siRNA treatment of diseases or conditions related to levels of IKK-gamma

[0001] This patent application claims priority from U.S. Ser. No. 60/294,412, filed May 29, 2001, entitled ‘ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF IKK-GAMMA AND PKR.” This application is hereby incorporated by reference herein in its entirety including the drawings.

[0002] The sequence listing submitted on compact discs, in compliance with 37 C. F. R. § 1.52(e)(5), is incorporated by reference. Two separate compact discs have been submitted, each containing the file “01-664-A_Seq.Listing”which is 1,821,582 bytes in size and was created on May 16, 2005.

[0003] The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders.

[0004] The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR. The discussion is provided only for understanding the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.

[0005] Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes. NFKB is a key component of the TNF signaling pathway. These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses. The REL-A gene product (a.k.a. RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation. NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits. NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli. NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates. In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop. NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.

[0006] The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells. NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NFKB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100). The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.

[0007] The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992, Proc. Natl. Acad. Sci USA, 89, 1529–1533). For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al, 1993, Mol. Cell. Biol., 13, 6283–6289). Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992, J. Virol., 66, 3883–3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB 1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993, Mol. Cell. Biol., 13, 3802–3810). Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989, Cell, 58, 227–229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family. Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.

[0008] A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member). These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.

[0009] NFKB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992, Science, 258, 1792–5) and IL-8 (Kunsch and Rosen, 1993, Mol. Cell. Biol., 13, 6137–46).

[0010] NFk is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993, Mol. Cell. Biol., 13, 6530–6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994, J. Exp. Med., 179, 503–512) on endothelial cells.

[0011] NFKB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).

[0012] HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IkB-alpha. Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001, Oncogene, 20, 1287–1299; Sovak et al., 1999, J. Clin. Invest., 100, 2952–2960).

[0013] Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997, Am. J. Pathol., 151,891–896).

[0014] NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001, Laboratory Investigation, 81, 349–360).

[0015] The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells. Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB. The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994, J. Biol. Chem., 269, 6185–6192). Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl. Acad. Sci USA, 91, 752–756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.

[0016] The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκB-epsilon, p105, and p100). The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals. Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway. Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling. In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1). IKK-gamma is a protein that is critical for the assembly of the IKK complex. IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA. Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti. Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001, J. Biol. Chem., 275, 29779–87).

[0017] The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1. PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001, J. Immunol., 166, 6170–6180). The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001, Oncogene, 20, 1900–1912). PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.

[0018] Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression. Krappmann et al, 2001, J. Biol. Chem., describe specific antisense oligonucleotides to IKK-gamma.

[0019] The present invention features a nucleic acid molecule, such as decoy RNA, dsRNA, siRNA, aptamers, antisense nucleic acid molecules, and enzymatic nucleic acid molecule which down regulates expression of a sequence encoding an IkappaB kinase (IKK) subunit. The invention also features an enzymatic nucleic acid molecule which down regulates expression of a sequence encoding protein kinase PKR.

[0020] In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 632–1261, 1762–2260, 2480–2698, 2904–3485, 3814–4360, 4555–4748, 5253–5756, 6034–6310, 6380–6789, 7142–7770, and 7884–8001.

[0021] In another embodiment, an enzymatic nucleic acid molecule of the invention comprises at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1–631, 1263–1761, 2261–2479, 2699–2903, 3486–3813, 4361–4554, 4749–5252, 5757–6033, 6311–6379, 6790–7141 and 7771–7883.

[0022] In another embodiment, an antisense nucleic acid molecule of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1–631, 1263–1761, 2261–2479, 2699–2903, 3486–3813, 4361–4554, 4749–5252, 5757–6033, 6311–6379, 6790–7141, and 7771–7883.

[0023] In another embodiment, an nucleic acid molecule of the invention is adapted to treat cancer. In yet another embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA having IKK-gamma or PKR nucleic acid sequence.

[0024] In one embodiment, an enzymatic nucleic acid molecule of the invention is in an Inozyme, Zinzyme, G-cleaver, Amberzyme, DNAzyme, or Hammerhead configuration.

[0025] In another embodiment, an Inozyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1263–1761, 4749–5252, 7781–7787, 7796–7800, 7822–7846, and 7866–7870.

[0026] In another embodiment, an Inozyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 1762–2260, 5253–5756, 7894–7900, 7909–7913, 7938–7962, and 7982–7986.

[0027] In another embodiment, a Zinzyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 2261–2479, 5757–6033, 7788–7790, 7847–7860, and 7871–7895.

[0028] In another embodiment, a Zinzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 2480–2698, 6034–6310, 7901–7903, 7963–7976, and 7987–7991.

[0029] In another embodiment, an Amberzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 3814–4360, 7142–7770, 7924–7928, and 7997–8001.

[0030] In another embodiment, a DNAzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 2904–3485, 6380–6789, 7919–7923, and 7992–7996.

[0031] In another embodiment, a Hammerhead of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1–631, 4361–4554, 7771–7780, 7791–7795, 7813–7821, and 7861–7865.

[0032] In another embodiment, a Hammerhead of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 632–1262, 4555–4748, 7884–7894, 7904–7908, 7929–7937, 7977–7981.

[0033] In one embodiment, a nucleic acid molecule of the invention comprises between 12 and 100 bases complementary to RNA having an IKK-gamma or PKR nucleic acid sequence. In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to RNA having anIKK-gamma or PKR nucleic acid sequence.

[0034] In yet another embodiment, a nucleic acid molecule of the invention is chemically synthesized.

[0035] In another embodiment, a nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least one 2′-sugar modification, at least one nucleic acid base modification, or at least one phosphate backbone modification.

[0036] In one embodiment, a siRNA molecule of the invention comprises a double stranded RNA wherein one strand of the RNA is complimentary to the RNA of IKK-gamma or PKR gene. In another embodiment, a siRNA molecule of the invention comprises a double stranded RNA wherein one strand of the RNA comprises a portion of a sequence of RNA having IKK-gamma or PKR gene sequence. In yet another embodiment, a siRNA molecule of the invention comprises a double stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker. Alternately, a siRNA molecule of the invention comprises a double stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.

[0037] In one embodiment, a single strand component of a siRNA molecule of the invention is from about 14 to about 50 nucleotides in length. In another embodiment, a single strand component of a siRNA molecule of the invention is about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, a single strand component of a siRNA molecule of the invention is about 23 nucleotides in length. In one embodiment, a siRNA molecule of the invention is from about 28 to about 56 nucleotides in length. In another embodiment, a siRNA molecule of the invention is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides in length. In yet another embodiment, a siRNA molecule of the invention is about 46 nucleotides in length.

[0038] In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, decoy RNA, dsRNA, siRNA, or aptarner molecules of the invention comprises at least one 2′-sugar modification.

[0039] In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, decoy RNA, dsRNA, siRNA, or aptamer, nucleic acids of the invention comprises at least one nucleic acid base modification.

[0040] In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, decoy RNA, dsRNA, siRNA, or aptamer, nucleic acids of the invention comprises at least one phosphate backbone modification.

[0041] In one embodiment, the invention features a mammalian cell, for example a human cell, including an nucleic acid molecule of the invention.

[0042] The present invention features method of down-regulating PKR activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule or antisense nucleic acid molecule, or other nucleic acid molecule of the invention, under conditions suitable for down-regulating of PKR activity.

[0043] The present invention also features method of treatment of a subject having a condition associated with the level of PKR, comprising contacting cells of the subject with an enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention under conditions suitable for the treatment.

[0044] The present invention features method of down-regulating IKK-gamma activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention, under conditions suitable for down-regulating of IKK-gamma activity.

[0045] The present invention also features method of treatment of a subject having a condition associated with the level of IKK-gamma, comprising contacting cells of the subject with the enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention, under conditions suitable for the treatment.

[0046] In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.

[0047] The present invention features methods of cleaving RNA comprising a PKR nucleic acid sequence comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA under conditions suitable for the cleavage.

[0048] The present invention also features methods of cleaving RNA comprising a IKK-gamma nucleic acid sequence comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA under conditions suitable for the cleavage.

[0049] In one embodiment, a method of cleavage of the invention is carried out in the presence of a divalent cation, for example Mg2+.

[0050] In another embodiment, an enzymatic nucleic acid or antisense nucleic acid molecule or other nucleic acid molecule of the invention comprises a cap structure, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end, for example a 3′, 3′-linked or 5′, 5′-linked deoxyabasic derivative.

[0051] The present invention also features an expression vector comprising a nucleic acid sequence encoding at least one enzymatic nucleic acid molecule, antisense, or other nucleic acid molecule of the invention in a manner which allows expression of the nucleic acid molecule.

[0052] In one embodiment, the invention features a mammalian cell, for example a human cell, including an expression vector contemplated by the invention.

[0053] In another embodiment, an expression vector of the invention further comprises an antisense nucleic acid molecule complementary to RNA of a subunit of IKK-gamma or PKR.

[0054] In yet another embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.

[0055] The present invention also features a method for treatment of cancer, for example breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a subject an enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention under conditions suitable for said treatment.

[0056] In one embodiment, a nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′- end modification such as a 3′-3′ inverted abasic moiety, and/or phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

[0057] In another embodiment, other drug therapies contemplated by the invention include monoclonal antibodies, IKI-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

[0058] Specific chemotherapy contemplated by the invention include paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, 5-fluoro uridine, Leucovorin, Irinotecan (CAMPTOSAR® or CPT-11 or Camptothecin-11 or Campto), Paclitaxel, Carboplatin doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine or a combination thereof.

[0059] The invention also features a method for treatment of an inflammatory disease, for example rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection, comprising the step of administering to a subject an enzymatic nucleic acid or antisense nucleic acid molecule of the invention under conditions suitable for the treatment.

[0060] The present invention features compositions comprising the enzymatic nucleic acid and/or antisense nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.

[0061] The invention also features a method of administering to a cell, such as mammalian cell (e.g. human cell), where the cell can be in culture or in a mammal, such as a human, an enzymatic nucleic acid molecule or antisense molecule of the instant invention, comprising contacting the cell with the enzymatic nucleic acid molecule or antisense molecule or other nucleic acid molecule of the invention under conditions suitable for such administration. The method of administration can be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipid, or liposome.

[0062] First the drawings will be described briefly.

[0067] The invention features nucleic acid molecules, for example enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2,5-A chimeras, decoys, double stranded RNA, triplex oligonucleotides, and/or aptamers, and methods to modulate gene expression, for example, genes encoding a member of the IκB kinase IKK complex, such as IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein. In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of IKK-gamma (IKKγ) and protein kinase PKR.

[0068] The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR. In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No. NM_—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No. AF080158), IKK-ganima (IKKγ) gene, for example (Genbank Accession No. NM_—003639) (SEQ ID NO:8014), and protein kinase PKR gene, for example (Genbank Accession No. NM_—002759).

[0069] The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes. IKK-gamma is also known as NEMO/IKKAP1. However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2). Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

[0070] In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH, G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to down-regulate the expression of IKK-gamma or PKR genes.

[0071] By “inhibit” or “down-regulate” it is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as IKK-gamma or PKR subunit(s), is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, inhibition or down-regulation with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA. In another embodiment, inhibition or down-regulation with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition or down-regulation of IKK-gamma or PKR with the nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.

[0072] By “up-regulate” is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as IKK-gamma or PKR subunit(s), is greater than that observed in the absence of the nucleic acid molecules of the invention. For example, the expression of a gene, such as IKK-gamma or PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.

[0073] By “modulate” is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunit(s) is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of the nucleic acid molecules of the invention.

[0074] By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50–75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092–2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25–31). The nucleic acids can be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

[0075] Several varieties of enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.

[0076] By “nucleic acid molecule” as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmnodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

[0077] By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS. 1–4).

[0078] By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid which is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092–2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25–31). Examples of such arms are shown generally in FIGS. 1–4. That is, these arms contain sequences within a enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to three nucleotides and of sufficient length to stably interact with the target RNA; preferably 12–100 nucleotides; more preferably 14–24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranz et al., 1993, EMBO J., 12, 2567–73). If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).

[0079] By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site. “I” in FIG. 1 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.

[0080] By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site. G-cleavers can be chemically modified as is generally shown in FIG. 1.

[0081] By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 2. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 2. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.

[0082] By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 3. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site. Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 3, including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure. Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.

[0083] By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linker(s) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in FIG. 4 and is generally reviewed in Usman et al., U.S. Pat. No. 6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422–423; and Santoro et. al., 2000, J. Am. Chem. Soc., 122, 2433–39. Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.

[0084] By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.

[0085] By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).

[0086] By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to TKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, flngi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.

[0087] By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring -RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins Awith similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.

[0088] By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.

[0089] By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA—RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783–21789, Delihas et al., 1997, Nature, 15, 751–753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3–45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121–157, Crooke, 1997, Ad. Pharmacol, 40, 1–49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.

[0090] By “RNase H activating region” is meant a region (generally greater than or equal to 4–25 nucleotides in length, preferably from 5–11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; more specifically, 4–11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.

[0091] By “2–5A chimera” is meant an oligonucleotide, for example an antisense nucleic acid molecule or enzymatic nucleic acid molecule, containing a 5′-phosphorylated 2′-5′-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2–5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522–533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55–113).

[0092] By “triplex forming oligonucleotides” or “triplex oligonucleotide” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17–37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181–206).

[0093] By “double stranded RNA” or “dsRNA” is meant a double stranded RNA that matches a predetermined gene sequence that is capable of activating cellular enzymes that degrade the corresponding messenger RNA transcripts of the gene. These dsRNAs are referred to as short intervening RNA (siRNA) and can be used to inhibit gene expression (see for example Elbashir et al., 2001, Nature, 411, 494–498; and Bass, 2001, Nature, 411, 428–429). The term “double stranded RNA” or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference “RNAi”, including short interfering RNA “siRNA” 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.

[0094] By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.

[0095] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA 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 target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. 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 which 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.

[0096] By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.

[0097] By “decoy” is meant a nucleic acid molecule, for example RNA or DNA, or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule. The decoy or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601–608). This is but a specific 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; Sun, 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. Similarly, a decoys can be designed to bind to IKK-gamma or PKR and block the binding of IKK-gamma or PKR or a decoy can be designed to bind to IKK-gamma or PKR and prevent interaction with the IKK-garma or PKR protein.

[0098] 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 is distinct from 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. Similarly, the nucleic acid molecules of the instant invention can bind to IKK-gamma or PKR to block activity of the respective proteins. 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., U.S. Pat. No. 5,475,096 and 5,270,163; Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 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.

[0099] The enzymatic nucleic acid molecule, antisense nucleic acid or other nucleic acid molecules of the invention that down regulate IKK-gamma or PKR gene expression represent a therapeutic approach to treat a variety of inflammatory-related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflanmmatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.

[0100] The enzymatic nucleic acid molecule, antisense nucleic acid or other nucleic acid molecules of the invention that down regulate IKK-gamma or PKR gene expression also represent a therapeutic approach to treat a variety of cancers, including but not limited to breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and/or other cancers which respond to the modulation of IKK-gamma or PKR function.

[0101] In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685–696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826–8830; Collins and Olive, 1993 Biochemistry 32, 2795–2799; Guo and Collins, 1995, EMBO. J 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al., International PCT Publication No. WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262, and Beigelman et al., International PCT publication No. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116–4120 and Eckstein et al., International PCT Publication No. WO 99/16871. Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG. 2; Beigelman et al., U.S. Ser. No. 09/301,511) and Zinzyme (FIG. 3) (Beigelman et al., U.S. Ser. No. 09/301,511), all included by reference herein including drawings, can also be used in the present invention. These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071).

[0102] In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between about 10 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III to VII. For example, enzymatic nucleic acid molecules of the invention are preferably between about 15 and 50 nucleotides in length, more preferably between about 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol. Chem., 271, 29107–29112). Exemplary DNAzymes of the invention are preferably between about 15 and 40 nucleotides in length, more preferably between about 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see for example Santoro et al., 1998, Biochemistry, 37, 13330–13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092–4096). Exemplary antisense molecules of the invention are preferably between about 15 and 75 nucleotides in length, more preferably between about 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305–7309; Milner et al., 1997, Nature Biotechnology, 15, 537–541). Exemplary triplex forming oligonucleotide molecules of the invention are preferably between about 10 and 40 nucleotides in length, more preferably between about 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al, 1990, Biochemistry, 29, 8820–8826; Strobel and Dervan, 1990, Science, 249, 73–75). Those skilled in the art will recognize that all that is required is that the nucleic acid molecule be of sufficient length and suitable conformation for the nucleic acid molecule to interact with its target and/or catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.

[0103] Preferably, a nucleic acid molecule that modulates, for example, down-regulates IKK-gamma or PKR expression comprises between 12 and 100 bases complementary to a RNA molecule of IKK-gamma or PKR. Even more preferably, a nucleic acid molecule that modulates, for example IKK-gamma or PKR expression comprises between 14 and 24 bases complementary to a RNA molecule of IKK-gamma or PKR.

[0104] The invention provides a method for producing a class of nucleic acid-based gene modulating agents which exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding IKK-gamma or PKR (specifically IKK-gamma or PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.

[0105] As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, 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).

[0106] By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.

[0107] By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.

[0108] 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.

[0109] Nucleic acid-based inhibitors of IKK-gamma or PKR function are useful for the prevention and/or treatment of cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of IKK-gamma or PKR in a cell or tissue, alone or in combination with other therapies.

[0110] Nucleic acid-based inhibitors of IKK-gamma or PKR function are also useful for the prevention and/or treatment of inflammatory related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.

[0111] The nucleic acid-based inhibitors 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 or infusion pump, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate sequences in Tables III to XIII. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XIII. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.

[0112] In another embodiment, the invention features antisense nucleic acid molecules and 2–5A chimera including sequences complementary to the substrate sequences shown in Tables III to XIII. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XIII. Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.

[0113] By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs. Other sequences can be present which do not interfere with such cleavage. Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity. Thus, the underlined regions in the sequences in Tables III, IV, VIII, and IX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”. For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO 8002), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker. Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2–5A antisense, triplex forming nucleic acid, and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.

[0114] Sequence X can be a linker of ≧2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferably be internally base-paired to form a stem of preferably ≧2 base pairs. In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

[0115] In yet another embodiment, alternatively or in addition, sequence X can be a non-nucleotide linker. Non-nucleotides as can include abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. 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 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 can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.

[0116] In another aspect of the invention, enzymatic nucleic acid molecules or antisense molecules that interact with target RNA molecules and down-regulate IKK-gamma or PKR (specifically IKK-gamma or PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression. Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from the patient or subject followed by reintroduction into the patient or subject, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.

[0117] By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

[0118] 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. Preferably, a subject is a mammal or mammalian cells. More preferably, a subject is a human or human cells.

[0119] By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention. In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.

[0120] 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 above. For example, to treat a disease or condition associated with the levels of IKK-gamma or PKR, the subject can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

[0121] In a further embodiment, the described nucleic acid molecules, such as antisense or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, incontinentia pigmenti, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other cancerous disease or inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR expression.

[0122] In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (eg; ribozymes), antisense nucleic acids, 2–5A antisense chimeras, triplex DNA, dsRNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., IKK-gamma or PKR) capable of progression and/or maintenance of cancer, inflammatory diseases, and/or other disease states which respond to the modulation of IKK-gamma or PKR expression.

[0123] By “comprising” is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and can or can not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements can be present.

[0124] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Mechanism of Action of Nucleic Acid Molecules of the Invention as Proposed in the Art

[0125] Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20–33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151–190).

[0126] In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently it has been reported that 2′-arabino and 2′-fluoro arabino-containing oligos can also activate RNase H activity.

[0127] A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., International PCT Publication No. WO 99/54459; Hartmann et al., U.S. Ser. No. 60/101,174 which was filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety.

[0128] In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.

[0129] Enzymatic Nucleic Acid: Several varieties of enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83–87; Beaudry et al., 1992, Science 257, 635–641; Joyce, 1992, Scientific American 267, 90–97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al.,1993, Science 261:1411–1418; Szostak, 1993, TIBS 17, 89–93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakacane & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.

[0130] Nucleic acid molecules of this invention will block to some extent IKK-gamma or PKR and/or IKK-gamma or PKR protein expression and can be used to treat disease or diagnose disease associated with the levels of IKK-gamma or PKR and/or IKK-gamma or PKR. Enzymatic nucleic acid sequences targeting IKK-gamma or PKR RNA and sequences that can be targeted with nucleic acid molecules of the invention to down-regulate IKK-gamma or PKR expression are shown in Tables III to XIII.

[0131] The enzymatic nature of an enzymatic nucleic acid molecule can allow the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment to be lower. This reflects the ability of the enzymatic nucleic acid molecule to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA. In addition, the enzymatic nucleic acid molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to greatly attenuate the catalytic activity of a enzymatic nucleic acid molecule.

[0132] Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and achieve efficient cleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).

[0133] Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285–294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023–2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237–250).

[0134] Enzymatic nucleic acid molecules of the invention that are allosterically regulated (“allozymes”) can be used to modulate IKK-gamma or PKR expression. These allosteric enzymatic nucleic acids or allozymes (see for example George et al, U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842) are designed to respond to a signaling agent, for example, mutant IKK-gamma protein, wild-type IKK-gamma protein, mutant IKK-gamma RNA, wild-type IKK-gainma RNA, other proteins and/or RNAs involved in IKK-gamma activity, compounds, metals, polymers, molecules and/or drugs that are targeted to IKK-gamma or an IKK subunit, such as IKK-alpha or IKK-beta, expressing cells etc., which in turn modulates the activity of the enzymatic nucleic acid molecule. In response to interaction with a predetermined signaling agent, the allosteric enzymatic nucleic acid molecule's activity is activated or inhibited such that the expression of a particular target is selectively down-regulated. The target can comprise wild-type IKK-gamma, mutant IKK-gamma, a component of IKK-gamma, and/or a predetermined cellular component that modulates IKK-gamnma activity. In a specific example, allosteric enzymatic nucleic acid molecules that are activated by interaction with a RNA encoding a mutant IKK-gamma protein are used as therapeutic agents in vivo. The presence of RNA encoding the mutant IKK-gamma activates the allosteric enzymatic nucleic acid molecule that subsequently cleaves the RNA encoding a mutant IKK-gamma protein resulting in the inhibition of mutant IKK-gamma protein expression. In this manner, cells that express the mutant form of the IKK-gamma protein are selectively targeted. Such an approach, can be used to treat, for example, incontinentia pigmenti.

[0135] In another non-limiting example, an allozyme can be activated by a IKK-gamma or PKR protein, peptide, or mutant polypeptide that caused the allozyme to inhibit the expression of IKK-gamma or PKR gene, by, for example, cleaving RNA encoded by IKK-gamma or PKR gene. In this non-limiting example, the allozyme acts as a decoy to inhibit the function of IKK-gamma or PKR and also inhibit the expression of IKK-gamma or PKR once activated by the IKK-gamma or PKR protein.

[0136] The nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (eg; ribozymes, antisense) capable of down-regulating gene expression.

Target Sites

[0137] Targets for useful enzymatic nucleic acid molecules and antisense nucleic acids can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat. No. 5,525,468, and hereby incorporated by reference herein in totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference herein. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Enzymatic nucleic acid molecules and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. The sequences of human IKK-gamma or PKR RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. These sites are shown in Tables III to XIII (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence “X” or linker X, the actual sequence is not relevant here). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule. While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted enzymatic nucleic acid molecules can be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.

[0138] Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.

[0139] Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and 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; Caruthers et al., 1992, Methods in Enzymology 211,3–19.

Synthesis of Nucleic Acid Molecules

[0140] Synthesis of nucleic acids greater than 100 nucleotides in length can be difficult using automated methods, and the therapeutic cost of such molecules can be prohibitive. In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs less than about 100 nucleotides in length, preferably less than about 80 nucleotides in length, and more preferably less than about 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

[0141] Oligonucleotides (eg; antisense, GeneBlocs) are synthesized using protocols known in the art 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 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides. Table II 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 calorimetric quantitation of the trityl fractions, are typically 97.5–99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I₂, 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.

[0142] Deprotection of the antisense 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% aq. methylamine (1 mL) at 65° C. for 10 min. 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.

[0143] The method of synthesis used for RNA and chemically modified RNA including certain enzymatic nucleic acid molecules 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 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table II 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; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 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.

[0144] 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 min. 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 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

[0145] 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 min. The vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

[0146] For purification of the trityl-on oligomers, the quenched NH₄HCO₃ solution 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 min. 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.

[0147] Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides can be synthesized by substituting a U for G₅ and a U for A₁₄ (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.

[0148] 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, with the ratio of chemicals being used in the reaction adjusted accordingly.

[0149] 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).

[0150] The nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 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). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.

[0151] The sequences of the nucleic acid molecules, including enzymatic nucleic acid molecules and antisense, that are chemically synthesized, are shown in Table XIII. The sequences of the enzymatic nucleic acid and antisense constructs that are chemically synthesized, are complementary to the Substrate sequences shown in Table XIII. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. The enzymatic nucleic acid and antisense construct sequences listed in Tables III to XIII can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such enzymatic nucleic acid molecules with enzymatic activity are equivalent to the enzymatic nucleic acid molecules described specifically in the Tables.

Optimizing Activity of the Nucleic Acid Molecule of the Invention

[0152] Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases 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; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein).

[0153] 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′-flouro, 2′-O-methyl, 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 ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.

[0154] While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications can cause some toxicity. 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.

