Method and reagent for the inhibition of grid

BACKGROUND OF THE INVENTION

[0002] The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of the T-cell co-stimulatory adapter protein GRID (Grb2-related with Insert Domain).

[0003] The following is a brief description of the current understanding of GRID. The discussion is not meant to be complete and is provided only for understanding the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

[0004] One of the emerging paradigms for signal transduction in lymphocytes is that receptors and other signaling molecules do not operate in isolation, but through the recruitment of a complex of other proteins (Pawson and Scott, 1997; Science, 278, 2075; Rudd, 1999, Cell, 96, 5). These other proteins serve to amplify and diversify the signal into a number of biochemical cascades. The archetypal adapter protein is Grb2, which serves to regulate downstream pathways such as Ras activation and Ca2+ mobilization (Lowenstein et al., 1992, Cell, 70, 431), and is ultimately responsible for modulating gene expression required for proliferation and differentiation. Grb2 is recruited to LAT and SLP-76 which are downstream targets in the signaling cascade initiated by ligation of the T-cell receptor by MHC-antigen. These functions are mediated by specialized domains which bind specific motifs and include the phosphotyrosine binding SH2 (Src homology) domain and SH3 domain which are associated with proline-rich PXXP motifs. Grb2, whose sole function appears to be the formation of bridges between other proteins, is entirely comprised of such domains having an SH3-SH2-SH3 structure (Peterson et al., 1998, Curr. Opin. Immunol., 10, 337; Koretzky, 1997, Immunol Today, 18, 401).

[0005] A novel member of the Grb2 family of adapter proteins termed GRID (Grb2-related with Insert Domain) has recently been identified (Asada et al, 1999, J. Exp. Med., 189, 1383; Liu et al., 1999, Curr. Biol., 9, 67; Liu et al., 1998, Oncogene, 17, 3073; Law et al., 1999, J. Exp. Med., 189, 1243; Qiu et al., 1998, Biochem. Biophys. Res. Commun., 253, 443; Bourette et al., 1998, Embo. J, 17, 7273). GRID is recruited to the T cell co-stimulatory receptor CD28 upon activation of this receptor by cross-linking antibodies. Although GRID shares significant similarity at the protein level with Grb2, possessing an SH3-SH2-SH3 domain structure, GRID also contains a unique proline-glutamine rich domain situated between the SH2 and C-terminal SH3 domain. The association of GRID with activated CD28 is absolutely dependent upon the integrity of the SH2 domain and phosphorylation of residue Y173 in the cytoplasmic tail of CD28. Although GRID has been shown to associate with other T cell signaling proteins including SLP-76 and LAT (Asada et al., supra; Liu et al., supra; Law et al., supra), it's role in T cell signaling pathways is not well defined.

[0006] Tari et al., 1999, Oncogene, 18(6), 1325-1332, describe the antisense inhibition of Grb2 in breast cancer cells in order to investigate the role of Grb2 in the proliferation of breast cancer cells. The resulting Grb2 inhibition led to MAP kinase inactivation in EGFR but not in ErbB2 expressing breast cancer cells.

[0007] Tari et al., 1998, J. Liposome Res., 8(2), 251-264, describe P-ethoxy antisense oligonucleotides targeting Bcr-Ab1, Grb2, Crk1, and Bc1-2 mRNA. Delivery of these antisense oligonucleotides via liposome transfection results in the inhibition of corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines.

[0008] Lopez-Berestein et al., 1998, International PCT publication No. WO 98/01547, describe inhibition of chronic myelogenous leukemic cell growth by liposomal-antisense oligodeoxynucleotides targeting Grb2 and Crk1.

[0009] Tari et al., 1997, Biochem. Biophys. Res. Commun., 235(2), 383-388, describe the antisense-based inhibition of Grb2 and Crk1 proteins results in growth inhbition of Philadelphia chromosome positive leukemic cells.

SUMMARY OF THE INVENTION

[0010] The invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (for example, ribozymes or DNAzymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods for their use to modulate the expression of GRID (Grb2-related with Insert Domain).

[0011] The description below of the various aspects and embodiments is provided with reference to the exemplary gene GRID. However, the various aspects and embodiments are also directed to other genes which express GRID-like adapter proteins involved in T-cell co-activation. Those additional genes can be analyzed for target sites using the methods described for GRID. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

[0012] In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding GRID. For example, the nucleic acid-based techniques of the present invention can be used to inhibit the expression of GRID gene sequences found at GenBank Accession NOS. AJ011736, NM—004810, Y18051, AF121002, AF042380, AF129476, AF090456).

[0013] In another preferred embodiment, the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of GRID gene.

[0014] By “inhibit” it is meant that the activity of GRID or level of GRID RNAs or equivalent RNAs encoding one or more protein subunits of GRID or GRID-like proteins is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, the inhibition 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 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 of GRID or GRID-like genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.

[0015] 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).

[0016] By “nucleic acid molecule” as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

[0017] By “enzymatic portion” or “catalytic domain” is meant that portion or region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example, see FIGS. 1-5).

