such as RNA encoding a MAP kinase protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

In one embodiment, a siNA molecule of the invention does not contain any ribonucleotides. In another embodiment, a siNA molecule of the invention comprises one or more ribonucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of the MAP kinase gene or a portion thereof, and wherein the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the MAP kinase gene or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of the MAP kinase gene or a portion thereof, and wherein the siNA further comprises a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the MAP kinase gene or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the antisense region and the sense region each comprise about 19 to about 23 nucleotides, and wherein the antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the MAP kinase gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the MAP kinase gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides, or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment of any of the above described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of RNA encoded by the MAP kinase gene and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In another embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. In an alternative embodiment, the antisense region comprises a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments each comprising 21 nucleotides, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the MAP kinase gene. In another embodiment, 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the MAP kinase gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a MAP kinase RNA sequence (e.g., wherein said target RNA sequence is encoded by a MAP kinase gene or a gene involved in the MAP kinase pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 21 nucleotides long.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to down-regulate expression of a MAP kinase gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long.

The invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of a MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, the nucleotide sequence of the antisense strand of the double-stranded siNA molecule is complementary to the nucleotide sequence of the MAP kinase RNA which encodes a protein or a portion thereof. In one embodiment, each strand of the siNA molecule comprises about 19 to about 29 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In another embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In one embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In one embodiment, wherein the sense strand comprises a 3′-end and a 5′-end, a terminal cap moiety (e.g., an inverted deoxy abasic moiety) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In one embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In one embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In one embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group. In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the 5′-untranslated region or a portion thereof of the MAP kinase RNA. In another embodiment, the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the MAP kinase RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein each of the two strands of the siNA molecule comprises 21 nucleotides. In one embodiment, about 19 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule and at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule are 2′-deoxy-pyrimidines, such as 2′-deoxy-thymidine. In another embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 19 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the MAP kinase RNA or a portion thereof. In another embodiment, 21 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the MAP kinase RNA or a portion thereof.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention and a pharmaceutically acceptable carrier or diluent.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to an RNA or DNA sequence encoding MAP kinase and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y, and Z are optionally not all O.

The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:

In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:

wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention.

In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0 and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the siNA comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and for example where one or more purine nucleotides present in the sense region are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides), and wherein inverted deoxy abasic modifications are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages.

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example, in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sunday, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclic phosphate). In another embodiment, the single stranded siNA molecule of the invention comprises about 19 to about 29 nucleotides. In yet another embodiment, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the MAP kinase genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the MAP kinase genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients. In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the MAP kinase gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the MAP kinase genes in the organism.

In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) contacting the siNA molecule with a cell in vitro or in vivo under conditions suitable to modulate the expression of the MAP kinase genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) contacting the siNA molecule with a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the MAP kinase gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the MAP kinase genes in the organism.

In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising contacting the organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the MAP kinase gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising contacting the organism with one or more siNA molecules of the invention under conditions suitable to modulate the expression of the MAP kinase genes in the organism.

The siNA molecules of the invention can be designed to down-regulate or inhibit target (MAP kinase) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as MAP kinase family genes. As such, siNA molecules targeting multiple MAP kinase targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down-regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example MAP kinase genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 419); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target MAP kinase RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 7 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of MAP kinase RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target MAP kinase RNA sequence. The target MAP kinase RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating a MAP kinase gene target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a MAP kinase target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the MAP kinase target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating a MAP kinase target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a MAP kinase target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the MAP kinase target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human, animal, plant, insect, bacterial, viral or other sources, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a MAP kinase target gene in a biological system, including, for example, in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one MAP kinase target gene in a biological system, including, for example, in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against a MAP kinase in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against MAP kinase comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a MAP kinase target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a MAP kinase target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In another embodiment, the invention features a method for generating siNA molecules against MAP kinase with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Tables II, III, and IV herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiment, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure to alter gene expression (see, for example, Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.

By “gene” or “target gene” is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

By “MAP kinase” is meant, any mitogen activated protein kinase (MAP kinase) polypeptide, protein and/or a polynucleotide encoding a MAP kinase protein (such as polynucleotides referred to by Genbank Accession number in Table I or any other MAP kinase transcript derived from a MAP kinase gene, e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38). As used herein, the term “MAP kinase gene” is meant to refer to any polynucleotide included in a group of MAP kinase genes, such as c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38).

By “MAP kinase protein” is meant, any mitogen activated protein kinase (MAP kinase) peptide or protein or a component thereof, wherein the peptide or protein is encoded by a MAP kinase gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38).

By “highly conserved sequence region” is meant a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

The siRNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications or other conditions, including oncology and proliferation related indications and conditions such as multidrug resistant cancers, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of the retina, cancers of the esophagus, multiple myeloma, ovarian cancer, melanoma, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, pancreatic cancer, prostate cancer, glioblastoma; obesity and insulin resistance (e.g. type I and II diabetes); inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other diseases or conditions that are related to or will respond to the levels of MAP kinase in a cell or tissue, alone or in combination with other therapies. The reduction of MAP kinase expression (specifically MAP kinase gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 17 to about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40, 41, 42, 43 or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Tables III and IV and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein (e.g., cancers and other proliferative conditions). For example, to treat a particular disease or condition, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4 A-F, the modified internucleotide linkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to a c-JUN siNA sequence. Such chemical modifications can be applied to any sequence herein, such as any MAP kinase sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined MAP kinase target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a MAP kinase target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined MAP kinase target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows a non-limiting example of reduction of p38 mRNA in A549 cells mediated by siNAs that target p38 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs comprising ribonucleotides and 3′-terminal dithymidine caps was compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, the siNA constructs significantly reduce p38 RNA expression.

FIG. 13 shows a non-limiting example of reduction of JNK1 mRNA in A549 cells mediated by siNAs that target JNK1 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs comprising ribonucleotides and 3′-terminal dithymidine caps was compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, the siNA constructs significantly reduce JNK1 RNA expression.

DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limited to siRNA only and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 minute coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5 minute coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 hours, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄.HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example, proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or can be present on both termini. Non-limiting examples of the 5′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR, where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to treat, for example, oncology and proliferation related indications and conditions such as multidrug resistant cancers, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of the retina, cancers of the esophagus, multiple myeloma, ovarian cancer, melanoma, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, pancreatic cancer, prostate cancer, glioblastoma; obesity and insulin resistance (e.g. type I and II diabetes); inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other diseases or conditions that are related to or will respond to the levels of MAP kinase in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cells producing excess MAP kinase.

By “pharmaceutically acceptable formulation” is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by an enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

MAP Kinase Biology and Biochemistry

The mitogen-activated protein kinases (MAPKs) have been at the forefront of a rapid advance in the understanding of cellular events in growth factor and cytokine receptor signaling. The MAP kinases (also referred to as extracellular signal-regulated protein kinases, or ERKs) are the terminal enzymes in a three-kinase cascade. The reiteration of three-kinase cascades for related but distinct signaling pathways gave rise to the concept of a MAPK pathway as a modular, multifunctional signaling element that acts sequentially within one pathway, where each enzyme phosphorylates and thereby activates the next member in the sequence. A typical MAPK pathway thus consists of three protein kinases: a MAPK kinase kinase (or MEKK) that activates a MAPK kinase (or MEK) which, in turn, activates a MAPK/ERK enzyme. Each of the MAPK/ERK, JNK and p38 cascades consists of a three-enzyme module that includes MEKK, MEK and an ERK or MAPK superfamily member. A variety of extracellular signals triggers initial events upon association with their respective cell surface receptors and this signal is then transmitted to the interior of the cell where it activates the appropriate cascades (see for example FIG. 12).

The identification of distinct MAPK cascades that are conserved across all eukaryotes indicates that the MAPK module has been adapted for interpretation of a diverse array of extracellular signals. Although mitogen activation of the MAPK subfamily (e.g., ERK1 and ERK2) has dominated efforts to understand MAPK signaling, increasing appreciation of the role of the stress-activated kinases, JNK and p38, illustrates the diverse nature of the MAPK superfamily of enzymes. Although sequence similarities among components of the individual MAPK modules used for activation of ERK1/2, JNKs and p38 are considerable, the fidelity that is maintained in order to translate specific extracellular signals into discrete physiological responses illustrates the selective adaptation of each MAPK pathway. The MAPK superfamily of enzymes is a critical component cellular regulative processes that coordinates incoming signals generated by a variety of extracellular and intracellular mediators. Specific phosphorylation and activation of enzymes in the MAPK pathway transmits the signal down the cascade, resulting in phosphorylation of many proteins with substantial regulatory functions throughout the cell, including other protein kinases, transcription factors, cytoskeletal proteins and other enzymes. The diversity of signals that culminates in MAPK activation indicates that these enzymes are not dedicated to regulation of any single growth factor, hormone or cytokine system. Instead, MAPKs—like cAMP-dependent protein kinase (PKA) and Ca²⁺- and phospholipid-dependent protein kinases (PKC) serve many signaling purposes. Because activation of the MAPK pathways are triggered to varying extents by a large number of receptor systems, temporal and spatial differences are critical to determining ligand- and cell-type-specific functions.

Following activation of cells with an appropriate extracellular stimuli, the signal is transmitted to the canonical MAPK module comprising three protein kinases. The progression of events for each enzyme cascade is the same, although specific isoforms of each enzyme appear to confer the required specificity within each pathway. The first enzyme in the module is a MEKK enzyme, of which Raf and its isoforms are one example. The MEKK enzymes comprise Ser/Thr protein kinases that activate the MEK enzymes by phosphorylating two serine or threonine residues within a Ser-X-X-X-Ser/Thr motif. Once activated, the MEK enzymes, which are hybrid function Ser/Thr/Tyr protein kinases, phosphorylate the MAPK/ERK enzymes on Thr and Tyr residues within the Thr-X-Tyr (TXY) consensus sequence. A critical and common feature of the MAPK superfamily of enzymes is that they are activated upon dual phosphorylation within a TXY consensus sequence present in the activation loop of the catalytic domain. The central amino acid differs for each MAPK superfamily member, corresponding to Glu for ERK1/2, Gly for p38/HOG and Pro for JNK/SAPK, although MEK specificity is not limited to these particular residues. Phosphorylation at only one of the two positions does not appear to activate the enzyme, although it may prime the kinase domain for receipt of the second phosphorylation event.

ERK1 and ERK2 were the first members of the MAPK superfamily whose cDNAs were cloned and the signaling cascades that lead to their activation characterized. Potent activation of ERK1 and ERK2 can be initiated through activation of transmembrane receptors with intrinsic protein tyrosine kinase (PTK) activity. Binding of extracellular ligands to their respective cell surface receptors results in receptor autophosphorylation and enhanced PTK activity. The subsequent association of the Src homology 2 (SH2) domains of adaptor proteins such as Grb2 and Shc with the autophosphorylated receptors, or with additional docking proteins, provides the molecular interactions that bring the required signal transduction molecules into close proximity with each other. Receptors without intrinsic PTK activity but which comprise sites for tyrosine phosphorylation can also activate the cascade via association of their phosphotyrosine residues with adaptor molecules. For example, the SH3 domain of Grb2 binds a proline-rich region of the guanine nucleotide-exchange protein SOS which, in turn, increases the association of Ras with GTP. The GTP-bound form of Ras binds to Raf (a MAPK kinase) isoforms, including C-Raf-1, B-Raf and A-Raf. This action targets Raf to the membrane, where its protein kinase activity is increased by phosphorylation. MAPK kinases (MEK1 and MEK2), are phosphorylated and activated by Raf. MEK1 and MEK2 are dual-specificity protein kinases that dually phosphorylate the ERK enzymes (corresponding to Thr¹⁸³and Tyr¹⁸⁵of p42ERK2), thereby increasing their enzymatic activity by approximately 1,000-fold over the activity found with the basal or monophosphorylated forms. Phosphorylation of these residues causes closure of the kinase active site and induces conformational changes necessary for high activity.

MAPK mutants, lacking either a lysine required for catalytic activity or the prerequisite TXY phosphorylation sites, can inhibit signaling by the native enzymes in cells. In the case of ERK1 and ERK2, these mutants have been used with repeated success. For example, mutant ERK2 completely blocks proliferation in response to epidermal growth factor (EGF) and v-Raf, and partially blocks induction by serum or small t antigen. ERK1 antisense mRNA and an ERK1 phosphorylation site mutants interfere with thrombin-induced transcription as well as serum-dependent proliferation. These findings suggest an essential role in proliferation and transformation for the ERK/MAPK pathway.

The JNK/SAPK and p38/HOG pathways are activated by ultraviolet light, cytokines, osmotic shock, inhibitors of DNA, RNA, and protein synthesis, and to a lesser extent by certain growth factors. This spectrum of regulators suggests that the enzymes are transducers of a variety of cellular stress responses. In contrast to activation of ERK1 and ERK2, upstream signal transduction mechanisms for the JNK and p38 cascades are less well understood. When transfected into mammalian cells, a diverse group of protein kinases including the mixed lineage kinases (MLKs) and relatives of the yeast Step 20p, such as the p21-activated kinases (PAKs) and germinal center kinase (GCK), cause activation of JNK/SAPK. Similarly, GTP-bound forms of the small GTP-binding proteins, Rac and Cdc42, activate the JNK/SAPK pathway and, to a lesser extent, the p38 pathway. Direct activation of both pathways by PAKs also has been demonstrated, suggesting that PAKs can be the relevant effectors for these small G proteins. The PAKs are homologs of the yeast kinases Step 20p and Shk1, enzymes upstream of the MAPK modules in yeast pheromone response pathways. Both yeast and mammalian protein kinases contain a binding site for Rac/Cdc42 and share the property of being activated in vitro through association with these small G proteins when in their GTP-bound states. In yeast, Step 20p is thought to phosphorylate and activate the MEKK isoform Ste11p, suggesting that MEKKs may be PAK targets. This summary of MAP kinase pathways has been adapted from Cobb and Schaefer, 1996, Promega Notes Magazine Number 59, page 37.

The regulation of c-Jun transcriptional activity by Jun N-terminal kinase (JNK), ERK1, ERK2, and p38 kinases has become a paradigm for the understanding of how mitogen-activated protein (MAP) kinase signaling pathways elicit specific changes in gene transcription through selective phosphorylation of nuclear transcription factors. Selective phosphorylation of c-Jun by JNK is detected by a specific docking motif in c-Jun, the delta region, which enables JNK to physically interact with c-Jun. Analogous MAP kinase docking motifs have subsequently been found in several other transcription factors, indicating that this is a general mechanism for ensuring the specificity of signal transduction. Furthermore, genetic and biochemical studies in mice, flies and cultured cells have provided evidence that signals relayed by JNK through c-Jun regulate a wide range of cellular processes including cell proliferation, tumorigenesis, apoptosis and embryonic development. Despite these advances, in most cases, the genes or programs of gene expression downstream of JNK and c-Jun, which control these processes, have yet to be defined. One important process that is associated with JNK gene expression is the development of insulin resistance in obese subjects.

Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus in mammals. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin activity in cultured cells and are activated by inflammatory cytokines and free fatty acids molecules that have been implicated in the development of type 2 diabetes. Hirosumi et al, 2002, Nature, 420, 333-336, demonstrate that JNK activity is abnormally elevated in obesity. Furthermore, Hirosumi et al, supra have shown that an absence of JNK1 results in decreased adiposity with significantly improved insulin sensitivity and enhanced insulin receptor capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and as such, provides a potential target for nucleic acid based therapeutics that modulate JNK gene expression.

The transcription factor and oncogene, c-JUN, is implicated in several critical cell processes including cell proliferation, cell survival, and oncogenic transformation. Although it is broadly expressed in a wide variety of cell types, it plays an especially important role in hepatocytes. However, the precise role played by c-JUN in hepatocytes seems to depend on the differentiation state of this cell type. Adult differentiated hepatocytes depend on c-JUN for progression through the cell cycle. Deletion of c-JUN reduces the proliferation capacity of hepatocytes following partial hepatectomy. c-JUN is thought to be major component in the development of human hepatocellular carcinoma (HCC). HCC is the most common form of primary liver cancer. Chronic HCV infection is a major risk factor for HCC.

The role of c-JUN in liver cancer has recently been investigated (Eferl et al., 2003, Cell, 112, 181). These investigators deleted c-JUN and then induced liver cancer by chemical carcinogenesis. They observed that deletion of c-JUN dramatically interfered with liver tumor formation. Animal survival was markedly worse in c-JUN wild-type animals relative to deletion mutants. In particular, the number of apoptotic cells increased about five fold in tumors in the c-JUN deletion strain relative to the wild-type animals. Importantly, levels of the pro-apoptotic gene products such as p53 and noxa were elevated in the c-JUN deletion strain. c-JUN is likely to antagonize other pro-apoptotic genes such as TNF-a. Thus, by blocking p53 and its large family of dependent genes, c-JUN seems to promote tumor formation. Since a large fraction of chronically infected HCV patients develop hepatocellular carcinoma, c-JUN provides an attractive target for treating HCV infected patients to prevent or ameliorate hepatocellular carcinoma.

Based upon the current understanding of MAP kinase pathways, the modulation of MAP kinase pathways is instrumental in the development of new therapeutics in, for example, the fields of inflammation, oncology, and metabolism. As such, modulation of a specific MAP kinase pathway using small interfering nucleic acid (siNA) mediated RNAi represents a novel approach to the treatment and study of diseases and conditions related to a specific MAP kinase activity and/or gene expression.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1
Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak Ig cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H₂O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H₂O followed by 1 CV 1M NaCl and additional H₂O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2
Identification of Potential siNA Target Sites in Any RNA Sequence

The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3
Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.

In an alternate approach, a pool of siNA constructs specific to a MAP kinase target sequence is used to screen for target sites in cells expressing MAP kinase (e.g., c-JUN) RNA, such as human kidney fibroblast (e.g., 293 cells), HeLa, or HepG2 cells. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such as pool is a pool comprising sequences having sense sequences comprising SEQ ID NOs. 1247-1427 and antisense sequences comprising SEQ ID NOs. 1428-1608 respectively. 293, HeLa, or HepG2 cells expressing MAP kinase (e.g., c-JUN) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with MAP kinase (e.g., c-JUN) inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased MAP kinase (e.g., c-JUN) mRNA levels or decreased MAP kinase (e.g., c-JUN) protein expression), are sequenced to determine the most suitable target site(s) within the target MAP kinase (e.g., c-JUN) RNA sequence.

Example 4
MAP Kinase Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the MAP kinase (e.g., c-JUN) RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5
Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.

Example 6
RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting MAP kinase (e.g., c-JUN) RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with MAP kinase target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate MAP kinase expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 μM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Inager® quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the MAP kinase RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the MAP kinase RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 7
Nucleic Acid Inhibition of MAP Kinase Target RNA In Vivo

siNA molecules targeted to the human MAP kinase (e.g., c-JUN) RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the MAP kinase (e.g., c-JUN) RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting MAP kinase (e.g., c-JUN). First, the reagents are tested in cell culture, for example using cultured human kidney fibroblast cells (e.g., 293, HeLa, or HepG2 cells), to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the MAP kinase (e.g., c-JUN) target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, 293, HeLa, or HepG2 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 Taqman®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., 293, HeLa, or HepG2 cells) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGM basal media (BioWhittaker) at 37° C. for 30 mins in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

Taqman and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1×TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 mM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AmpliTaq Gold (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA in parallel TaqMan reactions. For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control c RNA allularnd values are represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8
Models Useful to Evaluate the Down-Regulation of MAP Kinase Gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) expression
Cell Culture

There are numerous cell culture systems that can be used to analyze reduction of MAP kinase levels either directly or indirectly by measuring downstream effects. For example, cultured human kidney fibroblast cells (e.g., 293 cells), HeLa, or HepG2 cells can be used in cell culture experiments to assess the efficacy of nucleic acid molecules of the invention. As such, cells treated with nucleic acid molecules of the invention (e.g., siNA) targeting MAP kinase RNA would be expected to have decreased MAP kinase expression capacity compared to matched control nucleic acid molecules having a scrambled or inactive sequence. In a non-limiting example, 293, HeLa, or HepG2 cells are cultured and MAP kinase expression is quantified, for example by time-resolved immuno fluorometric assay. MAP kinase messenger-RNA expression is quantitated with RT-PCR in cultured cells. Untreated cells are compared to cells treated with siNA molecules transfected with a suitable reagent, for example a cationic lipid such as lipofectamine, and MAP kinase protein and RNA levels are quantitated. Dose response assays are then performed to establish dose dependent inhibition of MAP kinase expression. In another non-limiting example, cell culture experiments are carried out as described by Aguirre et al., 2000, J. Biol. Chem., 275, 9047-9054.

In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In one embodiment, siNA molecules of the invention are complexed with cationic lipids for cell culture experiments. siNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25° C.) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. siNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-MAP kinase agents in animal models is an important prerequisite to human clinical trials. Obesity and type 2 diabetes are the most prevalent and serious metabolic diseases in that they affect more than 50% of adults in the USA. These conditions are associated with a chronic inflammatory response characterized by abnormal inflammatory cytokine production, increased acute-phase reactants and other stress-induced molecules. Many of these alterations seem to be initiated and to reside within adipose tissue. Elevated production of tumor necrosis factor (TNF)-alpha by adipose tissue decreases sensitivity to insulin and has been detected in several experimental obesity models and obese humans. Free fatty acids (FFAs) are also implicated in the etiology of obesity-induced insulin resistance and diabetes. Because both TNF-alpha and FFAs are potent MAP kinase activators, Hirosumi et al., 2002, Nature, 420, 333-336 determined whether obesity is associated with alterations in stress-activated and inflammatory responses through this pathway and whether MAP kinases are causally linked to aberrant metabolic control in this state. In this study, Hirosumi et al., describe dietary and genetic (ob/ob) mouse models of obesity useful in evaluating MAP kinase gene expression. Such transgenic mice are useful as models for obesity and insulin resistance and can be used to identify nucleic acid molecules of the invention that modulate MAP kinase gene (e.g., ERK1, ERK2, JNK1, JNK2, and/or p38) expression and gene function toward therapeutic use in treating obesity and insulin resistance (e.g. type I and II diabetes).

Because mitogen activated protein kinases (MAP kinases) are constituents of numerous signal transduction pathways, and are activated by protein kinase cascades, intense efforts are under way to develop and evaluate compounds that target components of MAPK pathways. Several of these inhibitors are effective in animal models of disease and have advanced to clinical trials for the treatment of inflammatory diseases, metabolic diseases, autoimmune diseases and cancer. The clinical utility of specifically targeting MAP kinase genes (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) can be studied in animal models and clinical studies of inflammatory diseases, metabolic diseases, autoimmune diseases and cancer (see for example English et al., 2002, Trends in Pharmacological Sciences, 23, 40-45).

Example 9
RNAi Mediated Inhibition of p38 RNA Expression

siNA constructs are tested for efficacy in reducing p38 RNA expression in, for example in A549 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs was determined.

In a non-limiting example, siNA constructs were screened for activity (see FIG. 12) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in FIG. 12, the siNA constructs significantly reduce p38 RNA expression. Leads generated from such a screen are then further assayed. In a non-limiting example, siNA constructs comprising chemical modifications described herein (e.g., having modifications comprising Formulae I-VII and/or those modifications described in Table IV are assayed for activity. These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).

Example 10
RNAi Mediated Inhibition of p38 RNA Expression

siNA constructs are tested for efficacy in reducing JNK1 RNA expression in, for example in A549 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs was determined.

In a non-limiting example, siNA constructs were screened for activity (see FIG. 13) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in FIG. 13, the siNA constructs significantly reduce p38 RNA expression. Leads generated from such a screen are then further assayed. In a non-limiting example, siNA constructs comprise chemical modifications described herein (e.g., having modifications comprising Formulae I-VII and/or those modifications described in Table IV are assayed for activity). These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).

Example 11
Indications

The present body of knowledge in MAP kinase research indicates the need for methods and compounds that can regulate MAP kinase gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) product expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used to treat obesity and insulin resistance (e.g. type I and II diabetes), oncology and proliferation related indications and conditions, including cancers of the lung, bladder, colon, breast, prostate, retina, larynx, esophagus, liver (e.g., hepatocellular carcinoma), and ovary, along with lymphomas, melanomas and glioblastomas, inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other disease that responds to modulation of MAP kinase expression.