[0155] Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity can not be significantly lowered. Therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Nucleic acid molecules are preferably resistant to nucleases in order to function as effective intracellular therapeutic agents. 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.

[0156] Use of the nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules). The treatment of subjects with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.

[0157] Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. These nucleic acid molecules should be 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.

[0158] In one embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity of the nucleic acid can not be significantly lowered. As exemplified herein such enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced about 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such enzymatic nucleic acids herein are said to “maintain” the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.

[0159] In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.

[0160] By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al, WO 97/26270, 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′-terminus (3′-cap) or can be present on both terminus. In non-limiting examples, the 5′-cap includes inverted 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 (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).

[0161] In another embodiment the 3′-cap includes, for example 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).

[0162] 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.

[0163] The term “alkyl” as used herein refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain “isoalkyl”, and cyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1–C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from about 1 to 7 carbons, more preferably about 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1–C6 hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” also includes alkenyl groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has about 2 to 12 carbons. More preferably it is a lower alkenyl of from about 2 to 7 carbons, more preferably about 2 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C₁–C₆ hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” also includes alkynyl groups containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has about 2 to 12 carbons. More preferably it is a lower alkynyl of from about 2 to 7 carbons, more preferably about 2 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1–C6 hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moieties of the invention can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. 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 about 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.

[0164] The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example methoxyethyl or ethoxymethyl.

[0165] The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkyl thioether, for example methylthiomethyl or methylthioethyl.

[0166] The term “amino” as used herein refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms “aminoacyl” and “aminoalkyl” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.

[0167] The term “amination” as used herein refers to a process in which an amino group or substituted amine is introduced into an organic molecule.

[0168] The term “exocyclic amine protecting moiety” as used herein refers to a nucleobase amino protecting group compatible with oligonucleotide synthesis, for example an acyl or amide group.

[0169] The term “alkenyl” as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl, and 2-methyl-3-heptene.

[0170] The term “alkoxy” as used herein refers to an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.

[0171] The term “alkynyl” as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of “alkynyl” include propargyl, propyne, and 3-hexyne.

[0172] The term “aryl” as used herein refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring can optionally be fused or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.

[0173] The term “cycloalkenyl” as used herein refers to a C3–C8 cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0174] The term “cycloalkyl” as used herein refers to a C3–C8 cyclic hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0175] The term “cycloalkylalkyl,” as used herein, refers to a C3–C7 cycloalkyl group attached to the parent molecular moiety through an alkyl group, as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0176] The terms “halogen” or “halo” as used herein refers to indicate fluorine, chlorine, bromine, and iodine.

[0177] The term “heterocycloalkyl,” as used herein refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused to or otherwise attached to other heterocycloalkyl rings and/or non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups have from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.

[0178] The term “heteroaryl” as used herein refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heteroaryl ring can be filsed or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or heterocycloalkyl rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

[0179] The term “C1–C6 hydrocarbyl” as used herein refers to straight, branched, or cyclic alkyl groups having 1–6 carbon atoms, optionally containing one or more carbon-carbon double or triple bonds. Examples of hydrocarbyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene, cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl and propargyl. When reference is made herein to C1–C6 hydrocarbyl containing one or two double or triple bonds it is understood that at least two carbons are present in the alkyl for one double or triple bond, and at least four carbons for two double or triple bonds.

[0180] By “nucleotide” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, 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, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives 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; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.

[0181] By “nucleoside” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are 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 nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids 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, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al, 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.

[0182] In one embodiment, the invention features modified enzymatic nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, 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. These references are hereby incorporated by reference herein.

[0183] By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No. WO 97/26270).

[0184] By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.

[0185] 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.

[0186] In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH₂ or 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, WO 98/28317, respectively, which are both incorporated by reference in their entireties.

[0187] Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.

[0188] Use of these molecules can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and/or other chemical or biological molecules). The treatment of subjects with nucleic acid molecules can also include combinations of different types of nucleic acid molecules. Therapies can be devised which include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and/or 2–5A chimera molecules to one or more targets to alleviate symptoms of a disease.

Administration of Nucleic Acid Molecules

[0189] Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA 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 familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by a incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153–1158). Other approaches include the use of various transport and carrier systems, for example, through the use of conjugates and biodegradable polymers. For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336–343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J. NeuroVirol., 3, 387–400. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.

[0190] The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

[0191] The negatively charged 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.

[0192] 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.

[0193] 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, preferably 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 polymer is desired to be delivered to). 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 which prevent the composition or formulation from exerting its effect.

[0194] 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 which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, 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 which 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 cancer cells.

[0195] 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: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example 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 implantation (Emerich, DF 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, including CNS delivery of 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. All these references are hereby incorporated herein by reference.

[0196] 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). Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights. 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; all of which are incorporated by reference herein). 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. All of these references are incorporated by reference herein.

[0197] The present invention also includes compositions prepared for storage or administration which 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.

[0198] 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 which 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.

[0199] 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 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.

[0200] 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 monosterate or glyceryl distearate can be employed.

[0201] 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.

[0202] Aqueous suspensions contain the active materials in admixture 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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 patient or 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.

[0210] It is understood that the specific dose level for any particular patient or 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.

[0211] 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.

[0212] 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.

[0213] Alternatively, certain of the nucleic acid 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; all of these references are hereby incorporated in their totalities by reference herein). 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 a 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; all of these references are hereby incorporated in their totalities by reference herein). Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269–280; Peterson et al., 2000, Cent. Nerv. Syst. Dis., 485–508; Peel and Klein, 2000, J. Neurosci. Methods, 98, 95–104; Hagihara et al., 2000, Gene Ther., 7, 759–763; and Herrlinger et al., 2000, Methods Mol. Med., 35, 287–312. AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat. No. 6,180,613.

[0214] In another aspect of the invention, RNA molecules of the present invention are preferably expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are 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 nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient or subject followed by reintroduction into the patient or 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).

[0215] In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner which allows expression of that nucleic acid molecule.

[0216] 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); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid 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 nucleic acid catalyst of the invention; and/or an intron (intervening sequences).

[0217] Transcription of the nucleic acid molecule sequences are 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). All of these references are incorporated by reference herein. Several investigators have demonstrated that nucleic acid molecules, such as ribozymes 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 ribozymes 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; all of these publications are incorporated by reference herein. The above ribozyme 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).

[0218] In another aspect the invention features an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

[0219] In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

[0220] 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; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

[0221] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

[0222] The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver ribozyme molecules and binding/cleavage sites within IKK-gamma or PKR RNA.

[0223] The sequence of human IKK-gamma or PKR genes are screened for accessible sites using a computer-folding algorithm. Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified. The sequences of these binding/cleavage sites are shown in Tables III–XIII.

[0224] Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human IKK-gamma (Genbank accession No: NM_—003639) (SEQ ID NO:8014) and PKR (Genbank accession No: NM_—002759) and prioritizing the sites on the basis of folding. Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 19941 Mol. Struc. Theochern, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure. Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

[0225] Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above. The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimetboxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%.

[0226] Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table XIII. The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables III to XIII.

[0227] Enzymatic nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables III–XIII.

[0228] Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [a-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl₂) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1–5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.

[0229] Nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below. The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables III–XIII.

[0230] A variety of endpoints have been used in cell culture models to evaluate IKK-gamma or PKR-mediated effects after treatment with anti-IKK-gamma or PKR agents. Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of IKK-gamma or PKR protein expression, or a decrease in NFKB expression. Since IKK-gamma and PKR are both involved in the induction of NFKB, NFKB can be used as a surrogate marker in cell culture, animal, and clinical studies. Because overexpression of NFKB is directly associated with increased proliferation of tumor cells, a proliferation endpoint for cell culture assays is preferably used as a primary screen. There are several methods by which this endpoint can be measured. Following treatment of cells with nucleic acid molecules, cells are allowed to grow (typically 5 days) after which either the cell viability, the incorporation of [³H] thymidine into cellular DNA and/or the cell density can be measured. The assay of cell density is very straightforward and can be performed in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto® 13 or CyQuant®). An assay using CyQuant® is described herein.

[0231] As a secondary, confirmatory endpoint a nucleic acid-mediated decrease in the level of IKK-gamma or PKR RNA and/or IKK-gamma or PKR protein expression can be evaluated. Alternately, a decrease in the level of NFKB RNA can be evaluated.

Cell Culture

[0232] Cell types that express/over-express NFKB include HeLa, macrophages, peripheral blood lymphocytes, hepatocytes, fibroblasts, endothelial cells and epithelial cells. In culture, these cells can be stimulated to express/over-express NFKB by addition of TNF-alpha PMA or IL-1-beta to the culture medium. Some of these cell types also can respond with a similar activation of NFKB following LPS treatment. Activation of NFKB in cultured cells can be evaluated by electrophoretic mobility shift assay (EMSA). Delineation of alterations in the subunits can be determined by Western blot.

Primary Screen

[0233] A useful cell culture system in evaluating NFKB modulation is human colonic epithelial cells. One suitable cell line is SW620 colon carcinoma cells (CCL227). These cells respond to stimulation with TNF-alpha, LPS and/or IL-1-beta with an increase in NFKB activation. SW620 cells are grown in MEM supplemented with 10% heat-inactivated FBS and glutamine (2 mmol/L).

[0234] TNF-alpha dose-response curves in these cells are determined by incubating cells with various concentrations of recombinant human TNF-alpha (Sigma Chemical Co.). Maximal DNA binding activity induction can occur with 150 U/ml TNF-alpha in the culture medium. Induction is typically evident within 10 minutes of treatment with TNF-alpha reaches a peak at one hour post-treatment and persists for up to 4 hours post-treatment. The primary readout can be NFKB DNA activity in nuclear extracts of SW620 cells as determined by electrophoretic mobility shift assays (EMSA). Once the appropriate TNF-alpha dose/response profile has been determined, inhibition of IKK-gamma, PKR, or NFKB activation is evaluated using specific and non-specific inhibitors of activation, sulfasalazine and steroids, respectively. Cells are incubated with inhibitors or control media for 30 minutes prior to stimulation with TNF-alpha Nuclear extracts are prepared and evaluated for DNA binding activity by EMSA. Once the activity of positive controls has been established, enzymatic nucleic acids targeting the IKK-gamma or PKR are evaluated in this system. Supershift assays using polyclonal antibodies against the NFKB or PKR protein subunits can be performed to confirm down-regulation of NFKB.

Secondary Screens

[0235] SW620 cells can be transfected with the 3×Ig-kappa-B-Luc reporter construct 18 hours before challenge with TNF-alpha, LPS or PMA. The readout for this assay is luciferase activity. Test compounds are applied 17.5 hours after transfection (30 minutes before challenge). Cells are harvested 24 hours after challenge and relative changes in luciferase activity is used as the endpoint. Lastly, the activation of NFKB can be visualized fluorescently. Inactive NFKB heterodimers are held in the cytoplasm by inhibitory proteins. Once activated, the free heterodimers translocate to the nucleus. Thus, the relative change in cytoplasmic versus nuclear fluorescence can indicate the degree of NFKB activation. Cells can be grown on chamber slides, treated with TNF-alpha with and without test compounds), and the location of the NFKB subunit can be determined by immunofluorescence using a FITC-labeled antibody to NFKB.

Animal Models

[0236] Evaluating the efficacy of anti-IKK-gamma or PKR agents in animal models is an important prerequisite to human clinical trials. Studies have shown that human breast carcinoma cell lines express high levels of NFKB (Sovak et al., 1997, J. Clin. Invest., 100, 2952–2960). High levels of NFKB have also been observed in carcinogen-induced primary rat mammary tumors and in human breast cancer specimins. Additionally, HER2/neu overexpression has been shown to activate NFKB (Pianetti et al., 2001, Oncogene, 20, 1287–1299). As such, xenografts of cell lines that over-express NFKB can be used in animal models of tumorigenesis and/or inflammation to study the inhibition of NFKB.

Oncology Animal Model Development

[0237] Tumor cell lines are characterized to establish their growth curves in mice. These cell lines are implanted into both nude and SCID mice and primary tumor volumes are measured 3 times per week. Growth characteristics of these tumor lines using a Matrigel implantation format can also be established. The use of other cell lines that have been engineered to express high levels of NFKB can also be used in the described studies. The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead IKK-gamma or PKR nucleic acid(s). Nucleic acids are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration of the study. Group sizes of at least 10 animals are employed. Efficacy is determined by statistical comparison of tumor volume of nucleic acid-treated animals to a control group of animals treated with saline alone. Because the growth of these tumors is generally slow (45–60 days), an initial endpoint is the time in days it takes to establish an easily measurable primary tumor (i.e. 50–100 mm³) in the presence or absence of nucleic acid treatment.

Inflammation Animal Model Development

[0238] Chronic, sublethal administration of indomethacin to outbred rats produces an enteropathy characterized by thickening of the small intestine and mesentery, ulcerations, granulomatous inflammation, crypt abcesses and adhesions. These lesions are similar to those that are characteristic findings in human patients with Crohn's disease (CD). Thus, any beneficial therapeutic effects revealed using this model can be extrapolated to potential benefit for patients with CD.

[0239] Male Sprague-Dawley rats (200–275 g) are utilized for these studies. Chronic intestinal inflammation is induced by two subcutaneous injections of indomethacin (7.5 mg/kg in 5% NaHCO3) administered on subsequent days (Day-0 and Day-1). Animals are followed for four days following the first indomethacin injection. The mortality rate associated with this model is typically less than 10%. On the last day of the study, animals are euthanized by CO₂ asphyxiation, small intestines excised and gross pathologic findings ranked according to the following criteria: 0, normal ; 1, minimal abnormalities, slight thickening of the small intestine, no adhesions; 2, obvious thickening of small intestine with 1 adhesion; 3, obvious thickening of small intestine with 2 or 3 adhesions; 4, massive adhesions to the extent that the intestine cannot be separated, contents primarily fluid; 5, severe peritonitis resulting in death. A 10-cm portion of the most affected region of the small intestine is weighed, placed in 10% neutral buffered formalin and submitted for histopathologic evaluation.

[0240] The 10 cm portion of gut from each animal is cut into five equal sections. Transverse and longitudinal sections of each portion are cut and stained with hematoxylin and eosin. All slides are read in a blinded fashion and each section is scored for necrosis (% area of involvement) and inflammatory response according to the following scale:

• • [0241] Necrosis—1, 10%; 2, 10–25%; 3, 25–50%; 4, 50–75%; 5, 75–100%;
  • [0242] Inflammation—
    • [0243] 1=minimal in mesentery and muscle or lesion
    • [0244] 2=mild in mesentery and muscle or lesion
    • [0245] 3=moderate in mesentery and muscle or lesion
    • [0246] 4=marked in lesion
    • [0247] 5=severe in lesion

[0248] The scores for each of the five sections are averaged for necrosis and for inflammation.

NFKB Levels for Patient Screening and as a Potential Endpoint

[0249] Because elevated NFKB levels can be detected in cancers, cancer patients can be pre-screened for elevated NFKB prior to admission to initial clinical trials testing an anti-IKK-gamma or PKR nucleic acid. Initial NFKB levels can be determined (by ELISA) from tumor biopsies or resected tumor samples. During clinical trials, it can be possible to monitor circulating NFKB protein by ELISA. Evaluation of serial blood/serum samples over the course of the anti-IKK-gamma or PKR nucleic acid treatment period could be useful in determining early indications of efficacy.

[0250] Several nucleic acid molecules targeted against IKK-gamma or PKR RNA have been designed and synthesized. These nucleic acid molecules can be tested in cell proliferation and RNA reduction assays described herein.

Proliferation Assay

[0251] The model proliferation assay used in the study requires a cell-plating density of 2,000–10,000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day treatment period. Cells used in proliferation studies were either lung or ovarian cancer cells (A549 and SKOV-3 cells respectively). To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method known in the art was used. This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000–100,000 cells/well in 96-well format. Enzymatic nucleic acid molecules (50–200 nM) are delivered in the presence of cationic lipid at 2.5–5.0 μg/nL and inhibition of proliferation was determined on day 5 post-treatment.

RNA Assay

[0252] RNA is harvested 24 hours post-treatment using the Qiagen RNeasy® 96 procedure. Real time RT-PCR (TaqMan® assay) is performed on purified RNA samples using separate primer/probe sets specific for target IKK-gamma or PKR RNA.

Indications

[0253] Particular degenerative and disease states that can be associated with IKK-gamma or PKR expression modulation include but are not limited to cancerous and/or inflammatory diseases and conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, incontinentia pigmenti and any other diseases or conditions that are related to or respond to the levels of IKK-gamma or PKR in a cell or tissue. The present body of knowledge in IKI-gamma and PKR research indicates the need for methods to assay IKK-gamma and PKR activity and for compounds that can regulate IKK-gamma and PKR expression for research, diagnostic, and therapeutic use.

[0254] The use of monoclonal antibodies, chemotherapy, radiation therapy, analgesics, and/or anti-inflammatory compounds, are all non-limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells. These drugs include but are not limited to paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine 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. ribozymes and antisense molecules) are hence within the scope of the instant invention.

Diagnostic Uses

[0255] The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of IKK-gamma or PKR RNA in a cell. The close relationship between enzymatic nucleic acid molecule 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 enzymatic nucleic acid molecules described in this invention, one can 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 enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with IKK-gamma or PKR-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.

[0256] In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule 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 enzymatic nucleic acid 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., IKK-gamma or PKR) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease 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. The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842.

Additional Uses

[0257] Potential uses of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” can be replaced with either of the other two terms. 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 can be restored to by tose 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.

[0262] 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 groupl

[0263] Other embodiments are within the claims that follow.

[0264]

TABLE I
Characteristics of naturally occurring ribozymes
Group I Introns
Size: ~150 to >1000 nucleotides.
Requires a U in the target sequence immediately 5′ of the cleavage site.
Binds 4–6 nucleotides at the 5′-side of the cleavage site.
Reaction mechanism: attack by the 3′-OH of guanosine to generate cleavage
products with 3′-OH and 5′-guanosine.
Additional protein cofactors required in some cases to help folding and
maintenance of the active structure.
Over 300 known members of this class. Found as an intervening sequence in
Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-
green algae, and others.
Major structural features largely established through phylogenetic comparisons,
mutagenesis, and biochemical studies [i,ii].
Complete kinetic framework established for one ribozyme [iii,iv,v,vi].
Studies of ribozyme folding and substrate docking underway [vii,viii,ix].
Chemical modification investigation of important residues well established [x,xi].
The small (4–6 nt) binding site can make this ribozyme too non-specific for
targeted RNA cleavage, however, the Tetrahymena group I intron has been used
to repair a “defective” β-galactosidase message by the ligation of new β-
galactosidase sequences onto the defective message [xii].
RNAse P RNA (M1 RNA)
Size: ~290 to 400 nucleotides.
RNA portion of a ubiquitous ribonucleoprotein enzyme.
Cleaves tRNA precursors to form mature tRNA [xiii].
Reaction mechanism: possible attack by M2+-OH to generate cleavage products
with 3′-OH and 5′-phosphate.
RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit
has been sequenced from bacteria, yeast, rodents, and primates.
Recruitment of endogenous RNAse P for therapeutic applications is possible
through hybridization of an External Guide Sequence (EGS) to the target RNA
[xiv,xv]
Important phosphate and 2′ OH contacts recently identified [xvi,xvii]
Group II Introns
Size: >1000 nucleotides.
Trans cleavage of target RNAs recently demonstrated [xviii,xix].
Sequence requirements not fully determined.
Reaction mechanism: 2′-OH of an internal adenosine generates cleavage products
with 3′-OH and a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point.
Only natural ribozyme with demonstrated participation in DNA cleavage [xx,xxi] in
addition to RNA cleavage and ligation.
Major structural features largely established through phylogenetic comparisons
[xxii].
Important 2′OH contacts beginning to be identified [xxiii]
Kinetic framework under development [xxiv]
Neurospora VS RNA
Size: ~144 nucleotides.
Trans cleavage of hairpin target RNAs recently demonstrated [xxv].
Sequence requirements not fully determined.
Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage
products with 2′,3′-cyclic phosphate and 5′-OH ends.
Binding sites and structural requirements not fully determined.
Only 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme
(see text for references)
Size: ~13 to 40 nucleotides.
Requires the target sequence UH immediately 5′ of the cleavage site.
Binds a variable number nucleotides on both sides of the cleavage site.
Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage
products with 2′,3′-cyclic phosphate and 5′-OH ends.
14 known members of this class. Found in a number of plant pathogens
(virusoids) that use RNA as the infectious agent.
Essential structural features largely defined, including 2 crystal structures [xxvi,xxvii]
Minimal ligation activity demonstrated (for engineering through in vitro selection)
[xxviii]
Complete kinetic framework established for two or more ribozymes [xxix].
Chemical modification investigation of important residues well established [xxx].
Hairpin Ribozyme
Size: ~50 nucleotides.
Requires the target sequence GUC immediately 3′ of the cleavage site.
Binds 4–6 nucleotides at the 5′-side of the cleavage site and a variable number to
the 3′-side of the cleavage site.
Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage
products with 2′,3′-cyclic phosphate and 5′-OH ends.
3 known members of this class. Found in three plant pathogen (satellite RNAs of
the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus)
which uses RNA as the infectious agent.
Essential structural features largely defined [xxxi,xxxii,xxxiii,xxxiv]
Ligation activity (in addition to cleavage activity) makes ribozyme amenable to
engineering through in vitro selection [xxxv]
Complete kinetic framework established for one ribozyme [xxxvi].
Chemical modification investigation of important residues begun [xxxvii,xxxviii].
Hepatitis Delta Virus (HDV) Ribozyme
Size: ~60 nucleotides.
Trans cleavage of target RNAs demonstrated [xxxix].
Binding sites and structural requirements not fully determined, although no
sequences 5′ of cleavage site are required. Folded ribozyme contains a pseudoknot
structure [xl].
Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage
products with 2′,3′-cyclic phosphate and 5′-OH ends.
Only 2 known members of this class. Found in human HDV.
Circular form of HDV is active and shows increased nuclease stability [xli]
i. Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5–7.
ii. Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206–17.
iii. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159–71.
iv. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172–80.
v. Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560–70.
vi. Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648–58.
vii. Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394–9.
viii. Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504–12.
ix. Zarrinkar, Patrick P.; Williamson, James R.. The P9.1–P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854–8.
x. Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995), 267(5198), 675–9.
xi. Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5′-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201–11.
xii. Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371(6498), 619–22.
xiii. Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol. Chem., 247, 5243–5251 (1972).
xiv. Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C., 1883–) (1990), 249(4970), 783–6.
xv. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006–10.
xvi. Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210–18.
xvii. Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2′-hydroxyl-base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A. (1995), 92(26), 12510–14.
xviii. Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716–25.
xix. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965–77.
xx. Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529–38.
xxi. Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2′-hydroxyl groups. Chem. Biol. (1995), 2(11), 761–70.
xxii. Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435–61.
xxiii. Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of 2′-hydroxyl groups within a group II intron active site. Science (Washington, D. C.) (1996), 271(5254), 1410–13.
xxiv. Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1), 31–49.
xxv. Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368–76.
xxvi. Scott, W. G., Finch, J. T., Aaron, K. The crystal structure of an all RNA hammerhead ribozyme: Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991–1002.
xxvii. McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395–403.
xxviii. Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. U.S. Pat. No. 5,633,133.
xxix. Hertel, K. J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374–3385. Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702–25708.
xxx. Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702–25708.
xxxi. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. ‘Hairpin’ catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299–304.
xxxii. Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320–2.
xxxiii. Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel E.; Burke, John M.. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567–73.
xxxiv. Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel E.. Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1), 130–8.
xxxv. Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1), 129–34.
xxxvi. Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813–28.
xxxvii. Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068–76.
xxxviii. Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik S.; Gait, Michael J.. Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573–81.
xxxix. Perrotta, Anne T.; Been, Michael D.. Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16–21.
xl. Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434–6.
xli. Puttaraju, M.; Perrotta, Anne T.; Been, Michael D.. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253–8.