[0018] By “substrate binding arm” or “substrate binding domain” is meant that portion or 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. 1-5. That is, these arms contain sequences within an 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 four nucleotides and of sufficient length to stably interact with the target RNA. Preferably, the binding arm(s) are 12-100 nucleotides in length. More preferably, the binding arms are 14-24 nucleotides in length (see, for example, Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herrance et al., 1993, EMBOJ, 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).

[0019] By “Inozyme” or “NCH” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG. 2. 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. 2 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.

[0020] By “G-cleaver” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver in FIG. 2. 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 may be chemically modified as is generally shown in FIG. 2.

[0021] By “amberzyme” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 3. 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. 3. 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.

[0022] By “zinzyme” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 4. 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. 4, 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.

[0023] By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group for its 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. 5 and is generally reviewed in 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; 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.

[0024] 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.

[0025] 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.

[0026] 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).

[0027] By “equivalent” RNA to GRID is meant to include those naturally occurring RNA molecules having homology (partial or complete) to GRID proteins or encoding for proteins with similar function as GRID 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.

[0028] By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.

[0029] 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 cal., 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.

[0030] 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 preferably, 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.

[0031] By “2-5A antisense chimera” is meant an antisense oligonucleotide 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).

[0032] By “triplex forming oligonucleotides” 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).

[0033] By “gene” it is meant a nucleic acid that encodes RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.

[0034] “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, CSHSymp. 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.

[0035] 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.

[0036] By “decoy RNA” is meant a RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural 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.

[0037] Several varieties of naturally occurring 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.

[0038] The enzymatic nucleic acid molecule that cleave the specified sites in GRID-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to tissue/graft rejection and leukemia.

[0039] In one of the preferred embodiments 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. Examples of hairpin motifs are described 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, Hampel et al., 1990 Nucleic Acids Res. 18, 299; and Chowrira & McSwiggen, U.S. Pat. No. 5,631,359. The hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16. The RNase P motif is described by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; and Li and Altman, 1996, Nucleic Acids Res. 24, 835. The 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; and 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; and Pyle et al., International PCT Publication No. WO 96/22689. The Group I intron is described by Cech et al., U.S. Pat. No. 4,987,071. DNAzymes are described 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; and Santoro et al., 1997, PNAS 94, 4262. 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 include the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG. 3; Beigelman et al., International PCT publication No. WO 99/55857) and Zinzyme (Beigelman et al., International PCT publication No. WO 99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention. These specific motifs 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).

[0040] In preferred embodiments of the present invention, a nucleic acid molecule of the instant invention can be between 13 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-VIII and X. For example, enzymatic nucleic acid molecules of the invention are preferably between 15 and 50 nucleotides in length, more preferably between 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 15 and 40 nucleotides in length, more preferably between 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 and Cairns et al., 2000, Antisense & Nucleic Acid Drug Dev., 10, 323-332). Exemplary antisense molecules of the invention are preferably between 15 and 75 nucleotides in length, more preferably between 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 10 and 40 nucleotides in length, more preferably between 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 for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.

[0041] Preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 12 and 100 bases complementary to a GRID or GRID-like RNA. Even more preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 14 and 24 bases complementary to a GRID or GRID-like RNA.

[0042] In a preferred embodiment, the invention provides a method for producing a class of nucleic acid-based gene inhibiting 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 GRID or GRID-like proteins 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 target cells.

[0043] In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target genes that share homology with the GRID gene. For example, the invention describes the use of nucleic acid-based inhibitors to target the Grb2 (GenBank accession No. NM—002086) and GRAP (GenBank accession No. NM—006613) genes.

[0044] As used in herein “cell” is used in its usual biological sense and does not refer to an entire multicellular organism. The cell can be present in an organism which includes humans but is preferably a non-human multicellular organism, e.g., birds, plants and mammals such as 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).

[0045] By “GRID proteins” is meant, a protein or a mutant protein derivative thereof, comprising an adapter-protein type of association to the activated CD28 co-stimulatory receptor, and to other signaling proteins including but not limited to SLP-76 and LAT.

[0046] By “highly conserved sequence region” is meant a nucleotide sequence of one or more regions in a target gene that does not vary significantly from one generation to the other or from one biological system to the other.

[0047] The nucleic acid-based inhibitors of GRID expression are useful for the prevention and/or treatment of diseases and conditions that are related to or will respond to the levels of GRID in a cell or tissue, alone or in combination with other therapies. For example, the nucleic acid-based inhibitors of GRID expressions are useful for the prevention and/or treatment of tissue/graft rejection and cancer, such as leukemia, among other conditions.

[0048] By “related” is meant that the reduction of GRID expression (specifically GRID gene) RNA levels and thus reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.

[0049] In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target regions of GRID gene that are not homologous to Grb2 gene. Specifically, the invention describes the use of nucleic acid-based inhibitors to target sequences that are unique to GRID gene.

[0050] 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 using well-known methods described herein and generally known in the art. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate sequences in Tables III to X. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to VIII and X. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.

[0051] In yet 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 X. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to VIII and X and sequences shown as GeneBloc™ sequences in Table X. 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.

[0052] 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 and IV 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 a sequence X, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO 2236) 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 may be present that do not interfere with the function of the nucleic acid molecule.