Troglitazone, insulin, and PTP-1B modulators are non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention for treating obesity and diabetes. The use of radiation treatments and chemotherapeutics such as Gemcytabine and cyclophosphamide are non-limiting examples of chemotherapeutic agents that can also be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention for oncology therapeutic applications. Those skilled in the art will recognize that other anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention. Such compounds and therapies are well known in the art (see for example Cancer: Principles and Practice of Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J.B. Lippincott Company, Philadelphia, USA; incorporated herein by reference) and include, without limitations, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjunction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen, Herceptin; IMC C225; ABX-EGF: and combinations thereof are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA) of the instant invention. In addition, treatment of HCV infected subjects with siNA molecules of the invention targeting c-JUN or other MAP kinases involved in the maintenance or development of hepatocellular carcinoma can be combined with anti-viral compounds, such as siNA molecules targeting HCV RNA or other antiviral compounds known in the art (e.g., interferons, nucleoside analogs etc.). Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) are hence within the scope of the instant invention.

Example 12
Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one ca MAP nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE I

MAP kinase Accession Numbers

NM_002745

Homo sapiens mitogen-activated protein kinase 1 (MAPK1), transcript variant 1, mRNA.

NM_138957

Homo sapiens mitogen-activated protein kinase 1 (MAPK1), transcript variant 2, mRNA.

X60188
Human ERK1 mRNA for protein serine/threonine kinase (MAPK3).

XM_055766

Homo sapiens mitogen-activated protein kinase 3 (MAPK3), mRNA

NM_002747

Homo sapiens mitogen-activated protein kinase 4 (MAPK4), mRNA

XM_165662

Homo sapiens Mitogen-activated protein kinase 4 (Extracellular signal-regulated kinase 4) (ERK-4) (MAP kinase

isoform p63) (p63-MAPK) (LOC220131), mRNA

NM_002748

Homo sapiens mitogen-activated protein kinase 6 (MAPK6), mRNA.

XM_166057

Homo sapiens Mitogen-activated protein kinase 6 (Extracellular signal-regulated kinase 3) (ERK-3) (MAP kinase

isoform p97) (p97-MAPK) (LOC220839), mRNA

XM_035575

Homo sapiens mitogen-activated protein kinase 6 (MAPK6), mRNA

NM_139033

Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 1, mRNA

NM_139032

Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 2, mRNA

NM_002749

Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 3, mRNA

NM_139034

Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 4, mRNA

NM_139049

Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 1, mRNA.

NM_002750

Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 2, mRNA.

NM_139046

Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 3, mRNA.

NM_139047

Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 4, mRNA.

NM_002752

Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 1, mRNA.

NM_139068

Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 2, mRNA.

NM_139069

Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 3, mRNA.

NM_139070

Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 4, mRNA.

NM_002753

Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 1, mRNA

NM_138982

Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 2, mRNA

NM_138980

Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 3, mRNA

NM_138981

Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 4, mRNA

NM_002751

Homo sapiens mitogen-activated protein kinase 11 (MAPK11), transcript variant 1, mRNA.

NM_138993

Homo sapiens mitogen-activated protein kinase 11 (MAPK11), transcript variant 2, mRNA.

NM_002969

Homo sapiens mitogen-activated protein kinase 12 (MAPK12), mRNA.

NM_002754

Homo sapiens mitogen-activated protein kinase 13 (MAPK13), mRNA.

NM_001315

Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 1, mRNA.

NM_139012

Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 2, mRNA.

NM_139013

Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 3, mRNA.

NM_139014

Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 4, mRNA.

NM_002755

Homo sapiens mitogen-activated protein kinase kinase 1 (MAP2K1), mRNA

NM_030662

Homo sapiens mitogen-activated protein kinase kinase 2 (MAP2K2), mRNA

NM_002756

Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant A, mRNA

NM_145109

Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant B, mRNA

NM_145110

Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant C, mRNA

XM_008654

Homo sapiens mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA

NM_003010

Homo sapiens mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA

NM_145160

Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant A, mRNA

NM_002757

Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant B, mRNA

NM_145161

Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant C, mRNA

NM_145162

Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant D, mRNA

XM_113313

Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), mRNA

NM_002758

Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 1, mRNA

NM_031988

Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 2, mRNA

NM_005043

Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant A, mRNA

NM_145185

Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant B, mRNA

NM_145329

Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant C, mRNA

AF042838

Homo sapiens mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mRNA

NM_006609

Homo sapiens mitogen-activated protein kinase kinase kinase 2 (MAP3K2), mRNA

NM_002401

Homo sapiens mitogen-activated protein kinase kinase kinase 3 (MAP3K3), mRNA

NM_005922

Homo sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 1, mRNA

NM_006724

Homo sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 2, mRNA

NM_005923

Homo sapiens mitogen-activated protein kinase kinase kinase 5 (MAP3K5), mRNA

NM_004672

Homo sapiens mitogen-activated protein kinase kinase kinase 6 (MAP3K6), mRNA

NM_003188

Homo sapiens mitogen-activated protein kinase kinase kinase 7 (MAP3K7), mRNA

NM_005204

Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8), mRNA

AF251442

Homo sapiens mitogen-activated protein kinase kinase kinase 9 (MAP3K9), mRNA

NM_002446

Homo sapiens mitogen-activated protein kinase kinase kinase 10 (MAP3K10), mRNA

NM_002419

Homo sapiens mitogen-activated protein kinase kinase kinase 11 (MAP3K11), mRNA

NM_006301

Homo sapiens mitogen-activated protein kinase kinase kinase 12 (MAP3K12), mRNA

NM_004721

Homo sapiens mitogen-activated protein kinase kinase kinase 13 (MAP3K13), mRNA

NM_003954

Homo sapiens mitogen-activated protein kinase kinase kinase 14 (MAP3K14), mRNA

NM_007181

Homo sapiens mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), mRNA

NM_004579

Homo sapiens mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), mRNA

NM_003618

Homo sapiens mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3), mRNA

NM_004834

Homo sapiens mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), mRNA

NM_006575

Homo sapiens mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5), mRNA

NM_003668

Homo sapiens mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 1, mRNA

NM_139078

Homo sapiens mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 2, mRNA

NM_004635

Homo sapiens mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3), mRNA

NM_004759

Homo sapiens mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 1, mRNA

NM_032960

Homo sapiens mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 2, mRNA

NM_005373

Homo sapiens myeloproliferative leukemia virus oncogene (MPL), mRNA

NM_016848

Homo sapiens neuronal Shc (SHC3), mRNA

NM_002649

Homo sapiens phosphoinositide-3-kinase, catalytic, gamma polypeptide (PIK3CG), mRNA

NM_021003

Homo sapiens protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform (PPM1A), mRNA

NM_003942

Homo sapiens ribosomal protein S6 kinase, 90 kD, polypeptide 4 (RPS6KA4), mRNA

NM_004755

Homo sapiens ribosomal protein S6 kinase, 90 kD, polypeptide 5 (RPS6KA5), mRNA

NM_002228

Homo sapiens v-jun sarcoma virus 17 oncogene homolog (avian) (JUN), mRNA

TABLE II

MAP kinase siNA and Target Sequences

Seq

Seq

Seq

Pos
Target Sequence
ID
UPos
Upper seq
ID
LPos
Lower seq
ID

NM_002745 (MAPK1/ERK2)