[0265]

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

[0266]

TABLE III
Human IKK-gamma Hammerhead and Substrate Sequence
		Seq		Seq
Pos	Substrate	ID	Hammerhead	ID
18	AUGGCCCU U GUGAUCCA	4361	UGGAUCAC CUGAUGAGGCCGUUAGGCCGAA AGGGCCAU	4555
24	CUUGUGAU C CAGGUGGG	4362	CCCACCUG CUGAUGAGGCCGUUAGGCCGAA AUCACAAG	4556
39	GGGAAACU A AGGCCCAG	4363	CUGGGCCU CUGAUGAGGCCGUUAGGCCGAA AGUUUCCC	4557
70	CGCAGACU A UCAAUCCC	4364	GGGAUUGA CUGAUGAGGCCGUUAGGCCGAA AGUCUGCG	4558
72	CAGACUAU C AAUCCCAG	4365	CUGGGAUU CUGAUGAGGCCGUUAGGCCGAA AUAGUCUG	4559
76	CUAUCAAU C CCAGUCUC	4366	GAGACUGG CUGAUGAGGCCGUUAGGCCGAA AUUGAUAG	4560
82	AUCCCAGU C UCUUCCCC	4367	GGGGAAGA CUGAUGAGGCCGUUAGGCCGAA ACUGGGAU	4561
84	CCCAGUCU C UUCCCCUC	4368	GAGGGGAA CUGAUGAGGCCGUUAGGCCGAA AGACUGGG	4562
86	CAGUCUCU U CCCCUCAC	4369	GUGAGGGG CUGAUGAGGCCGUUAGGCCGAA AGAGACUG	4563
87	AGUCUCUU C CCCUCACU	4370	AGUGAGGG CUGAUGAGGCCGUUAGGCCGAA AAGAGACU	4564
92	CUUCCCCU C ACUCCCUG	4371	CAGGGAGU CUGAUGAGGCCGUUAGGCCGAA AGGGGAAG	4565
96	CCCUCACU C CCUGUGAA	4372	UUCACAGG CUGAUGAGGCCGUUAGGCCGAA AGUGAGGG	4566
108	GUGAAGCU C UCCAGCAU	4373	AUGCUGGA CUGAUGAGGCCGUUAGGCCGAA AGCUUCAC	4567
110	GAAGCUCU C CAGCAUCA	4374	UGAUGCUG CUGAUGAGGCCGUUAGGCCGAA AGAGCUUC	4568
117	UCCAGCAU C AUCGAGGU	4375	ACCUCGAU CUGAUGAGGCCGUUAGGCCGAA AUGCUGGA	4569
120	AGCAUCAU C GAGGUCCC	4376	GGGACCUC CUGAUGAGGCCGUUAGGCCGAA AUGAUGCU	4570
126	AUCGAGGU C CCAUCAGC	4377	GCUGAUGG CUGAUGAGGCCGUUAGGCCGAA ACCUCGAU	4571
131	GGUCCCAU C AGCCCUUG	4378	CAAGGGCU CUGAUGAGGCCGUUAGGCCGAA AUGGGACC	4572
138	UCAGCCCU U GCCCUGUU	4379	AACAGGGC CUGAUGAGGCCGUUAGGCCGAA AGGGCUGA	4573
146	UGCCCUGU U GGAUGAAU	4380	AUUCAUCC CUGAUGAGGCCGUUAGGCCGAA ACAGGGCA	4574
155	GGAUGAAU A GGCACCUC	4381	GAGGUGCC CUGAUGAGGCCGUUAGGCCGAA AUUCAUCC	4575
163	AGGCACCU C UGGAAGAG	4382	CUCUUCCA CUGAUGAGGCCGUUAGGCCGAA AGGUGCCU	4576
218	CAGCAGAU C AGGACGUA	4383	UACGUCCU CUGAUGAGGCCGUUAGGCCGAA AUCUGCUG	4577
226	CAGGACGU A CUGGGCGA	4384	UCGCCCAG CUGAUGAGGCCGUUAGGCCGAA ACGUCCUG	4578
240	CGAAGAGU C UCCUCUGG	4385	CCAGAGGA CUGAUGAGGCCGUUAGGCCGAA ACUCUUCG	4579
242	AAGAGUCU C CUCUGGGG	4386	CCCCAGAG CUGAUGAGGCCGUUAGGCCGAA AGACUCUU	4580
245	AGUCUCCU C UGGGGAAG	4387	CUUCCCCA CUGAUGAGGCCGUUAGGCCGAA AGGAGACU	4581
275	ACCUGCCU U CAGAACAG	4388	CUGUUCUG CUGAUGAGGCCGUUAGGCCGAA AGGCAGGU	4582
276	CCUGCCUU C AGAACAGG	4389	CCUGUUCU CUGAUGAGGCCGUUAGGCCGAA AAGGCAGG	4583
290	AGGGCGCU C CUGAGACC	4390	GGUCUCAG CUGAUGAGGCCGUUAGGCCGAA AGCGCCCU	4584
301	GAGACCCU C CAGCGCUG	4391	CAGCGCUG CUGAUGAGGCCGUUAGGCCGAA AGGGUCUC	4585
323	AGGAGAAU C AAGAGCUC	4392	GAGCUCUU CUGAUGAGGCCGUUAGGCCGAA AUUCUCCU	4586
331	CAAGAGCU C CGAGAUGC	4393	GCAUCUCG CUGAUGAGGCCGUUAGGCCGAA AGCUCUUG	4587
343	GAUGCCAU C CGGCAGAG	4394	CUCUGCCG CUGAUGAGGCCGUUAGGCCGAA AUGGCAUC	4588
361	AACCAGAU U CUGCGGGA	4395	UCCCGCAG CUGAUGAGGCCGUUAGGCCGAA AUCUGGUU	4589
362	ACCAGAUU C UGCGGGAG	4396	CUCCCGCA CUGAUGAGGCCGUUAGGCCGAA AAUCUGGU	4590
385	GAGGAGCU U CUGCAUUU	4397	AAAUGCAG CUGAUGAGGCCGUUAGGCCGAA AGCUCCUC	4591
386	AGGAGCUU C UGCAUUUC	4398	GAAAUGCA CUGAUGAGGCCGUUAGGCCGAA AAGCUCCU	4592
392	UUCUGCAU U UCCAAGCC	4399	GGCUUGGA CUGAUGAGGCCGUUAGGCCGAA AUGCAGAA	4593
393	UCUGCAUU U CCAAGCCA	4400	UGGCUUGG CUGAUGAGGCCGUUAGGCCGAA AAUGCAGA	4594
394	CUGCAUUU C CAAGCCAG	4401	CUGGCUUG CUGAUGAGGCCGUUAGGCCGAA AAAUGCAG	4595
423	GAAGGAGU U CCUCAUGU	4402	ACAUGAGG CUGAUGAGGCCGUUAGGCCGAA ACUCCUUC	4596
424	AAGGAGUU C CUCAUGUG	4403	CACAUGAG CUGAUGAGGCCGUUAGGCCGAA AACUCCUU	4597
427	GAGUUCCU C AUGUGCAA	4404	UUGCACAU CUGAUGAGGCCGUUAGGCCGAA AGGAACUC	4598
438	GUGCAAGU U CCAGGAGG	4405	CCUCCUGG CUGAUGAGGCCGUUAGGCCGAA ACUUGCAC	4599
439	UGCAAGUU C CAGGAGGC	4406	GCCUCCUG CUGAUGAGGCCGUUAGGCCGAA AACUUGCA	4600
469	GAGAGACU C GGCCUGGA	4407	UCCAGGCC CUGAUGAGGCCGUUAGGCCGAA AGUCUCUC	4601
484	GAGAAGCU C GAUCUGAA	4408	UUCAGAUC CUGAUGAGGCCGUUAGGCCGAA AGCUUCUC	4602
488	AGCUCGAU C UGAAGAGG	4409	CCUCUUCA CUGAUGAGGCCGUUAGGCCGAA AUCGAGCU	4603
512	AGCAGGCU C UGCGGGAG	4410	CUCCCGCA CUGAUGAGGCCGUUAGGCCGAA AGCCUGCU	4604
570	CAAGGCCU C UGUGAAAG	4411	CUUUCACA CUGAUGAGGCCGUUAGGCCGAA AGGCCUUG	4605
591	GGUGACGU C CUUGCUCG	4412	CGAGCAAG CUGAUGAGGCCGUUAGGCCGAA ACGUCACC	4606
594	GACGUCCU U GCUCGGGG	4413	CCCCGAGC CUGAUGAGGCCGUUAGGCCGAA AGGACGUC	4607
598	UCCUUGCU C GGGGAGCU	4414	AGCUCCCC CUGAUGAGGCCGUUAGGCCGAA AGCAAGGA	4608
623	GCCAGAGU C GCUUGGAG	4415	CUCCAAGC CUGAUGAGGCCGUUAGGCCGAA ACUCUGGC	4609
627	GAGUCGCU U GGAGGCUG	4416	CAGCCUCC CUGAUGAGGCCGUUAGGCCGAA AGCGACUC	4610
641	CUGCCACU A AGGAAUGC	4417	GCAUUCCU CUGAUGAGGCCGUUAGGCCGAA AGUGGCAG	4611
656	GCCAGGCU C UGGAGGGU	4418	ACCCUCCA CUGAUGAGGCCGUUAGGCCGAA AGCCUGGC	4612
665	UGGAGGGU C GGGCCCGG	4419	CCGGGCCC CUGAUGAGGCCGUUAGGCCGAA ACCCUCCA	4613
787	GCCGCGCU C CGCAUGGA	4420	UCCAUGCG CUGAUGAGGCCGUUAGGCCGAA AGCGCGGC	4614
810	GGCCGCCU C GGAGGAGA	4421	UCUCCUCC CUGAUGAGGCCGUUAGGCCGAA AGGCGGCC	4615
837	GGCCCAGU U GCAGGUGG	4422	CCACCUGC CUGAUGAGGCCGUUAGGCCGAA ACUGGGCC	4616
849	GGUGGCCU A UCACCAGC	4423	GCUGGUGA CUGAUGAGGCCGUUAGGCCGAA AGGCCACC	4617
851	UGGCCUAU C ACCAGCUC	4424	GAGCUGGU CUGAUGAGGCCGUUAGGCCGAA AUAGGCCA	4618
859	CACCAGCU C UUCCAAGA	4425	UCUUGGAA CUGAUGAGGCCGUUAGGCCGAA AGCUGGUG	4619
861	CCAGCUCU U CCAAGAAU	4426	AUUCUUGG CUGAUGAGGCCGUUAGGCCGAA AGAGCUGG	4620
862	CAGCUCUU C CAAGAAUA	4427	UAUUCUUG CUGAUGAGGCCGUUAGGCCGAA AAGAGCUG	4621
870	CCAAGAAU A CGACAACC	4428	GGUUGUCG CUGAUGAGGCCGUUAGGCCGAA AUUCUUGG	4622
883	AACCACAU C AAGAGCAG	4429	CUGCUCUU CUGAUGAGGCCGUUAGGCCGAA AUGUGGUU	4623
935	UGGAAGAU C UCAAACAG	4430	CUGUUUGA CUGAUGAGGCCGUUAGGCCGAA AUCUUCCA	4624
937	GAAGAUCU C AAACAGCA	4431	UGCUGUUU CUGAUGAGGCCGUUAGGCCGAA AGAUCUUC	4625
949	CAGCAGCU C CAGCAGGC	4432	GCCUGCUG CUGAUGAGGCCGUUAGGCCGAA AGCUGCUG	4626
991	GAGGUGAU C GAUAAGCU	4433	AGCUUAUC CUGAUGAGGCCGUUAGGCCGAA AUCACCUC	4627
995	UGAUCGAU A AGCUGAAG	4434	CUUCAGCU CUGAUGAGGCCGUUAGGCCGAA AUCGAUCA	4628
1027	CACAAGAU U GUGAUGGA	4435	UCCAUCAC CUGAUGAGGCCGUUAGGCCGAA AUCUUGUG	4629
1042	GAGACCGU U CCGGUGCU	4436	AGCACCGG CUGAUGAGGCCGUUAGGCCGAA ACGGUCUC	4630
1043	AGACCGUU C CGGUGCUG	4437	CAGCACCG CUGAUGAGGCCGUUAGGCCGAA AACGGUCU	4631
1067	AGGCGGAU A UCUACAAG	4438	CUUGUAGA CUGAUGAGGCCGUUAGGCCGAA AUCCGCCU	4632
1069	GCGGAUAU C UACAAGGC	4439	GCCUUGUA CUGAUGAGGCCGUUAGGCCGAA AUAUCCGC	4633
1071	GGAUAUCU A CAAGGCGG	4440	CCGCCUUG CUGAUGAGGCCGUUAGGCCGAA AGAUAUCC	4634
1083	GGCGGACU U CCAGGCUG	4441	CAGCCUGG CUGAUGAGGCCGUUAGGCCGAA AGUCCGCC	4635
1084	GCGGACUU C CAGGCUGA	4442	UCAGCCUG CUGAUGAGGCCGUUAGGCCGAA AAGUCCGC	4636
1132	AAGGAGCU C CUGCAGGA	4443	UCCUGCAG CUGAUGAGGCCGUUAGGCCGAA AGCUCCUU	4637
1167	GAGGGAGU A CAGCAAAC	4444	GUUUGCUG CUGAUGAGGCCGUUAGGCCGAA ACUCCCUC	4638
1190	CCAGCUGU C AGGAGUCG	4445	CGACUCCU CUGAUGAGGCCGUUAGGCCGAA ACAGCUGG	4639
1197	UCAGGAGU C GGCCAGGA	4446	UCCUGGCC CUGAUGAGGCCGUUAGGCCGAA ACUCCUGA	4640
1207	GCCAGGAU C GAGGACAU	4447	AUGUCCUC CUGAUGAGGCCGUUAGGCCGAA AUCCUGGC	4641
1231	CGGCAUGU C GAGGUCUC	4448	GAGACCUC CUGAUGAGGCCGUUAGGCCGAA ACAUGCCG	4642
1237	GUCGAGGU C UCCCAGGC	4449	GCCUGGGA CUGAUGAGGCCGUUAGGCCGAA ACCUCGAC	4643
1239	CGAGGUCU C CCAGGCCC	4450	GGGCCUGG CUGAUGAGGCCGUUAGGCCGAA AGACCUCG	4644
1251	GGCCCCCU U GCCCCCCG	4451	CGGGGGGC CUGAUGAGGCCGUUAGGCCGAA AGGGGGCC	4645
1269	CCCUGCCU A CCUCUCCU	4452	AGGAGAGG CUGAUGAGGCCGUUAGGCCGAA AGGCAGGG	4646
1273	GCCUACCU C UCCUCUCC	4453	GGAGAGGA CUGAUGAGGCCGUUAGGCCGAA AGGUAGGC	4647
1275	CUACCUCU C CUCUCCCC	4454	GGGGAGAG CUGAUGAGGCCGUUAGGCCGAA AGAGGUAG	4648
1278	CCUCUCCU C UCCCCUGG	4455	CCAGGGGA CUGAUGAGGCCGUUAGGCCGAA AGGAGAGG	4649
1280	UCUCCUCU C CCCUGGCC	4456	GGCCAGGG CUGAUGAGGCCGUUAGGCCGAA AGAGGAGA	4650
1332	ACCUGACU U CUGCUGUC	4457	GACAGCAG CUGAUGAGGCCGUUAGGCCGAA AGUCAGGU	4651
1333	CCUGACUU C UGCUGUCC	4458	GGACAGCA CUGAUGAGGCCGUUAGGCCGAA AAGUCAGG	4652
1340	UCUGCUGU C CCAAGUGC	4459	GCACUUGG CUGAUGAGGCCGUUAGGCCGAA ACAGCAGA	4653
1353	GUGCCAGU A UCAGGCCC	4460	GGGCCUGA CUGAUGAGGCCGUUAGGCCGAA ACUGGCAC	4654
1355	GCCAGUAU C AGGCCCCU	4461	AGGGGCCU CUGAUGAGGCCGUUAGGCCGAA AUACUGGC	4655
1367	CCCCUGAU A UGGACACC	4462	GGUGUCCA CUGAUGAGGCCGUUAGGCCGAA AUCAGGGG	4656
1384	CUGCAGAU A CAUGUCAU	4463	AUGACAUG CUGAUGAGGCCGUUAGGCCGAA AUCUGCAG	4657
1390	AUACAUGU C AUGGAGUG	4464	CACUCCAU CUGAUGAGGCCGUUAGGCCGAA ACAUGUAU	4658
1402	GAGUGCAU U GAGUAGGG	4465	CCCUACUC CUGAUGAGGCCGUUAGGCCGAA AUGCACUC	4659
1407	CAUUGAGU A GGGCCGGC	4466	GCCGGCCC CUGAUGAGGCCGUUAGGCCGAA ACUCAAUG	4660
1464	CGUGCAGU C UGCGCUUU	4467	AAAGCGCA CUGAUGAGGCCGUUAGGCCGAA ACUGCACG	4661
1471	UCUGCGCU U UCCUCUCC	4468	GGAGAGGA CUGAUGAGGCCGUUAGGCCGAA AGCGCAGA	4662
1472	CUGCGCUU U CCUCUCCC	4469	GGGAGAGG CUGAUGAGGCCGUUAGGCCGAA AAGCGCAG	4663
1473	UGCGCUUU C CUCUCCCG	4470	CGGGAGAG CUGAUGAGGCCGUUAGGCCGAA AAAGCGCA	4664
1476	GCUUUCCU C UCCCGCCU	4471	AGGCGGGA CUGAUGAGGCCGUUAGGCCGAA AGGAAAGC	4665
1478	UUUCCUCU C CCGCCUGC	4472	GCAGGCGG CUGAUGAGGCCGUUAGGCCGAA AGAGGAAA	4666
1489	GCCUGCCU A GCCCAGGA	4473	UCCUGGGC CUGAUGAGGCCGUUAGGCCGAA AGGCAGGC	4667
1565	CGGCACCU U ACGCUUCA	4474	UGAAGCGU CUGAUGAGGCCGUUAGGCCGAA AGGUGCCG	4668
1566	GGCACCUU A CGCUUCAG	4475	CUGAAGCG CUGAUGAGGCCGUUAGGCCGAA AAGGUGCC	4669
1571	CUUACGCU U CAGCUGUU	4476	AACAGCUG CUGAUGAGGCCGUUAGGCCGAA AGCGUAAG	4670
1572	UUACGCUU C AGCUGUUG	4477	CAACAGCU CUGAUGAGGCCGUUAGGCCGAA AAGCGUAA	4671
1579	UCAGCUGU U GAUCCGCU	4478	AGCGGAUC CUGAUGAGGCCGUUAGGCCGAA ACAGCUGA	4672
1583	CUGUUGAU C CGCUGGUC	4479	GACCAGCG CUGAUGAGGCCGUUAGGCCGAA AUCAACAG	4673
1591	CCGCUGGU C CCCUCUUU	4480	AAAGAGGG CUGAUGAGGCCGUUAGGCCGAA ACCAGCGG	4674
1596	GGUCCCCU C UUUUGGGG	4481	CCCCAAAA CUGAUGAGGCCGUUAGGCCGAA AGGGGACC	4675
1598	UCCCCUCU U UUGGGGUA	4482	UACCCCAA CUGAUGAGGCCGUUAGGCCGAA AGAGGGGA	4676
1599	CCCCUCUU U UGGGGUAG	4483	CUACCCCA CUGAUGAGGCCGUUAGGCCGAA AAGAGGGG	4677
1600	CCCUCUUU U GGGGUAGA	4484	UCUACCCC CUGAUGAGGCCGUUAGGCCGAA AAAGAGGG	4678
1606	UUUGGGGU A GAUGCGGC	4485	GCCGCAUC CUGAUGAGGCCGUUAGGCCGAA ACCCCAAA	4679
1621	GCCCCGAU C AGGCCUGA	4486	UCAGGCCU CUGAUGAGGCCGUUAGGCCGAA AUCGGGGC	4680
1632	GCCUGACU C GCUGCUCU	4487	AGAGCAGC CUGAUGAGGCCGUUAGGCCGAA AGUCAGGC	4681
1639	UCGCUGCU C UUUUUGUU	4488	AACAAAAA CUGAUGAGGCCGUUAGGCCGAA AGCAGCGA	4682
1641	GCUGCUCU U UUUGUUCC	4489	GGAACAAA CUGAUGAGGCCGUUAGGCCGAA AGAGCAGC	4683
1642	CUGCUCUU U UUGUUCCC	4490	GGGAACAA CUGAUGAGGCCGUUAGGCCGAA AAGAGCAG	4684
1643	UGCUCUUU U UGUUCCCU	4491	AGGGAACA CUGAUGAGGCCGUUAGGCCGAA AAAGAGCA	4685
1644	GCUCUUUU U GUUCCCUU	4492	AAGGGAAC CUGAUGAGGCCGUUAGGCCGAA AAAAGAGC	4686
1647	CUUUUUGU U CCCUUCUG	4493	CAGAAGGG CUGAUGAGGCCGUUAGGCCGAA ACAAAAAG	4687
1648	UUUUUGUU C CCUUCUGU	4494	ACAGAAGG CUGAUGAGGCCGUUAGGCCGAA AACAAAAA	4688
1652	UGUUCCCU U CUGUCUGC	4495	GCAGACAG CUGAUGAGGCCGUUAGGCCGAA AGGGAACA	4689
1653	GUUCCCUU C UGUCUGCU	4496	AGCAGACA CUGAUGAGGCCGUUAGGCCGAA AAGGGAAC	4690
1657	CCUUCUGU C UGCUCGAA	4497	UUCGAGCA CUGAUGAGGCCGUUAGGCCGAA ACAGAAGG	4691
1662	UGUCUGCU C GAACCACU	4498	AGUGGUUC CUGAUGAGGCCGUUAGGCCGAA AGCAGACA	4692
1671	GAACCACU U GCCUCGGG	4499	CCCGAGGC CUGAUGAGGCCGUUAGGCCGAA AGUGGUUC	4693
1676	ACUUGCCU C GGGCUAAU	4500	AUUAGCCC CUGAUGAGGCCGUUAGGCCGAA AGGCAAGU	4694
1682	CUCGGGCU A AUCCCUCC	4501	GGAGGGAU CUGAUGAGGCCGUUAGGCCGAA AGCCCGAG	4695
1685	GGGCUAAU C CCUCCCUC	4502	GAGGGAGG CUGAUGAGGCCGUUAGGCCGAA AUUAGCCC	4696
1689	UAAUCCCU C CCUCUUCC	4503	GGAAGAGG CUGAUGAGGCCGUUAGGCCGAA AGGGAUUA	4697
1693	CCCUCCCU C UUCCUCCA	4504	UGGAGGAA CUGAUGAGGCCGUUAGGCCGAA AGGGAGGG	4698
1695	CUCCCUCU U CCUCCACC	4505	GGUGGAGG CUGAUGAGGCCGUUAGGCCGAA AGAGGGAG	4699
1696	UCCCUCUU C CUCCACCC	4506	GGGUGGAG CUGAUGAGGCCGUUAGGCCGAA AAGAGGGA	4700
1699	CUCUUCCU C CACCCGGC	4507	GCCGGGUG CUGAUGAGGCCGUUAGGCCGAA AGGAAGAG	4701
1719	GGGGAAGU C AAGAAUGG	4508	CCAUUCUU CUGAUGAGGCCGUUAGGCCGAA ACUUCCCC	4702
1739	CUGGGCCU C UCAGGGAG	4509	CUCCCUGA CUGAUGAGGCCGUUAGGCCGAA AGCCCCAG	4703
1741	GGGGCUCU C AGGGAGAA	4510	UUCUCCCU CUGAUGAGGCCGUUAGGCCGAA AGAGCCCC	4704
1755	GAACUGCU U CCCCUGGC	4511	GCCAGGGG CUGAUGAGGCCGUUAGGCCGAA AGCAGUUC	4705
1756	AACUGCUU C CCCUGGCA	4512	UGCCAGGG CUGAUGAGGCCGUUAGGCCGAA AAGCAGUU	4706
1781	UGGCAGCU C UUCCUCCC	4513	GGGAGGAA CUGAUGAGGCCGUUAGGCCGAA AGCUGCCA	4707
1783	GCAGCUCU U CCUCCCAC	4514	GUGGGAGG CUGAUGAGGCCGUUAGGCCGAA AGAGCUGC	4708
1784	CAGCUCUU C CUCCCACC	4515	GGUGGGAG CUGAUGAGGCCGUUAGGCCGAA AAGAGCUG	4709
1787	CUCUUCCU C CCACCGGA	4516	UCCGGUGG CUGAUGAGGCCGUUAGGCCGAA AGGAAGAG	4710
1836	GCUGCCCU C UUACCAUG	4517	CAUGGUAA CUGAUGAGGCCGUUAGGCCGAA AGGGCAGC	4711
1838	UGCCCUCU U ACCAUGCA	4518	UGCAUGGU CUGAUGAGGCCGUUAGGCCGAA AGAGGGCA	4712
1839	GCCCUCUU A CCAUGCAC	4519	GUGCAUGG CUGAUGAGGCCGUUAGGCCGAA AAGAGGGC	4713
1857	CGGGUGCU C UCCUUUUG	4520	CAAAAGGA CUGAUGAGGCCGUUAGGCCGAA AGCACCCG	4714
1859	GGUGCUCU C CUUUUGGG	4521	CCCAAAAG CUGAUGAGGCCGUUAGGCCGAA AGAGCACC	4715
1862	GCUCUCCU U UUGGGCUG	4522	CAGCCCAA CUGAUGAGGCCGUUAGGCCGAA AGGAGAGC	4716
1863	CUCUCCUU U UGGGCUGC	4523	GCAGCCCA CUGAUGAGGCCGUUAGGCCGAA AAGGAGAG	4717
1864	UCUCCUUU U GGGCUGCA	4524	UGCAGCCC CUGAUGAGGCCGUUAGGCCGAA AAAGGAGA	4718
1877	UGCAUGCU A UUCCAUUU	4525	AAAUGGAA CUGAUGAGGCCGUUAGGCCGAA AGCAUGCA	4719
1879	CAUGCUAU U CCAUUUUG	4526	CAAAAUGG CUGAUGAGGCCGUUAGGCCGAA AUAGCAUG	4720
1880	AUGCUAUU C CAUUUUGC	4527	GCAAAAUG CUGAUGAGGCCGUUAGGCCGAA AAUAGCAU	4721
1884	UAUUCCAU U UUCCAGCC	4528	GGCUGCAA CUGAUGAGGCCGUUAGGCCGAA AUGGAAUA	4722
1885	AUUCCAUU U UGCAGCCA	4529	UGGCUGCA CUGAUGAGGCCGUUAGGCCGAA AAUGGAAU	4723
1886	UUCCAUUU U GCAGCCAG	4530	CUGGCUGC CUGAUGAGGCCGUUAGGCCGAA AAAUGGAA	4724
1905	CGAUGUGU A UUUAACCA	4531	UGGUUAAA CUGAUGAGGCCGUUAGGCCGAA ACACAUCG	4725
1907	AUGUGUAU U UAACCAGU	4532	ACUGGUUA CUGAUGAGGCCGUUAGGCCGAA AUACACAU	4726
1908	UGUGUAUU U AACCAGUC	4533	GACUGGUU CUGAUGAGGCCGUUAGGCCGAA AAUACACA	4727
1909	GUGUAUUU A ACCAGUCA	4534	UGACUGGU CUGAUGAGGCCGUUAGGCCGAA AAAUACAC	4728
1916	UAACCAGU C ACUAUUGA	4535	UCAAUAGU CUGAUGAGGCCGUUAGGCCGAA ACUGGUUA	4729
1920	CAGUCACU A UUGAUGGA	4536	UCCAUCAA CUGAUGAGGCCGUUAGGCCGAA AGUGACUG	4730
1922	GUCACUAU U GAUGGACA	4537	UGUCCAUC CUGAUGAGGCCGUUAGGCCGAA AUAGUGAC	4731
1932	AUGGACAU U UGGGUUGU	4538	ACAACCCA CUGAUGAGGCCGUUAGGCCGAA AUGUCCAU	4732
1933	UGGACAUU U GGGUUGUU	4539	AACAACCC CUGAUGAGGCCGUUAGGCCGAA AAUGUCCA	4733
1938	AUUUGGGU U GUUUCCCA	4540	UGGGAAAC CUGAUGAGGCCGUUAGGCCGAA ACCCAAAU	4734
1941	UGGGUUGU U UCCCAUCU	4541	AGAUGGGA CUGAUGAGGCCGUUAGGCCGAA ACAACCCA	4735
1942	GGGUUGUU C CCCAUCUU	4542	AAGAUGGG CUGAUGAGGCCGUUAGGCCGAA AACAACCC	4736
1943	GGUUGUUU C CCAUCUUU	4543	AAAGAUGG CUGAUGAGGCCGUUAGGCCGAA AAACAACC	4737
1948	UUUCCCAU C UUUUUGUU	4544	AACAAAAA CUGAUGAGGCCGUUAGGCCGAA AUGGGAAA	4738
1950	UCCCAUCU U UUUGUUAC	4545	GUAACAAA CUGAUGAGGCCGUUAGGCCGAA AGAUGGGA	4739
1951	CCCAUCUU U UUGUUACC	4546	GGUAACAA CUGAUGAGGCCGUUAGGCCGAA AAGAUGGG	4740
1952	CCAUCUUU U UGUUACCA	4547	UGGUAACA CUGAUGAGGCCGUUAGGCCGAA AAAGAUGG	4741
1953	CAUCUUUU U GUUACCAU	4548	AUGGUAAC CUGAUGAGGCCGUUAGGCCGAA AAAAGAUG	4742
1956	CUUUUUGU U ACCAUAAA	4549	UUUAUGGU CUGAUGAGGCCGUUAGGCCGAA ACAAAAAG	4743
1957	UUUUUGUU A CCAUAAAU	4550	AUUUAUGG CUGAUGAGGCCGUUAGGCCGAA AACAAAAA	4744
1962	GUUACCAU A AAUAAUGG	4551	CCAUUAUU CUGAUGAGGCCGUUAGGCCGAA AUGGUAAC	4745
1966	CCAUAAAU A AUGGCAUA	4552	UAUGCCAU CUGAUGAGGCCGUUAGGCCGAA AUUUAUGG	4746
1974	AAUGGCAU A GUAAAAAA	4553	UUUUUUAC CUGAUGAGGCCGUUAGGCCGAA AUGCCAUU	4747
1977	GGCAUAGU A AAAAAAAA	4554	UUUUUUUU CUGAUGAGGCCGUUAGGCCGAA ACUAUGCC	4748
Input Sequence = NM_003639. Cut Site = UH/.
Arm Length = 8. Core Sequence = CUGAUGAG GCCGUUAGGC CGAA
NM_003639 (Homo sapiens inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), mRNA.; 1994 bp)

Underlined region can be any X sequence or linker, as described herein.

[0267]

TABLE IV
Human IKK-gamma Inozyme and Substrate Sequence
		Seq		Seq
Pos	Substrate	ID	Inozyme	ID
10	GCACGAGC A UGGCCCUU	4749	AAGGGCCA CUGAUGAGGCCGUUAGGCCGAA ICUCGUGC	5253
15	AGCAUGGC C CUUGUGAU	4750	AUCACAAG CUGAUGAGGCCGUUAGGCCGAA ICCAUGCU	5254
16	GCAUGGCC C UUGUGAUC	4751	GAUCACAA CUGAUGAGGCCGUUAGGCCGAA IGCCAUGC	5255
17	CAUGGCCC U UGUGAUCC	4752	GGAUCACA CUGAUGAGGCCGUUAGGCCGAA IGGCCAUG	5256
25	UUGUGAUC C AGGUGGGG	4753	CCCCACCU CUGAUGAGGCCGUUAGGCCGAA IAUCACAA	5257
26	UGUGAUCC A GGUGGGGA	4754	UCCCCACC CUGAUGAGGCCGUUAGGCCGAA IGAUCACA	5258
38	GGGGAAAC U AAGGCCCA	4755	UGGGCCUU CUGAUGAGGCCGUUAGGCCGAA IUUUCCCC	5259
44	ACUAAGGC C CAGAGAAG	4756	CUUCUCUG CUGAUGAGGCCGUUAGGCCGAA ICCUUAGU	5260
45	CUAAGGCC C AGAGAAGU	4757	ACUUCUCU CUGAUGAGGCCGUUAGGCCGAA IGCCUUAG	5261
46	UAAGGCCC A GAGAAGUG	4758	CACUUCUC CUGAUGAGGCCGUUAGGCCGAA IGGCCUUA	5262
60	GUGAGGAC C CCGCAGAC	4759	GUCUGCGG CUGAUGAGGCCGUUAGGCCGAA IUCCUCAC	5263
61	UGAGGACC C CGCAGACU	4760	AGUCUGCG CUGAUGAGGCCGUUAGGCCGAA IGUCCUCA	5264
62	GAGGACCC C GCAGACUA	4761	UAGUCUGC CUGAUGAGGCCGUUAGGCCGAA IGGUCCUC	5265
65	GACCCCGC A GACUAUCA	4762	UGAUAGUC CUGAUGAGGCCGUUAGGCCGAA ICGGGGUC	5266
69	CCGCAGAC U AUCAAUCC	4763	GGAUUGAU CUGAUGAGGCCGUUAGGCCGAA IUCUGCGG	5267
73	AGACUAUC A AUCCCAGU	4764	ACUGGGAU CUGAUGAGGCCGUUAGGCCGAA IAUAGUCU	5268
77	UAUCAAUC C CAGUCUCU	4765	AGAGACUG CUGAUGAGGCCGUUAGGCCGAA IAUUGAUA	5269
78	AUCAAUCC C AGUCUCUU	4766	AAGAGACU CUGAUGAGGCCGUUAGGCCGAA IGAUUGAU	5270
79	UCAAUCCC A GUCUCUUC	4767	GAAGAGAC CUGAUGAGGCCGUUAGGCCGAA IGGAUUGA	5271
83	UCCCAGUC U CUUCCCCU	4768	AGGGGAAG CUGAUGAGGCCGUUAGGCCGAA IACUGGGA	5272
85	CCAGUCUC U UCCCCUCA	4769	UGAGGGGA CUGAUGAGGCCGUUAGGCCGAA IAGACUGG	5273
88	GUCUCUUC C CCUCACUC	4770	GAGUGAGG CUGAUGAGGCCGUUAGGCCGAA IAAGAGAC	5274
89	UCUCUUCC C CUCACUCC	4771	GGAGUGAG CUGAUGAGGCCGUUAGGCCGAA IGAAGAGA	5275
90	CUCUUCCC C UCACUCCC	4772	GGGAGUGA CUGAUGAGGCCGUUAGGCCGAA IGGAAGAG	5276
91	UCUUCCCC U CACUCCCU	4773	AGGGAGUG CUGAUGAGGCCGUUAGGCCGAA IGGGAAGA	5277
93	UUCCCCUC A CUCCCUGU	4774	ACAGGGAG CUGAUGAGGCCGUUAGGCCGAA IAGGGGAA	5278
95	CCCCUCAC U CCCUGUGA	4775	UCACAGGG CUGAUGAGGCCGUUAGGCCGAA IUGAGGGG	5279
97	CCUCACUC C CUGUGAAG	4776	CUUCACAG CUGAUGAGGCCGUUAGGCCGAA IAGUGAGG	5280
98	CUCACUCC C UGUGAAGC	4777	GCUUCACA CUGAUGAGGCCGUUAGGCCGAA IGAGUGAG	5281
99	UCACUCCC U GUGAAGCU	4778	AGCUUCAC CUGAUGAGGCCGUUAGGCCGAA IGGAGUGA	5282
107	UGUGAAGC U CUCCAGCA	4779	UGCUGGAG CUGAUGAGGCCGUUAGGCCGAA ICUUCACA	5283
109	UGAAGCUC U CCAGCAUC	4780	GAUGCUGG CUGAUGAGGCCGUUAGGCCGAA IAGCUUCA	5284
111	AAGCUCUC C AGCAUCAU	4781	AUGAUGCU CUGAUGAGGCCGUUAGGCCGAA IAGAGCUU	5285
112	AGCUCUCC A GCAUCAUC	4782	GAUGAUGC CUGAUGAGGCCGUUAGGCCGAA IGAGAGCU	5286
115	UCUCCAGC A UCAUCGAG	4783	CUCGAUGA CUGAUGAGGCCGUUAGGCCGAA ICUGGAGA	5287
118	CCAGCAUC A UCGAGGUC	4784	GACCUCGA CUGAUGAGGCCGUUAGGCCGAA IAUGCUGG	5288
127	UCGAGGUC C CAUCAGCC	4785	GGCUGAUG CUGAUGAGGCCGUUAGGCCGAA IACCUCGA	5289
128	CGAGGUCC C AUCAGCCC	4786	GGGCUGAU CUGAUGAGGCCGUUAGGCCGAA IGACCUCG	5290
129	GAGGUCCC A UCAGCCCU	4787	AGGGCUGA CUGAUGAGGCCGUUAGGCCGAA IGGACCUC	5291
132	GUCCCAUC A GCCCUUGC	4788	GCAAGGGC CUGAUGAGGCCGUUAGGCCGAA IAUGGGAC	5292
135	CCAUCAGC C CUUGCCCU	4789	AGGGCAAG CUGAUGAGGCCGUUAGGCCGAA ICUGAUGG	5293
136	CAUCAGCC C UUGCCCUG	4790	CAGGGCAA CUGAUGAGGCCGUUAGGCCGAA IGCUGAUG	5294
137	AUCAGCCC U UGCCCUGU	4791	ACAGGGCA CUGAUGAGGCCGUUAGGCCGAA IGGCUGAU	5295
141	GCCCUUGC C CUGUUGGA	4792	UCCAACAG CUGAUGAGGCCGUUAGGCCGAA ICAAGGGC	5296
142	CCCUUGCC C UGUUGGAU	4793	AUCCAACA CUGAUGAGGCCGUUAGGCCGAA IGCAAGGG	5297
143	CCUUGCCC U GUUGGAUG	4794	CAUCCAAC CUGAUGAGGCCGUUAGGCCGAA IGGCAAGG	5298
159	GAAUAGGC A CCUCUGGA	4795	UCCAGAGG CUGAUGAGGCCGUUAGGCCGAA ICCUAUUC	5299
161	AUAGGCAC C UCUGGAAG	4796	CUUCCAGA CUGAUGAGGCCGUUAGGCCGAA IUGCCUAU	5300
162	UAGGCACC U CUGGAAGA	4797	UCUUCCAG CUGAUGAGGCCGUUAGGCCGAA IGUGCCUA	5301
164	GGCACCUC U GGAAGAGC	4798	GCUCUUCC CUGAUGAGGCCGUUAGGCCGAA IAGGUGCC	5302
173	GGAAGAGC C AACUGUGU	4799	ACACAGUU CUGAUGAGGCCGUUAGGCCGAA ICUCUUCC	5303
174	GAAGAGCC A ACUGUGUG	4800	CACACAGU CUGAUGAGGCCGUUAGGCCGAA IGCUCUUC	5304
177	GAGCCAAC U GUGUGAGA	4801	UCUCACAC CUGAUGAGGCCGUUAGGCCGAA IUUGGCUC	5305
192	GAUGGUGC A GCCCAGUG	4802	CACUGGGC CUGAUGAGGCCGUUAGGCCGAA ICACCAUC	5306
195	GGUGCAGC C CAGUGGUG	4803	CACCACUG CUGAUGAGGCCGUUAGGCCGAA ICUGCACC	5307
196	GUGCAGCC C AGUGGUGG	4804	CCACCACU CUGAUGAGGCCGUUAGGCCGAA IGCUGCAC	5308
197	UGCAGCCC A GUGGUGGC	4805	GCCACCAC CUGAUGAGGCCGUUAGGCCGAA IGGCUGCA	5309
206	GUGGUGGC C CGGCAGCA	4806	UGCUGCCG CUGAUGAGGCCGUUAGGCCGAA ICCACCAC	5310
207	UGGUGGCC C GGCAGCAG	4807	CUGCUGCC CUGAUGAGGCCGUUAGGCCGAA IGCCACCA	5311
211	GGCCCGGC A GCAGAUCA	4808	UGAUCUGC CUGAUGAGGCCGUUAGGCCGAA ICCGGGCC	5312
214	CCGGCAGC A GAUCAGGA	4809	UCCUGAUC CUGAUGAGGCCGUUAGGCCGAA ICUGCCGG	5313
219	AGCAGAUC A GGACGUAC	4810	GUACGUCC CUGAUGAGGCCGUUAGGCCGAA IAUCUGCU	5314
228	GGACGUAC U GGGCGAAG	4811	CUUCGCCC CUGAUGAGGCCGUUAGGCCGAA IUACGUCC	5315
241	GAAGAGUC U CCUCUGGG	4812	CCCAGAGG CUGAUGAGGCCGUUAGGCCGAA IACUCUUC	5316
243	AGAGUCUC C UCUGGGGA	4813	UCCCCAGA CUGAUGAGGCCGUUAGGCCGAA IAGACUCU	5317
244	GAGUCUCC U CUGGGGAA	4814	UUCCCCAG CUGAUGAGGCCGUUAGGCCGAA IGAGACUC	5318
246	GUCUCCUC U GGGGAAGC	4815	GCUUCCCC CUGAUGAGGCCGUUAGGCCGAA IAGGAGAC	5319
255	GGGGAAGC C AGCCAUGC	4816	GCAUGGCU CUGAUGAGGCCGUUAGGCCGAA ICUUCCCC	5320
256	GGGAAGCC A GCCAUGCU	4817	AGCAUGGC CUGAUGAGGCCGUUAGGCCGAA IGCUUCCC	5321
259	AAGCCAGC C AUGCUGCA	4818	UGCAGCAU CUGAUGAGGCCGUUAGGCCGAA ICUGGCUU	5322
260	AGCCAGCC A UGCUGCAC	4819	GUGCAGCA CUGAUGAGGCCGUUAGGCCGAA IGCUGGCU	5323
264	AGCCAUGC U GCACCUGC	4820	GCAGGUGC CUGAUGAGGCCGUUAGGCCGAA ICAUGGCU	5324
267	CAUGCUGC A CCUGCCUU	4821	AAGGCAGG CUGAUGAGGCCGUUAGGCCGAA ICAGCAUG	5325
269	UGCUGCAC C UGCCUUCA	4822	UGAAGGCA CUGAUGAGGCCGUUAGGCCGAA IUGCAGCA	5326
270	GCUGCACC U GCCUUCAG	4823	CUGAAGGC CUGAUGAGGCCGUUAGGCCGAA IGUGCAGC	5327
273	GCACCUGC C UUCAGAAC	4824	GUUCUGAA CUGAUGAGGCCGUUAGGCCGAA ICAGGUGC	5328
274	CACCUGCC U UCAGAACA	4825	UGUUCUGA CUGAUGAGGCCGUUAGGCCGAA IGCAGGUG	5329
277	CUGCCUUC A GAACAGGG	4826	CCCUGUUC CUGAUGAGGCCGUUAGGCCGAA IAAGGCAG	5330
282	UUCAGAAC A GGGCGCUC	4827	GAGCGCCC CUGAUGAGGCCGUUAGGCCGAA IUUCUGAA	5331
289	CAGGGCGC U CCUGAGAC	4828	GUCUCAGG CUGAUGAGGCCGUUAGGCCGAA ICGCCCUG	5332
291	GGGCGCUC C UGAGACCC	4829	GGGUCUCA CUGAUGAGGCCGUUAGGCCGAA IAGCGCCC	5333
292	GGCGCUCC U GAGACCCU	4830	AGGGUCUC CUGAUGAGGCCGUUAGGCCGAA IGAGCGCC	5334
298	CCUGAGAC C CUCCAGCG	4831	CGCUGGAG CUGAUGAGGCCGUUAGGCCGAA IUCUCAGG	5335
299	CUGAGACC C UCCAGCGC	4832	GCGCUGGA CUGAUGAGGCCGUUAGGCCGAA IGUCUCAG	5336
300	UGAGACCC U CCAGCGCU	4833	AGCGCUGG CUGAUGAGGCCGUUAGGCCGAA IGGUCUCA	5337
302	AGACCCUC C AGCGCUGC	4834	GCAGCGCU CUGAUGAGGCCGUUAGGCCGAA IAGGGUCU	5338
303	GACCCUCC A GCGCUGCC	4835	GGCAGCGC CUGAUGAGGCCGUUAGGCCGAA IGAGGGUC	5339
308	UCCAGCGC U GCCUGGAG	4836	CUCCAGGC CUGAUGAGGCCGUUAGGCCGAA ICGCUGGA	5340
311	AGCGCUGC C UGGAGGAG	4837	CUCCUCCA CUGAUGAGGCCGUUAGGCCGAA ICAGCGCU	5341
312	GCGCUGCC U GGAGGAGA	4838	UCUCCUCC CUGAUGAGGCCGUUAGGCCGAA IGCAGCGC	5342
324	GGAGAAUC A AGAGCUCC	4839	GGAGCUCU CUGAUGAGGCCGUUAGGCCGAA IAUUCUCC	5343
330	UCAAGAGC U CCGAGAUG	4840	CAUCUCGG CUGAUGAGGCCGUUAGGCCGAA ICUCUUGA	5344
332	AAGAGCUC C GAGAUGCC	4841	GGCAUCUC CUGAUGAGGCCGUUAGGCCGAA IAGCUCUU	5345
340	CGAGAUGC C AUCCGGCA	4842	UGCCGGAU CUGAUGAGGCCGUUAGGCCGAA ICAUCUCG	5346
341	GAGAUGCC A UCCGGCAG	4843	CUGCCGGA CUGAUGAGGCCGUUAGGCCGAA IGCAUCUC	5347
344	AUGCCAUC C GGCAGAGC	4844	GCUCUGCC CUGAUGAGGCCGUUAGGCCGAA IAUGGCAU	5348
348	CAUCCGGC A GAGCAACC	4845	GGUUGCUC CUGAUGAGGCCGUUAGGCCGAA ICCGGAUG	5349
353	GGCAGAGC A ACCAGAUU	4846	AAUCUGGU CUGAUGAGGCCGUUAGGCCGAA ICUCUGCC	5350
356	AGAGCAAC C AGAUUCUG	4847	CAGAAUCU CUGAUGAGGCCGUUAGGCCGAA IUUGCUCU	5351
357	GAGCAACC A GAUUCUGC	4848	GCAGAAUC CUGAUGAGGCCGUUAGGCCGAA IGUUGCUC	5352
363	CCAGAUUC U GCGGGAGC	4849	GCUCCCGC CUGAUGAGGCCGUUAGGCCGAA IAAUCUGG	5353
374	GGGAGCGC U GCGAGGAG	4850	CUCCUCGC CUGAUGAGGCCGUUAGGCCGAA ICGCUCCC	5354
384	CGAGGAGC U UCUGCAUU	4851	AAUGCAGA CUGAUGAGGCCGUUAGGCCGAA ICUCCUCG	5355
387	GGAGCUUC U GCAUUUCC	4852	GGAAAUGC CUGAUGAGGCCGUUAGGCCGAA IAAGCUCC	5356
390	GCUUCUGC A UUUCCAAG	4853	CUUGGAAA CUGAUGAGGCCGUUAGGCCGAA ICAGAAGC	5357
395	UGCAUUUC C AAGCCAGC	4854	GCUGGCUU CUGAUGAGGCCGUUAGGCCGAA IAAAUGCA	5358
396	GCAUUUCC A AGCCAGCC	4855	GGCUGGCU CUGAUGAGGCCGUUAGGCCGAA IGAAAUGC	5359
400	UUCCAAGC C AGCCAGAG	4856	CUCUGGCU CUGAUGAGGCCGUUAGGCCGAA ICUUGGAA	5360
401	UCCAAGCC A GCCAGAGG	4857	CCUCUGGC CUGAUGAGGCCGUUAGGCCGAA IGCUUGGA	5361
404	AAGCCAGC C AGAGGGAG	4858	CUCCCUCU CUGAUGAGGCCGUUAGGCCGAA ICUGGCUU	5362
405	AGCCAGCC A GAGGGAGG	4859	CCUCCCUC CUGAUGAGGCCGUUAGGCCGAA IGCUGGCU	5363
425	AGGAGUUC C UCAUGUGC	4860	GCACAUGA CUGAUGAGGCCGUUAGGCCGAA IAACUCCU	5364
426	GGAGUUCC U CAUGUGCA	4861	UGCACAUG CUGAUGAGGCCGUUAGGCCGAA IGAACUCC	5365
428	AGUUCCUC A UGUGCAAG	4862	CUUGCACA CUGAUGAGGCCGUUAGGCCGAA IAGGAACU	5366
434	UCAUGUGC A AGUUCCAG	4863	CUGGAACU CUGAUGAGGCCGUUAGGCCGAA ICACAUGA	5367
440	GCAAGUUC C AGGAGGCC	4864	GGCCUCCU CUGAUGAGGCCGUUAGGCCGAA IAACUUGC	5368
441	CAAGUUCC A GGAGGCCA	4865	UGGCCUCC CUGAUGAGGCCGUUAGGCCGAA IGAACUUG	5369
448	CAGGAGGC C AGGAAACU	4866	AGUUUCCU CUGAUGAGGCCGUUAGGCCGAA ICCUCCUG	5370
449	AGGAGGCC A GGAAACUG	4867	CAGUUUCC CUGAUGAGGCCGUUAGGCCGAA IGCCUCCU	5371
456	CAGGAAAC U GGUGGAGA	4868	UCUCCACC CUGAUGAGGCCGUUAGGCCGAA IUUUCCUG	5372
468	GGAGAGAC U CGGCCUGG	4869	CCAGGCCG CUGAUGAGGCCGUUAGGCCGAA IUCUCUCC	5373
473	GACUCGGC C UGGAGAAG	4870	CUUCUCCA CUGAUGAGGCCGUUAGGCCGAA ICCGAGUC	5374
474	ACUCGGCC U GGAGAAGC	4871	GCUUCUCC CUGAUGAGGCCGUUAGGCCGAA IGCCGAGU	5375
483	GGAGAAGC U CGAUCUGA	4872	UCAGAUCG CUGAUGAGGCCGUUAGGCCGAA ICUUCUCC	5376
489	GCUCGAUC U GAAGAGGC	4873	GCCUCUUC CUGAUGAGGCCGUUAGGCCGAA IAUCGAGC	5377
498	GAAGAGGC A GAAGGAGC	4874	GCUCCUUC CUGAUGAGGCCGUUAGGCCGAA ICCUCUUC	5378
507	GAAGGAGC A GGCUCUGC	4875	GCAGAGCC CUGAUGAGGCCGUUAGGCCGAA ICUCCUUC	5379
511	GAGCAGGC U CUGCGGGA	4876	UCCCGCAG CUGAUGAGGCCGUUAGGCCGAA ICCUGCUC	5380
513	GCAGGCUC U GCGGGAGG	4877	CCUCCCGC CUGAUGAGGCCGUUAGGCCGAA IAGCCUGC	5381
528	GGUGGAGC A CCUGAAGA	4878	UCUUCAGG CUGAUGAGGCCGUUAGGCCGAA ICUCCACC	5382
530	UGGAGCAC C UGAAGAGA	4879	UCUCUUCA CUGAUGAGGCCGUUAGGCCGAA IUGCUCCA	5383
531	GGAGCACC U GAAGAGAU	4880	AUCUCUUC CUGAUGAGGCCGUUAGGCCGAA IGUGCUCC	5384
542	AGAGAUGC C AGCAGCAG	4881	CUGCUGCU CUGAUGAGGCCGUUAGGCCGAA ICAUCUCU	5385
543	GAGAUGCC A GCAGCAGA	4882	UCUGCUGC CUGAUGAGGCCGUUAGGCCGAA IGCAUCUC	5386
546	AUGCCAGC A GCAGAUGG	4883	CCAUCUGC CUGAUGAGGCCGUUAGGCCGAA ICUGGCAU	5387
549	CCAGCAGC A GAUGGCUG	4884	CAGCCAUC CUGAUGAGGCCGUUAGGCCGAA ICUGCUGG	5388
556	CAGAUGGC U GAGGACAA	4885	UUGUCCUC CUGAUGAGGCCGUUAGGCCGAA ICCAUCUG	5389
563	CUGAGGAC A AGGCCUCU	4886	AGAGGCCU CUGAUGAGGCCGUUAGGCCGAA IUCCUCAG	5390
568	GACAAGGC C UCUGUGAA	4887	UUCACAGA CUGAUGAGGCCGUUAGGCCGAA ICCUUGUC	5391
569	ACAAGGCC U CUGUGAAA	4888	UUUCACAG CUGAUGAGGCCGUUAGGCCGAA IGCCUUGU	5392
571	AAGGCCUC U GUGAAAGC	4889	GCUUUCAC CUGAUGAGGCCGUUAGGCCGAA IAGGCCUU	5393
580	GUGAAAGC C CAGGUGAC	4890	GUCACCUG CUGAUGAGGCCGUUAGGCCGAA ICUUUCAC	5394
581	UGAAAGCC C AGGUGACG	4891	CGUCACCU CUGAUGAGGCCGUUAGGCCGAA IGCUUUCA	5395
582	GAAAGCCC A GGUGACGU	4892	ACGUCACC CUGAUGAGGCCGUUAGGCCGAA IGGCUUUC	5396
592	GUGACGUC C UUGCUCGG	4893	CCGAGCAA CUGAUGAGGCCGUUAGGCCGAA IACGUCAC	5397
593	UGACGUCC U UGCUCGGG	4894	CCCGAGCA CUGAUGAGGCCGUUAGGCCGAA IGACGUCA	5398
597	GUCCUUGC U CGGGGAGC	4895	GCUCCCCG CUGAUGAGGCCGUUAGGCCGAA ICAAGGAC	5399
606	CGGGGAGC U GCAGGAGA	4896	UCUCCUGC CUGAUGAGGCCGUUAGGCCGAA ICUCCCCG	5400
609	GGAGCUGC A GGAGAGCC	4897	GGCUCUCC CUGAUGAGGCCGUUAGGCCGAA ICAGCUCC	5401
617	AGGAGAGC C AGAGUCGC	4898	GCGACUCU CUGAUGAGGCCGUUAGGCCGAA ICUCUCCU	5402
618	GGAGAGCC A GAGUCGCU	4899	AGCGACUC CUGAUGAGGCCGUUAGGCCGAA IGCUCUCC	5403
626	AGAGUCGC U UGGAGGCU	4900	AGCCUCCA CUGAUGAGGCCGUUAGGCCGAA ICGACUCU	5404
634	UUGGAGGC U GCCACUAA	4901	UUAGUGGC CUGAUGAGGCCGUUAGGCCGAA ICCUCCAA	5405
637	GAGGCUGC C ACUAAGGA	4902	UCCUUAGU CUGAUGAGGCCGUUAGGCCGAA ICAGCCUC	5406
638	AGGCUGCC A CUAAGGAA	4903	UUCCUUAG CUGAUGAGGCCGUUAGGCCGAA IGCAGCCU	5407
640	GCUGCCAC U AAGGAAUG	4904	CAUUCCUU CUGAUGAGGCCGUUAGGCCGAA IUGGCAGC	5408
650	AGGAAUGC C AGGCUCUG	4905	CAGAGCCU CUGAUGAGGCCGUUAGGCCGAA ICAUUCCU	5409
651	GGAAUCCC A GGCUCUGG	4906	CCAGAGCC CUGAUGAGGCCGUUAGGCCGAA IGCAUUCC	5410
655	UGCCAGGC U CUGGAGGG	4907	CCCUCCAG CUGAUGAGGCCGUUAGGCCGAA ICCUGGCA	5411
657	CCAGGCUC U GGAGGGUC	4908	GACCCUCC CUGAUGAGGCCGUUAGGCCGAA IAGCCUGG	5412
670	GGUCGGGC C CGGGCGGC	4909	GCCGCCCG CUGAUGAGGCCGUUAGGCCGAA ICCCGACC	5413
671	GUCGGGCC C GGGCGGCC	4910	GGCCGCCC CUGAUGAGGCCGUUAGGCCGAA IGCCCGAC	5414
679	CGGGCGGC C AGCGAGCA	4911	UGCUCGCU CUGAUGAGGCCGUUAGGCCGAA ICCGCCCG	5415
680	GGGCGGCC A GCGAGCAG	4912	CUGCUCGC CUGAUGAGGCCGUUAGGCCGAA IGCCGCCC	5416
687	CAGCGAGC A GGCGCGGC	4913	GCCGCGCC CUGAUGAGGCCGUUAGGCCGAA ICUCGCUG	5417
696	GGCGCGGC A GCUGGAGA	4914	UCUCCAGC CUGAUGAGGCCGUUAGGCCGAA ICCGCGCC	5418
699	GCGGCAGC U GGAGAGUG	4915	CACUCUCC CUGAUGAGGCCGUUAGGCCGAA ICUGCCGC	5419
720	CGAGGCGC U GCAGCAGC	4916	GCUGCUGC CUGAUGAGGCCGUUAGGCCGAA ICGCCUCG	5420
723	GGCGCUGC A GCAGCAGC	4917	GCUGCUGC CUGAUGAGGCCGUUAGGCCGAA ICAGCGCC	5421