3
CCCUCCCUCCGCCCGCCCG
1
3
CCCUCCCUCCGCCCGCCCG
1
21
CGGGCGGGCGGAGGGAGGG
164

21
GCCGGCCCGCCCGUCAGUC
2
21
GCCGGCCCGCCCGUCAGUC
2
39
GACUGACGGGCGGGCCGGC
165

39
CUGGCAGGCAGGCAGGCAA
3
39
CUGGCAGGCAGGCAGGCAA
3
57
UUGCCUGCCUGCCUGCCAG
166

57
AUCGGUCCGAGUGGCUGUC
4
57
AUCGGUCCGAGUGGCUGUC
4
75
GACAGCCACUCGGACCGAU
167

75
CGGCUCUUCAGCUCUCCCG
5
75
CGGCUCUUCAGCUCUCCCG
5
93
CGGGAGAGCUGAAGAGCCG
168

93
GCUCGGCGUCUUCCUUCCU
6
93
GCUCGGCGUCUUCCUUCCU
6
111
AGGAAGGAAGACGCCGAGC
169

111
UCCUCCCGGUCAGCGUCGG
7
111
UCCUCCCGGUCAGCGUCGG
7
129
CCGACGCUGACCGGGAGGA
170

129
GCGGCUGCACCGGCGGCGG
8
129
GCGGCUGCACCGGCGGCGG
8
147
CCGCCGCCGGUGCAGCCGC
171

147
GCGCAGUCCCUGCGGGAGG
9
147
GCGCAGUCCCUGCGGGAGG
9
165
CCUCCCGCAGGGACUGCGC
172

165
GGGCGACAAGAGCUGAGCG
10
165
GGGCGACAAGAGCUGAGCG
10
183
CGCUCAGCUCUUGUCGCCC
173

183
GGCGGCCGCCGAGCGUCGA
11
183
GGCGGCCGCCGAGCGUCGA
11
201
UCGACGCUCGGCGGCCGCC
174

201
AGCUCAGCGCGGCGGAGGC
12
201
AGCUCAGCGCGGCGGAGGC
12
219
GCCUCCGCCGCGCUGAGCU
175

219
CGGCGGCGGCCCGGCAGCC
13
219
CGGCGGCGGCCCGGCAGCC
13
237
GGCUGCCGGGCCGCCGCCG
176

237
CAACAUGGCGGCGGCGGCG
14
237
CAACAUGGCGGCGGCGGCG
14
255
CGCCGCCGCCGCCAUGUUG
177

255
GGCGGCGGGCGCGGGCCCG
15
255
GGCGGCGGGCGCGGGCCCG
15
273
CGGGCCCGCGCCCGCCGCC
178

273
GGAGAUGGUCCGCGGGCAG
16
273
GGAGAUGGUCCGCGGGCAG
16
291
CUGCCCGCGGACCAUCUCC
179

291
GGUGUUCGACGUGGGGCCG
17
291
GGUGUUCGACGUGGGGCCG
17
309
CGGCCCCACGUCGAACACC
180

309
GCGCUACACCAACCUCUCG
18
309
GCGCUACACCAACCUCUCG
18
327
CGAGAGGUUGGUGUAGCGC
181

327
GUACAUCGGCGAGGGCGCC
19
327
GUACAUCGGCGAGGGCGCC
19
345
GGCGCCCUCGCCGAUGUAC
182

345
CUACGGCAUGGUGUGCUCU
20
345
CUACGGCAUGGUGUGCUCU
20
363
AGAGCACACCAUGCCGUAG
183

363
UGCUUAUGAUAAUGUCAAC
21
363
UGCUUAUGAUAAUGUCAAC
21
381
GUUGACAUUAUCAUAAGCA
184

381
CAAAGUUCGAGUAGCUAUC
22
381
CAAAGUUCGAGUAGCUAUC
22
399
GAUAGCUACUCGAACUUUG
185

399
CAAGAAAAUCAGCCCCUUU
23
399
CAAGAAAAUCAGCCCCUUU
23
417
AAAGGGGCUGAUUUUCUUG
186

417
UGAGCACCAGACCUACUGC
24
417
UGAGCACCAGACCUACUGC
24
435
GCAGUAGGUCUGGUGCUCA
187

435
CCAGAGAACCCUGAGGGAG
25
435
CCAGAGAACCCUGAGGGAG
25
453
CUCCCUCAGGGUUCUCUGG
188

453
GAUAAAAAUCUUACUGCGC
26
453
GAUAAAAAUCUUACUGCGC
26
471
GCGCAGUAAGAUUUUUAUC
189

471
CUUCAGACAUGAGAACAUC
27
471
CUUCAGACAUGAGAACAUC
27
489
GAUGUUCUCAUGUCUGAAG
190

489
CAUUGGAAUCAAUGACAUU
28
489
CAUUGGAAUCAAUGACAUU
28
507
AAUGUCAUUGAUUCCAAUG
191

507
UAUUCGAGCACCAACCAUC
29
507
UAUUCGAGCACCAACCAUC
29
525
GAUGGUUGGUGCUCGAAUA
192

525
CGAGCAAAUGAAAGAUGUA
30
525
CGAGCAAAUGAAAGAUGUA
30
543
UACAUCUUUCAUUUGCUCG
193

543
AUAUAUAGUACAGGACCUC
31
543
AUAUAUAGUACAGGACCUC
31
561
GAGGUCCUGUACUAUAUAU
194

561
CAUGGAAACAGAUCUUUAC
32
561
CAUGGAAACAGAUCUUUAC
32
579
GUAAAGAUCUGUUUCCAUG
195

579
CAAGCUCUUGAAGACACAA
33
579
CAAGCUCUUGAAGACACAA
33
597
UUGUGUCUUCAAGAGCUUG
196

597
ACACCUCAGCAAUGACCAU
34
597
ACACCUCAGCAAUGACCAU
34
615
AUGGUCAUUGCUGAGGUGU
197

615
UAUCUGCUAUUUUCUCUAC
35
615
UAUCUGCUAUUUUCUCUAC
35
633
GUAGAGAAAAUAGCAGAUA
198

633
CCAGAUCCUCAGAGGGUUA
36
633
CCAGAUCCUCAGAGGGUUA
36
651
UAACCCUCUGAGGAUCUGG
199

651
AAAAUAUAUCCAUUCAGCU
37
651
AAAAUAUAUCCAUUCAGCU
37
669
AGCUGAAUGGAUAUAUUUU
200

669
UAACGUUCUGCACCGUGAC
38
669
UAACGUUCUGCACCGUGAC
38
687
GUCACGGUGCAGAACGUUA
201

687
CCUCAAGCCUUCCAACCUG
39
687
CCUCAAGCCUUCCAACCUG
39
705
CAGGUUGGAAGGCUUGAGG
202

705
GCUGCUCAACACCACCUGU
40
705
GCUGCUCAACACCACCUGU
40
723
ACAGGUGGUGUUGAGCAGC
203

723
UGAUCUCAAGAUCUGUGAC
41
723
UGAUCUCAAGAUCUGUGAC
41
741
GUCACAGAUCUUGAGAUCA
204

741
CUUUGGCCUGGCCCGUGUU
42
741
CUUUGGCCUGGCCCGUGUU
42
759
AACACGGGCCAGGCCAAAG
205

759
UGCAGAUCCAGACCAUGAU
43
759
UGCAGAUCCAGACCAUGAU
43
777
AUCAUGGUCUGGAUCUGCA
206

777
UCACACAGGGUUCCUGACA
44
777
UCACACAGGGUUCCUGACA
44
795
UGUCAGGAACCCUGUGUGA
207

795
AGAAUAUGUGGCCACACGU
45
795
AGAAUAUGUGGCCACACGU
45
813
ACGUGUGGCCACAUAUUCU
208

813
UUGGUACAGGGCUCCAGAA
46
813
UUGGUACAGGGCUCCAGAA
46
831
UUCUGGAGCCCUGUACCAA
209

831
AAUUAUGUUGAAUUCCAAG
47
831
AAUUAUGUUGAAUUCCAAG
47
849
CUUGGAAUUCAACAUAAUU
210

849
GGGCUACACCAAGUCCAUU
48
849
GGGCUACACCAAGUCCAUU
48
867
AAUGGACUUGGUGUAGCCC
211

867
UGAUAUUUGGUCUGUAGGC
49
867
UGAUAUUUGGUCUGUAGGC
49
885
GCCUACAGACCAAAUAUCA
212

885
CUGCAUUCUGGCAGAAAUG
50
885
CUGCAUUCUGGCAGAAAUG
50
903
CAUUUCUGCCAGAAUGCAG
213

903
GCUUUCUAACAGGCCCAUC
51
903
GCUUUCUAACAGGCCCAUC
51
921
GAUGGGCCUGUUAGAAAGC
214

921
CUUUCCAGGGAAGCAUUAU
52
921
CUUUCCAGGGAAGCAUUAU
52
939
AUAAUGCUUCCCUGGAAAG
215

939
UCUUGACCAGCUGAAACAC
53
939
UCUUGACCAGCUGAAACAC
53
957
GUGUUUCAGCUGGUCAAGA
216

957
CAUUUUGGGUAUUCUUGGA
54
957
CAUUUUGGGUAUUCUUGGA
54
975
UCCAAGAAUACCCAAAAUG
217

975
AUCCCCAUCACAAGAAGAC
55
975
AUCCCCAUCACAAGAAGAC
55
993
GUCUUCUUGUGAUGGGGAU
218

993
CCUGAAUUGUAUAAUAAAU
56
993
CCUGAAUUGUAUAAUAAAU
56
1011
AUUUAUUAUACAAUUCAGG
219

1011
UUUAAAAGCUAGGAACUAU
57
1011
UUUAAAAGCUAGGAACUAU
57
1029
AUAGUUCCUAGCUUUUAAA
220

1029
UUUGCUUUCUCUUCCACAC
58
1029
UUUGCUUUCUCUUCCACAC
58
1047
GUGUGGAAGAGAAAGCAAA
221

1047
CAAAAAUAAGGUGCCAUGG
59
1047
CAAAAAUAAGGUGCCAUGG
59
1065
CCAUGGCACCUUAUUUUUG
222

1065
GAACAGGCUGUUCCCAAAU
60
1065
GAACAGGCUGUUCCCAAAU
60
1083
AUUUGGGAACAGCCUGUUC
223

1083
UGCUGACUCCAAAGCUCUG
61
1083
UGCUGACUCCAAAGCUCUG
61
1101
CAGAGCUUUGGAGUCAGCA
224

1101
GGACUUAUUGGACAAAAUG
62
1101
GGACUUAUUGGACAAAAUG
62
1119
CAUUUUGUCCAAUAAGUCC
225

1119
GUUGACAUUCAACCCACAC
63
1119
GUUGACAUUCAACCCACAC
63
1137
GUGUGGGUUGAAUGUCAAC
226

1137
CAAGAGGAUUGAAGUAGAA
64
1137
CAAGAGGAUUGAAGUAGAA
64
1155
UUCUACUUCAAUCCUCUUG
227

1155
ACAGGCUCUGGCCCACCCA
65
1155
ACAGGCUCUGGCCCACCCA
65
1173
UGGGUGGGCCAGAGCCUGU
228

1173
AUAUCUGGAGCAGUAUUAC
66
1173
AUAUCUGGAGCAGUAUUAC
66
1191
GUAAUACUGCUCCAGAUAU
229

1191
CGACCCGAGUGACGAGCCC
67
1191
CGACCCGAGUGACGAGCCC
67
1209
GGGCUCGUCACUCGGGUCG
230

1209
CAUCGCCGAAGCACCAUUC
68
1209
CAUCGCCGAAGCACCAUUC
68
1227
GAAUGGUGCUUCGGCGAUG
231

1227
CAAGUUCGACAUGGAAUUG
69
1227
CAAGUUCGACAUGGAAUUG
69
1245
CAAUUCCAUGUCGAACUUG
232

1245
GGAUGACUUGCCUAAGGAA
70
1245
GGAUGACUUGCCUAAGGAA
70
1263
UUCCUUAGGCAAGUCAUCC
233

1263
AAAGCUCAAAGAACUAAUU
71
1263
AAAGCUCAAAGAACUAAUU
71
1281
AAUUAGUUCUUUGAGCUUU
234

1281
UUUUGAAGAGACUGCUAGA
72
1281
UUUUGAAGAGACUGCUAGA
72
1299
UCUAGCAGUCUCUUCAAAA
235

1299
AUUCCAGCCAGGAUACAGA
73
1299
AUUCCAGCCAGGAUACAGA
73
1317
UCUGUAUCCUGGCUGGAAU
236

1317
AUCUUAAAUUUGUCAGGAC
74
1317
AUCUUAAAUUUGUCAGGAC
74
1335
GUCCUGACAAAUUUAAGAU
237

1335
CAAGGGCUCAGAGGACUGG
75
1335
CAAGGGCUCAGAGGACUGG
75
1353
CCAGUCCUCUGAGCCCUUG
238

1353
GACGUGCUCAGACAUCGGU
76
1353
GACGUGCUCAGACAUCGGU
76
1371
ACCGAUGUCUGAGCACGUC
239

1371
UGUUCUUCUUCCCAGUUCU
77
1371
UGUUCUUCUUCCCAGUUCU
77
1389
AGAACUGGGAAGAAGAACA
240

1389
UUGACCCCUGGUCCUGUCU
78
1389
UUGACCCCUGGUCCUGUCU
78
1407
AGACAGGACCAGGGGUCAA
241

1407
UCCAGCCCGUCUUGGCUUA
79
1407
UCCAGCCCGUCUUGGCUUA
79
1425
UAAGCCAAGACGGGCUGGA
242

1425
AUCCACUUUGACUCCUUUG
80
1425
AUCCACUUUGACUCCUUUG
80
1443
CAAAGGAGUCAAAGUGGAU
243

1443
GAGCCGUUUGGAGGGGCGG
81
1443
GAGCCGUUUGGAGGGGCGG
81
1461
CCGCCCCUCCAAACGGCUC
244

1461
GUUUCUGGUAGUUGUGGCU
82
1461
GUUUCUGGUAGUUGUGGCU
82
1479
AGCCACAACUACCAGAAAC
245

1479
UUUUAUGCUUUCAAAGAAU
83
1479
UUUUAUGCUUUCAAAGAAU
83
1497
AUUCUUUGAAAGCAUAAAA
246

1497
UUUCUUCAGUCCAGAGAAU
84
1497
UUUCUUCAGUCCAGAGAAU
84
1515
AUUCUCUGGACUGAAGAAA
247

1515
UUCCUCCUGGCAGCCCUGU
85
1515
UUCCUCCUGGCAGCCCUGU
85
1533
ACAGGGCUGCCAGGAGGAA
248

1533
UGUGUGUCACCCAUUGGUG
86
1533
UGUGUGUCACCCAUUGGUG
86
1551
CACCAAUGGGUGACACACA
249

1551
GACCUGCGGCAGUAUGUAC
87
1551
GACCUGCGGCAGUAUGUAC
87
1569
GUACAUACUGCCGCAGGUC
250

1569
CUUCAGUGCACCUUACUGC
88
1569
CUUCAGUGCACCUUACUGC
88
1587
GCAGUAAGGUGCACUGAAG
251

1587
CUUACUGUUGCUUUAGUCA
89
1587
CUUACUGUUGCUUUAGUCA
89
1605
UGACUAAAGCAACAGUAAG
252

1605
ACUAAUUGCUUUCUGGUUU
90
1605
ACUAAUUGCUUUCUGGUUU
90
1623
AAACCAGAAAGCAAUUAGU
253

1623
UGAAAGAUGCAGUGGUUCC
91
1623
UGAAAGAUGCAGUGGUUCC
91
1641
GGAACCACUGCAUCUUUCA
254

1641
CUCCCUCUCCUGAAUCCUU
92
1641
CUCCCUCUCCUGAAUCCUU
92
1659
AAGGAUUCAGGAGAGGGAG
255

1659
UUUCUACAUGAUGCCCUGC
93
1659
UUUCUACAUGAUGCCCUGC
93
1677
GCAGGGCAUCAUGUAGAAA
256

1677
CUGACCAUGCAGCCGCACC
94
1677
CUGACCAUGCAGCCGCACC
94
1695
GGUGCGGCUGCAUGGUCAG
257

1695
CAGAGAGAGAUUCUUCCCC
95
1695
CAGAGAGAGAUUCUUCCCC
95
1713
GGGGAAGAAUCUCUCUCUG
258

1713
CAAUUGGCUCUAGUCACUG
96
1713
CAAUUGGCUCUAGUCACUG
96
1731
CAGUGACUAGAGCCAAUUG
259

1731
GGCAUCUCACUUUAUGAUA
97
1731
GGCAUCUCACUUUAUGAUA
97
1749
UAUCAUAAAGUGAGAUGCC
260

1749
AGGGAAGGCUACUACCUAG
98
1749
AGGGAAGGCUACUACCUAG
98
1767
CUAGGUAGUAGCCUUCCCU
261

1767
GGGCACUUUAAGUCAGUGA
99
1767
GGGCACUUUAAGUCAGUGA
99
1785
UCACUGACUUAAAGUGCCC
262

1785
ACAGCCCCUUAUUUGCACU
100
1785
ACAGCCCCUUAUUUGCACU
100
1803
AGUGCAAAUAAGGGGCUGU
263

1803
UUCACCUUUUGACCAUAAC
101
1803
UUCACCUUUUGACCAUAAC
101
1821
GUUAUGGUCAAAAGGUGAA
264

1821
CUGUUUCCCCAGAGCAGGA
102
1821
CUGUUUCCCCAGAGCAGGA
102
1839
UCCUGCUCUGGGGAAACAG
265

1839
AGCUUGUGGAAAUACCUUG
103
1839
AGCUUGUGGAAAUACCUUG
103
1857
CAAGGUAUUUCCACAAGCU
266

1857
GGCUGAUGUUGCAGCCUGC
104
1857
GGCUGAUGUUGCAGCCUGC
104
1875
GCAGGCUGCAACAUCAGCC
267

1875
CAGCAAGUGCUUCCGUCUC
105
1875
CAGCAAGUGCUUCCGUCUC
105
1893
GAGACGGAAGCACUUGCUG
268

1893
CCGGAAUCCUUGGGGAGCA
106
1893
CCGGAAUCCUUGGGGAGCA
106
1911
UGCUCCCCAAGGAUUCCGG
269

1911
ACUUGUCCACGUCUUUUCU
107
1911
ACUUGUCCACGUCUUUUCU
107
1929
AGAAAAGACGUGGACAAGU
270

1929
UCAUAUCAUGGUAGUCACU
108
1929
UCAUAUCAUGGUAGUCACU
108
1947
AGUGACUACCAUGAUAUGA
271

1947
UAACAUAUAUAAGGUAUGU
109
1947
UAACAUAUAUAAGGUAUGU
109
1965
ACAUACCUUAUAUAUGUUA
272

1965
UGCUAUUGGCCCAGCUUUU
110
1965
UGCUAUUGGCCCAGCUUUU
110
1983
AAAAGCUGGGCCAAUAGCA
273

1983
UAGAAAAUGCAGUCAUUUU
111
1983
UAGAAAAUGCAGUCAUUUU
111
2001
AAAAUGACUGCAUUUUCUA
274

2001
UUCUAAAUAAAAAGGAAGU
112
2001
UUCUAAAUAAAAAGGAAGU
112
2019
ACUUCCUUUUUAUUUAGAA
275

2019
UACUGCACCCAGCAGUGUC
113
2019
UACUGCACCCAGCAGUGUC
113
2037
GACACUGCUGGGUGCAGUA
276

2037
CACUCUGUAGUUACUGUGG
114
2037
CACUCUGUAGUUACUGUGG
114
2055
CCACAGUAACUACAGAGUG
277

2055
GUCACUUGUACCAUAUAGA
115
2055
GUCACUUGUACCAUAUAGA
115
2073
UCUAUAUGGUACAAGUGAC
278

2073
AGGUGUAACACUUGUCAAG
116
2073
AGGUGUAACACUUGUCAAG
116
2091
CUUGACAAGUGUUACACCU
279

2091
GAAGCGUUAUGUGCAGUAC
117
2091
GAAGCGUUAUGUGCAGUAC
117
2109
GUACUGCACAUAACGCUUC
280

2109
CUUAAUGUUUGUAAGACUU
118
2109
CUUAAUGUUUGUAAGACUU
118
2127
AAGUCUUACAAACAUUAAG
281

2127
UACAAAAAAAGAUUUAAAG
119
2127
UACAAAAAAAGAUUUAAAG
119
2145
CUUUAAAUCUUUUUUUGUA
282

2145
GUGGCAGCUUCACUCGACA
120
2145
GUGGCAGCUUCACUCGACA
120
2163
UGUCGAGUGAAGCUGCCAC
283

2163
AUUUGGUGAGAGAAGUACA
121
2163
AUUUGGUGAGAGAAGUACA
121
2181
UGUACUUCUCUCACCAAAU
284

2181
AAAGGUUGCAGUGCUGAGC
122
2181
AAAGGUUGCAGUGCUGAGC
122
2199
GCUCAGCACUGCAACCUUU
285

2199
CUGUGGGCGGUUUCUGGGG
123
2199
CUGUGGGCGGUUUCUGGGG
123
2217
CCCCAGAAACCGCCCACAG
286

2217
GAUGUCCCAGGGUGGAACU
124
2217
GAUGUCCCAGGGUGGAACU
124
2235
AGUUCCACCCUGGGACAUC
287

2235
UCCACAUGCUGGUGCAUAU
125
2235
UCCACAUGCUGGUGCAUAU
125
2253
AUAUGCACCAGCAUGUGGA
288

2253
UACGCCCUUGAGCUACUUC
126
2253
UACGCCCUUGAGCUACUUC
126
2271
GAAGUAGCUCAAGGGCGUA
289

2271
CAAAUGUGGUUUAUACCUC
127
2271
CAAAUGUGGUUUAUACCUC
127
2289
GAGGUAUAAACCACAUUUG
290

2289
CGCAGAUACAAGAAUCUUU
128
2289
CGCAGAUACAAGAAUCUUU
128
2307
AAAGAUUCUUGUAUCUGCG
291

2307
UAUGAAUAUACAAUUCUUU
129
2307
UAUGAAUAUACAAUUCUUU
129
2325
AAAGAAUUGUAUAUUCAUA
292

2325
UUUCCUUCUACAGCUUAGC
130
2325
UUUCCUUCUACAGCUUAGC
130
2343
GCUAAGCUGUAGAAGGAAA
293

2343
CUCCGUCUUUUCAACCACG
131
2343
CUCCGUCUUUUCAACCACG
131
2361
CGUGGUUGAAAAGACGGAG
294

2361
GAACAUUUAAAACCCGACC
132
2361
GAACAUUUAAAACCCGACC
132
2379
GGUCGGGUUUUAAAUGUUC
295

2379
CUACUAGCACUGUUCUGUC
133
2379
CUACUAGCACUGUUCUGUC
133
2397
GACAGAACAGUGCUAGUAG
296

2397
CCUCAAGUACUCAAAUAUU
134
2397
CCUCAAGUACUCAAAUAUU
134
2415
AAUAUUUGAGUACUUGAGG
297

2415
UUCUGAUACUGCUGAGUCA
135
2415
UUCUGAUACUGCUGAGUCA
135
2433
UGACUCAGCAGUAUCAGAA
298

2433
AGACUGUCAGAAAAAGCUA
136
2433
AGACUGUCAGAAAAAGCUA
136
2451
UAGCUUUUUCUGACAGUCU
299

2451
AGCACUAACUCGUGUUUGG
137
2451
AGCACUAACUCGUGUUUGG
137
2469
CCAAACACGAGUUAGUGCU
300

2469
GAGCUCUAUCCAUAUUUUA
138
2469
GAGCUCUAUCCAUAUUUUA
138
2487
UAAAAUAUGGAUAGAGCUC
301

2487
ACUGAUCUCUUUAAGUAUU
139
2487
ACUGAUCUCUUUAAGUAUU
139
2505
AAUACUUAAAGAGAUCAGU
302

2505
UUGUUCCUGCCACUGUGUA
140
2505
UUGUUCCUGCCACUGUGUA
140
2523
UACACAGUGGCAGGAACAA
303

2523
ACUGUGGAGUUGACUCGGU
141
2523
ACUGUGGAGUUGACUCGGU
141
2541
ACCGAGUCAACUCCACAGU
304

2541
UGUUCUGUCCCAGUGCGGU
142
2541
UGUUCUGUCCCAGUGCGGU
142
2559
ACCGCACUGGGACAGAACA
305

2559
UGCCUCCUCUUGACUUCCC
143
2559
UGCCUCCUCUUGACUUCCC
143
2577
GGGAAGUCAAGAGGAGGCA
306

2577
CCACUGCUCUCUGUGGUGA
144
2577
CCACUGCUCUCUGUGGUGA
144
2595
UCACCACAGAGAGCAGUGG
307

2595
AGAAAUUUGCCUUGUUCAA
145
2595
AGAAAUUUGCCUUGUUCAA
145
2613
UUGAACAAGGCAAAUUUCU
308

2613
AUAAUUACUGUACCCUCGC
146
2613
AUAAUUACUGUACCCUCGC
146
2631
GCGAGGGUACAGUAAUUAU
309

2631
CAUGACUGUUACAGCUUUC
147
2631
CAUGACUGUUACAGCUUUC
147
2649
GAAAGCUGUAACAGUCAUG
310

2649
CUGUGCAGAGAUGACUGUC
148
2649
CUGUGCAGAGAUGACUGUC
148
2667
GACAGUCAUCUCUGCACAG
311

2667
CCAAGUGCCACAUGCCUAC
149
2667
CCAAGUGCCACAUGCCUAC
149
2685
GUAGGCAUGUGGCACUUGG
312

2685
CGAUUGAAAUGAAAACUCU
150
2685
CGAUUGAAAUGAAAACUCU
150
2703
AGAGUUUUCAUUUCAAUCG
313

2703
UAUUGUUACCUCUGAGUUG
151
2703
UAUUGUUACCUCUGAGUUG
151
2721
CAACUCAGAGGUAACAAUA
314

2721
GUGUUCCACGGAAAAUGCU
152
2721
GUGUUCCACGGAAAAUGCU
152
2739
AGCAUUUUCCGUGGAACAC
315

2739
UAUCCAGCAGAUCAUUUAG
153
2739
UAUCCAGCAGAUCAUUUAG
153
2757
CUAAAUGAUCUGCUGGAUA
316

2757
GGAAAAAUAAUUCUAUUUU
154
2757
GGAAAAAUAAUUCUAUUUU
154
2775
AAAAUAGAAUUAUUUUUCC
317

2775
UUAGCUUUUCAUUUCUCAG
155
2775
UUAGCUUUUCAUUUCUCAG
155
2793
CUGAGAAAUGAAAAGCUAA
318

2793
GCUGUCCUUUUUUCUUGUU
156
2793
GCUGUCCUUUUUUCUUGUU
156
2811
AACAAGAAAAAAGGACAGC
319

2811
UUGAUUUUUGACAGCAAUG
157
2811
UUGAUUUUUGACAGCAAUG
157
2829
CAUUGCUGUCAAAAAUCAA
320

2829
GGAGAAUGGGUUAUAUAAA
158
2829
GGAGAAUGGGUUAUAUAAA
158
2847
UUUAUAUAACCCAUUCUCC
321

2847
AGACUGCCUGCUAAUAUGA
159
2847
AGACUGCCUGCUAAUAUGA
159
2865
UCAUAUUAGCAGGCAGUCU
322

2865
AACAGAAAUGCAUUUGUAA
160
2865
AACAGAAAUGCAUUUGUAA
160
2883
UUACAAAUGCAUUUCUGUU
323

2883
AUUCAUGAAAAUAAAUGUA
161
2883
AUUCAUGAAAAUAAAUGUA
161
2901
UACAUUUAUUUUCAUGAAU
324

2901
ACAUCUUCUAUCUUCAAAA
162
2901
ACAUCUUCUAUCUUCAAAA
162
2919
UUUUGAAGAUAGAAGAUGU
325

2913
UUCAAAAAAAAAAAAAAAA
163
2913
UUCAAAAAAAAAAAAAAAA
163
2931
UUUUUUUUUUUUUUUUGAA
326

Seq

Seq

Seq

Pos
Target Sequence
ID
UPos
Upper seq
ID
LPos
Lower seq
ID

XM_055766.6 (MAPK3/ERK1)