726	GCUGCAGC A GCAGCACA	4918	UGUGCUGC CUGAUGAGGCCGUUAGGCCGAA ICUGCAGC	5422
729	GCAGCAGC A GCACAGCG	4919	CGCUGUGC CUGAUGAGGCCGUUAGGCCGAA ICUGCUGC	5423
732	GCAGCAGC A CAGCGUGC	4920	GCACGCUG CUGAUGAGGCCGUUAGGCCGAA ICUGCUGC	5424
734	AGCAGCAC A GCGUGCAG	4921	CUGCACGC CUGAUGAGGCCGUUAGGCCGAA IUGCUGCU	5425
741	CAGCGUGC A GGUGGACC	4922	GGUCCACC CUGAUGAGGCCGUUAGGCCGAA ICACGCUG	5426
749	AGGUGGAC C AGCUGCGC	4923	GCGCAGCU CUGAUGAGGCCGUUAGGCCGAA IUCCACCU	5427
750	GGUGGACC A GCUGCGCA	4924	UGCGCAGC CUGAUGAGGCCGUUAGGCCGAA IGUCCACC	5428
753	GGACCAGC U GCGCAUGC	4925	GCAUGCGC CUGAUGAGGCCGUUAGGCCGAA ICUGGUCC	5429
758	AGCUGCGC A UGCAGGGC	4926	GCCCUGCA CUGAUGAGGCCGUUAGGCCGAA ICGCAGCU	5430
762	GCGCAUGC A GGGCCAGA	4927	UCUGGCCC CUGAUGAGGCCGUUAGGCCGAA ICAUGCGC	5431
767	UGCAGGGC C AGAGCGUG	4928	CACGCUCU CUGAUGAGGCCGUUAGGCCGAA ICCCUGCA	5432
768	GCAGGGCC A GAGCGUGG	4929	CCACGCUC CUGAUGAGGCCGUUAGGCCGAA IGCCCUGC	5433
781	GUGGAGGC C GCGCUCCG	4930	CGGAGCGC CUGAUGAGGCCGUUAGGCCGAA ICCUCCAC	5434
786	GGCCGCGC U CCGCAUGG	4931	CCAUGCGG CUGAUGAGGCCGUUAGGCCGAA ICGCGGCC	5435
788	CCGCGCUC C GCAUGGAG	4932	CUCCAUGC CUGAUGAGGCCGUUAGGCCGAA IAGCGCGG	5436
791	CGCUCCGC A UGGAGCGC	4933	GCGCUCCA CUGAUGAGGCCGUUAGGCCGAA ICGGAGCG	5437
800	UGGAGCGC C AGGCCGCC	4934	GGCGGCCU CUGAUGAGGCCGUUAGGCCGAA ICGCUCCA	5438
801	GGAGCGCC A GGCCGCCU	4935	AGGCGGCC CUGAUGAGGCCGUUAGGCCGAA IGCGCUCC	5439
805	CGCCAGGC C GCCUCGGA	4936	UCCGAGGC CUGAUGAGGCCGUUAGGCCGAA ICCUGGCG	5440
808	CAGGCCGC C UCGGAGGA	4937	UCCUCCGA CUGAUGAGGCCGUUAGGCCGAA ICGGCCUG	5441
809	AGGCCGCC U CGGAGGAG	4938	CUCCUCCG CUGAUGAGGCCGUUAGGCCGAA IGCGGCCU	5442
828	GAGGAAGC U GGCCCAGU	4939	ACUGGGCC CUGAUGAGGCCGUUAGGCCGAA ICUUCCUC	5443
832	AAGCUGGC C CAGUUGCA	4940	UGCAACUG CUGAUGAGGCCGUUAGGCCGAA ICCAGCUU	5444
833	AGCUGGCC C AGUUGCAG	4941	CUGCAACU CUGAUGAGGCCGUUAGGCCGAA IGCCAGCU	5445
834	GCUGGCCC A GUUGCAGG	4942	CCUGCAAC CUGAUGAGGCCGUUAGGCCGAA IGGCCAGC	5446
840	CCAGUUGC A GGUGGCCU	4943	AGGCCACC CUGAUGAGGCCGUUAGGCCGAA ICAACUGG	5447
847	CAGGUGGC C UAUCACCA	4944	UGGUGAUA CUGAUGAGGCCGUUAGGCCGAA ICCACCUG	5448
848	AGGUGGCC U AUCACCAG	4945	CUGGUGAU CUGAUGAGGCCGUUAGGCCGAA IGCCACCU	5449
852	GGCCUAUC A CCAGCUCU	4946	AGAGCUGG CUGAUGAGGCCGUUAGGCCGAA IAUAGGCC	5450
854	CCUAUCAC C AGCUCUUC	4947	GAAGAGCU CUGAUGAGGCCGUUAGGCCGAA IUGAUAGG	5451
855	CUAUCACC A GCUCUUCC	4948	GGAAGAGC CUGAUGAGGCCGUUAGGCCGAA IGUGAUAG	5452
858	UCACCAGC U CUUCCAAG	4949	CUUGGAAG CUGAUGAGGCCGUUAGGCCGAA ICUGGUGA	5453
860	ACCAGCUC U UCCAAGAA	4950	UUCUUGGA CUGAUGAGGCCGUUAGGCCGAA IAGCUGGU	5454
863	AGCUCUUC C AAGAAUAC	4951	GUAUUCUU CUGAUGAGGCCGUUAGGCCGAA IAAGAGCU	5455
864	GCUCUUCC A AGAAUACG	4952	CGUAUUCU CUGAUGAGGCCGUUAGGCCGAA IGAAGAGC	5456
875	AAUACGAC A ACCACAUC	4953	GAUGUGGU CUGAUGAGGCCGUUAGGCCGAA IUCGUAUU	5457
878	ACGACAAC C ACAUCAAG	4954	CUUGAUGU CUGAUGAGGCCGUUAGGCCGAA IUUGUCGU	5458
879	CGACAACC A CAUCAAGA	4955	UCUUGAUG CUGAUGAGGCCGUUAGGCCGAA IGUUGUCG	5459
881	ACAACCAC A UCAAGAGC	4956	GCUCUUGA CUGAUGAGGCCGUUAGGCCGAA IUGGUUGU	5460
884	ACCACAUC A AGAGCAGC	4957	GCUGCUCU CUGAUGAGGCCGUUAGGCCGAA IAUGUGGU	5461
890	UCAAGAGC A GCGUGGUG	4958	CACCACGC CUGAUGAGGCCGUUAGGCCGAA ICUCUUGA	5462
902	UGGUGGGC A GUGAGCGG	4959	CCGCUCAC CUGAUGAGGCCGUUAGGCCGAA ICCCACCA	5463
924	AGGAAUGC A GCUGGAAG	4960	CUUCCAGC CUGAUGAGGCCGUUAGGCCGAA ICAUUCCU	5464
927	AAUGCAGC U GGAAGAUC	4961	GAUCUUCC CUGAUGAGGCCGUUAGGCCGAA ICUGCAUU	5465
936	GGAAGAUC U CAAACAGC	4962	GCUGUUUG CUGAUGAGGCCGUUAGGCCGAA IAUCUUCC	5466
938	AAGAUCUC A AACAGCAG	4963	CUGCUGUU CUGAUGAGGCCGUUAGGCCGAA IAGAUCUU	5467
942	UCUCAAAC A GCAGCUCC	4964	GGAGCUGC CUGAUGAGGCCGUUAGGCCGAA IUUUGAGA	5468
945	CAAACAGC A GCUCCAGC	4965	GCUGGAGC CUGAUGAGGCCGUUAGGCCGAA ICUGUUUG	5469
948	ACAGCAGC U CCAGCAGG	4966	CCUGCUGG CUGAUGAGGCCGUUAGGCCGAA ICUGCUGU	5470
950	AGCAGCUC C AGCAGGCC	4967	GGCCUGCU CUGAUGAGGCCGUUAGGCCGAA IAGCUGCU	5471
951	GCAGCUCC A GCAGGCCG	4968	CGGCCUGC CUGAUGAGGCCGUUAGGCCGAA IGAGCUGC	5472
954	GCUCCAGC A GGCCGAGG	4969	CCUCGGCC CUGAUGAGGCCGUUAGGCCGAA ICUGGAGC	5473
958	CAGCAGGC C GAGGAGGC	4970	GCCUCCUC CUGAUGAGGCCGUUAGGCCGAA ICCUGCUG	5474
967	GAGGAGGC C CUGGUGGC	4971	GCCACCAG CUGAUGAGGCCGUUAGGCCGAA ICCUCCUC	5475
968	AGGAGGCC C UGGUGGCC	4972	GGCCACCA CUGAUGAGGCCGUUAGGCCGAA IGCCUCCU	5476
969	GGAGGCCC U GGUGGCCA	4973	UGGCCACC CUGAUGAGGCCGUUAGGCCGAA IGGCCUCC	5477
976	CUGGUGGC C AAACAGGA	4974	UCCUGUUU CUGAUGAGGCCGUUAGGCCGAA ICCACCAG	5478
977	UGGUGGCC A AACAGGAG	4975	CUCCUGUU CUGAUGAGGCCGUUAGGCCGAA IGCCACCA	5479
981	GGCCAAAC A GGAGGUGA	4976	UCACCUCC CUGAUGAGGCCGUUAGGCCGAA IUUUGGCC	5480
999	CGAUAAGC U GAAGGAGG	4977	CCUCCUUC CUGAUGAGGCCGUUAGGCCGAA ICUUAUCG	5481
1012	GAGGAGGC C GAGCAGCA	4978	UGCUGCUC CUGAUGAGGCCGUUAGGCCGAA ICCUCCUC	5482
1017	GGCCGAGC A GCACAAGA	4979	UCUUGUGC CUGAUGAGGCCGUUAGGCCGAA ICUCGGCC	5483
1020	CGAGCAGC A CAAGAUGG	4980	CAAUCUUG CUGAUGAGGCCGUUAGGCCGAA ICUGCUCG	5484
1022	AGCAGCAC A AGAUUGUG	4981	CACAAUCU CUGAUGAGGCCGUUAGGCCGAA IUGCUGCU	5485
1039	AUGGAGAC C GUUCCGGU	4982	ACCGGAAC CUGAUGAGGCCGUUAGGCCGAA IUCUCCAU	5486
1044	GACCGUUC C GGUGCUGA	4983	UCAGCACC CUGAUGAGGCCGUUAGGCCGAA IAACGGUC	5487
1050	UCCGGUGC U GAAGGCCC	4984	GGGCCUUC CUGAUGAGGCCGUUAGGCCGAA ICACCGGA	5488
1057	CUGAAGGC C CAGGCGGA	4985	UCCGCCUG CUGAUGAGGCCGUUAGGCCGAA ICCUUCAG	5489
1058	UGAAGGCC C AGGCGGAU	4986	AUCCGCCU CUGAUGAGGCCGUUAGGCCGAA IGCCUUCA	5490
1059	GAAGGCCC A GGCGGAUA	4987	UAUCCGCC CUGAUGAGGCCGUUAGGCCGAA IGGCCUUC	5491
1070	CGGAUAUC U ACAAGGCG	4988	CGCCUUGU CUGAUGAGGCCGUUAGGCCGAA IAUAUCCG	5492
1073	AUAUCUAC A AGGCGGAC	4989	GUCCGCCU CUGAUGAGGCCGUUAGGCCGAA IUAGAUAU	5493
1082	AGGCGGAC U UCCAGGCU	4990	AGCCUGGA CUGAUGAGGCCGUUAGGCCGAA IUCCGCCU	5494
1085	CGGACUUC C AGGCUGAG	4991	CUCAGCCU CUGAUGAGGCCGUUAGGCCGAA IAAGUCCG	5495
1086	GGACUUCC A GGCUGAGA	4992	UCUCAGCC CUGAUGAGGCCGUUAGGCCGAA IGAAGUCC	5496
1090	UUCCAGGC U GAGAGGCA	4993	UGCCUCUC CUGAUGAGGCCGUUAGGCCGAA ICCUGGAA	5497
1098	UGAGAGGC A GGCCCGGG	4994	CCCGGGCC CUGAUGAGGCCGUUAGGCCGAA ICCUCUCA	5498
1102	AGGCAGGC C CGGGAGAA	4995	UUCUCCCG CUGAUGAGGCCGUUAGGCCGAA ICCUGCCU	5499
1103	GGCAGGCC C GGGAGAAG	4996	CUUCUCCC CUGAUGAGGCCGUUAGGCCGAA IGCCUGCC	5500
1113	GGAGAAGC U GGCCGAGA	4997	UCUCGGCC CUGAUGAGGCCGUUAGGCCGAA ICUUCUCC	5501
1117	AAGCUGGC C GAGAAGAA	4998	UUCUUCUC CUGAUGAGGCCGUUAGGCCGAA ICCAGCUU	5502
1131	GAAGGAGC U CCUGCAGG	4999	CCUGCAGG CUGAUGAGGCCGUUAGGCCGAA ICUCCUUC	5503
1133	AGGAGCUC C UGCAGGAG	5000	CUCCUGCA CUGAUGAGGCCGUUAGGCCGAA IAGCUCCU	5504
1134	GGAGCUCC U GCAGGAGC	5001	GCUCCUGC CUGAUGAGGCCGUUAGGCCGAA IGAGCUCC	5505
1137	GCUCCUGC A GGAGCAGC	5002	GCUGCUCC CUGAUGAGGCCGUUAGGCCGAA ICAGGAGC	5506
1143	GCAGGAGC A GCUGGAGC	5003	GCUCCAGC CUGAUGAGGCCGUUAGGCCGAA ICUCCUGC	5507
1146	GGAGCAGC U GGAGCAGC	5004	GCUGCUCC CUGAUGAGGCCGUUAGGCCGAA ICUGCUCC	5508
1152	GCUGGAGC A GCUGCAGA	5005	UCUGCAGC CUGAUGAGGCCGUUAGGCCGAA ICUCCAGC	5509
1155	GGAGCAGC U GCAGAGGG	5006	CCCUCUGC CUGAUGAGGCCGUUAGGCCGAA ICUGCUCC	5510
1158	GCAGCUGC A GAGGGAGU	5007	ACUCCCUC CUGAUGAGGCCGUUAGGCCGAA ICAGCUGC	5511
1169	GGGAGUAC A GCAAACUG	5008	CAGUUUGC CUGAUGAGGCCGUUAGGCCGAA IUACUCCC	5512
1172	AGUACAGC A AACUGAAG	5009	CUUCAGUU CUGAUGAGGCCGUUAGGCCGAA ICUGUACU	5513
1176	CAGCAAAC U GAAGGCCA	5010	UGGCCUUC CUGAUGAGGCCGUUAGGCCGAA IUUUGCUG	5514
1183	CUGAAGGC C AGCUGUCA	5011	UGACAGCU CUGAUGAGGCCGUUAGGCCGAA ICCUUCAG	5515
1184	UGAAGGCC A GCUGUCAG	5012	CUGACAGC CUGAUGAGGCCGUUAGGCCGAA IGCCUUCA	5516
1187	AGGCCAGC U GUCAGGAG	5013	CUCCUGAC CUGAUGAGGCCGUUAGGCCGAA ICUGGCCU	5517
1191	CAGCUGUC A GGAGUCGG	5014	CCGACUCC CUGAUGAGGCCGUUAGGCCGAA IACAGCUG	5518
1201	GAGUCGGC C AGGAUCGA	5015	UCGAUCCU CUGAUGAGGCCGUUAGGCCGAA ICCGACUC	5519
1202	AGUCGGCC A GGAUCGAG	5016	CUCGAUCC CUGAUGAGGCCGUUAGGCCGAA IGCCGACU	5520
1214	UCGAGGAC A UGAGGAAG	5017	CUUCCUCA CUGAUGAGGCCGUUAGGCCGAA IUCCUCGA	5521
1227	GAAGCGGC A UGUCGAGG	5018	CCUCGACA CUGAUGAGGCCGUUAGGCCGAA ICCGCUUC	5522
1238	UCGAGGUC U CCCAGGCC	5019	GGCCUGGG CUGAUGAGGCCGUUAGGCCGAA IACCUCGA	5523
1240	GAGGUCUC C CAGGCCCC	5020	GGGGCCUG CUGAUGAGGCCGUUAGGCCGAA IAGACCUC	5524
1241	AGGUCUCC C AGGCCCCC	5021	GGGGGCCU CUGAUGAGGCCGUUAGGCCGAA IGAGACCU	5525
1242	GGUCUCCC A GGCCCCCU	5022	AGGGGGCC CUGAUGAGGCCGUUAGGCCGAA IGGAGACC	5526
1246	UCCCAGGC C CCCUUGCC	5023	GGCAAGGG CUGAUGAGGCCGUUAGGCCGAA ICCUGGGA	5527
1247	CCCAGGCC C CCUUGCCC	5024	GGGCAAGG CUGAUGAGGCCGUUAGGCCGAA IGCCUGGG	5528
1248	CCAGGCCC C CUUGCCCC	5025	GGGGCAAG CUGAUGAGGCCGUUAGGCCGAA IGGCCUGG	5529
1249	CAGGCCCC C UUGCCCCC	5026	GGGGGCAA CUGAUGAGGCCGUUAGGCCGAA IGGGCCUG	5530
1250	AGGCCCCC U UGCCCCCC	5027	GGGGGGCA CUGAUGAGGCCGUUAGGCCGAA IGGGGCCU	5531
1254	CCCCUUGC C CCCCGCCC	5028	GGGCGGGG CUGAUGAGGCCGUUAGGCCGAA ICAAGGGG	5532
1255	CCCUUGCC C CCCGCCCC	5029	GGGGCGGG CUGAUGAGGCCGUUAGGCCGAA IGCAAGGG	5533
1256	CCUUGCCC C CCGCCCCU	5030	AGGGGCGG CUGAUGAGGCCGUUAGGCCGAA IGGCAAGG	5534
1257	CUUGCCCC C CGCCCCUG	5031	CAGGGGCG CUGAUGAGGCCGUUAGGCCGAA IGGGCAAG	5535
1258	UUGCCCCC C GCCCCUGC	5032	GCAGGGGC CUGAUGAGGCCGUUAGGCCGAA IGGGGCAA	5536
1261	CCCCCCGC C CCUGCCUA	5033	UAGGCAGG CUGAUGAGGCCGUUAGGCCGAA ICGGGGGG	5537
1262	CCCCCGCC C CUGCCUAC	5034	GUAGGCAG CUGAUGAGGCCGUUAGGCCGAA IGCGGGGG	5538
1263	CCCCGCCC C UGCCUACC	5035	GGUAGGCA CUGAUGAGGCCGUUAGGCCGAA IGGCGGGG	5539
1264	CCCGCCCC U GCCUACCU	5036	AGGUAGGC CUGAUGAGGCCGUUAGGCCGAA IGGGCGGG	5540
2267	GCCCCUGC C UACCUCUC	5037	GAGAGGUA CUGAUGAGGCCGUUAGGCCGAA ICAGGGGC	5541
1268	CCCCUGCC U ACCUCUCC	5038	GGAGAGGU CUGAUGAGGCCGUUAGGCCGAA IGCAGGGG	5542
1271	CUGCCUAC C UCUCCUCU	5039	AGAGGAGA CUGAUGAGGCCGUUAGGCCGAA IUAGGCAG	5543
1272	UGCCUACC U CUCCUCUC	5040	GAGAGGAG CUGAUGAGGCCGUUAGGCCGAA IGUAGGCA	5544
1274	CCUACCUC U CCUCUCCC	5041	GGGAGAGG CUGAUGAGGCCGUUAGGCCGAA IAGGUAGG	5545
1276	UACCUCUC C UCUCCCCU	5042	AGGGGAGA CUGAUGAGGCCGUUAGGCCGAA IAGAGGUA	5546
1277	ACCUCUCC U CUCCCCUG	5043	CAGGGGAG CUGAUGAGGCCGUUAGGCCGAA IGAGAGGU	5547
1279	CUCUCCUC U CCCCUGGC	5044	GCCAGGGG CUGAUGAGGCCGUUAGGCCGAA IAGGAGAG	5548
1281	CUCCUCUC C CCUGGCCC	5045	GGGCCAGG CUGAUGAGGCCGUUAGGCCGAA IAGAGGAG	5549
1282	UCCUCUCC C CUGGCCCU	5046	AGGGCCAG CUGAUGAGGCCGUUAGGCCGAA IGAGAGGA	5550
1283	CCUCUCCC C UGGCCCUG	5047	CAGGGCCA CUGAUGAGGCCGUUAGGCCGAA IGGAGAGG	5551
1284	CUCUCCCC U GGCCCUGC	5048	GCAGGGCC CUGAUGAGGCCGUUAGGCCGAA IGGGAGAG	5552
1288	CCCCUGGC C CUGCCCAG	5049	CUGGGCAG CUGAUGAGGCCGUUAGGCCGAA ICCAGGGG	5553
1289	CCCUGGCC C UGCCCAGC	5050	GCUGGGCA CUGAUGAGGCCGUUAGGCCGAA IGCCAGGG	5554
1290	CCUGGCCC U GCCCAGCC	5051	GGCUGGGC CUGAUGAGGCCGUUAGGCCGAA IGGCCAGG	5555
1293	GGCCCUGC C CAGCCAGA	5052	UCUGGCUG CUGAUGAGGCCGUUAGGCCGAA ICAGGGCC	5556
1294	GCCCUGCC C AGCCAGAG	5053	CUCUGGCU CUGAUGAGGCCGUUAGGCCGAA IGCAGGGC	5557
1295	CCCUGCCC A GCCAGAGG	5054	CCUCUGGC CUGAUGAGGCCGUUAGGCCGAA IGGCAGGG	5558
1298	UGCCCAGC C AGAGGAGG	5055	CCUCCUCU CUGAUGAGGCCGUUAGGCCGAA ICUGGGCA	5559
1299	GCCCAGCC A GAGGAGGA	5056	UCCUCCUC CUGAUGAGGCCGUUAGGCCGAA IGCUGGGC	5560
1310	GGAGGAGC C CCCCCGAG	5057	CUCGGGGG CUGAUGAGGCCGUUAGGCCGAA ICUCCUCC	5561
1311	GAGGAGCC C CCCCGAGG	5058	CCUCGGGG CUGAUGAGGCCGUUAGGCCGAA IGCUCCUC	5562
1312	AGGAGCCC C CCCGAGGA	5059	UCCUCGGG CUGAUGAGGCCGUUAGGCCGAA IGGCUCCU	5563
1313	GGAGCCCC C CCGAGGAG	5060	CUCCUCGG CUGAUGAGGCCGUUAGGCCGAA IGGGCUCC	5564
1314	GAGCCCCC C CGAGGAGC	5061	GCUCCUCG CUGAUGAGGCCGUUAGGCCGAA IGGGGCUC	5565
1315	AGCCCCCC C GAGGAGCC	5062	GGCUCCUC CUGAUGAGGCCGUUAGGCCGAA IGGGGGCU	5566
1323	CGAGGAGC C ACCUGACU	5063	AGUCAGGU CUGAUGAGGCCGUUAGGCCGAA ICUCCUCG	5567
1324	GAGGAGCC A CCUGACUU	5064	AAGUCAGG CUGAUGAGGCCGUUAGGCCGAA IGCUCCUC	5568
1326	GGAGCCAC C UGACUUCU	5065	AGAAGUCA CUGAUGAGGCCGUUAGGCCGAA IUGGCUCC	5569
1327	GAGCCACC U GACUUCUG	5066	CAGAAGUC CUGAUGAGGCCGUUAGGCCGAA IGUGGCUC	5570
1331	CACCUGAC U UCUGCUGU	5067	ACAGCAGA CUGAUGAGGCCGUUAGGCCGAA IUCAGGUG	5571
1334	CUGACUUC U GCUGUCCC	5068	GGGACAGC CUGAUGAGGCCGUUAGGCCGAA IAAGUCAG	5572
1337	ACUUCUGC U GUCCCAAG	5069	CUUGGGAC CUGAUGAGGCCGUUAGGCCGAA ICAGAAGU	5573
1341	CUGCUGUC C CAAGUGCC	5070	GGCACUUG CUGAUGAGGCCGUUAGGCCGAA IACAGCAG	5574
1342	UGCUGUCC C AAGUGCCA	5071	UGGCACUU CUGAUGAGGCCGUUAGGCCGAA IGACAGCA	5575
1343	GCUGUCCC A AGUGCCAG	5072	CUGGCACU CUGAUGAGGCCGUUAGGCCGAA IGGACAGC	5576
1349	CCAAGUGC C AGUAUCAG	5073	CUGAUACU CUGAUGAGGCCGUUAGGCCGAA ICACUUGG	5577
1350	CAAGUGCC A GUAUCAGG	5074	CCUGAUAC CUGAUGAGGCCGUUAGGCCGAA IGCACUUG	5578
1356	CCAGUAUC A GGCCCCUG	5075	CAGGGGCC CUGAUGAGGCCGUUAGGCCGAA IAUACUGG	5579
1360	UAUCAGGC C CCUGAUAU	5076	AUAUCAGG CUGAUGAGGCCGUUAGGCCGAA ICCUGAUA	5580
1361	AUCAGGCC C CUGAUAUG	5077	CAUAUCAG CUGAUGAGGCCGUUAGGCCGAA IGCCUGAU	5581
1362	UCAGGCCC C UGAUAUGG	5078	CCAUAUCA CUGAUGAGGCCGUUAGGCCGAA IGGCCUGA	5582
1363	CAGGCCCC U GAUAUGGA	5079	UCCAUAUC CUGAUGAGGCCGUUAGGCCGAA IGGGCCUG	5583
1373	AUAUGGAC A CCCUGCAG	5080	CUGCAGGG CUGAUGAGGCCGUUAGGCCGAA IUCCAUAU	5584
1375	AUGGACAC C CUGCAGAU	5081	AUCUGCAG CUGAUGAGGCCGUUAGGCCGAA IUGUCCAU	5585
1376	UGGACACC C UGCAGAUA	5082	UAUCUGCA CUGAUGAGGCCGUUAGGCCGAA IGUGUCCA	5586
1377	GGACACCC U GCAGAUAC	5083	GUAUCUGC CUGAUGAGGCCGUUAGGCCGAA IGGUGUCC	5587
1380	CACCCUGC A GAUACAUG	5084	CAUGUAUC CUGAUGAGGCCGUUAGGCCGAA ICAGGGUG	5588
1386	GCAGAUAC A UGUCAUGG	5085	CCAUGACA CUGAUGAGGCCGUUAGGCCGAA IUAUCUGC	5589
1391	UACAUGUC A UGGAGUGC	5086	GCACUCCA CUGAUGAGGCCGUUAGGCCGAA IACAUGUA	5590