3
CGGGGCCUCGGGCGGGGCC
327
3
CGGGGCCUCGGGCGGGGCC
327
21
GGCCCCGCCCGAGGCCCCG
432

21
CGCCGUGGGGAGGAGGGCG
328
21
CGCCGUGGGGAGGAGGGCG
328
39
CGCCCUCCUCCCCACGGCG
433

39
GGUGGGAGGGGAGGAGUGG
329
39
GGUGGGAGGGGAGGAGUGG
329
57
CCACUCCUCCCCUCCCACC
434

57
GAGAUGGCGGCGGCGGCGG
330
57
GAGAUGGCGGCGGCGGCGG
330
75
CCGCCGCCGCCGCCAUCUC
435

75
GCUCAGGGGGGCGGGGGCG
331
75
GCUCAGGGGGGCGGGGGCG
331
93
CGCCCCCGCCCCCCUGAGC
436

93
GGGGAGCCCCGUAGAACCG
332
93
GGGGAGCCCCGUAGAACCG
332
111
CGGUUCUACGGGGCUCCCC
437

111
GAGGGGGUCGGCCCGGGGG
333
111
GAGGGGGUCGGCCCGGGGG
333
129
CCCCCGGGCCGACCCCCUC
438

129
GUCCCGGGGGAGGUGGAGA
334
129
GUCCCGGGGGAGGUGGAGA
334
147
UCUCCACCUCCCCCGGGAC
439

147
AUGGUGAAGGGGCAGCCGU
335
147
AUGGUGAAGGGGCAGCCGU
335
165
ACGGCUGCCCCUUCACCAU
440

165
UUCGACGUGGGCCCGCGCU
336
165
UUCGACGUGGGCCCGCGCU
336
183
AGCGCGGGCCCACGUCGAA
441

183
UACACGCAGUUGCAGUACA
337
183
UACACGCAGUUGCAGUACA
337
201
UGUACUGCAACUGCGUGUA
442

201
AUCGGCGAGGGCGCGUACG
338
201
AUCGGCGAGGGCGCGUACG
338
219
CGUACGCGCCCUCGCCGAU
443

219
GGCAUGGUCAGCUCGGCCU
339
219
GGCAUGGUCAGCUCGGCCU
339
237
AGGCCGAGCUGACCAUGCC
444

237
UAUGACCACGUGCGCAAGA
340
237
UAUGACCACGUGCGCAAGA
340
255
UCUUGCGCACGUGGUCAUA
445

255
ACUCGCGUGGCCAUCAAGA
341
255
ACUCGCGUGGCCAUCAAGA
341
273
UCUUGAUGGCCACGCGAGU
446

273
AAGAUCAGCCCCUUCGAAC
342
273
AAGAUCAGCCCCUUCGAAC
342
291
GUUCGAAGGGGCUGAUCUU
447

291
CAUCAGACCUACUGCCAGC
343
291
CAUCAGACCUACUGCCAGC
343
309
GCUGGCAGUAGGUCUGAUG
448

309
CGCACGCUCCGGGAGAUCC
344
309
CGCACGCUCCGGGAGAUCC
344
327
GGAUCUCCCGGAGCGUGCG
449

327
CAGAUCCUGCUGCGCUUCC
345
327
CAGAUCCUGCUGCGCUUCC
345
345
GGAAGCGCAGCAGGAUCUG
450

345
CGCCAUGAGAAUGUCAUCG
346
345
CGCCAUGAGAAUGUCAUCG
346
363
CGAUGACAUUCUCAUGGCG
451

363
GGCAUCCGAGACAUUCUGC
347
363
GGCAUCCGAGACAUUCUGC
347
381
GCAGAAUGUCUCGGAUGCC
452

381
CGGGCGUCCACCCUGGAAG
348
381
CGGGCGUCCACCCUGGAAG
348
399
CUUCCAGGGUGGACGCCCG
453

399
GCCAUGAGAGAUGUCUACA
349
399
GCCAUGAGAGAUGUCUACA
349
417
UGUAGACAUCUCUCAUGGC
454

417
AUUGUGCAGGACCUGAUGG
350
417
AUUGUGCAGGACCUGAUGG
350
435
CCAUCAGGUCCUGCACAAU
455

435
GAGACUGACCUGUACAAGU
351
435
GAGACUGACCUGUACAAGU
351
453
ACUUGUACAGGUCAGUCUC
456

453
UUGCUGAAAAGCCAGCAGC
352
453
UUGCUGAAAAGCCAGCAGC
352
471
GCUGCUGGCUUUUCAGCAA
457

471
CUGAGCAAUGACCAUAUCU
353
471
CUGAGCAAUGACCAUAUCU
353
489
AGAUAUGGUCAUUGCUCAG
458

489
UGCUACUUCCUCUACCAGA
354
489
UGCUACUUCCUCUACCAGA
354
507
UCUGGUAGAGGAAGUAGCA
459

507
AUCCUGCGGGGCCUCAAGU
355
507
AUCCUGCGGGGCCUCAAGU
355
525
ACUUGAGGCCCCGCAGGAU
460

525
UACAUCCACUCCGCCAACG
356
525
UACAUCCACUCCGCCAACG
356
543
CGUUGGCGGAGUGGAUGUA
461

543
GUGCUCCACCGAGAUCUAA
357
543
GUGCUCCACCGAGAUCUAA
357
561
UUAGAUCUCGGUGGAGCAC
462

561
AAGCCCUCCAACCUGCUCA
358
561
AAGCCCUCCAACCUGCUCA
358
579
UGAGCAGGUUGGAGGGCUU
463

579
AUCAACACCACCUGCGACC
359
579
AUCAACACCACCUGCGACC
359
597
GGUCGCAGGUGGUGUUGAU
464

597
CUUAAGAUUUGUGAUUUCG
360
597
CUUAAGAUUUGUGAUUUCG
360
615
CGAAAUCACAAAUCUUAAG
465

615
GGCCUGGCCCGGAUUGCCG
361
615
GGCCUGGCCCGGAUUGCCG
361
633
CGGCAAUCCGGGCCAGGCC
466

633
GAUCCUGAGCAUGACCACA
362
633
GAUCCUGAGCAUGACCACA
362
651
UGUGGUCAUGCUCAGGAUC
467

651
ACCGGCUUCCUGACGGAGU
363
651
ACCGGCUUCCUGACGGAGU
363
669
ACUCCGUCAGGAAGCCGGU
468

669
UAUGUGGCUACGCGCUGGU
364
669
UAUGUGGCUACGCGCUGGU
364
687
ACCAGCGCGUAGCCACAUA
469

687
UACCGGGCCCCAGAGAUCA
365
687
UACCGGGCCCCAGAGAUCA
365
705
UGAUCUCUGGGGCCCGGUA
470

705
AUGCUGAACUCCAAGGGCU
366
705
AUGCUGAACUCCAAGGGCU
366
723
AGCCCUUGGAGUUCAGCAU
471

723
UAUACCAAGUCCAUCGACA
367
723
UAUACCAAGUCCAUCGACA
367
741
UGUCGAUGGACUUGGUAUA
472

741
AUCUGGUCUGUGGGCUGCA
368
741
AUCUGGUCUGUGGGCUGCA
368
759
UGCAGCCCACAGACCAGAU
473

759
AUUCUGGCUGAGAUGCUCU
369
759
AUUCUGGCUGAGAUGCUCU
369
777
AGAGCAUCUCAGCCAGAAU
474

777
UCUAACCGGCCCAUCUUCC
370
777
UCUAACCGGCCCAUCUUCC
370
795
GGAAGAUGGGCCGGUUAGA
475

795
CCUGGCAAGCACUACCUGG
371
795
CCUGGCAAGCACUACCUGG
371
813
CCAGGUAGUGCUUGCCAGG
476

813
GAUCAGCUCAACCACAUUC
372
813
GAUCAGCUCAACCACAUUC
372
831
GAAUGUGGUUGAGCUGAUC
477

831
CUGGGCAUCCUGGGCUCCC
373
831
CUGGGCAUCCUGGGCUCCC
373
849
GGGAGCCCAGGAUGCCCAG
478

849
CCAUCCCAGGAGGACCUGA
374
849
CCAUCCCAGGAGGACCUGA
374
867
UCAGGUCCUCCUGGGAUGG
479

867
AAUUGUAUCAUCAACAUGA
375
867
AAUUGUAUCAUCAACAUGA
375
885
UCAUGUUGAUGAUACAAUU
480

885
AAGGCCCGAAACUACCUAC
376
885
AAGGCCCGAAACUACCUAC
376
903
GUAGGUAGUUUCGGGCCUU
481

903
CAGUCUCUGCCCUCCAAGA
377
903
CAGUCUCUGCCCUCCAAGA
377
921
UCUUGGAGGGCAGAGACUG
482

921
ACCAAGGUGGCUUGGGCCA
378
921
ACCAAGGUGGCUUGGGCCA
378
939
UGGCCCAAGCCACCUUGGU
483

939
AAGCUUUUCCCCAAGUCAG
379
939
AAGCUUUUCCCCAAGUCAG
379
957
CUGACUUGGGGAAAAGCUU
484

957
GACUCCAAAGCCCUUGACC
380
957
GACUCCAAAGCCCUUGACC
380
975
GGUCAAGGGCUUUGGAGUC
485

975
CUGCUGGACCGGAUGUUAA
381
975
CUGCUGGACCGGAUGUUAA
381
993
UUAACAUCCGGUCCAGCAG
486

993
ACCUUUAACCCCAAUAAAC
382
993
ACCUUUAACCCCAAUAAAC
382
1011
GUUUAUUGGGGUUAAAGGU
487

1011
CGGAUCACAGUGGAGGAAG
383
1011
CGGAUCACAGUGGAGGAAG
383
1029
CUUCCUCCACUGUGAUCCG
488

1029
GCGCUGGCUCACCCCUACC
384
1029
GCGCUGGCUCACCCCUACC
384
1047
GGUAGGGGUGAGCCAGCGC
489

1047
CUGGAGCAGUACUAUGACC
385
1047
CUGGAGCAGUACUAUGACC
385
1065
GGUCAUAGUACUGCUCCAG
490

1065
CCGACGGAUGAGCCAGUGG
386
1065
CCGACGGAUGAGCCAGUGG
386
1083
CCACUGGCUCAUCCGUCGG
491

1083
GCCGAGGAGCCCUUCACCU
387
1083
GCCGAGGAGCCCUUCACCU
387
1101
AGGUGAAGGGCUCCUCGGC
492

1101
UUCGCCAUGGAGCUGGAUG
388
1101
UUCGCCAUGGAGCUGGAUG
388
1119
CAUCCAGCUCCAUGGCGAA
493

1119
GACCUACCUAAGGAGCGGC
389
1119
GACCUACCUAAGGAGCGGC
389
1137
GCCGCUCCUUAGGUAGGUC
494

1137
CUGAAGGAGCUCAUCUUCC
390
1137
CUGAAGGAGCUCAUCUUCC
390
1155
GGAAGAUGAGCUCCUUCAG
495

1155
CAGGAGACAGCACGCUUCC
391
1155
CAGGAGACAGCACGCUUCC
391
1173
GGAAGCGUGCUGUCUCCUG
496

1173
CAGCCCGGAGUGCUGGAGG
392
1173
CAGCCCGGAGUGCUGGAGG
392
1191
CCUCCAGCACUCCGGGCUG
497

1191
GCCCCCUAGCCCAGACAGA
393
1191
GCCCCCUAGCCCAGACAGA
393
1209
UCUGUCUGGGCUAGGGGGC
498

1209
ACAUCUCUGCACCCUGGGG
394
1209
ACAUCUCUGCACCCUGGGG
394
1227
CCCCAGGGUGCAGAGAUGU
499

1227
GCCUGGAACAGAACUGGCA
395
1227
GCCUGGAACAGAACUGGCA
395
1245
UGCCAGUUCUGUUCCAGGC
500

1245
AAAGAGGCAAGAGGUCACU
396
1245
AAAGAGGCAAGAGGUCACU
396
1263
AGUGACCUCUUGCCUCUUU
501

1263
UGAGGGCCUCUGUCACCCA
397
1263
UGAGGGCCUCUGUCACCCA
397
1281
UGGGUGACAGAGGCCCUCA
502

1281
AGGACCUGCCUCCUGCCUG
398
1281
AGGACCUGCCUCCUGCCUG
398
1299
CAGGCAGGAGGCAGGUCCU
503

1299
GCCCCUCUCCCGCCAGACU
399
1299
GCCCCUCUCCCGCCAGACU
399
1317
AGUCUGGCGGGAGAGGGGC
504

1317
UGUUAGAAAAUGGACACUG
400
1317
UGUUAGAAAAUGGACACUG
400
1335
CAGUGUCCAUUUUCUAACA
505

1335
GUGCCCAGCCCGGACCUUG
401
1335
GUGCCCAGCCCGGACCUUG
401
1353
CAAGGUCCGGGCUGGGCAC
506

1353
GGCAGCCCAGGCCGGGGUG
402
1353
GGCAGCCCAGGCCGGGGUG
402
1371
CACCCCGGCCUGGGCUGCC
507

1371
GGAGCAUGGGCCUGGCCAC
403
1371
GGAGCAUGGGCCUGGCCAC
403
1389
GUGGCCAGGCCCAUGCUCC
508

1389
CCUCUCUCCUUUGCUGAGG
404
1389
CCUCUCUCCUUUGCUGAGG
404
1407
CCUCAGCAAAGGAGAGAGG
509

1407
GCCUCCAGCUUCAGGCAGG
405
1407
GCCUCCAGCUUCAGGCAGG
405
1425
CCUGCCUGAAGCUGGAGGC
510

1425
GCCAAGGCCUUCUCCUCCC
406
1425
GCCAAGGCCUUCUCCUCCC
406
1443
GGGAGGAGAAGGCCUUGGC
511

1443
CCACCCGCCCUCCCCACGG
407
1443
CCACCCGCCCUCCCCACGG
407
1461
CCGUGGGGAGGGCGGGUGG
512

1461
GGGCCUCGGGACCUCAGGU
408
1461
GGGCCUCGGGACCUCAGGU
408
1479
ACCUGAGGUCCCGAGGCCC
513

1479
UGGCCCCAGUUCAAUCUCC
409
1479
UGGCCCCAGUUCAAUCUCC
409
1497
GGAGAUUGAACUGGGGCCA
514

1497
CCGCUGCUGCUGCUGCGCC
410
1497
CCGCUGCUGCUGCUGCGCC
410
1515
GGCGCAGCAGCAGCAGCGG
515

1515
CCUUACCUUCCCCAGCGUC
411
1515
CCUUACCUUCCCCAGCGUC
411
1533
GACGCUGGGGAAGGUAAGG
516

1533
CCCAGUCUCUGGCAGUUCU
412
1533
CCCAGUCUCUGGCAGUUCU
412
1551
AGAACUGCCAGAGACUGGG
517

1551
UGGAAUGGAAGGGUUCUGG
413
1551
UGGAAUGGAAGGGUUCUGG
413
1569
CCAGAACCCUUCCAUUCCA
518

1569
GCUGCCCCAACCUGCUGAA
414
1569
GCUGCCCCAACCUGCUGAA
414
1587
UUCAGCAGGUUGGGGCAGC
519

1587
AGGGCAGAGGUGGAGGGUG
415
1587
AGGGCAGAGGUGGAGGGUG
415
1605
CACCCUCCACCUCUGCCCU
520

1605
GGGGGGCGCUGAGUAGGGA
416
1605
GGGGGGCGCUGAGUAGGGA
416
1623
UCCCUACUCAGCGCCCCCC
521

1623
ACUCAGGGCCAUGCCUGCC
417
1623
ACUCAGGGCCAUGCCUGCC
417
1641
GGCAGGCAUGGCCCUGAGU
522

1641
CCCCCUCAUCUCAUUCAAA
418
1641
CCCCCUCAUCUCAUUCAAA
418
1659
UUUGAAUGAGAUGAGGGGG
523

1659
ACCCCACCCUAGUUUCCCU
419
1659
ACCCCACCCUAGUUUCCCU
419
1677
AGGGAAACUAGGGUGGGGU
524

1677
UGAAGGAACAUUCCUUAGU
420
1677
UGAAGGAACAUUCCUUAGU
420
1695
ACUAAGGAAUGUUCCUUCA
525

1695
UCUCAAGGGCUAGCAUCCC
421
1695
UCUCAAGGGCUAGCAUCCC
421
1713
GGGAUGCUAGCCCUUGAGA
526

1713
CUGAGGAGCCAGGCCGGGC
422
1713
CUGAGGAGCCAGGCCGGGC
422
1731
GCCCGGCCUGGCUCCUCAG
527

1731
CCGAAUCCCCUCCCUGUCA
423
1731
CCGAAUCCCCUCCCUGUCA
423
1749
UGACAGGGAGGGGAUUCGG
528

1749
AAAGCUGUCACUUCGCGUG
424
1749
AAAGCUGUCACUUCGCGUG
424
1767
CACGCGAAGUGACAGCUUU
529

1767
GCCCUCGCUGCUUCUGUGU
425
1767
GCCCUCGCUGCUUCUGUGU
425
1785
ACACAGAAGCAGCGAGGGC
530

1785
UGUGGUGAGCAGAAGUGGA
426
1785
UGUGGUGAGCAGAAGUGGA
426
1803
UCCACUUCUGCUCACCACA
531

1803
AGCUGGGGGGCGUGGAGAG
427
1803
AGCUGGGGGGCGUGGAGAG
427
1821
CUCUCCACGCCCCCCAGCU
532

1821
GCCCGGCGCCCCUGCCACC
428
1821
GCCCGGCGCCCCUGCCACC
428
1839
GGUGGCAGGGGCGCCGGGC
533

1839
CUCCCUGACCCGUCUAAUA
429
1839
CUCCCUGACCCGUCUAAUA
429
1857
UAUUAGACGGGUCAGGGAG
534

1857
AUAUAAAUAUAGAGAUGUG
430
1857
AUAUAAAUAUAGAGAUGUG
430
1875
CACAUCUCUAUAUUUAUAU
535

1865
AUAGAGAUGUGUCUAUGGC
431
1865
AUAGAGAUGUGUCUAUGGC
431
1883
GCCAUAGACACAUCUCUAU
536

ID

Seq

Seq

Pos
Target Sequence
Seq
UPos
Upper seq
ID
LPos
Lower seq
ID

NM_002750 (MAPK8/JNK1)

3
UAAUUGCUUGCCAUCAUGA
537
3
UAAUUGCUUGCCAUCAUGA
537
21
UCAUGAUGGCAAGCAAUUA
616

21
AGCAGAAGCAAGCGUGACA
538
21
AGCAGAAGCAAGCGUGACA
538
39
UGUCACGCUUGCUUCUGCU
617

39
AACAAUUUUUAUAGUGUAG
539
39
AACAAUUUUUAUAGUGUAG
539
57
CUACACUAUAAAAAUUGUU
618

57
GAGAUUGGAGAUUCUACAU
540
57
GAGAUUGGAGAUUCUACAU
540
75
AUGUAGAAUCUCCAAUCUC
619

75
UUCACAGUCCUGAAACGAU
541
75
UUCACAGUCCUGAAACGAU
541
93
AUCGUUUCAGGACUGUGAA
620

93
UAUCAGAAUUUAAAACCUA
542
93
UAUCAGAAUUUAAAACCUA
542
111
UAGGUUUUAAAUUCUGAUA
621

111
AUAGGCUCAGGAGCUCAAG
543
111
AUAGGCUCAGGAGCUCAAG
543
129
CUUGAGCUCCUGAGCCUAU
622

129
GGAAUAGUAUGCGCAGCUU
544
129
GGAAUAGUAUGCGCAGCUU
544
147
AAGCUGCGCAUACUAUUCC
623

147
UAUGAUGCCAUUCUUGAAA
545
147
UAUGAUGCCAUUCUUGAAA
545
165
UUUCAAGAAUGGCAUCAUA
624

165
AGAAAUGUUGCAAUCAAGA
546
165
AGAAAUGUUGCAAUCAAGA
546
183
UCUUGAUUGCAACAUUUCU
625

183
AAGCUAAGCCGACCAUUUC
547
183
AAGCUAAGCCGACCAUUUC
547
201
GAAAUGGUCGGCUUAGCUU
626

201
CAGAAUCAGACUCAUGCCA
548
201
CAGAAUCAGACUCAUGCCA
548
219
UGGCAUGAGUCUGAUUCUG
627

219
AAGCGGGCCUACAGAGAGC
549
219
AAGCGGGCCUACAGAGAGC
549
237
GCUCUCUGUAGGCCCGCUU
628

237
CUAGUUCUUAUGAAAUGUG
550
237
CUAGUUCUUAUGAAAUGUG
550
255
CACAUUUCAUAAGAACUAG
629

255
GUUAAUCACAAAAAUAUAA
551
255
GUUAAUCACAAAAAUAUAA
551
273
UUAUAUUUUUGUGAUUAAC
630

273
AUUGGCCUUUUGAAUGUUU
552
273
AUUGGCCUUUUGAAUGUUU
552
291
AAACAUUCAAAAGGCCAAU
631

291
UUCACACCACAGAAAUCCC
553
291
UUCACACCACAGAAAUCCC
553
309
GGGAUUUCUGUGGUGUGAA
632

309
CUAGAAGAAUUUCAAGAUG
554
309
CUAGAAGAAUUUCAAGAUG
554
327
CAUCUUGAAAUUCUUCUAG
633

327
GUUUACAUAGUCAUGGAGC
555
327
GUUUACAUAGUCAUGGAGC
555
345
GCUCCAUGACUAUGUAAAC
634

345
CUCAUGGAUGCAAAUCUUU
556
345
CUCAUGGAUGCAAAUCUUU
556
363
AAAGAUUUGCAUCCAUGAG
635

363
UGCCAAGUGAUUCAGAUGG
557
363
UGCCAAGUGAUUCAGAUGG
557
381
CCAUCUGAAUCACUUGGCA
636

381
GAGCUAGAUCAUGAAAGAA
558
381
GAGCUAGAUCAUGAAAGAA
558
399
UUCUUUCAUGAUCUAGCUC
637

399
AUGUCCUACCUUCUCUAUC
559
399
AUGUCCUACCUUCUCUAUC
559
417
GAUAGAGAAGGUAGGACAU
638

417
CAGAUGCUGUGUGGAAUCA
560
417
CAGAUGCUGUGUGGAAUCA
560
435
UGAUUCCACACAGCAUCUG
639

435
AAGCACCUUCAUUCUGCUG
561
435
AAGCACCUUCAUUCUGCUG
561
453
CAGCAGAAUGAAGGUGCUU
640

453
GGAAUUAUUCAUCGGGACU
562
453
GGAAUUAUUCAUCGGGACU
562
471
AGUCCCGAUGAAUAAUUCC
641

471
UUAAAGCCCAGUAAUAUAG
563
471
UUAAAGCCCAGUAAUAUAG
563
489
CUAUAUUACUGGGCUUUAA
642

489
GUAGUAAAAUCUGAUUGCA
564
489
GUAGUAAAAUCUGAUUGCA
564
507
UGCAAUCAGAUUUUACUAC
643

507
ACUUUGAAGAUUCUUGACU
565
507
ACUUUGAAGAUUCUUGACU
565
525
AGUCAAGAAUCUUCAAAGU
644

525
UUCGGUCUGGCCAGGACUG
566
525
UUCGGUCUGGCCAGGACUG
566
543
CAGUCCUGGCCAGACCGAA
645

543
GCAGGAACGAGUUUUAUGA
567
543
GCAGGAACGAGUUUUAUGA
567
561
UCAUAAAACUCGUUCCUGC
646

561
AUGACGCCUUAUGUAGUGA
568
561
AUGACGCCUUAUGUAGUGA
568
579
UCACUACAUAAGGCGUCAU
647

579
ACUCGCUACUACAGAGCAC
569
579
ACUCGCUACUACAGAGCAC
569
597
GUGCUCUGUAGUAGCGAGU
648

597
CCCGAGGUCAUCCUUGGCA
570
597
CCCGAGGUCAUCCUUGGCA
570
615
UGCCAAGGAUGACCUCGGG
649

615
AUGGGCUACAAGGAAAACG
571
615
AUGGGCUACAAGGAAAACG
571
633
CGUUUUCCUUGUAGCCCAU
650

633
GUGGAUUUAUGGUCUGUGG
572
633
GUGGAUUUAUGGUCUGUGG
572
651
CCACAGACCAUAAAUCCAC
651

651
GGGUGCAUUAUGGGAGAAA
573
651
GGGUGCAUUAUGGGAGAAA
573
669
UUUCUCCCAUAAUGCACCC
652

669
AUGGUUUGCCACAAAAUCC
574
669
AUGGUUUGCCACAAAAUCC
574
687
GGAUUUUGUGGCAAACCAU
653

687
CUCUUUCCAGGAAGGGACU
575
687
CUCUUUCCAGGAAGGGACU
575
705
AGUCCCUUCCUGGAAAGAG
654

705
UAUAUUGAUCAGUGGAAUA
576
705
UAUAUUGAUCAGUGGAAUA
576
723
UAUUCCACUGAUCAAUAUA
655

723
AAAGUUAUUGAACAGCUUG
577
723
AAAGUUAUUGAACAGCUUG
577
741
CAAGCUGUUCAAUAACUUU
656

741
GGAACACCAUGUCCUGAAU
578
741
GGAACACCAUGUCCUGAAU
578
759
AUUCAGGACAUGGUGUUCC
657

759
UUCAUGAAGAAACUGCAAC
579
759
UUCAUGAAGAAACUGCAAC
579
777
GUUGCAGUUUCUUCAUGAA
658

777
CCAACAGUAAGGACUUACG
580
777
CCAACAGUAAGGACUUACG
580
795
CGUAAGUCCUUACUGUUGG
659

795
GUUGAAAACAGACCUAAAU
581
795
GUUGAAAACAGACCUAAAU
581
813
AUUUAGGUCUGUUUUCAAC
660

813
UAUGCUGGAUAUAGCUUUG
582
813
UAUGCUGGAUAUAGCUUUG
582
831
CAAAGCUAUAUCCAGCAUA
661

831
GAGAAACUCUUCCCUGAUG
583
831
GAGAAACUCUUCCCUGAUG
583
849
CAUCAGGGAAGAGUUUCUC
662

849
GUCCUUUUCCCAGCUGACU
584
849
GUCCUUUUCCCAGCUGACU
584
867
AGUCAGCUGGGAAAAGGAC
663

867
UCAGAACACAACAAACUUA
585
867
UCAGAACACAACAAACUUA
585
885
UAAGUUUGUUGUGUUCUGA
664

885
AAAGCCAGUCAGGCAAGGG
586
885
AAAGCCAGUCAGGCAAGGG
586
903
CCCUUGCCUGACUGGCUUU
665

903
GAUUUGUUAUCCAAAAUGC
587
903
GAUUUGUUAUCCAAAAUGC
587
921
GCAUUUUGGAUAACAAAUC
666

921
CUGGUAAUAGAUGCAUCUA
588
921
CUGGUAAUAGAUGCAUCUA
588
939
UAGAUGCAUCUAUUACCAG
667

939
AAAAGGAUCUCUGUAGAUG
589
939
AAAAGGAUCUCUGUAGAUG
589
957
CAUCUACAGAGAUCCUUUU
668

957
GAAGCUCUCCAACACCCGU
590
957
GAAGCUCUCCAACACCCGU
590
975
ACGGGUGUUGGAGAGCUUC
669

975
UACAUCAAUGUCUGGUAUG
591
975
UACAUCAAUGUCUGGUAUG
591
993
CAUACCAGACAUUGAUGUA
670

993
GAUCCUUCUGAAGCAGAAG
592
993
GAUCCUUCUGAAGCAGAAG
592
1011
CUUCUGCUUCAGAAGGAUC
671

1011
GCUCCACCACCAAAGAUCC
593
1011
GCUCCACCACCAAAGAUCC
593
1029
GGAUCUUUGGUGGUGGAGC
672

1029
CCUGACAAGCAGUUAGAUG
594
1029
CCUGACAAGCAGUUAGAUG
594
1047
CAUCUAACUGCUUGUCAGG
673

1047
GAAAGGGAACACACAAUAG
595
1047
GAAAGGGAACACACAAUAG
595
1065
CUAUUGUGUGUUCCCUUUC
674

1065
GAAGAGUGGAAAGAAUUGA
596
1065
GAAGAGUGGAAAGAAUUGA
596
1083
UCAAUUCUUUCCACUCUUC
675

1083
AUAUAUAAGGAAGUUAUGG
597
1083
AUAUAUAAGGAAGUUAUGG
597
1101
CCAUAACUUCCUUAUAUAU
676

1101
GACUUGGAGGAGAGAACCA
598
1101
GACUUGGAGGAGAGAACCA
598
1119
UGGUUCUCUCCUCCAAGUC
677

1119
AAGAAUGGAGUUAUACGGG
599
1119
AAGAAUGGAGUUAUACGGG
599
1137
CCCGUAUAACUCCAUUCUU
678

1137
GGGCAGCCCUCUCCUUUAG
600
1137
GGGCAGCCCUCUCCUUUAG
600
1155
CUAAAGGAGAGGGCUGCCC
679

1155
GCACAGGUGCAGCAGUGAU
601
1155
GCACAGGUGCAGCAGUGAU
601
1173
AUCACUGCUGCACCUGUGC
680

1173
UCAAUGGCUCUCAGCAUCC
602
1173
UCAAUGGCUCUCAGCAUCC
602
1191
GGAUGCUGAGAGCCAUUGA
681

1191
CAUCAUCAUCGUCGUCUGU
603
1191
CAUCAUCAUCGUCGUCUGU
603
1209
ACAGACGACGAUGAUGAUG
682

1209
UCAAUGAUGUGUCUUCAAU
604
1209
UCAAUGAUGUGUCUUCAAU
604
1227
AUUGAAGACACAUCAUUGA
683

1227
UGUCAACAGAUCCGACUUU
605
1227
UGUCAACAGAUCCGACUUU
605
1245
AAAGUCGGAUCUGUUGACA
684

1245
UGGCCUCUGAUACAGACAG
606
1245
UGGCCUCUGAUACAGACAG
606
1263
CUGUCUGUAUCAGAGGCCA
685

1263
GCAGUCUAGAAGCAGCAGC
607
1263
GCAGUCUAGAAGCAGCAGC
607
1281
GCUGCUGCUUCUAGACUGC
686

1281
CUGGGCCUCUGGGCUGCUG
608
1281
CUGGGCCUCUGGGCUGCUG
608
1299
CAGCAGCCCAGAGGCCCAG
687

1299
GUAGAUGACUACUUGGGCC
609
1299
GUAGAUGACUACUUGGGCC
609
1317
GGCCCAAGUAGUCAUCUAC
688

1317
CAUCGGGGGGUGGGAGGGA
610
1317
CAUCGGGGGGUGGGAGGGA
610
1335
UCCCUCCCACCCCCCGAUG
689

1335
AUGGGGAGUCGGUUAGUCA
611
1335
AUGGGGAGUCGGUUAGUCA
611
1353
UGACUAACCGACUCCCCAU
690

1353
AUUGAUAGAACUACUUUGA
612
1353
AUUGAUAGAACUACUUUGA
612
1371
UCAAAGUAGUUCUAUCAAU
691

1371
AAAACAAUUCAGUGGUCUU
613
1371
AAAACAAUUCAGUGGUCUU
613
1389
AAGACCACUGAAUUGUUUU
692

1389
UAUUUUUGGGUGAUUUUUC
614
1389
UAUUUUUGGGUGAUUUUUC
614
1407
GAAAAAUCACCCAAAAAUA
693

1397
GGUGAUUUUUCAAAAAAUG
615
1397
GGUGAUUUUUCAAAAAAUG
615
1415
CAUUUUUUGAAAAAUCACC
694

ID

Seq

Seq

Pos
Target Sequence
Seq
UPos
Upper seq
ID
LPos
Lower seq
ID

NM_139012 (MAPK14/p38)

3
AACCGCGACCACUGGAGCC
695
3
AACCGCGACCACUGGAGCC
695
21
GGCUCCAGUGGUCGCGGUU
904

21
CUUAGCGGGCGCAGCAGCU
696
21
CUUAGCGGGCGCAGCAGCU
696
39
AGCUGCUGCGCCCGCUAAG
905

39
UGGAACGGGAGUACUGCGA
697
39
UGGAACGGGAGUACUGCGA
697
57
UCGCAGUACUCCCGUUCCA
906

57
ACGCAGCCCGGAGUCGGCC
698
57
ACGCAGCCCGGAGUCGGCC
698
75
GGCCGACUCCGGGCUGCGU
907

75
CUUGUAGGGGCGAAGGUGC
699
75
CUUGUAGGGGCGAAGGUGC
699
93
GCACCUUCGCCCCUACAAG
908

93
CAGGGAGAUCGCGGCGGGC
700
93
CAGGGAGAUCGCGGCGGGC
700
111
GCCCGCCGCGAUCUCCCUG
909

111
CGCAGUCUUGAGCGCCGGA
701
111
CGCAGUCUUGAGCGCCGGA
701
129
UCCGGCGCUCAAGACUGCG
910

129
AGCGCGUCCCUGCCCUUAG
702
129
AGCGCGUCCCUGCCCUUAG
702
147
CUAAGGGCAGGGACGCGCU
911

147
GCGGGGCUUGCCCCAGUCG
703
147
GCGGGGCUUGCCCCAGUCG
703
165
CGACUGGGGCAAGCCCCGC
912

165
GCAGGGGCACAUCCAGCCG
704
165
GCAGGGGCACAUCCAGCCG
704
183
CGGCUGGAUGUGCCCCUGC
913

183
GCUGCGGCUGACAGCAGCC
705
183
GCUGCGGCUGACAGCAGCC
705
201
GGCUGCUGUCAGCCGCAGC
914

201
CGCGCGCGCGGGAGUCUGC
706
201
CGCGCGCGCGGGAGUCUGC
706
219
GCAGACUCCCGCGCGCGCG
915

219
CGGGGUCGCGGCAGCCGCA
707
219
CGGGGUCGCGGCAGCCGCA
707
237
UGCGGCUGCCGCGACCCCG
916

237
ACCUGCGCGGGCGACCAGC
708
237
ACCUGCGCGGGCGACCAGC
708
255
GCUGGUCGCCCGCGCAGGU
917

255
CGCAAGGUCCCCGCCCGGC
709
255
CGCAAGGUCCCCGCCCGGC
709
273
GCCGGGCGGGGACCUUGCG
918

273
CUGGGCGGGCAGCAAGGGC
710
273
CUGGGCGGGCAGCAAGGGC
710
291
GCCCUUGCUGCCCGCCCAG
919

291
CCGGGGAGAGGGUGCGGGU
711
291
CCGGGGAGAGGGUGCGGGU
711
309
ACCCGCACCCUCUCCCCGG
920

309
UGCAGGCGGGGGCCCCACA
712
309
UGCAGGCGGGGGCCCCACA
712
327
UGUGGGGCCCCCGCCUGCA
921

327
AGGGCCACCUUCUUGCCCG
713
327
AGGGCCACCUUCUUGCCCG
713
345
CGGGCAAGAAGGUGGCCCU
922

345
GGCGGCUGCCGCUGGAAAA
714
345
GGCGGCUGCCGCUGGAAAA
714
363
UUUUCCAGCGGCAGCCGCC
923

363
AUGUCUCAGGAGAGGCCCA
715
363
AUGUCUCAGGAGAGGCCCA
715
381
UGGGCCUCUCCUGAGACAU
924

381
ACGUUCUACCGGCAGGAGC
716
381
ACGUUCUACCGGCAGGAGC
716
399
GCUCCUGCCGGUAGAACGU
925

399
CUGAACAAGACAAUCUGGG
717
399
CUGAACAAGACAAUCUGGG
717
417
CCCAGAUUGUCUUGUUCAG
926

417
GAGGUGCCCGAGCGUUACC
718
417
GAGGUGCCCGAGCGUUACC
718
435
GGUAACGCUCGGGCACCUC
927

435
CAGAACCUGUCUCCAGUGG
719
435
CAGAACCUGUCUCCAGUGG
719
453
CCACUGGAGACAGGUUCUG
928

453
GGCUCUGGCGCCUAUGGCU
720
453
GGCUCUGGCGCCUAUGGCU
720
471
AGCCAUAGGCGCCAGAGCC
929

471
UCUGUGUGUGCUGCUUUUG
721
471
UCUGUGUGUGCUGCUUUUG
721
489
CAAAAGCAGCACACACAGA
930

489
GACACAAAAACGGGGUUAC
722
489
GACACAAAAACGGGGUUAC
722
507
GUAACCCCGUUUUUGUGUC
931

507
CGUGUGGCAGUGAAGAAGC
723
507
CGUGUGGCAGUGAAGAAGC
723
525
GCUUCUUCACUGCCACACG
932

525
CUCUCCAGACCAUUUCAGU
724
525
CUCUCCAGACCAUUUCAGU
724
543
ACUGAAAUGGUCUGGAGAG
933

543
UCCAUCAUUCAUGCGAAAA
725
543
UCCAUCAUUCAUGCGAAAA
725
561
UUUUCGCAUGAAUGAUGGA
934

561
AGAACCUACAGAGAACUGC
726
561
AGAACCUACAGAGAACUGC
726
579
GCAGUUCUCUGUAGGUUCU
935

579
CGGUUACUUAAACAUAUGA
727
579
CGGUUACUUAAACAUAUGA
727
597
UCAUAUGUUUAAGUAACCG
936

597
AAACAUGAAAAUGUGAUUG
728
597
AAACAUGAAAAUGUGAUUG
728
615
CAAUCACAUUUUCAUGUUU
937

615
GGUCUGUUGGACGUUUUUA
729
615
GGUCUGUUGGACGUUUUUA
729
633
UAAAAACGUCCAACAGACC
938

633
ACACCUGCAAGGUCUCUGG
730
633
ACACCUGCAAGGUCUCUGG
730
651
CCAGAGACCUUGCAGGUGU
939

651
GAGGAAUUCAAUGAUGUGU
731
651
GAGGAAUUCAAUGAUGUGU
731
669
ACACAUCAUUGAAUUCCUC
940

669
UAUCUGGUGACCCAUCUCA
732
669
UAUCUGGUGACCCAUCUCA
732
687
UGAGAUGGGUCACCAGAUA
941

687
AUGGGGGCAGAUCUGAACA
733
687
AUGGGGGCAGAUCUGAACA
733
705
UGUUCAGAUCUGCCCCCAU
942

705
AACAUUGUGAAAUGUCAGA
734
705
AACAUUGUGAAAUGUCAGA
734
723
UCUGACAUUUCACAAUGUU
943

723
AAGCUUACAGAUGACCAUG
735
723
AAGCUUACAGAUGACCAUG
735
741
CAUGGUCAUCUGUAAGCUU
944

741
GUUCAGUUCCUUAUCUACC
736
741
GUUCAGUUCCUUAUCUACC
736
759
GGUAGAUAAGGAACUGAAC
945

759
CAAAUUCUCCGAGGUCUAA
737
759
CAAAUUCUCCGAGGUCUAA
737
777
UUAGACCUCGGAGAAUUUG
946

777
AAGUAUAUACAUUCAGCUG
738
777
AAGUAUAUACAUUCAGCUG
738
795
CAGCUGAAUGUAUAUACUU
947

795
GACAUAAUUCACAGGGACC
739
795
GACAUAAUUCACAGGGACC
739
813
GGUCCCUGUGAAUUAUGUC
948

813
CUAAAACCUAGUAAUCUAG
740
813
CUAAAACCUAGUAAUCUAG
740
831
CUAGAUUACUAGGUUUUAG
949

831
GCUGUGAAUGAAGACUGUG
741
831
GCUGUGAAUGAAGACUGUG
741
849
CACAGUCUUCAUUCACAGC
950

849
GAGCUGAAGAUUCUGGAUU
742
849
GAGCUGAAGAUUCUGGAUU
742
867
AAUCCAGAAUCUUCAGCUC
951

867
UUUGGACUGGCUCGGCACA
743
867
UUUGGACUGGCUCGGCACA
743
885
UGUGCCGAGCCAGUCCAAA
952

885
ACAGAUGAUGAAAUGACAG
744
885
ACAGAUGAUGAAAUGACAG
744
903
CUGUCAUUUCAUCAUCUGU
953

903
GGCUACGUGGCCACUAGGU
745
903
GGCUACGUGGCCACUAGGU
745
921
ACCUAGUGGCCACGUAGCC
954

921
UGGUACAGGGCUCCUGAGA
746
921
UGGUACAGGGCUCCUGAGA
746
939
UCUCAGGAGCCCUGUACCA
955

939
AUCAUGCUGAACUGGAUGC
747
939
AUCAUGCUGAACUGGAUGC
747
957
GCAUCCAGUUCAGCAUGAU
956

957
CAUUACAACCAGACAGUUG
748
957
CAUUACAACCAGACAGUUG
748
975
CAACUGUCUGGUUGUAAUG
957

975
GAUAUUUGGUCAGUGGGAU
749
975
GAUAUUUGGUCAGUGGGAU
749
993
AUCCCACUGACCAAAUAUC
958

993
UGCAUAAUGGCCGAGCUGU
750
993
UGCAUAAUGGCCGAGCUGU
750
1011
ACAGCUCGGCCAUUAUGCA
959

1011
UUGACUGGAAGAACAUUGU
751
1011
UUGACUGGAAGAACAUUGU
751
1029
ACAAUGUUCUUCCAGUCAA
960

1029
UUUCCUGGUACAGACCAUA
752
1029
UUUCCUGGUACAGACCAUA
752
1047
UAUGGUCUGUACCAGGAAA
961

1047
AUUGAUCAGUUGAAGCUCA
753
1047
AUUGAUCAGUUGAAGCUCA
753
1065
UGAGCUUCAACUGAUCAAU
962

1065
AUUUUAAGACUCGUUGGAA
754
1065
AUUUUAAGACUCGUUGGAA
754
1083
UUCCAACGAGUCUUAAAAU
963

1083
ACCCCAGGGGCUGAGCUUU
755
1083
ACCCCAGGGGCUGAGCUUU
755
1101
AAAGCUCAGCCCCUGGGGU
964

1101
UUGAAGAAAAUCUCCUCAG
756
1101
UUGAAGAAAAUCUCCUCAG
756
1119
CUGAGGAGAUUUUCUUCAA
965

1119
GAGUCUGCAAGAAACUAUA
757
1119
GAGUCUGCAAGAAACUAUA
757
1137
UAUAGUUUCUUGCAGACUC
966

1137
AUUCAGUCUUUGACUCAGA
758
1137
AUUCAGUCUUUGACUCAGA
758
1155
UCUGAGUCAAAGACUGAAU
967

1155
AUGCCGAAGAUGAACUUUG
759
1155
AUGCCGAAGAUGAACUUUG
759
1173
CAAAGUUCAUCUUCGGCAU
968

1173
GCGAAUGUAUUUAUUGGUG
760
1173
GCGAAUGUAUUUAUUGGUG
760
1191
CACCAAUAAAUACAUUCGC
969

1191
GCCAAUCCCCUGGCUGUCG
761
1191
GCCAAUCCCCUGGCUGUCG
761
1209
CGACAGCCAGGGGAUUGGC
970

1209
GACUUGCUGGAGAAGAUGC
762
1209
GACUUGCUGGAGAAGAUGC
762
1227
GCAUCUUCUCCAGCAAGUC
971

1227
CUUGUAUUGGACUCAGAUA
763
1227
CUUGUAUUGGACUCAGAUA
763
1245
UAUCUGAGUCCAAUACAAG
972

1245
AAGAGAAUUACAGCGGCCC
764
1245
AAGAGAAUUACAGCGGCCC
764
1263
GGGCCGCUGUAAUUCUCUU
973

1263
CAAGCCCUUGCACAUGCCU
765
1263
CAAGCCCUUGCACAUGCCU
765
1281
AGGCAUGUGCAAGGGCUUG
974

1281
UACUUUGCUCAGUACCACG
766
1281
UACUUUGCUCAGUACCACG
766
1299
CGUGGUACUGAGCAAAGUA
975

1299
GAUCCUGAUGAUGAACCAG
767
1299
GAUCCUGAUGAUGAACCAG
767
1317
CUGGUUCAUCAUCAGGAUC
976

1317
GUGGCCGAUCCUUAUGAUC
768
1317
GUGGCCGAUCCUUAUGAUC
768
1335
GAUCAUAAGGAUCGGCCAC
977

1335
CAGUCCUUUGAAAGCAGGG
769
1335
CAGUCCUUUGAAAGCAGGG
769
1353
CCCUGCUUUCAAAGGACUG
978

1353
GACCUCCUUAUAGAUGAGU
770
1353
GACCUCCUUAUAGAUGAGU
770
1371
ACUCAUCUAUAAGGAGGUC
979

1371
UGGAAAAGCCUGACCUAUG
771
1371
UGGAAAAGCCUGACCUAUG
771
1389
CAUAGGUCAGGCUUUUCCA
980

1389
GAUGAAGUCAUCAGCUUUG
772
1389
GAUGAAGUCAUCAGCUUUG
772
1407
CAAAGCUGAUGACUUCAUC
981

1407
GUGCCACCACCCCUUGACC
773
1407
GUGCCACCACCCCUUGACC
773
1425
GGUCAAGGGGUGGUGGCAC
982

1425
CAAGAAGAGAUGGAGUCCU
774
1425
CAAGAAGAGAUGGAGUCCU
774
1443
AGGACUCCAUCUCUUCUUG
983

1443
UGAGCACCUGGUUUCUGUU
775
1443
UGAGCACCUGGUUUCUGUU
775
1461
AACAGAAACCAGGUGCUCA
984

1461
UCUGUUGAUCCCACUUCAC
776
1461
UCUGUUGAUCCCACUUCAC
776
1479
GUGAAGUGGGAUCAACAGA
985

1479
CUGUGAGGGGAAGGCCUUU
777
1479
CUGUGAGGGGAAGGCCUUU
777
1497
AAAGGCCUUCCCCUCACAG
986

1497
UUCACGGGAACUCUCCAAA
778
1497
UUCACGGGAACUCUCCAAA
778
1515
UUUGGAGAGUUCCCGUGAA
987

1515
AUAUUAUUCAAGUGCCUCU
779
1515
AUAUUAUUCAAGUGCCUCU
779
1533
AGAGGCACUUGAAUAAUAU
988

1533
UUGUUGCAGAGAUUUCCUC
780
1533
UUGUUGCAGAGAUUUCCUC
780
1551
GAGGAAAUCUCUGCAACAA
989

1551
CCAUGGUGGAAGGGGGUGU
781
1551
CCAUGGUGGAAGGGGGUGU
781
1569
ACACCCCCUUCCACCAUGG
990

1569
UGCGUGCGUGUGCGUGCGU
782
1569
UGCGUGCGUGUGCGUGCGU
782
1587
ACGCACGCACACGCACGCA
991

1587
UGUUAGUGUGUGUGCAUGU
783
1587
UGUUAGUGUGUGUGCAUGU
783
1605
ACAUGCACACACACUAACA
992

1605
UGUGUGUCUGUCUUUGUGG
784
1605
UGUGUGUCUGUCUUUGUGG
784
1623
CCACAAAGACAGACACACA
993

1623
GGAGGGUAAGACAAUAUGA
785
1623
GGAGGGUAAGACAAUAUGA
785
1641
UCAUAUUGUCUUACCCUCC
994

1641
AACAAACUAUGAUCACAGU
786
1641
AACAAACUAUGAUCACAGU
786
1659
ACUGUGAUCAUAGUUUGUU
995

1659
UGACUUUACAGGAGGUUGU
787
1659
UGACUUUACAGGAGGUUGU
787
1677
ACAACCUCCUGUAAAGUCA
996

1677
UGGAUGCUCCAGGGCAGCC
788
1677
UGGAUGCUCCAGGGCAGCC
788
1695
GGCUGCCCUGGAGCAUCCA
997

1695
CUCCACCUUGCUCUUCUUU
789
1695
CUCCACCUUGCUCUUCUUU
789
1713
AAAGAAGAGCAAGGUGGAG
998

1713
UCUGAGAGUUGGCUCAGGC
790
1713
UCUGAGAGUUGGCUCAGGC
790
1731
GCCUGAGCCAACUCUCAGA
999

1731
CAGACAAGAGCUGCUGUCC
791
1731
CAGACAAGAGCUGCUGUCC
791
1749
GGACAGCAGCUCUUGUCUG
1000

1749
CUUUUAGGAAUAUGUUCAA
792
1749
CUUUUAGGAAUAUGUUCAA
792
1767
UUGAACAUAUUCCUAAAAG
1001

1767
AUGCAAAGUAAAAAAAUAU
793
1767
AUGCAAAGUAAAAAAAUAU
793
1785
AUAUUUUUUUACUUUGCAU
1002

1785
UGAAUUGUCCCCAAUCCCG
794
1785
UGAAUUGUCCCCAAUCCCG
794
1803
CGGGAUUGGGGACAAUUCA
1003

1803
GGUCAUGCUUUUGCCACUU
795
1803
GGUCAUGCUUUUGCCACUU
795
1821
AAGUGGCAAAAGCAUGACC
1004

1821
UUGGCUUCUCCUGUGACCC
796
1821
UUGGCUUCUCCUGUGACCC
796
1839
GGGUCACAGGAGAAGCCAA
1005

1839
CCACCUUGACGGUGGGGCG
797
1839
CCACCUUGACGGUGGGGCG
797
1857
CGCCCCACCGUCAAGGUGG
1006

1857
GUAGACUUGACAACAUCCC
798
1857
GUAGACUUGACAACAUCCC
798
1875
GGGAUGUUGUCAAGUCUAC
1007

1875
CACAGUGGCACGGAGAGAA
799
1875
CACAGUGGCACGGAGAGAA
799
1893
UUCUCUCCGUGCCACUGUG
1008

1893
AGGCCCAUACCUUCUGGUU
800
1893
AGGCCCAUACCUUCUGGUU
800
1911
AACCAGAAGGUAUGGGCCU
1009

1911
UGCUUCAGACCUGACACCG
801
1911
UGCUUCAGACCUGACACCG
801
1929
CGGUGUCAGGUCUGAAGCA
1010

1929
GUCCCUCAGUGAUACGUAC
802
1929
GUCCCUCAGUGAUACGUAC
802
1947
GUACGUAUCACUGAGGGAC
1011

1947
CAGCCAAAAAGGACCAACU
803
1947
CAGCCAAAAAGGACCAACU
803
1965
AGUUGGUCCUUUUUGGCUG
1012

1965
UGGCUUCUGUGCACUAGCC
804
1965
UGGCUUCUGUGCACUAGCC
804
1983
GGCUAGUGCACAGAAGCCA
1013

1983
CUGUGAUUAACUUGCUUAG
805
1983
CUGUGAUUAACUUGCUUAG
805
2001
CUAAGCAAGUUAAUCACAG
1014

2001
GUAUGGUUCUCAGAUCUUG
806
2001
GUAUGGUUCUCAGAUCUUG
806
2019
CAAGAUCUGAGAACCAUAC
1015

2019
GACAGUAUAUUUGAAACUG
807
2019
GACAGUAUAUUUGAAACUG
807
2037
CAGUUUCAAAUAUACUGUC
1016

2037
GUAAAUAUGUUUGUGCCUU
808
2037
GUAAAUAUGUUUGUGCCUU
808
2055
AAGGCACAAACAUAUUUAC
1017

2055
UAAAAGGAGAGAAGAAAGU
809
2055
UAAAAGGAGAGAAGAAAGU
809
2073
ACUUUCUUCUCUCCUUUUA
1018

2073
UGUAGAUAGUUAAAAGACU
810
2073
UGUAGAUAGUUAAAAGACU
810
2091
AGUCUUUUAACUAUCUACA
1019

2091
UGCAGCUGCUGAAGUUCUG
811
2091
UGCAGCUGCUGAAGUUCUG
811
2109
CAGAACUUCAGCAGCUGCA
1020

2109
GAGCCGGGCAAGUCGAGAG
812
2109
GAGCCGGGCAAGUCGAGAG
812
2127
CUCUCGACUUGCCCGGCUC
1021

2127
GGGCUGUUGGACAGCUGCU
813
2127
GGGCUGUUGGACAGCUGCU
813
2145
AGCAGCUGUCCAACAGCCC
1022

2145
UUGUGGGCCCGGAGUAAUC
814
2145
UUGUGGGCCCGGAGUAAUC
814
2163
GAUUACUCCGGGCCCACAA
1023

2163
CAGGCAGCCUUCAUAGGCG
815
2163
CAGGCAGCCUUCAUAGGCG
815
2181
CGCCUAUGAAGGCUGCCUG
1024

2181
GGUCAUGUGUGCAUGUGAG
816
2181
GGUCAUGUGUGCAUGUGAG
816
2199
CUCACAUGCACACAUGACC
1025

2199
GCACAUGCGUAUAUGUGCG
817
2199
GCACAUGCGUAUAUGUGCG
817
2217
CGCACAUAUACGCAUGUGC
1026

2217
GUCUCUCUUUCUCCCUCAC
818
2217
GUCUCUCUUUCUCCCUCAC
818
2235
GUGAGGGAGAAAGAGAGAC
1027

2235
CCCCCAGGUGUUGCCAUUU
819
2235
CCCCCAGGUGUUGCCAUUU
819
2253
AAAUGGCAACACCUGGGGG
1028

2253
UCUCUGCUUACCCUUCACC
820
2253
UCUCUGCUUACCCUUCACC
820
2271
GGUGAAGGGUAAGCAGAGA
1029

2271
CUUUGGUGCAGAGGUUUCU
821
2271
CUUUGGUGCAGAGGUUUCU
821
2289
AGAAACCUCUGCACCAAAG
1030

2289
UUGAAUAUCUGCCCCAGUA
822
2289
UUGAAUAUCUGCCCCAGUA
822
2307
UACUGGGGCAGAUAUUCAA
1031

2307
AGUCAGAAGCAGGUUCUUG
823
2307
AGUCAGAAGCAGGUUCUUG
823
2325
CAAGAACCUGCUUCUGACU
1032

2325
GAUGUCAUGUACUUCCUGU
824
2325
GAUGUCAUGUACUUCCUGU
824
2343
ACAGGAAGUACAUGACAUC
1033

2343
UGUACUCUUUAUUUCUAGC
825
2343
UGUACUCUUUAUUUCUAGC
825
2361
GCUAGAAAUAAAGAGUACA
1034

2361
CAGAGUGAGGAUGUGUUUU
826
2361
CAGAGUGAGGAUGUGUUUU
826
2379
AAAACACAUCCUCACUCUG
1035

2379
UGCACGUCUUGCUAUUUGA
827
2379
UGCACGUCUUGCUAUUUGA
827
2397
UCAAAUAGCAAGACGUGCA
1036

2397
AGCAUGCACAGCUGCUUGU
828
2397
AGCAUGCACAGCUGCUUGU
828
2415
ACAAGCAGCUGUGCAUGCU
1037

2415
UCCUGCUCUCUUCAGGAGG
829
2415
UCCUGCUCUCUUCAGGAGG
829
2433
CCUCCUGAAGAGAGCAGGA
1038

2433
GCCCUGGUGUCAGGCAGGU
830
2433
GCCCUGGUGUCAGGCAGGU
830
2451
ACCUGCCUGACACCAGGGC
1039

2451
UUUGCCAGUGAAGACUUCU
831
2451
UUUGCCAGUGAAGACUUCU
831
2469
AGAAGUCUUCACUGGCAAA
1040

2469
UUGGGUAGUUUAGAUCCCA
832
2469
UUGGGUAGUUUAGAUCCCA
832
2487
UGGGAUCUAAACUACCCAA
1041

2487
AUGUCACCUCAGCUGAUAU
833
2487
AUGUCACCUCAGCUGAUAU
833
2505
AUAUCAGCUGAGGUGACAU
1042

2505
UUAUGGCAAGUGAUAUCAC
834
2505
UUAUGGCAAGUGAUAUCAC
834
2523
GUGAUAUCACUUGCCAUAA
1043

2523
CCUCUCUUCAGCCCCUAGU
835
2523
CCUCUCUUCAGCCCCUAGU
835
2541
ACUAGGGGCUGAAGAGAGG
1044

2541
UGCUAUUCUGUGUUGAACA
836
2541
UGCUAUUCUGUGUUGAACA
836
2559
UGUUCAACACAGAAUAGCA
1045

2559
ACAAUUGAUACUUCAGGUG
837
2559
ACAAUUGAUACUUCAGGUG
837
2577
CACCUGAAGUAUCAAUUGU
1046

2577
GCUUUUGAUGUGAAAAUCA
838
2577
GCUUUUGAUGUGAAAAUCA
838
2595
UGAUUUUCACAUCAAAAGC
1047

2595
AUGAAAAGAGGAACAGGUG
839
2595
AUGAAAAGAGGAACAGGUG
839
2613
CACCUGUUCCUCUUUUCAU
1048

2613
GGAUGUAUAGCAUUUUUAU
840
2613
GGAUGUAUAGCAUUUUUAU
840
2631
AUAAAAAUGCUAUACAUCC
1049

2631
UUCAUGCCAUCUGUUUUCA
841
2631
UUCAUGCCAUCUGUUUUCA
841
2649
UGAAAACAGAUGGCAUGAA
1050

2649
AACCAACUAUUUUUGAGGA
842
2649
AACCAACUAUUUUUGAGGA
842
2667
UCCUCAAAAAUAGUUGGUU
1051

2667
AAUUAUCAUGGGAAAAGAC
843
2667
AAUUAUCAUGGGAAAAGAC
843
2685
GUCUUUUCCCAUGAUAAUU
1052

2685
CCAGGGCUUUUCCCAGGAA
844
2685
CCAGGGCUUUUCCCAGGAA
844
2703
UUCCUGGGAAAAGCCCUGG
1053

2703
AUAUCCCAAACUUCGGAAA
845
2703
AUAUCCCAAACUUCGGAAA
845
2721
UUUCCGAAGUUUGGGAUAU
1054

2721
ACAAGUUAUUCUCUUCACU
846
2721
ACAAGUUAUUCUCUUCACU
846
2739
AGUGAAGAGAAUAACUUGU
1055

2739
UCCCAAUAACUAAUGCUAA
847
2739
UCCCAAUAACUAAUGCUAA
847
2757
UUAGCAUUAGUUAUUGGGA
1056