1400	UGGAGUGC A UUGAGUAG	5087	CUACUCAA CUGAUGAGGCCGUUAGGCCGAA ICACUCCA	5591
1412	AGUAGGGC C GGCCAGUG	5088	CACUGGCC CUGAUGAGGCCGUUAGGCCGAA ICCCUACU	5592
1416	GGGCCGGC C AGUGCAAG	5089	CUUGCACU CUGAUGAGGCCGUUAGGCCGAA ICCGGCCC	5593
1417	GGCCGGCC A GUGCAAGG	5090	CCUUGCAC CUGAUGAGGCCGUUAGGCCGAA IGCCGGCC	5594
1422	GCCAGUGC A AGGCCACU	5091	AGUGGCCU CUGAUGAGGCCGUUAGGCCGAA ICACUGGC	5595
1427	UGCAAGGC C ACUGCCUG	5092	CAGGCAGU CUGAUGAGGCCGUUAGGCCGAA ICCUUGCA	5596
1428	GCAAGGCC A CUGCCUGC	5093	GCAGGCAG CUGAUGAGGCCGUUAGGCCGAA IGCCUUGC	5597
1430	AAGGCCAC U GCCUGCCC	5094	GGGCAGGC CUGAUGAGGCCGUUAGGCCGAA IUGGCCUU	5598
1433	GCCACUGC C UGCCCGAG	5095	CUCGGGCA CUGAUGAGGCCGUUAGGCCGAA ICAGUGGC	5599
1434	CCACUGCC U GCCCGAGG	5096	CCUCGGGC CUGAUGAGGCCGUUAGGCCGAA IGCAGUGG	5600
1437	CUGCCUGC C CGAGGACG	5097	CGUCCUCG CUGAUGAGGCCGUUAGGCCGAA ICAGGCAG	5601
1438	UGCCUGCC C GAGGACGU	5098	ACGUCCUC CUGAUGAGGCCGUUAGGCCGAA IGCAGGCA	5602
1449	GGACGUGC C CGGGACCG	5099	CGGUCCCG CUGAUGAGGCCGUUAGGCCGAA ICACGUCC	5603
1450	GACGUGCC C GGGACCGU	5100	ACGGUCCC CUGAUGAGGCCGUUAGGCCGAA IGCACGUC	5604
1456	CCCGGGAC C GUGCAGUC	5101	GACUGCAC CUGAUGAGGCCGUUAGGCCGAA IUCCCGGG	5605
1461	GACCGUGC A GUCUGCGC	5102	GCGCAGAC CUGAUGAGGCCGUUAGGCCGAA ICACGGUC	5606
1465	GUGCAGUC U GCGCUUUC	5103	GAAAGCGC CUGAUGAGGCCGUUAGGCCGAA IACUGCAC	5607
1470	GUCUGCGC U UUCCUCUC	5104	GAGAGGAA CUGAUGAGGCCGUUAGGCCGAA ICGCAGAC	5608
1474	GCGCUUUC C UCUCCCGC	5105	GCGGGAGA CUGAUGAGGCCGUUAGGCCGAA IAAAGCGC	5609
1475	CGCUUUCC U CUCCCGCC	5106	GGCGGGAG CUGAUGAGGCCGUUAGGCCGAA IGAAAGCG	5610
1477	CUUUCCUC U CCCGCCUG	5107	CAGGCGGG CUGAUGAGGCCGUUAGGCCGAA IAGGAAAG	5611
1479	UUCCUCUC C CGCCUGCC	5108	GGCAGGCG CUGAUGAGGCCGUUAGGCCGAA IAGAGGAA	5612
1480	UCCUCUCC C GCCUGCCU	5109	AGGCAGGC CUGAUGAGGCCGUUAGGCCGAA IGAGAGGA	5613
1483	UCUCCCGC C UGCCUAGC	5110	GCUAGGCA CUGAUGAGGCCGUUAGGCCGAA ICGGGAGA	5614
1484	CUCCCGCC U GCCUAGCC	5111	GGCUAGGC CUGAUGAGGCCGUUAGGCCGAA IGCGGGAG	5615
1487	CCGCCUGC C UAGCCCAG	5112	CUGGGCUA CUGAUGAGGCCGUUAGGCCGAA ICAGGCGG	5616
1488	CGCCUGCC U AGCCCAGG	5113	CCUGGGCU CUGAUGAGGCCGUUAGGCCGAA IGCAGGCG	5617
1492	UGCCUAGC C CAGGAUGA	5114	UCAUCCUG CUGAUGAGGCCGUUAGGCCGAA ICUAGGCA	5618
1493	GCCUAGCC C AGGAUGAA	5115	UUCAUCCU CUGAUGAGGCCGUUAGGCCGAA IGCUAGGC	5619
1494	CCUAGCCC A GGAUGAAG	5116	CUUCAUCC CUGAUGAGGCCGUUAGGCCGAA IGGCUAGG	5620
1506	UGAAGGGC U GGGUGGCC	5117	GGCCACCC CUGAUGAGGCCGUUAGGCCGAA ICCCUUCA	5621
1514	UGGGUGGC C ACAACUGG	5118	CCAGUUGU CUGAUGAGGCCGUUAGGCCGAA ICCACCCA	5622
1515	GGGUGGCC A CAACUGGG	5119	CCCAGUUG CUGAUGAGGCCGUUAGGCCGAA IGCCACCC	5623
1517	GUGGCCAC A ACUGGGAU	5120	AUCCCAGU CUGAUGAGGCCGUUAGGCCGAA IUGGCCAC	5624
1520	GCCACAAC U GGGAUGCC	5121	GGCAUCCC CUGAUGAGGCCGUUAGGCCGAA IUUGUGGC	5625
1528	UGGGAUGC C ACCUGGAG	5122	CUCCAGGU CUGAUGAGGCCGUUAGGCCGAA ICAUCCCA	5626
1529	GGGAUGCC A CCUGGAGC	5123	GCUCCAGG CUGAUGAGGCCGUUAGGCCGAA IGCAUCCC	5627
1531	GAUGCCAC C UGGAGCCC	5124	GGGCUCCA CUGAUGAGGCCGUUAGGCCGAA IUGGCAUC	5628
1532	AUGCCACC U GGAGCCCC	5125	GGGGCUCC CUGAUGAGGCCGUUAGGCCGAA IGUGGCAU	5629
1538	CCUGGAGC C CCACCCAG	5126	CUGGGUGG CUGAUGAGGCCGUUAGGCCGAA ICUCCAGG	5630
1539	CUGGAGCC C CACCCAGG	5127	CCUGGGUG CUGAUGAGGCCGUUAGGCCGAA IGCUCCAG	5631
1540	UGGAGCCC C ACCCAGGA	5128	UCCUGGGU CUGAUGAGGCCGUUAGGCCGAA IGGCUCCA	5632
1541	GGAGCCCC A CCCAGGAG	5129	CUCCUGGG CUGAUGAGGCCGUUAGGCCGAA IGGGCUCC	5633
1543	AGCCCCAC C CAGGAGCU	5130	AGCUCCUG CUGAUGAGGCCGUUAGGCCGAA IUGGGGCU	5634
1544	GCCCCACC C AGGAGCUG	5131	CAGCUCCU CUGAUGAGGCCGUUAGGCCGAA IGUGGGGC	5635
1545	CCCCACCC A GGAGCUGG	5132	CCAGCUCC CUGAUGAGGCCGUUAGGCCGAA IGGUGGGG	5636
1551	CCAGGAGC U GGCCGCGG	5133	CCGCGGCC CUGAUGAGGCCGUUAGGCCGAA ICUCCUGG	5637
1555	GAGCUGGC C GCGGCACC	5134	GGUGCCGC CUGAUGAGGCCGUUAGGCCGAA ICCAGCUC	5638
1561	GCCGCGGC A CCUUACGC	5135	GCGUAAGG CUGAUGAGGCCGUUAGGCCGAA ICCGCGGC	5639
1563	CGCGGCAC C UUACGCUU	5136	AAGCGUAA CUGAUGAGGCCGUUAGGCCGAA IUGCCGCG	5640
1564	GCGGCACC U UACGCUUC	5137	GAAGCGUA CUGAUGAGGCCGUUAGGCCGAA IGUGCCGC	5641
1570	CCUUACGC U UCAGCUGU	5138	ACAGCUGA CUGAUGAGGCCGUUAGGCCGAA ICGUAAGG	5642
1573	UACGCUUC A GCUGUUGA	5139	UCAACAGC CUGAUGAGGCCGUUAGGCCGAA IAAGCGUA	5643
1576	GCUUCAGC U GUUGAUCC	5140	GGAUCAAC CUGAUGAGGCCGUUAGGCCGAA ICUGAAGC	5644
1584	UGUUGAUC C GCUGGUCC	5141	GGACCAGC CUGAUGAGGCCGUUAGGCCGAA IAUCAACA	5645
1587	UGAUCCGC U GGUCCCCU	5142	AGGGGACC CUGAUGAGGCCGUUAGGCCGAA ICGGAUCA	5646
1592	CGCUGGUC C CCUCUUUU	5143	AAAAGAGG CUGAUGAGGCCGUUAGGCCGAA IACCAGCG	5647
1593	GCUGGUCC C CUCUUUUG	5144	CAAAAGAG CUGAUGAGGCCGUUAGGCCGAA IGACCAGC	5648
1594	CUGGUCCC C UCUUUUGG	5145	CCAAAAGA CUGAUGAGGCCGUUAGGCCGAA IGGACCAG	5649
1595	UGGUCCCC U CUUUUGGG	5146	CCCAAAAG CUGAUGAGGCCGUUAGGCCGAA IGGGACCA	5650
1597	GUCCCCUC U UUUGGGGU	5147	ACCCCAAA CUGAUGAGGCCGUUAGGCCGAA IAGGGGAC	5651
1615	GAUGCGGC C CCGAUCAG	5148	CUGAUCGG CUGAUGAGGCCGUUAGGCCGAA ICCGCAUC	5652
1616	AUGCGGCC C CGAUCAGG	5149	CCUGAUCG CUGAUGAGGCCGUUAGGCCGAA IGCCGCAU	5653
1617	UGCGGCCC C GAUCAGGC	5150	GCCUGAUC CUGAUGAGGCCGUUAGGCCGAA IGGCCGCA	5654
1622	CCCCGAUC A GGCCUGAC	5151	GUCAGGCC CUGAUGAGGCCGUUAGGCCGAA IAUCGGGG	5655
1626	GAUCAGGC C UGACUCGC	5152	GCGAGUCA CUGAUGAGGCCGUUAGGCCGAA ICCUGAUC	5656
1627	AUCAGGCC U GACUCGCU	5153	AGCGAGUC CUGAUGAGGCCGUUAGGCCGAA IGCCUGAU	5657
1631	GGCCUGAC U CGCUGCUC	5154	GAGCAGCG CUGAUGAGGCCGUUAGGCCGAA IUCAGGCC	5658
1635	UGACUCGC U GCUCUUUU	5155	AAAAGAGC CUGAUGAGGCCGUUAGGCCGAA ICGAGUCA	5659
1638	CUCGCUGC U CUUUUUGU	5156	ACAAAAAG CUGAUGAGGCCGUUAGGCCGAA ICAGCGAG	5660
1640	CGCUGCUC U UUUUGUUC	5157	GAACAAAA CUGAUGAGGCCGUUAGGCCGAA IAGCAGCG	5661
1649	UUUUGUUC C CUUCUGUC	5158	GACAGAAG CUGAUGAGGCCGUUAGGCCGAA IAACAAAA	5662
1650	UUUGUUCC C UUCUGUCU	5159	AGACAGAA CUGAUGAGGCCGUUAGGCCGAA IGAACAAA	5663
1651	UUGUUCCC U UCUGUCUG	5160	CAGACAGA CUGAUGAGGCCGUUAGGCCGAA IGGAACAA	5664
1654	UUCCCUUC U GUCUGCUC	5161	GAGCAGAC CUGAUGAGGCCGUUAGGCCGAA IAAGGGAA	5665
1658	CUUCUGUC U GCUCGAAC	5162	GUUCGAGC CUGAUGAGGCCGUUAGGCCGAA IACAGAAG	5666
1661	CUGUCUGC U CGAACCAC	5163	GUGGUUCG CUGAUGAGGCCGUUAGGCCGAA ICAGACAG	5667
1667	GCUCGAAC C ACUUGCCU	5164	AGGCAAGU CUGAUGAGGCCGUUAGGCCGAA IUUCGAGC	5668
1668	CUCGAACC A CUUGCCUC	5165	GAGGCAAG CUGAUGAGGCCGUUAGGCCGAA IGUUCGAG	5669
1670	CGAACCAC U UGCCUCGG	5166	CCGAGGCA CUGAUGAGGCCGUUAGGCCGAA IUGGUUCG	5670
1674	CCACUUGC C UCGGGCUA	5167	UAGCCCGA CUGAUGAGGCCGUUAGGCCGAA ICAAGUGG	5671
1675	CACUUGCC U CGGGCUAA	5168	UUAGCCCG CUGAUGAGGCCGUUAGGCCGAA IGCAAGUG	5672
1681	CCUCGGGC U AAUCCCUC	5169	GAGGGAUU CUGAUGAGGCCGUUAGGCCGAA ICCCGAGG	5673
1686	GGCUAAUC C CUCCCUCU	5170	AGAGGGAG CUGAUGAGGCCGUUAGGCCGAA IAUUAGCC	5674
1687	GCUAAUCC C UCCCUCUU	5171	AAGAGGGA CUGAUGAGGCCGUUAGGCCGAA IGAUUAGC	5675
1688	CUAAUCCC U CCCUCUUC	5172	GAAGAGGG CUGAUGAGGCCGUUAGGCCGAA IGGAUUAG	5676
1690	AAUCCCUC C CUCUUCCU	5173	AGGAAGAG CUGAUGAGGCCGUUAGGCCGAA IAGGGAUU	5677
1691	AUCCCUCC C UCUUCCUC	5174	GAGGAAGA CUGAUGAGGCCGUUAGGCCGAA IGAGGGAU	5678
1692	UCCCUCCC U CUUCCUCC	5175	GGAGGAAG CUGAUGAGGCCGUUAGGCCGAA IGGAGGGA	5679
1694	CCUCCCUC U UCCUCCAC	5176	GUGGAGGA CUGAUGAGGCCGUUAGGCCGAA IAGGGAGG	5680
1697	CCCUCUUC C UCCACCCG	5177	CGGGUGGA CUGAUGAGGCCGUUAGGCCGAA IAAGAGGG	5681
1698	CCUCUUCC U CCACCCGG	5178	CCGGGUGG CUGAUGAGGCCGUUAGGCCGAA IGAAGAGG	5682
1700	UCUUCCUC C ACCCGGCA	5179	UGCCGGGU CUGAUGAGGCCGUUAGGCCGAA IAGGAAGA	5683
1701	CUUCCUCC A CCCGGCAC	5180	GUGCCGGG CUGAUGAGGCCGUUAGGCCGAA IGAGGAAG	5684
1703	UCCUCCAC C CGGCACUG	5181	CAGUGCCG CUGAUGAGGCCGUUAGGCCGAA IUGGAGGA	5685
1704	CCUCCACC C GGCACUGG	5182	CCAGUGCC CUGAUGAGGCCGUUAGGCCGAA IGUGGAGG	5686
1708	CACCCGGC A CUGGGGAA	5183	UUCCCCAG CUGAUGAGGCCGUUAGGCCGAA ICCGGGUG	5687
1710	CCCGGCAC U GGGGAAGU	5184	ACUUCCCC CUGAUGAGGCCGUUAGGCCGAA IUGCCGGG	5688
1720	GGGAAGUC A AGAAUGGG	5185	CCCAUUCU CUGAUGAGGCCGUUAGGCCGAA IACUUCCC	5689
1731	AAUGGGGC C UGGGGCUC	5186	GAGCCCCA CUGAUGAGGCCGUUAGGCCGAA ICCCCAUU	5690
1732	AUGGGGCC U GGGGCUCU	5187	AGAGCCCC CUGAUGAGGCCGUUAGGCCGAA IGCCCCAU	5691
1738	CCUGGGGC U CUCAGGGA	5188	UCCCUGAG CUGAUGAGGCCGUUAGGCCGAA ICCCCAGG	5692
1740	UGGGGCUC U CAGGGAGA	5189	UCUCCCUG CUGAUGAGGCCGUUAGGCCGAA IAGCCCCA	5693
1742	GGGCUCUC A GGGAGAAC	5190	GUUCUCCC CUGAUGAGGCCGUUAGGCCGAA IAGAGCCC	5694
1751	GGGAGAAC U GCUUCCCC	5191	GGGGAAGC CUGAUGAGGCCGUUAGGCCGAA IUUCUCCC	5695
1754	AGAACUGC U UCCCCUGG	5192	CCAGGGGA CUGAUGAGGCCGUUAGGCCGAA ICAGUUCU	5696
1757	ACUGCUUC C CCUGGCAG	5193	CUGCCAGG CUGAUGAGGCCGUUAGGCCGAA IAAGCAGU	5697
1758	CUGCUUCC C CUGGCAGA	5194	UCUGCCAG CUGAUGAGGCCGUUAGGCCGAA IGAAGCAG	5698
1759	UGCUUCCC C UGGCAGAG	5195	CUCUGCCA CUGAUGAGGCCGUUAGGCCGAA IGGAAGCA	5699
1760	GCUUCCCC U GGCAGAGC	5196	GCUCUGCC CUGAUGAGGCCGUUAGGCCGAA IGGGAAGC	5700
1764	CCCCUGGC A GAGCUGGG	5197	CCCAGCUC CUGAUGAGGCCGUUAGGCCGAA ICCAGGGG	5701
1769	GGCAGAGC U GGGUGGCA	5198	UGCCACCC CUGAUGAGGCCGUUAGGCCGAA ICUCUGCC	5702
1777	UGGGUGGC A GCUCUUCC	5199	GGAAGAGC CUGAUGAGGCCGUUAGGCCGAA ICCACCCA	5703
1780	GUGGCAGC U CUUCCUCC	5200	GGAGGAAG CUGAUGAGGCCGUUAGGCCGAA ICUGCCAC	5704
1782	GGCAGCUC U UCCUCCCA	5201	UGGGAGGA CUGAUGAGGCCGUUAGGCCGAA IAGCUGCC	5705
1785	AGCUCUUC C UCCCACCG	5202	CGGUGGGA CUGAUGAGGCCGUUAGGCCGAA IAAGAGCU	5706
1786	GCUCUUCC U CCCACCGG	5203	CCGGUGGG CUGAUGAGGCCGUUAGGCCGAA IGAAGAGC	5707
1788	UCUUCCUC C CACCGGAC	5204	GUCCGGUG CUGAUGAGGCCGUUAGGCCGAA IAGGAAGA	5708
1789	CUUCCUCC C ACCGGACA	5205	UGUCCGGU CUGAUGAGGCCGUUAGGCCGAA IGAGGAAG	5709
1790	UUCCUCCC A CCGGACAC	5206	GUGUCCGG CUGAUGAGGCCGUUAGGCCGAA IGGACGAA	5710
1792	CCUCCCAC C GGACACCG	5207	CGGUGUCC CUGAUGAGGCCGUUAGGCCGAA IUGGGAGG	5711
1797	CACCGGAC A CCGACCCG	5208	CGGGUCGG CUGAUGAGGCCGUUAGGCCGAA IUCCGGUG	5712
1799	CCGGACAC C GACCCGCC	5209	GGCGGGUC CUGAUGAGGCCGUUAGGCCGAA IUGUCCGG	5713
1803	ACACCGAC C CGCCCGCC	5210	GGCGGGCG CUGAUGAGGCCGUUAGGCCGAA IUCGGUGU	5714
1804	CACCGACC C GCCCGCCG	5211	CGGCGGGC CUGAUGAGGCCGUUAGGCCGAA IGUCGGUG	5715
1807	CGACCCGC C CGCCGCUG	5212	CAGCGGCG CUGAUGAGGCCGUUAGGCCGAA ICGGGUCG	5716
1808	GACCCGCC C GCCGCUGU	5213	ACAGCGGC CUGAUGAGGCCGUUAGGCCGAA IGCGGGUC	5717
1811	CCGCCCGC C GCUGUGCC	5214	GGCACAGC CUGAUGAGGCCGUUAGGCCGAA ICGGGCGG	5718
1814	CCCGCCGC U GUGCCCUG	5215	CAGGGCAC CUGAUGAGGCCGUUAGGCCGAA ICGGCGGG	5719
1819	CGCUGUGC C CUGGGAGU	5216	ACUCCCAG CUGAUGAGGCCGUUAGGCCGAA ICACAGCG	5720
1820	GCUGUGCC C UGGGAGUG	5217	CACUCCCA CUGAUGAGGCCGUUAGGCCGAA IGCACAGC	5721
1821	CUGUGCCC U GGGAGUGC	5218	GCACUCCC CUGAUGAGGCCGUUAGGCCGAA IGGCACAG	5722
1830	GGGAGUGC U GCCCUCUU	5219	AAGAGGGC CUGAUGAGGCCGUUAGGCCGAA ICACUCCC	5723
1833	AGUGCUGC C CUCUUACC	5220	GGUAAGAG CUGAUGAGGCCGUUAGGCCGAA ICAGCACU	5724
1834	GUGCUGCC C UCUUACCA	5221	UGGUAAGA CUGAUGAGGCCGUUAGGCCGAA IGCAGCAC	5725
1835	UGCUGCCC U CUUACCAU	5222	AUGGUAAG CUGAUGAGGCCGUUAGGCCGAA IGGCAGCA	5726
1837	CUGCCCUC U UACCAUGC	5223	GCAUGGUA CUGAUGAGGCCGUUAGGCCGAA IAGGGCAG	5727
1841	CCUCUUAC C AUGCACAC	5224	GUGUGCAU CUGAUGAGGCCGUUAGGCCGAA IUAAGAGG	5728
1842	CUCUUACC A UGCACACG	5225	CGUGUGCA CUGAUGAGGCCGUUAGGCCGAA IGUAAGAG	5729
1846	UACCAUGC A CACGGGUG	5226	CACCCGUG CUGAUGAGGCCGUUAGGCCGAA ICAUGGUA	5730
1848	CCAUGCAC A CGGGUGCU	5227	AGCACCCG CUGAUGAGGCCGUUAGGCCGAA IUGCAUGG	5731
1856	ACGGGUGC U CUCCUUUU	5228	AAAAGGAG CUGAUGAGGCCGUUAGGCCGAA ICACCCGU	5732
1858	GGGUGCUC U CCUUUUGG	5229	CCAAAAGG CUGAUGAGGCCGUUAGGCCGAA IAGCACCC	5733
1860	GUGCUCUC C UUUUGGGC	5230	GCCCAAAA CUGAUGAGGCCGUUAGGCCGAA IAGAGCAC	5734
1861	UGCUCUCC U UUUGGGCU	5231	AGCCCAAA CUGAUGAGGCCGUUAGGCCGAA IGAGAGCA	5735
1869	UUUUGGGC U GCAUGCUA	5232	UAGCAUGC CUGAUGAGGCCGUUAGGCCGAA ICCCAAAA	5736
1872	UGGGCUGC A UGCUAUUC	5233	GAAUAGCA CUGAUGAGGCCGUUAGGCCGAA ICAGCCCA	5737
1876	CUGCAUGC U AUUCCAUU	5234	AAUGGAAU CUGAUGAGGCCGUUAGGCCGAA ICAUGCAG	5738
1881	UGCUAUUC C AUUUUGCA	5235	UGCAAAAU CUGAUGAGGCCGUUAGGCCGAA IAAUAGCA	5739
1882	GCUAUUCC A UUUUGCAG	5236	CUGCAAAA CUGAUGAGGCCGUUAGGCCGAA IGAAUAGC	5740
1889	CAUUUUGC A GCCAGACC	5237	GGUCUGGC CUGAUGAGGCCGUUAGGCCGAA ICAAAAUG	5741
1892	UUUGCAGC C AGACCGAU	5238	AUCGGUCU CUGAUGAGGCCGUUAGGCCGAA ICUGCAAA	5742
1893	UUGCAGCC A GACCGAUG	5239	CAUCGGUC CUGAUGAGGCCGUUAGGCCGAA IGCUGCAA	5743
1897	AGCCAGAC C GAUGUGUA	5240	UACACAUC CUGAUGAGGCCGUUAGGCCGAA IUCUGGCU	5744
1912	UAUUUAAC C AGUCACUA	5241	UAGUCACU CUGAUGAGGCCGUUAGGCCGAA IUUAAAUA	5745
1913	AUUUAACC A GUCACUAU	5242	AUAGUGAC CUGAUGAGGCCGUUAGGCCGAA IGUUAAAU	5746
1917	AACCAGUC A CUAUUGAU	5243	AUCAAUAG CUGAUGAGGCCGUUAGGCCGAA IACUGGUU	5747
1919	CCAGUCAC U AUUGAUGG	5244	CCAUCAAU CUGAUGAGGCCGUUAGGCCGAA IUGACUGG	5748
1930	UGAUGGAC A UUUGGGUU	5245	AACCCAAA CUGAUGAGGCCGUUAGGCCGAA IUCCAUCA	5749
1944	GUUGUUUC C CAUCUUUU	5246	AAAAGAUG CUGAUGAGGCCGUUAGGCCGAA IAAACAAC	5750
1945	UUGUUUCC C AUCUUUUU	5247	AAAAAGAU CUGAUGAGGCCGUUAGGCCGAA IGAAACAA	5751
1946	UGUUUCCC A UCUUUUUG	5248	CAAAAAGA CUGAUGAGGCCGUUAGGCCGAA IGGAAACA	5752
1949	UUCCCAUC U UUUUGUUA	5249	UAACAAAA CUGAUGAGGCCGUUAGGCCGAA IAUGGGAA	5753
1959	UUUGUUAC C AUAAAUAA	5250	UUAUUUAU CUGAUGAGGCCGUUAGGCCGAA IUAACAAA	5754
1960	UUGUUACC A UAAAUAAU	5251	AUUAUUUA CUGAUGAGGCCGUUAGGCCGAA IGUAACAA	5755
1972	AUAAUGGC A UAGUAAAA	5252	UUUUACUA CUGAUGAGGCCGUUAGGCCGAA ICCAUUAU	5756
Input Sequence NM_003639. Cut Site = CH/.
Arm Length = 8. Core Sequence CUGAUGAG GCCGUUAGGC CGAA
NM_003639 (Homo sapiens inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), mRNA.; 1994 bp)