2757
AGAAAUGCUGAAAAUCAAA
848
2757
AGAAAUGCUGAAAAUCAAA
848
2775
UUUGAUUUUCAGCAUUUCU
1057

2775
AGUAAAAAAUUAAAGCCCA
849
2775
AGUAAAAAAUUAAAGCCCA
849
2793
UGGGCUUUAAUUUUUUACU
1058

2793
AUAAGGCCAGAAACUCCUU
850
2793
AUAAGGCCAGAAACUCCUU
850
2811
AAGGAGUUUCUGGCCUUAU
1059

2811
UUUGCUGUCUUUCUCUAAA
851
2811
UUUGCUGUCUUUCUCUAAA
851
2829
UUUAGAGAAAGACAGCAAA
1060

2829
AUAUGAUUACUUUAAAAUA
852
2829
AUAUGAUUACUUUAAAAUA
852
2847
UAUUUUAAAGUAAUCAUAU
1061

2847
AAAAAAGUAACAAGGUGUC
853
2847
AAAAAAGUAACAAGGUGUC
853
2865
GACACCUUGUUACUUUUUU
1062

2865
CUUUUCCACUCCUAUGGAA
854
2865
CUUUUCCACUCCUAUGGAA
854
2883
UUCCAUAGGAGUGGAAAAG
1063

2883
AAAGGGUCUUCUUGGCAGC
855
2883
AAAGGGUCUUCUUGGCAGC
855
2901
GCUGCCAAGAAGACCCUUU
1064

2901
CUUAACAUUGACUUCUUGG
856
2901
CUUAACAUUGACUUCUUGG
856
2919
CCAAGAAGUCAAUGUUAAG
1065

2919
GUUUGGGGAGAAAUAAAUU
857
2919
GUUUGGGGAGAAAUAAAUU
857
2937
AAUUUAUUUCUCCCCAAAC
1066

2937
UUUGUUUCAGAAUUUUGUA
858
2937
UUUGUUUCAGAAUUUUGUA
858
2955
UACAAAAUUCUGAAACAAA
1067

2955
AUAUUGUAGGAAUCCCUUU
859
2955
AUAUUGUAGGAAUCCCUUU
859
2973
AAAGGGAUUCCUACAAUAU
1068

2973
UGAGAAUGUGAUUCCUUUU
860
2973
UGAGAAUGUGAUUCCUUUU
860
2991
AAAAGGAAUCACAUUCUCA
1069

2991
UGAUGGGGAGAAAGGGCAA
861
2991
UGAUGGGGAGAAAGGGCAA
861
3009
UUGCCCUUUCUCCCCAUCA
1070

3009
AAUUAUUUUAAUAUUUUGU
862
3009
AAUUAUUUUAAUAUUUUGU
862
3027
ACAAAAUAUUAAAAUAAUU
1071

3027
UAUUUUCAACUUUAUAAAG
863
3027
UAUUUUCAACUUUAUAAAG
863
3045
CUUUAUAAAGUUGAAAAUA
1072

3045
GAUAAAAUAUCCUCAGGGG
864
3045
GAUAAAAUAUCCUCAGGGG
864
3063
CCCCUGAGGAUAUUUUAUC
1073

3063
GUGGAGAAGUGUCGUUUUC
865
3063
GUGGAGAAGUGUCGUUUUC
865
3081
GAAAACGACACUUCUCCAC
1074

3081
CAUAACUUGCUGAAUUUCA
866
3081
CAUAACUUGCUGAAUUUCA
866
3099
UGAAAUUCAGCAAGUUAUG
1075

3099
AGGCAUUUUGUUCUACAUG
867
3099
AGGCAUUUUGUUCUACAUG
867
3117
CAUGUAGAACAAAAUGCCU
1076

3117
GAGGACUCAUAUAUUUAAG
868
3117
GAGGACUCAUAUAUUUAAG
868
3135
CUUAAAUAUAUGAGUCCUC
1077

3135
GCCUUUUGUGUAAUAAGAA
869
3135
GCCUUUUGUGUAAUAAGAA
869
3153
UUCUUAUUACACAAAAGGC
1078

3153
AAGUAUAAAGUCACUUCCA
870
3153
AAGUAUAAAGUCACUUCCA
870
3171
UGGAAGUGACUUUAUACUU
1079

3171
AGUGUUGGCUGUGUGACAG
871
3171
AGUGUUGGCUGUGUGACAG
871
3189
CUGUCACACAGCCAACACU
1080

3189
GAAUCUUGUAUUUGGGCCA
872
3189
GAAUCUUGUAUUUGGGCCA
872
3207
UGGCCCAAAUACAAGAUUC
1081

3207
AAGGUGUUUCCAUUUCUCA
873
3207
AAGGUGUUUCCAUUUCUCA
873
3225
UGAGAAAUGGAAACACCUU
1082

3225
AAUCAGUGCAGUGAUACAU
874
3225
AAUCAGUGCAGUGAUACAU
874
3243
AUGUAUCACUGCACUGAUU
1083

3243
UGUACUCCAGAGGGACAGG
875
3243
UGUACUCCAGAGGGACAGG
875
3261
CCUGUCCCUCUGGAGUACA
1084

3261
GGUGGACCCCCUGAGUCAA
876
3261
GGUGGACCCCCUGAGUCAA
876
3279
UUGACUCAGGGGGUCCACC
1085

3279
ACUGGAGCAAGAAGGAAGG
877
3279
ACUGGAGCAAGAAGGAAGG
877
3297
CCUUCCUUCUUGCUCCAGU
1086

3297
GAGGCAGACUGAUGGCGAU
878
3297
GAGGCAGACUGAUGGCGAU
878
3315
AUCGCCAUCAGUCUGCCUC
1087

3315
UUCCCUCUCACCCGGGACU
879
3315
UUCCCUCUCACCCGGGACU
879
3333
AGUCCCGGGUGAGAGGGAA
1088

3333
UCUCCCCCUUUCAAGGAAA
880
3333
UCUCCCCCUUUCAAGGAAA
880
3351
UUUCCUUGAAAGGGGGAGA
1089

3351
AGUGAACCUUUAAAGUAAA
881
3351
AGUGAACCUUUAAAGUAAA
881
3369
UUUACUUUAAAGGUUCACU
1090

3369
AGGCCUCAUCUCCUUUAUU
882
3369
AGGCCUCAUCUCCUUUAUU
882
3387
AAUAAAGGAGAUGAGGCCU
1091

3387
UGCAGUUCAAAUCCUCACC
883
3387
UGCAGUUCAAAUCCUCACC
883
3405
GGUGAGGAUUUGAACUGCA
1092

3405
CAUCCACAGCAAGAUGAAU
884
3405
CAUCCACAGCAAGAUGAAU
884
3423
AUUCAUCUUGCUGUGGAUG
1093

3423
UUUUAUCAGCCAUGUUUGG
885
3423
UUUUAUCAGCCAUGUUUGG
885
3441
CCAAACAUGGCUGAUAAAA
1094

3441
GUUGUAAAUGCUCGUGUGA
886
3441
GUUGUAAAUGCUCGUGUGA
886
3459
UCACACGAGCAUUUACAAC
1095

3459
AUUUCCUACAGAAAUACUG
887
3459
AUUUCCUACAGAAAUACUG
887
3477
CAGUAUUUCUGUAGGAAAU
1096

3477
GCUCUGAAUAUUUUGUAAU
888
3477
GCUCUGAAUAUUUUGUAAU
888
3495
AUUACAAAAUAUUCAGAGC
1097

3495
UAAAGGUCUUUGCACAUGU
889
3495
UAAAGGUCUUUGCACAUGU
889
3513
ACAUGUGCAAAGACCUUUA
1098

3513
UGACCACAUACGUGUUAGG
890
3513
UGACCACAUACGUGUUAGG
890
3531
CCUAACACGUAUGUGGUCA
1099

3531
GAGGCUGCAUGCUCUGGAA
891
3531
GAGGCUGCAUGCUCUGGAA
891
3549
UUCCAGAGCAUGCAGCCUC
1100

3549
AGCCUGGACUCUAAGCUGG
892
3549
AGCCUGGACUCUAAGCUGG
892
3567
CCAGCUUAGAGUCCAGGCU
1101

3567
GAGCUCUUGGAAGAGCUCU
893
3567
GAGCUCUUGGAAGAGCUCU
893
3585
AGAGCUCUUCCAAGAGCUC
1102

3585
UUCGGUUUCUGAGCAUAAU
894
3585
UUCGGUUUCUGAGCAUAAU
894
3603
AUUAUGCUCAGAAACCGAA
1103

3603
UGCUCCCAUCUCCUGAUUU
895
3603
UGCUCCCAUCUCCUGAUUU
895
3621
AAAUCAGGAGAUGGGAGCA
1104

3621
UCUCUGAACAGAAAACAAA
896
3621
UCUCUGAACAGAAAACAAA
896
3639
UUUGUUUUCUGUUCAGAGA
1105

3639
AAGAGAGAAUGAGGGAAAU
897
3639
AAGAGAGAAUGAGGGAAAU
897
3657
AUUUCCCUCAUUCUCUCUU
1106

3657
UUGCUAUUUUAUUUGUAUU
898
3657
UUGCUAUUUUAUUUGUAUU
898
3675
AAUACAAAUAAAAUAGCAA
1107

3675
UCAUGAACUUGGCUGUAAU
899
3675
UCAUGAACUUGGCUGUAAU
899
3693
AUUACAGCCAAGUUCAUGA
1108

3693
UCAGUUAUGCCGUAUAGGA
900
3693
UCAGUUAUGCCGUAUAGGA
900
3711
UCCUAUACGGCAUAACUGA
1109

3711
AUGUCAGACAAUACCACUG
901
3711
AUGUCAGACAAUACCACUG
901
3729
CAGUGGUAUUGUCUGACAU
1110

3729
GGUUAAAAUAAAGCCUAUU
902
3729
GGUUAAAAUAAAGCCUAUU
902
3747
AAUAGGCUUUAUUUUAACC
1111

3737
UAAAGCCUAUUUUUCAAAU
903
3737
UAAAGCCUAUUUUUCAAAU
903
3755
AUUUGAAAAAUAGGCUUUA
1112

Seq

Seq

Seq

Pos
Seq
ID
UPos
Upper seq
ID
LPos
Lower seq
ID

hJUN NM_002228

3
AGUUGCACUGAGUGUGGCU
1247
3
AGUUGCACUGAGUGUGGCU
1247
21
AGCCACACUCAGUGCAACU
1428

21
UGAAGCAGCGAGGCGGGAG
1248
21
UGAAGCAGCGAGGCGGGAG
1248
39
CUCCCGCCUCGCUGCUUCA
1429

39
GUGGAGGUGCGCGGAGUCA
1249
39
GUGGAGGUGCGCGGAGUCA
1249
57
UGACUCCGCGCACCUCCAC
1430

57
AGGCAGACAGACAGACACA
1250
57
AGGCAGACAGACAGACACA
1250
75
UGUGUCUGUCUGUCUGCCU
1431

75
AGCCAGCCAGCCAGGUCGG
1251
75
AGCCAGCCAGCCAGGUCGG
1251
93
CCGACCUGGCUGGCUGGCU
1432

93
GCAGUAUAGUCCGAACUGC
1252
93
GCAGUAUAGUCCGAACUGC
1252
111
GCAGUUCGGACUAUACUGC
1433

111
CAAAUCUUAUUUUCUUUUC
1253
111
CAAAUCUUAUUUUCUUUUC
1253
129
GAAAAGAAAAUAAGAUUUG
1434

129
CACCUUCUCUCUAACUGCC
1254
129
CACCUUCUCUCUAACUGCC
1254
147
GGCAGUUAGAGAGAAGGUG
1435

147
CCAGAGCUAGCGCCUGUGG
1255
147
CCAGAGCUAGCGCCUGUGG
1255
165
CCACAGGCGCUAGCUCUGG
1436

165
GCUCCCGGGCUGGUGGUUC
1256
165
GCUCCCGGGCUGGUGGUUC
1256
183
GAACCACCAGCCCGGGAGC
1437

183
CGGGAGUGUCCAGAGAGCC
1257
183
CGGGAGUGUCCAGAGAGCC
1257
201
GGCUCUCUGGACACUCCCG
1438

201
CUUGUCUCCAGCCGGCCCC
1258
201
CUUGUCUCCAGCCGGCCCC
1258
219
GGGGCCGGCUGGAGACAAG
1439

219
CGGGAGGAGAGCCCUGCUG
1259
219
CGGGAGGAGAGCCCUGCUG
1259
237
CAGCAGGGCUCUCCUCCCG
1440

237
GCCCAGGCGCUGUUGACAG
1260
237
GCCCAGGCGCUGUUGACAG
1260
255
CUGUCAACAGCGCCUGGGC
1441

255
GCGGCGGAAAGCAGCGGUA
1261
255
GCGGCGGAAAGCAGCGGUA
1261
273
UACCGCUGCUUUCCGCCGC
1442

273
ACCCCACGCGCCCGCCGGG
1262
273
ACCCCACGCGCCCGCCGGG
1262
291
CCCGGCGGGCGCGUGGGGU
1443

291
GGGACGUCGGCGAGCGGCU
1263
291
GGGACGUCGGCGAGCGGCU
1263
309
AGCCGCUCGCCGACGUCCC
1444

309
UGCAGCAGCAAAGAACUUU
1264
309
UGCAGCAGCAAAGAACUUU
1264
327
AAAGUUCUUUGCUGCUGCA
1445

327
UCCCGGCGGGGAGGACCGG
1265
327
UCCCGGCGGGGAGGACCGG
1265
345
CCGGUCCUCCCCGCCGGGA
1446

345
GAGACAAGUGGCAGAGUCC
1266
345
GAGACAAGUGGCAGAGUCC
1266
363
GGACUCUGCCACUUGUCUC
1447

363
CCGGAGCGAACUUUUGCAA
1267
363
CCGGAGCGAACUUUUGCAA
1267
381
UUGCAAAAGUUCGCUCCGG
1448

381
AGCCUUUCCUGCGUCUUAG
1268
381
AGCCUUUCCUGCGUCUUAG
1268
399
CUAAGACGCAGGAAAGGCU
1449

399
GGCUUCUCCACGGCGGUAA
1269
399
GGCUUCUCCACGGCGGUAA
1269
417
UUACCGCCGUGGAGAAGCC
1450

417
AAGACCAGAAGGCGGCGGA
1270
417
AAGACCAGAAGGCGGCGGA
1270
435
UCCGCCGCCUUCUGGUCUU
1451

435
AGAGCCACGCAAGAGAAGA
1271
435
AGAGCCACGCAAGAGAAGA
1271
453
UCUUCUCUUGCGUGGCUCU
1452

453
AAGGACGUGCGCUCAGCUU
1272
453
AAGGACGUGCGCUCAGCUU
1272
471
AAGCUGAGCGCACGUCCUU
1453

471
UCGCUCGCACCGGUUGUUG
1273
471
UCGCUCGCACCGGUUGUUG
1273
489
CAACAACCGGUGCGAGCGA
1454

489
GAACUUGGGCGAGCGCGAG
1274
489
GAACUUGGGCGAGCGCGAG
1274
507
CUCGCGCUCGCCCAAGUUC
1455

507
GCCGCGGCUGCCGGGCGCC
1275
507
GCCGCGGCUGCCGGGCGCC
1275
525
GGCGCCCGGCAGCCGCGGC
1456

525
CCCCUCCCCCUAGCAGCGG
1276
525
CCCCUCCCCCUAGCAGCGG
1276
543
CCGCUGCUAGGGGGAGGGG
1457

543
GAGGAGGGGACAAGUCGUC
1277
543
GAGGAGGGGACAAGUCGUC
1277
561
GACGACUUGUCCCCUCCUC
1458

561
CGGAGUCCGGGCGGCCAAG
1278
561
CGGAGUCCGGGCGGCCAAG
1278
579
CUUGGCCGCCCGGACUCCG
1459

579
GACCCGCCGCCGGCCGGCC
1279
579
GACCCGCCGCCGGCCGGCC
1279
597
GGCCGGCCGGCGGCGGGUC
1460

597
CACUGCAGGGUCCGCACUG
1280
597
CACUGCAGGGUCCGCACUG
1280
615
CAGUGCGGACCCUGCAGUG
1461

615
GAUCCGCUCCGCGGGGAGA
1281
615
GAUCCGCUCCGCGGGGAGA
1281
633
UCUCCCCGCGGAGCGGAUC
1462

633
AGCCGCUGCUCUGGGAAGU
1282
633
AGCCGCUGCUCUGGGAAGU
1282
651
ACUUCCCAGAGCAGCGGCU
1463

651
UGAGUUCGCCUGCGGACUC
1283
651
UGAGUUCGCCUGCGGACUC
1283
669
GAGUCCGCAGGCGAACUCA
1464

669
CCGAGGAACCGCUGCGCCC
1284
669
CCGAGGAACCGCUGCGCCC
1284
687
GGGCGCAGCGGUUCCUCGG
1465

687
CGAAGAGCGCUCAGUGAGU
1285
687
CGAAGAGCGCUCAGUGAGU
1285
705
ACUCACUGAGCGCUCUUCG
1466

705
UGACCGCGACUUUUCAAAG
1286
705
UGACCGCGACUUUUCAAAG
1286
723
CUUUGAAAAGUCGCGGUCA
1467

723
GCCGGGUAGCGCGCGCGAG
1287
723
GCCGGGUAGCGCGCGCGAG
1287
741
CUCGCGCGCGCUACCCGGC
1468

741
GUCGACAAGUAAGAGUGCG
1288
741
GUCGACAAGUAAGAGUGCG
1288
759
CGCACUCUUACUUGUCGAC
1469

759
GGGAGGCAUCUUAAUUAAC
1289
759
GGGAGGCAUCUUAAUUAAC
1289
777
GUUAAUUAAGAUGCCUCCC
1470

777
CCCUGCGCUCCCUGGAGCG
1290
777
CCCUGCGCUCCCUGGAGCG
1290
795
CGCUCCAGGGAGCGCAGGG
1471

795
GAGCUGGUGAGGAGGGCGC
1291
795
GAGCUGGUGAGGAGGGCGC
1291
813
GCGCCCUCCUCACCAGCUC
1472

813
CAGCGGGGACGACAGCCAG
1292
813
CAGCGGGGACGACAGCCAG
1292
831
CUGGCUGUCGUCCCCGCUG
1473

831
GCGGGUGCGUGCGCUCUUA
1293
831
GCGGGUGCGUGCGCUCUUA
1293
849
UAAGAGCGCACGCACCCGC
1474

849
AGAGAAACUUUCCCUGUCA
1294
849
AGAGAAACUUUCCCUGUCA
1294
867
UGACAGGGAAAGUUUCUCU
1475

867
AAAGGCUCCGGGGGGCGCG
1295
867
AAAGGCUCCGGGGGGCGCG
1295
885
CGCGCCCCCCGGAGCCUUU
1476

885
GGGUGUCCCCCGCUUGCCA
1296
885
GGGUGUCCCCCGCUUGCCA
1296
903
UGGCAAGCGGGGGACACCC
1477

903
AGAGCCCUGUUGCGGCCCC
1297
903
AGAGCCCUGUUGCGGCCCC
1297
921
GGGGCCGCAACAGGGCUCU
1478

921
CGAAACUUGUGCGCGCACG
1298
921
CGAAACUUGUGCGCGCACG
1298
939
CGUGCGCGCACAAGUUUCG
1479

939
GCCAAACUAACCUCACGUG
1299
939
GCCAAACUAACCUCACGUG
1299
957
CACGUGAGGUUAGUUUGGC
1480

957
GAAGUGACGGACUGUUCUA
1300
957
GAAGUGACGGACUGUUCUA
1300
975
UAGAACAGUCCGUCACUUC
1481

975
AUGACUGCAAAGAUGGAAA
1301
975
AUGACUGCAAAGAUGGAAA
1301
993
UUUCCAUCUUUGCAGUCAU
1482

993
ACGACCUUCUAUGACGAUG
1302
993
ACGACCUUCUAUGACGAUG
1302
1011
CAUCGUCAUAGAAGGUCGU
1483

1011
GCCCUCAACGCCUCGUUCC
1303
1011
GCCCUCAACGCCUCGUUCC
1303
1029
GGAACGAGGCGUUGAGGGC
1484

1029
CUCCCGUCCGAGAGCGGAC
1304
1029
CUCCCGUCCGAGAGCGGAC
1304
1047
GUCCGCUCUCGGACGGGAG
1485

1047
CCUUAUGGCUACAGUAACC
1305
1047
CCUUAUGGCUACAGUAACC
1305
1065
GGUUACUGUAGCCAUAAGG
1486

1065
CCCAAGAUCCUGAAACAGA
1306
1065
CCCAAGAUCCUGAAACAGA
1306
1083
UCUGUUUCAGGAUCUUGGG
1487

1083
AGCAUGACCCUGAACCUGG
1307
1083
AGCAUGACCCUGAACCUGG
1307
1101
CCAGGUUCAGGGUCAUGCU
1488

1101
GCCGACCCAGUGGGGAGCC
1308
1101
GCCGACCCAGUGGGGAGCC
1308
1119
GGCUCCCCACUGGGUCGGC
1489

1119
CUGAAGCCGCACCUCCGCG
1309
1119
CUGAAGCCGCACCUCCGCG
1309
1137
CGCGGAGGUGCGGCUUCAG
1490

1137
GCCAAGAACUCGGACCUCC
1310
1137
GCCAAGAACUCGGACCUCC
1310
1155
GGAGGUCCGAGUUCUUGGC
1491

1155
CUCACCUCGCCCGACGUGG
1311
1155
CUCACCUCGCCCGACGUGG
1311
1173
CCACGUCGGGCGAGGUGAG
1492

1173
GGGCUGCUCAAGCUGGCGU
1312
1173
GGGCUGCUCAAGCUGGCGU
1312
1191
ACGCCAGCUUGAGCAGCCC
1493

1191
UCGCCCGAGCUGGAGCGCC
1313
1191
UCGCCCGAGCUGGAGCGCC
1313
1209
GGCGCUCCAGCUCGGGCGA
1494

1209
CUGAUAAUCCAGUCCAGCA
1314
1209
CUGAUAAUCCAGUCCAGCA
1314
1227
UGCUGGACUGGAUUAUCAG
1495

1227
AACGGGCACAUCACCACCA
1315
1227
AACGGGCACAUCACCACCA
1315
1245
UGGUGGUGAUGUGCCCGUU
1496

1245
ACGCCGACCCCCACCCAGU
1316
1245
ACGCCGACCCCCACCCAGU
1316
1263
ACUGGGUGGGGGUCGGCGU
1497

1263
UUCCUGUGCCCCAAGAACG
1317
1263
UUCCUGUGCCCCAAGAACG
1317
1281
CGUUCUUGGGGCACAGGAA
1498

1281
GUGACAGAUGAGCAGGAGG
1318
1281
GUGACAGAUGAGCAGGAGG
1318
1299
CCUCCUGCUCAUCUGUCAC
1499

1299
GGGUUCGCCGAGGGCUUCG
1319
1299
GGGUUCGCCGAGGGCUUCG
1319
1317
CGAAGCCCUCGGCGAACCC
1500

1317
GUGCGCGCCCUGGCCGAAC
1320
1317
GUGCGCGCCCUGGCCGAAC
1320
1335
GUUCGGCCAGGGCGCGCAC
1501

1335
CUGCACAGCCAGAACACGC
1321
1335
CUGCACAGCCAGAACACGC
1321
1353
GCGUGUUCUGGCUGUGCAG
1502

1353
CUGCCCAGCGUCACGUCGG
1322
1353
CUGCCCAGCGUCACGUCGG
1322
1371
CCGACGUGACGCUGGGCAG
1503

1371
GCGGCGCAGCCGGUCAACG
1323
1371
GCGGCGCAGCCGGUCAACG
1323
1389
CGUUGACCGGCUGCGCCGC
1504

1389
GGGGCAGGCAUGGUGGCUC
1324
1389
GGGGCAGGCAUGGUGGCUC
1324
1407
GAGCCACCAUGCCUGCCCC
1505

1407
CCCGCGGUAGCCUCGGUGG
1325
1407
CCCGCGGUAGCCUCGGUGG
1325
1425
CCACCGAGGCUACCGCGGG
1506

1425
GCAGGGGGCAGCGGCAGCG
1326
1425
GCAGGGGGCAGCGGCAGCG
1326
1443
CGCUGCCGCUGCCCCCUGC
1507

1443
GGCGGCUUCAGCGCCAGCC
1327
1443
GGCGGCUUCAGCGCCAGCC
1327
1461
GGCUGGCGCUGAAGCCGCC
1508

1461
CUGCACAGCGAGCCGCCGG
1328
1461
CUGCACAGCGAGCCGCCGG
1328
1479
CCGGCGGCUCGCUGUGCAG
1509

1479
GUCUACGCAAACCUCAGCA
1329
1479
GUCUACGCAAACCUCAGCA
1329
1497
UGCUGAGGUUUGCGUAGAC
1510

1497
AACUUCAACCCAGGCGCGC
1330
1497
AACUUCAACCCAGGCGCGC
1330
1515
GCGCGCCUGGGUUGAAGUU
1511

1515
CUGAGCAGCGGCGGCGGGG
1331
1515
CUGAGCAGCGGCGGCGGGG
1331
1533
CCCCGCCGCCGCUGCUCAG
1512

1533
GCGCCCUCCUACGGCGCGG
1332
1533
GCGCCCUCCUACGGCGCGG
1332
1551
CCGCGCCGUAGGAGGGCGC
1513

1551
GCCGGCCUGGCCUUUCCCG
1333
1551
GCCGGCCUGGCCUUUCCCG
1333
1569
CGGGAAAGGCCAGGCCGGC
1514

1569
GCGCAACCCCAGCAGCAGC
1334
1569
GCGCAACCCCAGCAGCAGC
1334
1587
GCUGCUGCUGGGGUUGCGC
1515

1587
CAGCAGCCGCCGCACCACC
1335
1587
CAGCAGCCGCCGCACCACC
1335
1605
GGUGGUGCGGCGGCUGCUG
1516

1605
CUGCCCCAGCAGAUGCCCG
1336
1605
CUGCCCCAGCAGAUGCCCG
1336
1623
CGGGCAUCUGCUGGGGCAG
1517

1623
GUGCAGCACCCGCGGCUGC
1337
1623
GUGCAGCACCCGCGGCUGC
1337
1641
GCAGCCGCGGGUGCUGCAC
1518

1641
CAGGCCCUGAAGGAGGAGC
1338
1641
CAGGCCCUGAAGGAGGAGC
1338
1659
GCUCCUCCUUCAGGGCCUG
1519

1659
CCUCAGACAGUGCCCGAGA
1339
1659
CCUCAGACAGUGCCCGAGA
1339
1677
UCUCGGGCACUGUCUGAGG
1520

1677
AUGCCCGGCGAGACACCGC
1340
1677
AUGCCCGGCGAGACACCGC
1340
1695
GCGGUGUCUCGCCGGGCAU
1521

1695
CCCCUGUCCCCCAUCGACA
1341
1695
CCCCUGUCCCCCAUCGACA
1341
1713
UGUCGAUGGGGGACAGGGG
1522

1713
AUGGAGUCCCAGGAGCGGA
1342
1713
AUGGAGUCCCAGGAGCGGA
1342
1731
UCCGCUCCUGGGACUCCAU
1523

1731
AUCAAGGCGGAGAGGAAGC
1343
1731
AUCAAGGCGGAGAGGAAGC
1343
1749
GCUUCCUCUCCGCCUUGAU
1524

1749
CGCAUGAGGAACCGCAUCG
1344
1749
CGCAUGAGGAACCGCAUCG
1344
1767
CGAUGCGGUUCCUCAUGCG
1525

1767
GCUGCCUCCAAGUGCCGAA
1345
1767
GCUGCCUCCAAGUGCCGAA
1345
1785
UUCGGCACUUGGAGGCAGC
1526

1785
AAAAGGAAGCUGGAGAGAA
1346
1785
AAAAGGAAGCUGGAGAGAA
1346
1803
UUCUCUCCAGCUUCCUUUU
1527

1803
AUCGCCCGGCUGGAGGAAA
1347
1803
AUCGCCCGGCUGGAGGAAA
1347
1821
UUUCCUCCAGCCGGGCGAU
1528

1821
AAAGUGAAAACCUUGAAAG
1348
1821
AAAGUGAAAACCUUGAAAG
1348
1839
CUUUCAAGGUUUUCACUUU
1529

1839
GCUCAGAACUCGGAGCUGG
1349
1839
GCUCAGAACUCGGAGCUGG
1349
1857
CCAGCUCCGAGUUCUGAGC
1530

1857
GCGUCCACGGCCAACAUGC
1350
1857
GCGUCCACGGCCAACAUGC
1350
1875
GCAUGUUGGCCGUGGACGC
1531

1875
CUCAGGGAACAGGUGGCAC
1351
1875
CUCAGGGAACAGGUGGCAC
1351
1893
GUGCCACCUGUUCCCUGAG
1532

1893
CAGCUUAAACAGAAAGUCA
1352
1893
CAGCUUAAACAGAAAGUCA
1352
1911
UGACUUUCUGUUUAAGCUG
1533

1911
AUGAACCACGUUAACAGUG
1353
1911
AUGAACCACGUUAACAGUG
1353
1929
CACUGUUAACGUGGUUCAU
1534

1929
GGGUGCCAACUCAUGCUAA
1354
1929
GGGUGCCAACUCAUGCUAA
1354
1947
UUAGCAUGAGUUGGCACCC
1535

1947
ACGCAGCAGUUGCAAACAU
1355
1947
ACGCAGCAGUUGCAAACAU
1355
1965
AUGUUUGCAACUGCUGCGU
1536

1965
UUUUGAAGAGAGACCGUCG
1356
1965
UUUUGAAGAGAGACCGUCG
1356
1983
CGACGGUCUCUCUUCAAAA
1537

1983
GGGGGCUGAGGGGCAACGA
1357
1983
GGGGGCUGAGGGGCAACGA
1357
2001
UCGUUGCCCCUCAGCCCCC
1538

2001
AAGAAAAAAAAUAACACAG
1358
2001
AAGAAAAAAAAUAACACAG
1358
2019
CUGUGUUAUUUUUUUUCUU
1539

2019
GAGAGACAGACUUGAGAAC
1359
2019
GAGAGACAGACUUGAGAAC
1359
2037
GUUCUCAAGUCUGUCUCUC
1540

2037
CUUGACAAGUUGCGACGGA
1360
2037
CUUGACAAGUUGCGACGGA
1360
2055
UCCGUCGCAACUUGUCAAG
1541

2055
AGAGAAAAAAGAAGUGUCC
1361
2055
AGAGAAAAAAGAAGUGUCC
1361
2073
GGACACUUCUUUUUUCUCU
1542

2073
CGAGAACUAAAGCCAAGGG
1362
2073
CGAGAACUAAAGCCAAGGG
1362
2091
CCCUUGGCUUUAGUUCUCG
1543

2091
GUAUCCAAGUUGGACUGGG
1363
2091
GUAUCCAAGUUGGACUGGG
1363
2109
CCCAGUCCAACUUGGAUAC
1544

2109
GUUCGGUCUGACGGCGCCC
1364
2109
GUUCGGUCUGACGGCGCCC
1364
2127
GGGCGCCGUCAGACCGAAC
1545

2127
CCCAGUGUGCACGAGUGGG
1365
2127
CCCAGUGUGCACGAGUGGG
1365
2145
CCCACUCGUGCACACUGGG
1546

2145
GAAGGACUUGGUCGCGCCC
1366
2145
GAAGGACUUGGUCGCGCCC
1366
2163
GGGCGCGACCAAGUCCUUC
1547

2163
CUCCCUUGGCGUGGAGCCA
1367
2163
CUCCCUUGGCGUGGAGCCA
1367
2181
UGGCUCCACGCCAAGGGAG
1548

2181
AGGGAGCGGCCGCCUGCGG
1368
2181
AGGGAGCGGCCGCCUGCGG
1368
2199
CCGCAGGCGGCCGCUCCCU
1549

2199
GGCUGCCCCGCUUUGCGGA
1369
2199
GGCUGCCCCGCUUUGCGGA
1369
2217
UCCGCAAAGCGGGGCAGCC
1550

2217
ACGGGCUGUCCCCGCGCGA
1370
2217
ACGGGCUGUCCCCGCGCGA
1370
2235
UCGCGCGGGGACAGCCCGU
1551

2235
AACGGAACGUUGGACUUUC
1371
2235
AACGGAACGUUGGACUUUC
1371
2253
GAAAGUCCAACGUUCCGUU
1552

2253
CGUUAACAUUGACCAAGAA
1372
2253
CGUUAACAUUGACCAAGAA
1372
2271
UUCUUGGUCAAUGUUAACG
1553

2271
ACUGCAUGGACCUAACAUU
1373
2271
ACUGCAUGGACCUAACAUU
1373
2289
AAUGUUAGGUCCAUGCAGU
1554

2289
UCGAUCUCAUUCAGUAUUA
1374
2289
UCGAUCUCAUUCAGUAUUA
1374
2307
UAAUACUGAAUGAGAUCGA
1555

2307
AAAGGGGGGAGGGGGAGGG
1375
2307
AAAGGGGGGAGGGGGAGGG
1375
2325
CCCUCCCCCUCCCCCCUUU
1556

2325
GGGUUACAAACUGCAAUAG
1376
2325
GGGUUACAAACUGCAAUAG
1376
2343
CUAUUGCAGUUUGUAACCC
1557

2343
GAGACUGUAGAUUGCUUCU
1377
2343
GAGACUGUAGAUUGCUUCU
1377
2361
AGAAGCAAUCUACAGUCUC
1558

2361
UGUAGUACUCCUUAAGAAC
1378
2361
UGUAGUACUCCUUAAGAAC
1378
2379
GUUCUUAAGGAGUACUACA
1559

2379
CACAAAGCGGGGGGAGGGU
1379
2379
CACAAAGCGGGGGGAGGGU
1379
2397
ACCCUCCCCCCGCUUUGUG
1560

2397
UUGGGGAGGGGCGGCAGGA
1380
2397
UUGGGGAGGGGCGGCAGGA
1380
2415
UCCUGCCGCCCCUCCCCAA
1561

2415
AGGGAGGUUUGUGAGAGCG
1381
2415
AGGGAGGUUUGUGAGAGCG
1381
2433
CGCUCUCACAAACCUCCCU
1562

2433
GAGGCUGAGCCUACAGAUG
1382
2433
GAGGCUGAGCCUACAGAUG
1382
2451
CAUCUGUAGGCUCAGCCUC
1563

2451
GAACUCUUUCUGGCCUGCU
1383
2451
GAACUCUUUCUGGCCUGCU
1383
2469
AGCAGGCCAGAAAGAGUUC
1564

2469
UUUCGUUAACUGUGUAUGU
1384
2469
UUUCGUUAACUGUGUAUGU
1384
2487
ACAUACACAGUUAACGAAA
1565

2487
UACAUAUAUAUAUUUUUUA
1385
2487
UACAUAUAUAUAUUUUUUA
1385
2505
UAAAAAAUAUAUAUAUGUA
1566

2505
AAUUUGAUUAAAGCUGAUU
1386
2505
AAUUUGAUUAAAGCUGAUU
1386
2523
AAUCAGCUUUAAUCAAAUU
1567

2523
UACUGUCAAUAAACAGCUU
1387
2523
UACUGUCAAUAAACAGCUU
1387
2541
AAGCUGUUUAUUGACAGUA
1568

2541
UCAUGCCUUUGUAAGUUAU
1388
2541
UCAUGCCUUUGUAAGUUAU
1388
2559
AUAACUUACAAAGGCAUGA
1569

2559
UUUCUUGUUUGUUUGUUUG
1389
2559
UUUCUUGUUUGUUUGUUUG
1389
2577
CAAACAAACAAACAAGAAA
1570

2577
GGGUAUCCUGCCCAGUGUU
1390
2577
GGGUAUCCUGCCCAGUGUU
1390
2595
AACACUGGGCAGGAUACCC
1571

2595
UGUUUGUAAAUAAGAGAUU
1391
2595
UGUUUGUAAAUAAGAGAUU
1391
2613
AAUCUCUUAUUUACAAACA
1572

2613
UUGGAGCACUCUGAGUUUA
1392
2613
UUGGAGCACUCUGAGUUUA
1392
2631
UAAACUCAGAGUGCUCCAA
1573

2631
ACCAUUUGUAAUAAAGUAU
1393
2631
ACCAUUUGUAAUAAAGUAU
1393
2649
AUACUUUAUUACAAAUGGU
1574

2649
UAUAAUUUUUUUAUGUUUU
1394
2649
UAUAAUUUUUUUAUGUUUU
1394
2667
AAAACAUAAAAAAAUUAUA
1575

2667
UGUUUCUGAAAAUUCCAGA
1395
2667
UGUUUCUGAAAAUUCCAGA
1395
2685
UCUGGAAUUUUCAGAAACA
1576

2685
AAAGGAUAUUUAAGAAAAU
1396
2685
AAAGGAUAUUUAAGAAAAU
1396
2703
AUUUUCUUAAAUAUCCUUU
1577

2703
UACAAUAAACUAUUGGAAA
1397
2703
UACAAUAAACUAUUGGAAA
1397
2721
UUUCCAAUAGUUUAUUGUA
1578

2721
AGUACUCCCCUAACCUCUU
1398
2721
AGUACUCCCCUAACCUCUU
1398
2739
AAGAGGUUAGGGGAGUACU
1579

2739
UUUCUGCAUCAUCUGUAGA
1399
2739
UUUCUGCAUCAUCUGUAGA
1399
2757
UCUACAGAUGAUGCAGAAA
1580

2757
AUCCUAGUCUAUCUAGGUG
1400
2757
AUCCUAGUCUAUCUAGGUG
1400
2775
CACCUAGAUAGACUAGGAU
1581

2775
GGAGUUGAAAGAGUUAAGA
1401
2775
GGAGUUGAAAGAGUUAAGA
1401
2793
UCUUAACUCUUUCAACUCC
1582

2793
AAUGCUCGAUAAAAUCACU
1402
2793
AAUGCUCGAUAAAAUCACU
1402
2811
AGUGAUUUUAUCGAGCAUU
1583

2811
UCUCAGUGCUUCUUACUAU
1403
2811
UCUCAGUGCUUCUUACUAU
1403
2829
AUAGUAAGAAGCACUGAGA
1584

2829
UUAAGCAGUAAAAACUGUU
1404
2829
UUAAGCAGUAAAAACUGUU
1404
2847
AACAGUUUUUACUGCUUAA
1585

2847
UCUCUAUUAGACUUAGAAA
1405
2847
UCUCUAUUAGACUUAGAAA
1405
2865
UUUCUAAGUCUAAUAGAGA
1586

2865
AUAAAUGUACCUGAUGUAC
1406
2865
AUAAAUGUACCUGAUGUAC
1406
2883
GUACAUCAGGUACAUUUAU
1587

2883
CCUGAUGCUAUGUCAGGCU
1407
2883
CCUGAUGCUAUGUCAGGCU
1407
2901
AGCCUGACAUAGCAUCAGG
1588

2901
UUCAUACUCCACGCUCCCC
1408
2901
UUCAUACUCCACGCUCCCC
1408
2919
GGGGAGCGUGGAGUAUGAA
1589

2919
CCAGCGUAUCUAUAUGGAA
1409
2919
CCAGCGUAUCUAUAUGGAA
1409
2937
UUCCAUAUAGAUACGCUGG
1590

2937
AUUGCUUACCAAAGGCUAG
1410
2937
AUUGCUUACCAAAGGCUAG
1410
2955
CUAGCCUUUGGUAAGCAAU
1591

2955
GUGCGAUGUUUCAGGAGGC
1411
2955
GUGCGAUGUUUCAGGAGGC
1411
2973
GCCUCCUGAAACAUCGCAC
1592

2973
CUGGAGGAAGGGGGGUUGC
1412
2973
CUGGAGGAAGGGGGGUUGC
1412
2991
GCAACCCCCCUUCCUCCAG
1593

2991
CAGUGGAGAGGGACAGCCC
1413
2991
CAGUGGAGAGGGACAGCCC
1413
3009
GGGCUGUCCCUCUCCACUG
1594

3009
CACUGAGAAGUCAAACAUU
1414
3009
CACUGAGAAGUCAAACAUU
1414
3027
AAUGUUUGACUUCUCAGUG
1595

3027
UUCAAAGUUUGGAUUGCAU
1415
3027
UUCAAAGUUUGGAUUGCAU
1415
3045
AUGCAAUCCAAACUUUGAA
1596

3045
UCAAGUGGCAUGUGCUGUG
1416
3045
UCAAGUGGCAUGUGCUGUG
1416
3063
CACAGCACAUGCCACUUGA
1597

3063
GACCAUUUAUAAUGUUAGA
1417
3063
GACCAUUUAUAAUGUUAGA
1417
3081
UCUAACAUUAUAAAUGGUC
1598

3081
AAAUUUUACAAUAGGUGCU
1418
3081
AAAUUUUACAAUAGGUGCU
1418
3099
AGCACCUAUUGUAAAAUUU
1599

3099
UUAUUCUCAAAGCAGGAAU
1419
3099
UUAUUCUCAAAGCAGGAAU
1419
3117
AUUCCUGCUUUGAGAAUAA
1600

3117
UUGGUGGCAGAUUUUACAA
1420
3117
UUGGUGGCAGAUUUUACAA
1420
3135
UUGUAAAAUCUGCCACCAA
1601

3135
AAAGAUGUAUCCUUCCAAU
1421
3135
AAAGAUGUAUCCUUCCAAU
1421
3153
AUUGGAAGGAUACAUCUUU
1602

3153
UUUGGAAUCUUCUCUUUGA
1422
3153
UUUGGAAUCUUCUCUUUGA
1422
3171
UCAAAGAGAAGAUUCCAAA
1603

3171
ACAAUUCCUAGAUAAAAAG
1423
3171
ACAAUUCCUAGAUAAAAAG
1423
3189
CUUUUUAUCUAGGAAUUGU
1604

3189
GAUGGCCUUUGUCUUAUGA
1424
3189
GAUGGCCUUUGUCUUAUGA
1424
3207
UCAUAAGACAAAGGCCAUC
1605

3207
AAUAUUUAUAACAGCAUUC
1425
3207
AAUAUUUAUAACAGCAUUC
1425
3225
GAAUGCUGUUAUAAAUAUU
1606

3225
CUGUCACAAUAAAUGUAUU
1426
3225
CUGUCACAAUAAAUGUAUU
1426
3243
AAUACAUUUAUUGUGACAG
1607

3234
UAAAUGUAUUCAAAUACCA
1427
3234
UAAAUGUAUUCAAAUACCA
1427
3252
UGGUAUUUGAAUACAUUUA
1608

The 3-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence.

The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand.

The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I-VII or any combination thereof.

TABLE III

MAP Kinase Synthetic Modified siNA constructs

Target

Seq

Seq

Pos
Target
ID
Aliases
Sequence
ID

MAPK1/ERK2

3302
ACCAGACCUACUGCCAGAGAACC
1113
MAPK1:424U21 siRNA sense
CAGACCUACUGCCAGAGAATT
1129

3852
AUCACACAGGGUUCCUGACAGAA
1114
MAPK1:778U21 siRNA sense
CACACAGGGUUCCUGACAGTT
1130

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MAPK1:1718U21 siRNA sense
GGCUCUAGUCACUGGCAUCTT
1131

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MAPK1:2525U21 siRNA sense
UGUGGAGUUGACUCGGUGUTT
1132

3302
ACCAGACCUACUGCCAGAGAACC
1113
MAPK1:442L21 siRNA (424C)
UUCUCUGGCAGUAGGUCUGTT
1133

antisense

3852
AUCACACAGGGUUCCUGACAGAA
1114
MAPK1:796L21 siRNA (778C)
CUGUCAGGAACCCUGUGUGTT
1134

antisense

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MAPK1:1736L21 siRNA (1718C)
GAUGCCAGUGACUAGAGCCTT
1135

antisense

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MAPK1:2543L21 siRNA (2525C)
ACACCGAGUCAACUCCACATT
1136

antisense

3302
ACCAGACCUACUGCCAGAGAACC
1113
MAPK1:424U21 siRNA stab04
B cAGAccuAcuGccAGAGAATT B
1137

sense RPI 30817

3852
AUCACACAGGGUUCCUGACAGAA
1114
MAPK1:778U21 siRNA stab04
B cAcAcAGGGuuccuGAcAGTT B
1138

sense RPI 30818

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MAPK1:1718U21 siRNA stab04
B GGcucuAGucAcuGGcAucTT B
1139

sense RPI 30819

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MAPK1:2525U21 siRNA stab04
B uGuGGAGuuGAcucGGuGuTT B
1140

sense RPI 30820

3302
ACCAGACCUACUGCCAGAGAACC
1113
MAPK1:442L21 siRNA (424C)
uucucuGGcAGuAGGucuGTsT
1141

stab05 antisense RPI 30821

3852
AUCACACAGGGUUCCUGACAGAA
1114
MAPK1:796L21 siRNA (778C)
cuGucAGGAAcccuGuGuGTsT
1142

stab05 antisense RPI 30822

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MAPK1:1736L21 siRNA (1718C)
GAuGccAGuGAcuAGAGccTsT
1143

stab05 antisense RPI 30823

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MAPK1:2543L21 siRNA (2525C)
AcAccGAGucAAcuCCAcATsT
1144

stab05 antisense RPI 30824

3302
ACCAGACCUA0UGCCAGAGAACC
1113
MARK1:424U21 siRNA stab07
B cAGAccuAcuGccAGAGAATT B
1145

sense

3852
AUCACACAGGGUUCCUGACAGAA
1114
MARK1:778U21 siRNA stabo7
B cAcAcAGGGuuccuGAcAGTT B
1146

sense

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MAPK1:1718U21 siRNA stabo7
B GGcucuAGucAcuGGcAucTTB 1147

sense

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MARK1:2525U21 siRNA stabo7
B uGuGGAGuuGAcucGGuGuTT B
1148

sense

3302
ACCAGACCUACUGCCAGAGAACC
1113
MARK1:442L21 siRNA (424C)
uucucuGGcAGuAGGucuGTsT
1149

stab11 antisense

3852
AUCACACAGGGUUCCUGACAGAA
1114
MARK1:796L21 siRNA (778C)
cuGucAGGAAcccuGuGuGTsT
1150

stab11 antisense

3892
UUGGCUCUAGUCACUGGCAUCUC
1115
MARK1:1736L21 siRNA (1718C)

GAuGccAGuGAcuAGAGccTsT
1151

stab11 antisense

3946
ACUGUGGAGUUGACUCGGUGUUC
1116
MARK1:2543L21 siRNA (25250)

AcAccGAGucAAcuccAcATsT
1152

stab11 antisense

Target

Seq

Seq

Pos
Target
ID
Aliases
Sequence
ID

MAPK3/ERK1

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:285U21 siRNA sense
UUCGAACAUCAGACCUACUTT
1153

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:711U21 siRNA sense
AACUCCAAGGGCUAUACCATT
1154

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:720U21 siRNA sense
GGCUAUACCAAGUCCAUCGTT
1155

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1780U21 siRNA sense
CUGUGUGUGGUGAGCAGAATT
1156

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:303L21 siRNA (285C)
AGUAGGUCUGAUGUUCGAATT
1157

antisense

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:729L21 siRNA (711C)
UGGUAUAGCCCUUGGAGUUTT
1158

antisense

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:738L21 siRNA (720C)
CGAUGGACUUGGUAUAGCCTT
1159

antisense

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1798L21 siRNA (1780C)
UUCUGCUCACCACACACAGTT
1160

antisense

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:285U21 siRNA stab04 sense
B uucGAAcAucAGAccuAcuTT B
1161

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:711U21 siRNA stab04 sense
B AAcuccAAGGGcuAuAccATT B
1162

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:720U21 siRNA stab04 sense
B GGcuAuAccAAGuccAucGTT B
1163

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1780U21 siRNA stab04 sense
B cuGuGuGuGGuGAGcAGAATT B
1164

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:303L21 siRNA (285C) stab05
AGuAGGucuGAuGuucGAATsT
1165

antisense

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:729L21 siRNA (711C) stab05
uGGuAuAGcccuuGGAGuuTsT
1166

antisense

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:738L21 siRNA (720C) stab05
cGAuGGAcuuGGuAuAGccTsT
1167

antisense

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1798L21 siRNA (1780C)
uucuGcucAccAcAcAcAGTsT
1168

stab05 antisense

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:285U21 siRNA stab07 sense
B uucGAAcAucAGAccuAcuTT B
1169

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:711U21 siRNA stab07 sense
B AAcuccAAGGGcuAuAccATT B
1170

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:720U21 siRNA stab07 sense
B GGcuAuAccAAGuccAucGTT B
1171

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1780U21 siRNA stab07 sense
B cuGuGuGuGGuGAGcAGAATT B
1172