Underlined region can be any X sequence or linker, as described herein. “I” stands for Inosine.

[0268]

TABLE V
Human IKK-gamma Zinzyme and Substrate Sequence
		Seq		Seq
Pos	Substrate	ID	Zinzyme	ID
13	CGAGCAUG G CCCUUGUG	5757	CACAAGGG GCCGAAAGGCGAGUGAGGUCU CAUGCUCG	6034
19	UGGCCCUU G UGAUCCAG	5758	CUGGAUCA GCCGAAAGGCGAGUGAGGUCU AAGGGCCA	6035
28	UGAUCCAG G UGGGGAAA	5759	UUUCCCCA GCCGAAAGGCGAGUGAGGUCU CUGGAUCA	6036
42	AAACUAAG G CCCAGAGA	5760	UCUCUGGG GCCGAAAGGCGAGUGAGGUCU CUUAGUUU	6037
52	CCAGAGAA G UGAGGACC	5761	GGUCCUCA GCCGAAAGGCGAGUGAGGUCU UUCUCUGG	6038
63	AGGACCCC G CAGACUAU	5762	AUAGUCUG GCCGAAAGGCGAGUGAGGUCU GGGGUCCU	6039
80	CAAUCCCA G UCUCUUCC	5763	GGAAGAGA GCCGAAAGGCGAGUGAGGUCU UGGGAUUG	6040
100	CACUCCCU G UGAAGCUC	5764	GAGCUUCA GCCGAAAGGCGAGUGAGGUCU AGGGAGUG	6041
105	CCUGUGAA G CUCUCCAG	5765	CUGGAGAG GCCGAAAGGCGAGUGAGGUCU UUCACAGG	6042
113	GCUCUCCA G CAUCAUCG	5766	CGAUGAUG GCCGAAAGGCGAGUGAGGUCU UGGAGAGC	6043
124	UCAUCGAG G UCCCAUCA	5767	UGAUGGGA GCCGAAAGGCGAGUGAGGUCU CUCGAUGA	6044
133	UCCCAUCA G CCCUUGCC	5768	GGCAAGGG GCCGAAAGGCGAGUGAGGUCU UGAUGGGA	6045
139	CAGCCCUU G CCCUGUUG	5769	CAACAGGG GCCGAAAGGCGAGUGAGGUCU AAGGGCUG	6046
144	CUUGCCCU G UUGGAUGA	5770	UCAUCCAA GCCGAAAGGCGAGUGAGGUCU AGGGCAAG	6047
157	AUGAAUAG G CACCUCUG	5771	CAGAGGUG GCCGAAAGGCGAGUGAGGUCU CUAUUCAU	6048
171	CUGGAAGA G CCAACUGU	5772	ACAGUUGG GCCGAAAGGCGAGUGAGGUCU UCUUCCAG	6049
178	AGCCAACU G UGUGAGAU	5773	AUCUCACA GCCGAAAGGCGAGUGAGGUCU AGUUGGCU	6050
180	CCAACUGU G UGAGAUGG	5774	CCAUCUCA GCCGAAAGGCGAGUGAGGUCU ACAGUUGG	6051
188	GUGAGAUG G UGCAGCCC	5775	GGGCUGCA GCCGAAAGGCGAGUGAGGUCU CAUCUCAC	6052
190	GAGAUGGU G CAGCCCAG	5776	CUGGGCUG GCCGAAAGGCGAGUGAGGUCU ACCAUCUC	6053
193	AUGGUGCA G CCCAGUGG	5777	CCACUGGG GCCGAAAGGCGAGUGAGGUCU UGCACCAU	6054
198	GCAGCCCA G UGGUGGCC	5778	GGCCACCA GCCGAAAGGCGAGUGAGGUCU UGGGCUGC	6055
201	GCCCAGUG G UGGCCCGG	5779	CCGGGCCA GCCGAAAGGCGAGUGAGGUCU CACUGGGC	6056
204	CAGUGGUG G CCCGGCAG	5780	CUGCCGGG GCCGAAAGGCGAGUGAGGUCU CACCACUG	6057
209	GUGGCCCG G CAGCAGAU	5781	AUCUGCUG GCCGAAAGGCGAGUGAGGUCU CGGGCCAC	6058
212	GCCCGGCA G CAGAUCAG	5782	CUGAUCUG GCCGAAAGGCGAGUGAGGUCU UGCCGGGC	6059
224	AUCAGGAC G UACUGGGC	5783	GCCCAGUA GCCGAAAGGCGAGUGAGGUCU GUCCUGAU	6060
231	CGUACUGG G CGAAGAGU	5784	ACUCUUCG GCCGAAAGGCGAGUGAGGUCU CCAGUACG	6061
238	GGCGAAGA G UCUCCUCU	5785	AGAGGAGA GCCGAAAGGCGAGUGAGGUCU UCUUCGCC	6062
253	CUGGGGAA G CCAGCCAU	5786	AUGGCUGG GCCGAAAGGCGAGUGAGGUCU UUCCCCAG	6063
257	GGAAGCCA G CCAUGCUG	5787	CAGCAUGG GCCGAAAGGCGAGUGAGGUCU UGGCUUCC	6064
262	CCAGCCAU G CUGCACCU	5788	AGGUGCAG GCCGAAAGGCGAGUGAGGUCU AUGGCUGG	6065
265	GCCAUGCU G CACCUGCC	5789	GGCAGGUG GCCGAAAGGCGAGUGAGGUCU AGCAUGGC	6066
271	CUGCACCU G CCUUCAGA	5790	UCUGAAGG GCCGAAAGGCGAGUGAGGUCU AGGUGCAG	6067
285	AGAACAGG G CGCUCCUG	5791	CAGGAGCG GCCGAAAGGCGAGUGAGGUCU CCUGUUCU	6068
287	AACAGGGC G CUCCUGAG	5792	CUCAGGAG GCCGAAAGGCGAGUGAGGUCU GCCCUGUU	6069
304	ACCCUCCA G CGCUGCCU	5793	AGGCAGCG GCCGAAAGGCGAGUGAGGUCU UGGAGGGU	6070
306	CCUCCAGC G CUGCCUGG	5794	CCAGGCAG GCCGAAAGGCGAGUGAGGUCU GCUGGAGG	6071
309	CCAGCGCU G CCUGGAGG	5795	CCUCCAGG GCCGAAAGGCGAGUGAGGUCU AGCGCUGG	6072
328	AAUCAAGA G CUCCGAGA	5796	UCUCGGAG GCCGAAAGGCGAGUGAGGUCU UCUUGAUU	6073
338	UCCGAGAU G CCAUCCGG	5797	CCGGAUGG GCCGAAAGGCGAGUGAGGUCU AUCUCGGA	6074
346	GCCAUCCG G CAGAGCAA	5798	UUGCUCUG GCCGAAAGGCGAGUGAGGUCU CGGAUGGC	6075
351	CCGGCAGA G CAACCAGA	5799	UCUGGUUG GCCGAAAGGCGAGUGAGGUCU UCUGCCGG	6076
364	CAGAUUCU G CGGGAGCG	5800	CGCUCCCG GCCGAAAGGCGAGUGAGGUCU AGAAUCUG	6077
370	CUGCGGGA G CGCUGCGA	5801	UCGCAGCG GCCGAAAGGCGAGUGAGGUCU UCCCGCAG	6078
372	GCGGGAGC G CUGCGAGG	5802	CCUCGCAG GCCGAAAGGCGAGUGAGGUCU GCUCCCGC	6079
375	GGAGCGCU G CGAGGAGC	5803	GCUCCUCG GCCGAAAGGCGAGUGAGGUCU AGCGCUCC	6080
382	UGCGAGGA G CUUCUGCA	5804	UGCAGAAG GCCGAAAGGCGAGUGAGGUCU UCCUCGCA	6081
388	GAGCUUCU G CAUUUCCA	5805	UGGAAAUG GCCGAAAGGCGAGUGAGGUCU AGAAGCUC	6082
398	AUUUCCAA G CCAGCCAG	5806	CUGGCUGG GCCGAAAGGCGAGUGAGGUCU UUGGAAAU	6083
402	CCAAGCCA G CCAGAGGG	5807	CCCUCUGG GCCGAAAGGCGAGUGAGGUCU UGGCUUGG	6084
421	GAGAAGGA G UUCCUCAU	5808	AUGAGGAA GCCGAAAGGCGAGUGAGGUCU UCCUUCUC	6085
430	UUCCUCAU G UGCAAGUU	5809	AACUUGCA GCCGAAAGGCGAGUGAGGUCU AUGAGGAA	6086
432	CCUCAUGU G CAAGUUCC	5810	GGAACUUG GCCGAAAGGCGAGUGAGGUCU ACAUGAGG	6087
436	AUGUGCAA G UUCCAGGA	5811	UCCUGGAA GCCGAAAGGCGAGUGAGGUCU UUGCACAU	6088
446	UCCAGGAG G CCAGGAAA	5812	UUUCCUGG GCCGAAAGGCGAGUGAGGUCU CUCCUGGA	6089
458	GGAAACUG G UGGAGAGA	5813	UCUCUCCA GCCGAAAGGCGAGUGAGGUCU CAGUUUCC	6090
471	GAGACUCG G CCUGGAGA	5814	UCUCCAGG GCCGAAAGGCGAGUGAGGUCU CGAGUCUC	6091
481	CUGGAGAA G CUCGAUCU	5815	AGAUCGAG GCCGAAAGGCGAGUGAGGUCU UUCUCCAG	6092
496	CUGAAGAG G CAGAAGGA	5816	UCCUUCUG GCCGAAAGGCGAGUGAGGUCU CUCUUCAG	6093
505	CAGAAGGA G CAGGCUCU	5817	AGAGCCUG GCCGAAAGGCGAGUGAGGUCU UCCUUCUG	6094
509	AGGAGCAG G CUCUGCGG	5818	CCGCAGAG GCCGAAAGGCGAGUGAGGUCU CUGCUCCU	6095
514	CAGGCUCU G CGGGAGGU	5819	ACCUCCCG GCCGAAAGGCGAGUGAGGUCU AGAUCCUG	6096
521	UGCGGGAG G UGGAUCAC	5820	GUGCUCCA GCCGAAAGGCGAGUGAGGUCU CUCCCGCA	6097
526	GAGGUGGA G CACCUGAA	5821	UUCAGGUG GCCGAAAGGCGAGUGAGGUCU UCCACCUC	6098
540	GAAGAGAU G CCAGCAGC	5822	GCUGCUGG GCCGAAAGGCGAGUGAGGUCU AUCUCUUC	6099
544	AGAUGCCA G CAGCAGAU	5823	AUCUGCUG GCCGAAAGGCGAGUGAGGUCU UGGCAUCU	6100
547	UGCCAGCA G CAGAUGGC	5824	GCCAUCUG GCCGAAAGGCGAGUGAGGUCU UGCUGGCA	6101
554	AUCAGAUG G CUGAGGAC	5825	GUCCUCAG GCCGAAAGGCGAGUGAGGUCU CAUCUGCU	6102
566	AGGACAAG G CCUCUGUG	5826	CACAGAGG GCCGAAAGGCGAGUGAGGUCU CUUGUCCU	6103
572	AGGCCUCU G UGAAAGCC	5827	GGCUUUCA GCCGAAAGGCGAGUGAGGUCU AGAGGCCU	6104
578	CUGUGAAA G CCCAGGUG	5828	CACCUGGG GCCGAAAGGCGAGUGAGGUCU UUUCACAG	6105
584	AAGCCCAG G UGACGUCC	5829	GGACGUCA GCCGAAAGGCGAGUGAGGUCU CUGGGCUU	6106
589	CAGGUGAC G UCCUUGCU	5830	AGCAAGGA GCCGAAAGGCGAGUGAGGUCU GUCACCUG	6107
595	ACGUCCUU G CUCGGGGA	5831	UCCCCGAG GCCGAAAGGCGAGUGAGGUCU AAGGACGU	6108
604	CUCGGGGA G CUGCAGGA	5832	UCCUGCAG GCCGAAAGGCGAGUGAGGUCU UCCCCGAG	6109
607	GGGGAGCU G CAGGAGAG	5833	CUCUCCUG GCCGAAAGGCGAGUGAGGUCU AGCUCCCC	6110
615	GCAGGAGA G CCAGAGUC	5834	GACUCUGG GCCGAAAGGCGAGUGAGGUCU UCUCCUGC	6111
621	GAGCCAGA G UCGCUUGG	5835	CCAAGCGA GCCGAAAGGCGAGUGAGGUCU UCUGUCUC	6112
624	CCAGAGUC G CUUGGAGG	5836	CCUCCAAG GCCGAAAGGCGAGUGAGGUCU GACUCUGG	6113
632	GCUUGGAG G CUGCCACU	5837	AGUGUCAG GCCGAAAGGCGAGUGAGGUCU CUCCAAGC	6114
635	UGGAGGCU G CCACUAAG	5838	CUUAGUGG GCCGAAAGGCGAGUGAGGUCU AGCCUCCA	6115
648	UAAGGAAU G CCAGGCUC	5839	GAGCCUGG GCCGAAAGGCGAGUGAGGUCU AUUCCUUA	6116
653	AAUGCCAG G CUCUGGAG	5840	CUCCAGAG GCCGAAAGGCGAGUGAGGUCU CUGGCAUU	6117
663	UCUGGAGG G UCGGGCCC	5841	GGGCCCGA GCCGAAAGGCGAGUGAGGUCU CCUCCAGA	6118
668	AGGGUCGG G CCCGGGCG	5842	CGCCCGGG GCCGAAAGGCGAGUGAGGUCU CCGACCCU	6119
674	GGGCCCGG G CGGCCAGC	5843	GCUGGCCG GCCGAAAGGCGAGUGAGGUCU CCGGGCCC	6120
677	CCCGGGCG G CCACGCAG	5844	CUCGCUGG GCCGAAAGGCGAGUGAGGUCU CGCCCGGG	6121
681	GGCGGCCA G CGAGCAGG	5845	CCUGCUCG GCCGAAAGGCGAGUGAGGUCU UGGCCGCC	6122
685	GCCAGCGA G CAGGCGCG	5846	CGCGCCUG GCCGAAAGGCGAGUGAGGUCU UCGCUGGC	6123
689	GCGAGCAG G CGCGGCAG	5847	CUGCCGCG GCCGAAAGGCGAGUGAGGUCU CUGCUCGC	6124
691	GAGCAGGC G CGGCAGCU	5848	AGCUGCCG GCCGAAAGGCGAGUGAGGUCU GCCUGCUC	6125
694	CAGGCGCG G CAGCUGGA	5849	UCCAGCUG GCCGAAAGGCGAGUGAGGUCU CGCGCCUG	6126
697	GCGCGGCA G CUGGAGAG	5850	CUCUCCAG GCCGAAAGGCGAGUGAGGUCU UGCCGCGC	6127
705	GCUGGAGA G UGAGCGCG	5851	CGCGCUCA GCCGAAAGGCGAGUGAGGUCU UCUCCAGC	6128
709	GAGAGUGA G CGCGAGGC	5852	GCCUCGCG GCCGAAAGGCGAGUGAGGUCU UCACUCUC	6129
711	GAGUGAGC G CGAGGCGC	5853	GCGCCUCG GCCGAAAGGCGAGUGAGGUCU GCUCACUC	6130
716	AGCGCGAG G CGCUGCAG	5854	CUGCAGCG GCCGAAAGGCGAGUGAGGUCU CUCGCGCU	6131
718	CGCGAGGC G CUGCAGCA	5855	UGCUGCAG GCCGAAAGGCGAGUGAGGUCU GCCUCGCG	6132
721	GAGGCGCU G CAGCAGCA	5856	UGCUGCUG GCCGAAAGGCGAGUGAGGUCU AGCGCCUC	6133
724	GCGCUGCA G CAGCAGCA	5857	UGCUGCUG GCCGAAAGGCGAGUGAGGUCU UGCAGCGC	6134
727	CUGCAGCA G CAGCACAG	5858	CUGUGCUG GCCGAAAGGCGAGUGAGGUCU UGCUGCAG	6135
730	CAGCAGCA G CACAGCGU	5859	ACGCUGUG GCCGAAAGGCGAGUGAGGUCU UGCUGCUG	6136
735	GCAGCACA G CGUGCAGG	5860	CCUGCACG GCCGAAAGGCGAGUGAGGUCU UGUGCUGC	6137
737	AGCACAGC G UGCAGGUG	5861	CACCUGCA GCCGAAAGGCGAGUGAGGUCU GCUGUGCU	6138
739	CACAGCGU G CAGGUGGA	5862	UCCACCUG GCCGAAAGGCGAGUGAGGUCU ACGCUGUG	6139
743	GCGUGCAG G UGGACCAG	5863	CUGGUCCA GCCGAAAGGCGAGUGAGGUCU CUGCACGC	6140
751	GUGGACCA G CUGCGCAU	5864	AUGCGCAG GCCGAAAGGCGAGUGAGGUCU UGGUCCAC	6141
754	GACCAGCU G CGCAUGCA	5865	UGCAUGCG GCCGAAAGGCGAGUGAGGUCU AGCUGGUC	6142
756	CCAGCUGC G CAUGCAGG	5866	CCUGCAUG GCCGAAAGGCGAGUGAGGUCU GCAGCUGG	6143
760	CUGCGCAU G CAGGGCCA	5867	UGGCCCUG GCCGAAAGGCGAGUGAGGUCU AUGCGCAG	6144
765	CAUGCAGG G CCAGAGCG	5868	CGCUCUGG GCCGAAAGGCGAGUGAGGUCU CCUGCAUG	6145
771	GGGCCAGA G CGUGGAGG	5869	CCUCCACG GCCGAAAGGCGAGUGAGGUCU UCUGGCCC	6146
773	GCCAGAGC G UGGAGGCC	5870	GGCCUCCA GCCGAAAGGCGAGUGAGGUCU GCUCUGGC	6147
779	GCGUGGAG G CCGCGCUC	5871	GAGCGCGG GCCGAAAGGCGAGUGAGGUCU CUCCACGC	6148
782	UGGAGGCC G CGCUCCGC	5872	GCGGAGCG GCCGAAAGGCGAGUGAGGUCU GGCCUCCA	6149
784	GACGCCGC G CUCCGCAU	5873	AUGCGGAG GCCGAAAGGCGAGUGAGGUCU GCGGCCUC	6150
789	CGCGCUCC G CAUGGAGC	5874	GCUCCAUG GCCGAAAGGCGAGUGAGGUCU GGACCGCG	6151
796	CGCAUGGA G CGCCAGGC	5875	GCCUGGCG GCCGAAAGGCGAGUGAGGUCU UCCAUCCG	6152
798	CAUGGAGC G CCAGCCCG	5876	CGGCCUGG GCCGAAAGGCGAGUGAGGUCU GCUCCAUG	6153
803	AGCGCCAC G CCGCCUCG	5877	CGAGCCGG GCCGAAAGGCGAGUGAGGUCU CUGGCGCU	6154
806	GCCAGGCC G CCUCGGAG	5878	CUCCGAGG GCCGAAAGGCGAGUGAGGUCU GCCCUGGC	6155
826	AAGAGGAA G CUGGCCCA	5879	UGGGCCAG GCCGAAAGGCGAGUGAGGUCU UUCCUCUU	6156
830	GGAAGCUG G CCCAGUUG	5880	CAACUGGG GCCGAAAGGCGAGUGAGGUCU CAGCUUCC	6157
835	CUGGCCCA G UUGCAGGU	5881	ACCUGCAA GCCGAAAGGCGAGUGAGGUCU UGGGCCAG	6158
838	GCCCAGUU G CAGGUGGC	5882	GCCACCUG GCCGAAAGGCGAGUGAGGUCU AACUGGGC	6159
842	AGUUGCAG G UGGCCUAU	5883	AUAGGCCA GCCGAAAGGCGAGUGAGGUCU CUGCAACU	6160
845	UGCAGGUG G CCUAUCAC	5884	GUGAUAGG GCCGAAAGGCGAGUGAGGUCU CACCUGCA	6161
856	UAUCACCA G CUCUUCCA	5885	UGGAAGAG GCCGAAAGGCGAGUGAGGUCU UGGUGAUA	6162
888	CAUCAAGA G CAGCGUGG	5886	CCACCCUG GCCGAAAGGCGAGUGAGGUCU UCUUGAUG	6163
891	CAAGAGCA G CGUGCUGG	5887	CCACCACG GCCGAAAGGCGAGUGAGGUCU UGCUCUUG	6164
893	AGACCAGC G UGGUGCCC	5888	GCCCACCA GCCGAAAGGCGAGUGAGGUCU GCUGCUCU	6165
896	GCACCGUG G UGGGCAGU	5889	ACUGCCCA GCCGAAAGGCGAGUGAGGUCU CACGCUGC	6166
900	CGUGGUCG G CAGUGAGC	5890	GCUCACUG GCCGAAAGGCGAGUGAGGUCU CCACCACG	6167
903	GGUGGGCA G UCAGCGGA	5891	UCCGCUCA GCCGAAAGGCGAGUGAGGUCU UGCCCACC	6168
907	GGCAGUGA G CGGAAGCG	5892	CGCUUCCG GCCGAAAGGCGAGUGAGGUCU UCACUGCC	6169
913	GAGCGGAA G CGAGGAAU	5893	AUUCCUCG GCCGAAAGGCGAGUGAGGUCU UUCCGCUC	6170
922	CGAGGAAU G CAGCUGGA	5894	UCCAGCUG GCCGAAAGGCGAGUGAGGUCU AUUCCUCG	6171
925	GGAAUGCA G CUGGAAGA	5895	UCUUCCAG GCCGAAAGGCGAGUGAGGUCU UGCAUUCC	6172