283
CCUUCGAACAUCAGACCUACUGC
1117
MAPK3:303L21 siRNA (285C) stab11

AGuAGGucuGAuGuucGAATsT
1173

antisense

709
UGAACUCCAAGGGCUAUACCAAG
1118
MAPK3:729L21 siRNA (711C) stab11
uGGuAuAGcccuuGGAGuuTsT
1174

antisense

718
AGGGCUAUACCAAGUCCAUCGAC
1119
MAPK3:738L21 siRNA (720C) stab11
cGAuGGAcuuGGuAuAGccTsT
1175

antisense

1778
UUCUGUGUGUGGUGAGCAGAAGU
1120
MAPK3:1798L21 siRNA (178C0)
uucuGcucAccAcAcAcAGTsT
1176

stab11 antisense

Target

Seq

Seq

Pos
Target
ID
RPI #
Aliases
Sequence
ID

MAPK8/JNK1

733
AACAGCUUGGAACACCAUGUCCU
1121
31517
MAPK8:735U21 siRNA sense
CAGCUUGGAACACCAUGUCTT
1177

853
UUUUCCCAGCUGACUCAGAACAC
1122
31518
MAPK8:855U21 siRNA sense
UUCCCAGCUGACUCAGAACTT
1178

1224
CAAUGUCAACAGAUCCGACUUUG
1123
31519
MAPK8:1226U21 siRNA sense
AUGUCAACAGAUCCGACUUTT
1179

1242
CUUUGGCCUCUGAUACAGACAGC
1124
31520
MAPK8:1244U21 siRNA sense
UUGGCCUCUGAUACAGACATT
1180

733
AACAGCUUGGAACACCAUGUCCU
1121
31521
MAPK8:753L21 siRNA (735C)
GACAUGGUGUUCCAAGCUGTT
1181

antisense

853
UUUUCCCAGCUGACUCAGAACAC
1122
31522
MAPK8:873L21 siRNA (855C)
GUUCUGAGUCAGCUGGGAATT
1182

antisense

1224
CAAUGUCAACAGAUCCGACUUUG
1123
31523
MAPK8:1244L21 siRNA (1226C)
AAGUCGGAUCUGUUGACAUTT
1183

antisense

1242
CUUUGGCCUCUGAUACAGACAGC
1124
31524
MAPK8:1262L21 siRNA (1244C)
UGUCUGUAUCAGAGGCCAATT
1184

antisense

733
AACAGCUUGGAACACCAUGUCCU
1121

MAPK8:735U21 siRNA stab4
B cAGcuuGGAAcAccAuGucTT B
1185

sense

853
UUUUCCCAGCUGACUCAGAACAC
1122

MAPK8:855U21 siRNA stab4
B uucccAGcuGAcucAGAAcTT B
1186

sense

1224
CAAUGUCAACAGAUCCGACUUUG
1123

MAPK8:1226U21 siRNA stab4
BAuGucAAcAGAuccGAcuuTTB
1187

sense

1242
CUUUGGCCUCUGAUACAGACAGC
1124

MAPK8:1244U21 siRNA stab4
B uuGGccucuGAuAcAGAcATT B
1188

sense

733
AACAGCUUGGAACACCAUGUCCU
1121

MAPK8:753L21 siRNA (735C)
GAcAuGGuGuuccAAGcuGTsT
1189

stab5 antisense

853
UUUUCCCAGCUGACUCAGAACAC
1122

MAPK8:873L21 siRNA (855C)
GuucuGAGucAGcuGGGAATsT
1190

stab5 antisense

1224
CAAUGUCAACAGAUCCGACUUUG
1123

MAPK8:1244L21 siRNA (1226C)
AAGucGGAucuGuuGAcAuTsT
1191

stab5 antisense

1242
CUUUGGCCUCUGAUACAGACAGC
1124

MAPK8:1262L21 siRNA (1244C)
uGucuGuAucAGAGGccAATsT
1192

stab5 antisense

733
AACAGCUUGGAACACCAUGUCCU
1121

MAPK8:735U21 siRNA stab7
BcAGcuuGGAAcAccAuGucTTB
1193

sense

853
UUUUCCCAGCUGACUCAGAACAC
1122

MAPK8:855U21 siRNA stab7
B uucccAGcuGAcucAGAAcTT B
1194

sense

1224
CAAUGUCAACAGAUCCGACUUUG
1123

MAPK8:1226U21 siRNA stab7
B AuGucAAcAGAuccGAcuuTT B
1195

sense

1242
CUUUGGCCUCUGAUACAGACAGC
1124

MAPK8:1244U21 siRNA stab7
B uuGGccucuGAuAcAGAcATT B
1196

sense

733
AACAGCUUGGAACACCAUGUCCU
1121

MAPK8:753L21 siRNA (735C)

GAcAuGGuGuuccAAGcuGTsT
1197

stab11 antisense

853
UUUUCCCAGCUGACUCAGAACAC
1122

MAPK8:873L21 siRNA (855C)

GuucuGAGucAGcuGGGAATsT
1198

stab11 antisense

1224
CAAUGUCAACAGAUCCGACUUUG
1123

MAPK8:1244L21 siRNA (1226C)

AAGucGGAucuGuuGAcAuTsT
1199

stab11 antisense

1242
CUUUGGCCUCUGAUACAGACAGC
1124

MAPK8:1262L21 siRNA (1244C)
uGucuGuAucAGAGGccAATsT
1200

stab11 antisense

Target

Seq

Pos
Target
ID
RPI #
Aliases
Sequence
ID

MAPK14/p38

1278
GCCUACUUUGCUCAGUACCACGA
1125
31586
MAPK14:1280U21 siRNA sense
CUACUUUGCUCAGUACCACTT
1201

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126
31587
MARK14:1611U21 siRNA sense
UCUGUCUUUGUGGGAGGGUTT
1202

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127
31588
MAPK14:2884U21 siRNA sense
AAGGGUCUUCUUGGCAGCUTT
1203

3554
GGACUCUAAGCUGGAGCUCUUGG
1128
31589
MAPK14:3556U21 siRNA sense
ACUCUAAGCUGGAGCUCUUTT
1204

1278
GCCUACUUUGCUCAGUACCACGA
1125
31590
MAPK14:1298L21 siRNA (1280C)
GUGGUACUGAGCAAAGUAGTT
1205

antisense

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126
31591
MAPK14:1629L21 siRNA (1611C)
ACCCUCCCACAAAGACAGATT
1206

antisense

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127
31592
MARK14:2902L21 siRNA (2884C)
AGCUGCCAAGAAGACCCUUTT
1207

antisense

3554
GGACUCUAAGCUGGAGCUCUUGG
1128
31593
MAPK14:3574L21 siRNA (3556C)
AAGAGCUCCAGCUUAGAGUTT
1208

antisense

1278
GCCUACUUUGCUCAGUACCACGA
1125

MAPK14:1280U21 siRNA stab04
B cuAcuuuGcucAGuAccAcTT B
1209

sense

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126

MARK14:1611U21 siRNA stab04
B ucuGucuuuGuGGGAGGGuTT B
1210

sense

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127

MAPK14:2884U21 siRNA stab04
B AAGGGucuucuuGGcAGcuTT B
1211

sense

3554
GGACUCUAAGCUGGAGCUCUUGG
1128

MAPK14:3556U21 siRNA stab04
B AcucuAAGcuGGAGcucuuTT B
1212

sense

1278
GCCUACUUUGCUCAGUACCACGA
1125

MAPK14:1298L21 siRNA (1280C)
GuGGuAcuGAGcAAAGuAGTsT
1213

stab05 antisense

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126

MARK14:1629L21 siRNA (1611C)
AcccucccAcAAAGAcAGATsT
1214

stab05 antisense

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127

MAPK14:2902L21 siRNA (2884C)
AGcuGccAAGAAGAcccuuTsT
1215

stab05 antisense

3554
GGACUCUAAGCUGGAGCUCUUGG
1128

MARK14:3574L21 siRNA (3556C)
AAGAGcuccAGcuuAGAGuTsT
1216

stab05 antisense

1278
GCCUACUUUGCUCAGUACCACGA
1125

MAPK14:1280U21 siRNA stab07
B cuAcuuuGcucAGuAccAcTT B
1217

sense

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126

MARK14:1611U21 siRNA stab07
B ucuGucuuuGuGGGAGGGuTT B
1218

sense

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127

MAPK14:2884U21 siRNA stab07
B AAGGGucuucuuGGcAGcuTT B
1219

sense

3554
GGACUCUAAGCUGGAGCUCUUGG
1128

MAPK14:3556U21 siRNA stab07
B AcucuAAGcuGGAGcucuuTT B
1220

sense

1278
GCCUACUUUGCUCAGUACCACGA
1125

MAPK14:1298L21 siRNA (1280C)

GuGGuAcuGAGcAAAGuAGTsT
1221

stab11 antisense

1609
UGUCUGUCUUUGUGGGAGGGUAA
1126

MARK14:1629L21 siRNA (1611C)

AcccucccAcAAAGAcAGATsT
1222

stab11 antisense

2882
AAAAGGGUCUUCUUGGCAGCUUA
1127

MAPK14:2902L21 siRNA (2884C)

AGcuGccAAGAAGAcccuuTsT
1223

stab11 antisense

3554
GGACUCUAAGCUGGAGCUCUUGG
1128

MAPK14:3574L21 siRNA (3556C)

AAGAGcuccAGcuuAGAGuTsT
1224

stab11 antisense

Target

Seq

Seq

Pos
Target
ID
RPI#
Aliases
Sequence
ID

c-JUN

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1819U21 siRNA sense
AAAAAGUGAAAACCUUGAATT
1617

1935
CAACUCAUGCUAACGCAGCAGUU
1610

JUN:1937U21 siRNA sense
ACUCAUGCUAACGCAGCAGTT
1618

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2261U21 siRNA sense
UUGACCAAGAACUGCAUGGTT
1619

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2266U21 siRNA sense
CAAGAACUGCAUGGACCUATT
1620

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2271U21 siRNA sense
ACUGCAUGGACCUAACAUUTT
1621

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2272U21 siRNA sense
CUGCAUGGACCUAACAUUCTT
1622

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2274U21 siRNA sense
GCAUGGACCUAACAUUCGATT
1623

2274
GCAUGGACCUAACAUUCGAUCUC
1616

JUN:2276U21 siRNA sense
AUGGACCUAACAUUCGAUCTT
1624

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1837L21 siRNA (1819C)
UUCAAGGUUUUCACUUUUUTT
1625

antisense

1935
CAACUCAUGCUAACGCAGCAGUU
1610

JUN:1955L21 siRNA (1937C)
CUGCUGCGUUAGCAUGAGUTT
1626

antisense

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2279L21 siRNA (2261C)
CCAUGCAGUUCUUGGUCAATT
1627

antisense

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2284L21 siRNA (2266C)
UAGGUCCAUGCAGUUCUUGTT
1628

antisense

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2289L21 siRNA (2271C)
AAUGUUAGGUCCAUGCAGUTT
1629

antisense

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2290L21 siRNA (2272C)
GAAUGUUAGGUCCAUGCAGTT
1630

antisense

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2292L21 siRNA (2274C)
UCGAAUGUUAGGUCCAUGCTT
1631

antisense

2274
GCAUGGACCUAACAUUCGAUCUC
1616

JUN:2294L21 siRNA (2276C)
GAUCGAAUGUUAGGUCCAUTT
1632

antisense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1819U21 siRNA stab04
B AAAAAGuGAAAAccuuGAATT B
1633

sense

1935
CAACUGAUGCUAACGCAGCAGUU
1610

JUN:1937U21 siRNA stab04
B AcucAuGcuAAcGcAGcAGTT B
1634

sense

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2261U21 siRNA stab04
B uuGAccAAGAAcuGcAuGGTT B
1635

sense

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2266U21 siRNA stab04
B cAAGAAcuGcAuGGAccuATT B
1636

sense

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2271U21 siRNA stab04
B AcuGcAuGGAccuAAcAuuTT B
1637

sense

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2272U21 siRNA stab04
B cuGcAuGGAccuAAcAuucTT B
1638

sense

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2274U21 siRNA stab04
B GcAuGGAccuAAcAuucGATT B
1639

sense

2274
GCAUGGACCUAACAUUCGAUCUC
1616

JUN:2276U21 siRNA stab04
B AuGGAccuAAcAuucGAucTT B
1640

sense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT
1641

stab05 antisense

1935
CAACUCAUGCUAACGCAGCAGUU
1610

JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT
1642

stab05 antisense

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2279L21 siRNA (2261C)
ccAuGcAGuucuuGGucAATsT
1643

stab05 antisense

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT
1644

stab05 antisense

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2289L21 siRNA (2271C)
AAuGuuAGGuccAuGcAGuTsT
1645

stab05 antisense

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2290L21 siRNA (2272C)
GAAuGuuAGGuccAuGcAGTsT
1646

stab05 antisense

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT
1647

stab05 antisense

2274
GCAUGGACCUAACAUUCGAUCUG
1616

JUN:2294L21 siRNA (2276C)
GAucGAAuGuuAGGuccAuTsT
1648

stab05 antisense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609
31818
JUN:1819U21 siRNA stab07
B AAAAAGuGAAAAccuuGAATT B
1649

sense

1935
CAACUCAUGCUAACGCAGCAGUU
1610
31819
JUN:1937U21 siRNA stab07
B AcucAuGcuAAcGcAGcAGTT B
1650

sense

2259
CAUUGACCAAGAACUGCAUGGAC
1611
31820
JUN:2261U21 siRNA stab07
B uuGAccAAGAAcuGcAuGGTT B
1651

sense

2264
ACCAAGAACUGCAUGGACCUAAC
1612
31821
JUN:2266U21 siRNA stab07
B cAAGAAcuGcAuGGAccuATT B
1652

sense

2269
GAACUGCAUGGACCUAACAUUCG
1613
31822
JUN:2271U21 siRNA stab07
B AcuGcAuGGAccuAAcAuuTT B
1653

sense

2270
AACUGCAUGGACCUAACAUUCGA
1614
31823
JUN:2272U21 siRNA stab07
B cuGcAuGGAccuAAcAuucTT B
1654

sense

2272
CUGCAUGGACCUAACAUUCGAUC
1615
31824
JUN:2274U21 siRNA stab07
B GcAuGGAccuAAcAuucGATT B
1655

sense

2274
GCAUGGACCUAACAUUCGAUCUG
1616
31825
JUN:2276U21 siRNA stab07
B AuGGAccuAAcAuucGAucTT B
1656

sense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT
1657

stab11 antisense

1935
CAACUCAUGCUAACGCAGCAGUU
1610

JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT
1658

stab11 antisense

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2279L21 siRNA (2261C)
ccAucAGuucuuGGucAATsT
1659

stab11 antisense

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT
1660

stab11 antisense

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2289L21 siRNA (2271C)

AAuGuuAGGuccAuGcAGuTsT
1661

stab11 antisense

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2290L21 siRNA (2272C)

GAAuGuuAGGuccAuGcAGTsT
1662

stab11 antisense

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT
1663

stab11 antisense

2274
GCAUGGACCUAACAUUCGAUCUC
1616

JUN:2294L21 siRNA (2276C)

GAucGAAuGuuAGGuccAuTsT
1664

stab11 antisense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609

JUN:1819U21 siRNA stab08

AAAAAGuGAAAAccuuGAATsT
1665

sense

1935
CAACUCAUGCUAACGCAGCAGUU
1610

JUN:1937U21 siRNA stab08

AcucAuGcuAAcGcAGcAGTsT
1666

sense

2259
CAUUGACCAAGAACUGCAUGGAC
1611

JUN:2261U21 siRNA stab08
uuGAccAAGAAcuGcAuGGTsT
1667

sense

2264
ACCAAGAACUGCAUGGACCUAAC
1612

JUN:2266U21 siRNA stab08
cAAGAAcuGcAuGGAccuATsT
1668

sense

2269
GAACUGCAUGGACCUAACAUUCG
1613

JUN:2271U21 siRNA stab08

AcuGcAuGGAccuAAcAuuTsT
1669

sense

2270
AACUGCAUGGACCUAACAUUCGA
1614

JUN:2272U21 siRNA stab08
cuGcAuGGAccuAAcAuucTsT
1670

sense

2272
CUGCAUGGACCUAACAUUCGAUC
1615

JUN:2274U21 siRNA stabo8

GcAuGGAccuAAcAuucGATsT
1671

sense

2274
GCAUGGACCUAACAUUCGAUCUC
1616

JUN:2276U21 siRNA stabo8

AuGGAccuAAcAuucGAucTsT
1672

sense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609
31826
JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT
1673

stab08 antisense

1935
CAACUCAUGCUAACGCAGCAGUU
1610
31827
JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT
1674

stab08 antisense

2259
CAUUGACCAAGAACUGCAUGGAC
1611
31828
JUN:2279L21 siRNA (2261C)
ccAuGcAGuucuuGGucAATsT
1675

stab08 antisense

2264
ACCAAGAACUGCAUGGACCUAAC
1612
31829
JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT
1676

stab08 antisense

2269
GAACUGCAUGGACCUAACAUUCG
1613
31830
JUN:2289L21 siRNA (2271C)

AAuGuuAGGuccAuGcAGuTsT
1677

stab08 antisense

2270
AACUGCAUGGACCUAACAUUCGA
1614
31831
JUN:2290L21 siRNA (2272C)

GAAuGuuAGGuccAuGcAGTsT
1678

stab08 antisense

2272
CUGCAUGGACCUAACAUUCGAUC
1615
31832
JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT
1679

stab08 antisense

2274
GCAUGGACCUAACAUUCGAUCUC
1616
31833
JUN:2294L21 siRNA (2276C)

GAucGAAuGuuAGGuccAuTsT
1680

stab08 antisense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609
31834
JUN:1819U21 siRNA inv stab07
B AAGuuccAAAAGuGAAAAATT B
1681

sense

1935
CAACUCAUGCUAACGCAGCAGUU
1610
31835
JUN:1937U21 siRNA inv stab07
B GAcGAcGcAAucGuAcucATT B
1682

sense

2259
CAUUGACCAAGAACUGCAUGGAC
1611
31836
JUN:2261U21 siRNA inv stab07
B GGuAcGucAAGAAccAGuuTT B
1683

sense

2264
ACCAAGAACUGCAUGGACCUAAC
1612
31837
JUN:2266U21 siRNA inv stab07
B AuccAGGuAcGucAAGAAcTT B
1684

sense

2269
GAACUGCAUGGACCUAACAUUCG
1613
31838
JUN:2271U21 siRNA inv stab07
B uuAcAAuccAGGuAcGucATT B
1685

sense

2270
AACUGCAUGGACCUAACAUUCGA
1614
31839
JUN:2272U21 siRNA inv stab07
B cuuAcAAuccAGGuAcGucTT B
1686

sense

2272
CUGCAUGGACCUAACAUUCGAUC
1615
31840
JUN:2274U21 siRNA inv stab07
B AGcuuAcAAuccAGGuAcGTT B
1687

sense

2274
GCAUGGACCUAACAUUCGAUCUC
1616
31841
JUN:2276U21 siRNA inv stab07
B cuAGcuuAcAAuccAGGuATT B
1688

sense

1817
GGAAAAAGUGAAAACCUUGAAAG
1609
31842
JUN:1837L21 siRNA (1819C)
uuuuucAcuuuuGGAAcuuTsT
1689

inv stab08 antisense

1935
CAACUCAUGCUAACGCAGCAGUU
1610
31843
JUN:1955L21 siRNA (1937C)
uGAGuAcGAuuGcGucGucTsT
1690

inv stab08 antisense

2259
CAUUGACCAAGAACUGCAUGGAC
1611
31844
JUN:2279L21 siRNA (2261C)

AAcuGGuucuuGAcGuAccTsT
1691

inv stab08 antisense

2264
ACCAAGAACUGCAUGGACCUAAC
1612
31845
JUN:2284L21 siRNA (2266C)

GuucuuGAcGuAccuGGAuTsT
1692

inv stab08 antisense

2269
GAACUGCAUGGACCUAACAUUCG
1613
31846
JUN:2289L21 siRNA (2271C)
uGAcGuAccuGGAuuGuAATsT
1693

inv stab08 antisense

2270
AACUGCAUGGACCUAACAUUCGA
1614
31847
JUN:2290L21 siRNA (2272C)

GAcGuAccuGGAuuGuAAGTsT
1694

inv stab08 antisense

2272
CUGCAUGGACCUAACAUUCGAUC
1615
31848
JUN:2292L21 siRNA (2274C)
cGuAccuGGAuuGuAAGcuTsT
1695

inv stab08 antisense

2274
GCAUGGACCUAACAUUCGAUCUC
1616
31849
JUN:2294L21 siRNA (2276C)
uAccuGGAuuGuAAGcuAGTsT
1696

inv stab08 antisense

Uppercase = ribonucleotide

u,c = 2′-deoxy-2′-fluoro U, C

A = 2′-O-methyl Adenosine

G = 2′-O-methyl Guanosine

T = thymidine

B = inverted deoxy abasic

s = phosphorothioate linkage

A = deoxy Adenosine

G = deoxy Guanosine

TABLE IV

Non-limiting examples of Stabilization Chemistries

for chemically modified siNA constructs

Chemistry
pyrimidine
Purine
cap
p = S
Strand

“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS

1 at 3′-end

“Stab 2”
Ribo
Ribo
—
All linkages
Usually AS

“Stab 3”
2′-fluoro
Ribo
—
4 at 5′-end
Usually S

4 at 3′-end

“Stab 4”
2′-fluoro
Ribo
5′ and 3′-ends
—
Usually S

“Stab 5”
2′-fluoro
Ribo
—
1 at 3′-end
Usually AS

“Stab 6”
2′-O-Methyl
Ribo
5′ and 3′-ends
—
Usually S

“Stab 7”
2′-fluoro
2′-deoxy
5′ and 3′-ends
—
Usually S

“Stab 8”
2′-fluoro
2′-O-Methyl
—
1 at 3′-end
S or AS

“Stab 9”
Ribo
Ribo
5′ and 3′-ends
—
Usually S

“Stab 10”
Ribo
Ribo
—
1 at 3′-end
Usually AS

“Stab 11”
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually AS

Stab 12
2′-fluoro
LNA
5′ and 3′-ends

Usually S

“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually AS

“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 16
Ribo
2′-O-Methyl
5′ and 3′-ends

Usually S

“Stab 17”
2′-O-Methyl
2′-O-Methyl
5′ and 3′-ends

Usually S

“Stab 18”
2′-fluoro
2′-O-Methyl

1 at 3′-end
Usually AS

CAP = any terminal cap, see for example FIG. 10.

All Stab 1-18 chemistries can comprise 3′-terminal thymidine (TT) residues

All Stab 1-18 chemistries typically comprise 21 nucleotides, but can vary as described herein.

S = sense strand

AS = antisense strand

TABLE V

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time*RNA

Phosphoramidites
6.5
163
μL
45
sec
2.5
min
7.5
min

S-Ethyl Tetrazole
23.8
238
μL
45
sec
2.5
min
7.5
min

Acetic Anhydride
100
233
μL
5
sec
5
sec
5
sec

N-Methyl
186
233
μL
5
sec
5
sec
5
sec

Imidazole

TCA
176
2.3
mL
21
sec
21
sec
21
sec

Iodine
11.2
1.7
mL
45
sec
45
sec
45
sec

Beaucage
12.9
645
μL
100
sec
300
sec
300
sec

Acetonitrile
NA
6.67
mL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time*RNA

Phosphoramidites
15
31
μL
45
sec
233
sec
465
sec

S-Ethyl Tetrazole
38.7
31
μL
45
sec
233
min
465
sec

Acetic Anhydride
655
124
μL
5
sec
5
sec
5
sec

N-Methyl
1245
124
μL
5
sec
5
sec
5
sec

Imidazole

TCA
700
732
μL
10
sec
10
sec
10
sec

Iodine
20.6
244
μL
15
sec
15
sec
15
sec

Beaucage
7.7
232
μL
100
sec
300
sec
300
sec

Acetonitrile
NA
2.64
mL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2′-O-

Wait Time* 2′-O-

Reagent
2′-O-methyl/Ribo
methyl/Ribo
Wait Time* DNA
methyl
Wait Time* Ribo

Phosphoramidites
22/33/66
40/60/120
μL
60
sec
180
sec
360
sec

S-Ethyl Tetrazole
70/105/210
40/60/120
μL
60
sec
180
min
360
sec

Acetic Anhydride
265/265/265
50/50/50
μL
10
sec
10
sec
10
sec

N-Methyl
502/502/502
50/50/50
μL
10
sec
10
sec
10
sec

Imidazole

TCA
238/475/475
250/500/500
μL
15
sec
15
sec
15
sec

Iodine
6.8/6.8/6.8
80/80/80
μL
30
sec
30
sec
30
sec

Beaucage
34/51/51
80/120/120

100
sec
200
sec
200
sec

Acetonitrile
NA
1150/1150/1150
μL
NA
NA
NA

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

Number	Date	Country
60358580	Feb 2002	US
60363124	Mar 2002	US
60386782	Jun 2002	US
60406784	Aug 2002	US
60408378	Sep 2002	US
60409293	Sep 2002	US
60440129	Jan 2003	US

	Number	Date	Country
Parent	10424339	Apr 2003	US
Child	12201759		US

	Number	Date	Country
Parent	PCT/US03/02510	Jan 2003	US
Child	10424339		US
Parent	PCT/US03/05346	Feb 2003	US
Child	PCT/US03/02510		US
Parent	PCT/US03/05028	Feb 2003	US
Child	PCT/US03/05346		US

RNA Interference Mediated Inhibition of MAP Kinase Gene Expression or Expression of Genes Involved in MAP Kinase Pathway Using Short Interfering Nucleic Acid (SiNA)

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Parent Case Info

Provisional Applications (7)

Continuations (1)

Continuation in Parts (3)