943	CUCAAACA G CAGCUCCA	5896	UGGAGCUG GCCGAAAGGCGAGUGAGGUCU UGUUUGAC	6173
946	AAACAGCA G CUCCAGCA	5897	UGCUGGAC GCCGAAAGGCGAGUGAGGUCU UGCUGUUU	6174
952	CAGCUCCA G CAGCCCGA	5898	UCGGCCUG GCCGAAAGGCGAGUGAGGUCU UGGAGCUG	6175
956	UCCAGCAC G CCGAGGAG	5899	CUCCUCGG GCCGAAAGGCGAGUGAGGUCU CUGCUGGA	6176
965	CCGAGGAG G CCCUGGUG	5900	CACCAGGG GCCGAAAGGCGAGUGAGGUCU CUCCUCGG	6177
971	AGGCCCUG G UGGCCAAA	5901	UUUGGCCA GCCGAAAGGCGAGUGAGGUCU CAGGGCCU	6178
974	CCCUGGUG G CCAAACAG	5902	CUGUUUGG GCCGAAAGGCGAGUGAGGUCU CACCAGGG	6179
986	AACAGGAG G UGAUCGAU	5903	AUCGAUCA GCCGAAAGGCGAGUGAGGUCU CUCCUGUU	6180
997	AUCGAUAA G CUGAAGGA	5904	UCCUUCAG GCCGAAAGGCGAGUGAGGUCU UUAUCGAU	6181
1010	AGGAGGAG G CCGAGCAG	5905	CUGCUCGG GCCGAAAGGCGAGUGAGGUCU CUCCUCCU	6182
1015	GAGGCCGA G CAGCACAA	5906	UUGUGCUG GCCGAAAGGCGAGUGAGGUCU UCGGCCUC	6183
1018	GCCGAGCA G CACAAGAU	5907	AUCUUGUG GCCGAAAGGCGAGUGAGGUCU UGCUCGGC	6184
1028	ACAAGAUU G UGAUGGAG	5908	CUCCAUCA GCCGAAAGGCGAGUGAGGUCU AAUCUUGU	6185
1040	UGGAGACC G UUCCGGUG	5909	CACCGGAA GCCGAAAGGCGAGUGAGGUCU GGUCUCCA	6186
1046	CCGUUCCG G UGCUGAAG	5910	CUUCAGCA GCCGAAAGGCGAGUGAGGUCU CGGAACGG	6187
1048	GUUCCGGU G CUGAAGGC	5911	GCCUUCAG GCCGAAAGGCGAGUGAGGUCU ACCGGAAC	6188
1055	UGCUGAAG G CCCAGGCG	5912	CGCCUGGG GCCGAAAGGCGAGUGAGGUCU CUUCAGCA	6189
1061	AGGCCCAG G CGGAUAUC	5913	GAUAUCCG GCCGAAAGGCGAGUGAGGUCU CUGGGCCU	6190
1076	UCUACAAG G CGGACUUC	5914	GAAGUCCG GCCGAAAGGCGAGUGAGGUCU CUUGUAGA	6191
1088	ACUUCCAG G CUGAGAGG	5915	CCUCUCAG GCCGAAAGGCGAGUGAGGUCU CUGGAAGU	6192
1096	GCUGAGAG G CAGGCCCG	5916	CGGGCCUG GCCGAAAGGCGAGUGAGGUCU CUCUCAGC	6193
1100	AGAGGCAG G CCCGGGAG	5917	CUCCCGGG GCCGAAAGGCGAGUGAGGUCU CUGCCUCU	6194
1111	CGGGAGAA G CUGGCCGA	5918	UCGGCCAG GCCGAAAGGCGAGUGAGGUCU UUCUCCCG	6195
1115	AGAAGCUG G CCGAGAAG	5919	CUUCUCGG GCCGAAAGGCGAGUGAGGUCU CAGCUUCU	6196
1129	AAGAAGGA G CUCCUGCA	5920	UGCAGGAG GCCGAAAGGCGAGUGAGGUCU UCCUUCUU	6197
1135	GAGCUCCU G CAGGAGCA	5921	UGCUCCUG GCCGAAAGGCGAGUGAGGUCU AGGAGCUC	6198
1141	CUGCAGGA G CAGCUGGA	5922	UCCAGCUG GCCGAAAGGCGAGUGAGGUCU UCCUGCAG	6199
1144	CAGGACCA G CUGGAGCA	5923	UGCUCCAG GCCGAAAGGCGAGUGAGGUCU UGCUCCUG	6200
1150	CAGCUGGA G CAGCUGCA	5924	UGCAGCUG GCCGAAAGGCGAGUGAGGUCU UCCAGCUG	6201
1153	CUGGAGCA G CUGCAGAG	5925	CUCUGCAG GCCGAAAGGCGAGUGAGGUCU UGCUCCAG	6202
1156	GAGCAGCU G CAGAGGGA	5926	UCCCUCUG GCCGAAAGGCGAGUGAGGUCU AGCUGCUC	6203
1165	CAGAGGGA G UACAGCAA	5927	UUGCUGUA GCCGAAAGGCGAGUGAGGUCU UCCCUCUG	6204
1170	GGAGUACA G CAAACUGA	5928	UCAGUUUG GCCGAAAGGCGAGUGAGGUCU UGUACUCC	6205
1181	AACUGAAG G CCAGCUGU	5929	ACAGCUGG GCCGAAAGGCGAGUGAGGUCU CUUCAGUU	6206
1185	GAAGGCCA G CUGUCAGG	5930	CCUGACAG GCCGAAAGGCGAGUGAGGUCU UGGCCUUC	6207
1188	GGCCAGCU G UCAGGAGU	5931	ACUCCUGA GCCGAAAGGCGAGUGAGGUCU AGCUGGCC	6208
1195	UGUCAGGA G UCGGCCAG	5932	CUGGCCGA GCCGAAAGGCGAGUGAGGUCU UCCUGACA	6209
1199	AGGAGUCG G CCAGGAUC	5933	GAUCCUGG GCCGAAAGGCGAGUGAGGUCU CGACUCCU	6210
1222	AUGAGGAA G CGGCAUGU	5934	ACAUGCCG GCCGAAAGGCGAGUGAGGUCU UUCCUCAU	6211
1225	AGGAAGCG G CAUGUCGA	5935	UCGACAUG GCCGAAAGGCGAGUGAGGUCU CGCUUCCU	6212
1229	AGCGGCAU G UCGAGGUC	5936	GACCUCGA GCCGAAAGGCGAGUGAGGUCU AUGCCGCU	6213
1235	AUGUCGAG G UCUCCCAG	5937	CUGGGAGA GCCGAAAGGCGAGUGAGGUCU CUCGACAU	6214
1244	UCUCCCAG G CCCCCUUG	5938	CAAGGGGG GCCGAAAGGCGAGUGAGGUCU CUGGGAGA	6215
1252	GCCCCCUU G CCCCCCGC	5939	GCGGGGGG GCCGAAAGGCGAGUGAGGUCU AAGGGGGC	6216
1259	UGCCCCCC G CCCCUGCC	5940	GGCAGGGG GCCGAAAGGCGAGUGAGGUCU GGGGGGCA	6217
1265	CCGCCCCU G CCUACCUC	5941	GAGGUAGG GCCGAAAGGCGAGUGAGGUCU AGGGGCGG	6218
1286	CUCCCCUG G CCCUGCCC	5942	GGGCAGGG GCCGAAAGGCGAGUGAGGUCU CAGGGGAG	6219
1291	CUGGCCCU G CCCAGCCA	5943	UGGCUGGG GCCGAAAGGCGAGUGAGGUCU AGGGCCAG	6220
1296	CCUGCCCA G CCAGAGGA	5944	UCCUCUGG GCCGAAAGGCGAGUGAGGUCU UGGGCAGG	6221
1308	GAGGAGGA G CCCCCCCG	5945	CGGGGGGG GCCGAAAGGCGAGUGAGGUCU UCCUCCUC	6222
1321	CCCGAGGA G CCACCUGA	5946	UCAGGUGG GCCGAAAGGCGAGUGAGGUCU UCCUCGGG	6223
1335	UGACUUCU G CUGUCCCA	5947	UGGGACAG GCCGAAAGGCGAGUGAGGUCU AGAAGUCA	6224
1338	CUUCUGCU G UCCCAAGU	5948	ACUUGGGA GCCGAAAGGCGAGUGAGGUCU AGCAGAAG	6225
1345	UGUCCCAA G UGCCAGUA	5949	UACUGGCA GCCGAAAGGCGAGUGAGGUCU UUGGGACA	6226
1347	UCCCAAGU G CCAGUAUC	5950	GAUACUGG GCCGAAAGGCGAGUGAGGUCU ACUUGGGA	6227
1351	AAGUGCCA G UAUCAGGC	5951	GCCUGAUA GCCGAAAGGCGAGUGAGGUCU UGGCACUU	6228
1358	AGUAUCAG G CCCCUGAU	5952	AUCAGGGG GCCGAAAGGCGAGUGAGGUCU CUGAUACU	6229
1378	GACACCCU G CAGAUACA	5953	UGUAUCUG GCCGAAAGGCGAGUGAGGUCU AGGGUGUC	6230
1388	AGAUACAU G UCAUGGAG	5954	CUCCAUGA GCCGAAAGGCGAGUGAGGUCU AUGUAUCU	6231
1396	GUCAUGGA G UGCAUUGA	5955	UCAAUGCA GCCGAAAGGCGAGUGAGGUCU UCCAUGAC	6232
1398	CAUGGAGU G CAUUGAGU	5956	ACUCAAUG GCCGAAAGGCGAGUGAGGUCU ACUCCAUG	6233
1405	UGCAUUGA G UAGGGCCG	5957	CGGCCCUA GCCGAAAGGCGAGUGAGGUCU UCAAUGCA	6234
1410	UGAGUAGG G CCGGCCAG	5958	CUGGCCGG GCCGAAAGGCGAGUGAGGUCU CCUACUCA	6235
1414	UAGGGCCG G CCAGUGCA	5959	UGCACUGG GCCGAAAGGCGAGUGAGGUCU CGGCCCUA	6236
1418	GCCGGCCA G UGCAAGGC	5960	GCCUUGCA GCCGAAAGGCGAGUGAGGUCU UGGCCGGC	6237
1420	CGGCCAGU G CAAGGCCA	5961	UGGCCUUG GCCGAAAGGCGAGUGAGGUCU ACUGGCCG	6238
1425	AGUGCAAG G CCACUGCC	5962	GGCAGUGG GCCGAAAGGCGAGUGAGGUCU CUUGCACU	6239
1431	AGGCCACU G CCUGCCCG	5963	CGGGCAGG GCCGAAAGGCGAGUGAGGUCU AGUGGCCU	6240
1435	CACUGCCU G CCCGAGGA	5964	UCCUCGGG GCCGAAAGGCGAGUGAGGUCU AGGCAGUG	6241
1445	CCGAGGAC G UGCCCGGG	5965	CCCGGGCA GCCGAAAGGCGAGUGAGGUCU GUCCUCGG	6242
1447	GAGGACGU G CCCGGGAC	5966	GUCCCGGG GCCGAAAGGCGAGUGAGGUCU ACGUCCUC	6243
1457	CCGGGACC G UGCAGUCU	5967	AGACUGCA GCCGAAAGGCGAGUGAGGUCU GGUCCCGG	6244
1459	GGGACCGU G CAGUCUGC	5968	GCAGACUG GCCGAAAGGCGAGUGAGGUCU ACGGUCCC	6245
1462	ACCGUGCA G UCUGCGCU	5969	AGCGCAGA GCCGAAAGGCGAGUGAGGUCU UGCACGGU	6246
1466	UGCAGUCU G CGCUUUCC	5970	GGAAAGCG GCCGAAAGGCGAGUGAGGUCU AGACUGCA	6247
1468	CAGUCUGC G CUUUCCUC	5971	GAGGAAAG GCCGAAAGGCGAGUGAGGUCU GCAGACUG	6248
1481	CCUCUCCC G CCUGCCUA	5972	UAGGCAGG GCCGAAAGGCGAGUGAGGUCU GGGAGAGG	6249
1485	UCCCGCCU G CCUAGCCC	5973	GGGCUAGG GCCGAAAGGCGAGUGAGGUCU AGGCGGGA	6250
1490	CCUGCCUA G CCCAGGAU	5974	AUCCUGGG GCCGAAAGGCGAGUGAGGUCU UAGGCAGG	6251
1504	GAUGAAGG G CUGGGUGG	5975	CCACCCAG GCCGAAAGGCGAGUGAGGUCU CCUUCAUC	6252
1509	AGGGCUGG G UGGCCACA	5976	UGUGCCCA GCCGAAAGGCGAGUGAGGUCU CCAGCCCU	6253
1512	GCUGGGUG G CCACAACU	5977	AGUUGUGG GCCGAAAGGCGAGUGAGGUCU CACCCAGC	6254
1526	ACUGGGAU G CCACCUGG	5978	CCAGGUGG GCCGAAAGGCGAGUGAGGUCU AUCCCAGU	6255
1536	CACCUGGA G CCCCACCC	5979	GGGUGGGG GCCGAAAGGCGAGUGAGGUCU UCCAGGUG	6256
1549	ACCCAGGA G CUGGCCGC	5980	GCGGCCAG GCCGAAAGGCGAGUGAGGUCU UCCUGGGU	6257
1553	AGGAGCUG G CCGCGGCA	5981	UGCCGCGG GCCGAAAGGCGAGUGAGGUCU CAGCUCCU	6258
1556	AGCUGGCC G CGGCACCU	5982	AGGUGCCG GCCGAAAGGCGAGUGAGGUCU GGCCAGCU	6259
1559	UGGCCGCG G CACCUUAC	5983	GUAAGGUG GCCGAAAGGCGAGUGAGGUCU CGCGGCCA	6260
1568	CACCUUAC G CUUCAGCU	5984	AGCUGAAG GCCGAAAGGCGAGUGAGGUCU GUAAGGUG	6261
1574	ACGCUUCA G CUGUUGAU	5985	AUCAACAG GCCGAAAGGCGAGUGAGGUCU UGAAGCGU	6262
1577	CUUCAGCU G UUGAUCCG	5986	CGGAUCAA GCCGAAAGGCGAGUGAGGUCU AGCUGAAG	6263
1585	GUUGAUCC G CUGGUCCC	5987	GGGACCAG GCCGAAAGGCGAGUGAGGUCU GGAUCAAC	6264
1589	AUCCGCUG G UCCCCUCU	5988	AGAGGGGA GCCGAAAGGCGAGUGAGGUCU CAGCGGAU	6265
1604	CUUUUGGG G UAGAUGCG	5989	CGCAUCUA GCCGAAAGGCGAGUGAGGUCU CCCAAAAG	6266
1610	GGGUAGAU G CGGCCCCG	5990	CGGGGCCG GCCGAAAGGCGAGUGAGGUCU AUCUACCC	6267
1613	UAGAUGCG G CCCCGAUC	5991	GAUCGGGG GCCGAAAGGCGAGUGAGGUCU CGCAUCUA	6268
1624	CCGAUCAG G CCUGACUC	5992	GAGUCAGG GCCGAAAGGCGAGUGAGGUCU CUGAUCGG	6269
1633	CCUGACUC G CUGCUCUU	5993	AAGAGCAG GCCGAAAGGCGAGUGAGGUCU GAGUCAGG	6270
1636	GACUCGCU G CUCUUUUU	5994	AAAAAGAG GCCGAAAGGCGAGUGAGGUCU AGCGAGUC	6271
1645	CUCUUUUU G UUCCCUUC	5995	GAAGGGAA GCCGAAAGGCGAGUGAGGUCU AAAAAGAG	6272
1655	UCCCUUCU G UCUGCUCG	5996	CGAGCAGA GCCGAAAGGCGAGUGAGGUCU AGAAGGGA	6273
1659	UUCUGUCU G CUCGAACC	5997	GGUUCGAG GCCGAAAGGCGAGUGAGGUCU AGACAGAA	6274
1672	AACCACUU G CCUCGGGC	5998	GCCCGAGG GCCGAAAGGCGAGUGAGGUCU AAGUGGUU	6275
1679	UGCCUCGG G CUAAUCCC	5999	GGGAUUAG GCCGAAAGGCGAGUGAGGUCU CCGAGGCA	6276
1706	UCCACCCG G CACUGGGG	6000	CCCCAGUG GCCGAAAGGCGAGUGAGGUCU CGGGUGGA	6277
1717	CUGGGGAA G UCAAOAAU	6001	AUUCUUGA GCCGAAAGGCGAGUGAGGUCU UUCCCCAG	6278
1729	AGAAUGGG G CCUGGGGC	6002	GCCCCAGG GCCGAAAGGCGAGUGAGGUCU CCCAUUCU	6279
1736	GGCCUGGG G CUCUCAGG	6003	CCUGAGAG GCCGAAAGGCGAGUGAGGUCU CCCAGGCC	6280
1752	GGAGAACU G CUUCCCCU	6004	AGGGGAAG GCCGAAAGGCGAGUGAGGUCU AGUUUCUC	6281
1762	UUCCCCUG G CAGAGCUG	6005	CAGCUCUG GCCGAAAGGCGAGUGAGGUCU CAGGGGAA	6282
1767	CUGGCAGA G CUGGGUGG	6006	CCACCCAG GCCGAAAGGCGAGUGAGGUCU UCUGCCAG	6283
1772	AGAGCUGG G UGGCAGCU	6007	AGCUGCCA GCCGAAAGGCGAGUGAGGUCU CCAGCUCU	6284
1775	GCUGGGUG G CAGCUCUU	6008	AAGAGCUG GCCGAAAGGCGAGUGAGGUCU CACCCAGC	6285
1778	GGGUGGCA G CUCUUCCU	6009	AGGAAGAG GCCGAAAGGCGAGUGAGGUCU UGCCACCC	6286
1805	ACCGACCC G CCCGCCGC	6010	GCGGCGGG GCCGAAAGGCGAGUGAGGUCU GGGUCGGU	6287
1809	ACCCGCCC G CCGCUGUG	6011	CACAGCGG GCCGAAAGGCGAGUGAGGUCU GGGCGGGU	6288
1812	CGCCCGCC G CUGUGCCC	6012	CGGCACAG GCCGAAAGGCGAGUGAGGUCU CGCGCGCG	6289
1815	CCGCCGCU G UGCCCUGG	6013	CCACGGCA GCCGAAAGGCGAGUGAGGUCU AGCGGCCG	6290
1817	GCCGCUGU G CCCUGGGA	6014	UCCCAGGG GCCGAAAGGCGAGUGAGGUCU ACAGCGGC	6291
1826	CCCUGGGA G UGCUGCCC	6015	GGGCAGCA GCCGAAAGGCGAGUGAGGUCU UCCCAGGC	6292
1828	CUGGGAGU G CUGCCCUC	6016	GAGCGCAG GCCGAAAGGCGAGUGAGGUCU ACUCCCAG	6293
1831	GGAGUGCU G CCCUCUUA	6017	UAACAGGG GCCGAAAGGCGAGUGAGGUCU AGCACUCC	6294
1844	CUUACCAU G CACACGGG	6018	CCCGUGUG GCCGAAAGGCGAGUGAGGUCU AUGGUAAG	6295
1852	GCACACGG G UGCUCUCC	6019	GGAGAGCA GCCGAAAGGCGAGUGAGGUCU CCGUGUGC	6296
1854	ACACGGGU G CUCUCCUU	6020	AAGCAGAG GCCGAAAGGCGAGUGAGGUCU ACCCGUGU	6297
1867	CCUUUUGG G CUGCAUGC	6021	GCAUGCAC GCCGAAAGGCGAGUGAGGUCU CCAAAACG	6298
1870	UUUGGGCU G CAUGCUAU	6022	AUAGCAUG GCCGAAAGGCGAGUGAGGUCU AGCCCAAA	6299
1874	GGCUGCAU G CUAUUCCA	6023	UGGAAUAG GCCGAAAGGCGAGUGAGGUCU AUGCAGCC	6300
1887	UCCAUUUU G CAGCCAGA	6024	UCUGGCUG GCCGAAAGGCGAGUGAGGUCU AAAAUGGA	6301
1890	AUUUUGCA G CCAGACCG	6025	CGGUCUGC GCCGAAAGGCGAGUGAGGUCU UGCAAAAU	6302
1901	AGACCGAU G UGUAUUUA	6026	UAAAUACA GCCGAAAGGCGAGUGAGGUCU AUCGGUCU	6303
1903	ACCGAUGU G UAUUUAAC	6027	GUUAAAUA GCCGAAAGGCGAGUGAGGUCU ACAUCGGU	6304
1914	UUUAACCA G UCACUAUU	6028	AAUAGUCA GCCGAAAGGCGAGUGAGGUCU UGGUUAAA	6305
1936	ACAUUUGG G UUGUUUCC	6029	GGAAACAA GCCGAAAGGCGAGUGAGGUCU CCAAAUGU	6306
1939	UUUGGGUU G UUUCCCAU	6030	AUGGGAAA GCCGAAAGGCGAGUGAGGUCU AACCCAAA	6307
1954	AUCUUUUU G UUACCAUA	6031	UAUGGUAA GCCGAAAGGCGAGUGAGGUCU AAAAAGAU	6308
1970	AAAUAAUG G CAUAGUAA	6032	UUACUAUG GCCGAAAGGCGAGUGAGGUCU CAUUAUUU	6309
1975	AUGCCAUA G UAAAAAAA	6033	UUUUUUUA GCCGAAAGGCGAGUGAGGUCU UAUGCCAU	6310
Input Sequence = NM_003639. Cut Site = G/Y
Arm Length = 8. Core Sequence GCcgaaagGCGaGuCaaGGuCu
NM_003639 (Homo sapiens inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), mRNA.; 1994 bp)