[0053] 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. Alternatively or in addition, sequence X can be a non-nucleotide linker.

[0054] 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.

[0055] In yet another embodiment, the non-nucleotide linker X is as defined herein. The term “non-nucleotide linker” as used herein include either 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. The term “non-nucleotide” further refers to 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.

[0056] In another aspect of the invention, ribozymes or antisense molecules that interact with target RNA molecules and inhibit GRID activity (e.g., inhibit GRID gene) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme 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 ribozymes or antisense are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of ribozymes or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression. Delivery of ribozyme or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed endogenously via the use of a single stranded DNA intracellular expression vector.

[0057] By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

[0058] By “patient” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.

[0059] 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 individual catalytic 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.

[0060] 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 GRID, the patient 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.

[0061] In a further embodiment, the described 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 tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.

[0062] In another preferred embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., GRID) related to the progression and/or maintenance of tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.

[0063] In another aspect, the invention provides mammalian cells containing one or more nucleic acid molecules and/or expression vectors of this invention. The one or more nucleic acid molecules can independently be targeted to the same or different sites.

[0064] 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 may or may 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 may be present.

[0065] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] First the drawings will be described briefly.

[0067] Drawings

[0068]
FIG. 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage . . . indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions.—is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al., 1994, Nature Struc. Bio., 1, 273). RNase P (M1RNA): EGS represents external guide sequence (Forster et al., 1990, Science, 249, 783; Pace et al., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5′SS means 5′ splice site; 3′SS means 3′-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577). HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al., U.S. Pat. No. 5,625,047). Hammerhead Ribozyme:: I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and can be symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527). Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 can be covalently linked by one or more bases (i.e., r is >1 base). Helix 1, 4 or 5 can also be extended by 2 or more base pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N′ independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides can be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more can be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present can be a ribonucleotide with or without modifications to its base, sugar or phosphate. “q”≧is 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. “______” refers to a covalent bond. (Burke et al, 1996, Nucleic Acids & Mol. Biol., 10, 129; Chowrira et al., U.S. Pat. No. 5,631,359).

[0069]
FIG. 2 shows examples of chemically stabilized ribozyme motifs. HH Rz, represents hammerhead ribozyme motif (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT Publication No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research 26, 4116-4120). N or n, represent independently a nucleotide which can be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is shown as having 2′-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.

[0070]
FIG. 3 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al., International PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class I Motif). The Amberzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2′-OH) group for its activity.

[0071]
FIG. 4 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al., International PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class A or Class II Motif). The Zinzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2′-OH) group for its activity.

[0072]
FIG. 5 shows an example of a DNAzyme motif described by Santoro et al., 1997, PNAS, 94, 4262.

[0073]
FIG. 6 shows a graph of optimization of GeneBloc concentration. A fluoresceinated randomized antisense GeneBloc (fGB) was used as a marker for uptake using a fixed concentration of lipid. Cells were either untreated (A) or treated continuously for 24hrs with 10-200 nM antisense GeneBloc (B-F). Following treatment, cells were analyzed by flow cytometry. Gate M1 represents either untransfected cells or cells refractory to transfection. Gate M2 represents the transfected cells.

[0074]
FIG. 7 shows a bar graph of a primary screen of twelve GRID GeneBlocs. Taqman mRNA assay was used to quantify the level of GRID transcript in Jurkat cells treated continuously for 24 hours with 100 nM antisense GeneBloc and 5.0 μgml−1 cationic lipid. For comparison, all data was normalized to the level of β-actin. Error bars represent the standard error of the mean of triplicate points.

[0075]
FIG. 8 shows a graph demonstrating that flow cytometric sorting of transfected cells improves antisense GeneBloc mediated inhibition of GRID mRNA expression. Jurkat cells were treated continuously for 24 and 72 hours with GB14540 (75 nM) or control GeneBloc GBC3.3 (75 nM) spiked with 25 nM fluorescent randomized GeneBloc (A) to facilitate the identification of transfected cells. After transfection, the 10% most and least fluorescent cells (gates M2 and M1 respectively) were sorted on a FACStar Plus. Post-sort low transfecting (B) and high transfecting (C) fractions were re-analyzed for purity. Histograms A-D are representative of results obtained in all experiments and were taken from cells treated for 72 hours. The GRID mRNA content of all samples was quantified by Taqman RNA assay and normalized to the β-actin content. For the purposes of inter-experiment comparison, all GB14540 values were also normalized to the appropriate control GBC3.3 value. (D) Normalized GRID mRNA levels in pre-sort samples; (E) Normalized GRID mRNA levels in the post-sort low transfecting fraction; (F) Normalized GRID mRNA levels in the post-sort high transfecting fraction. Error bars represent the range of duplicate points.

[0076]
FIG. 9 shows a graph representing the phenotypic analysis of antisense GeneBloc treated Jurkat cells following activation with anti-CD3 and anti-CD28 anti-sera. Jurkat cells were treated continuously for 72 hours with the anti-GRID reagent GB14540 (A, C) and the mismatch control reagent GB17477 (B, D), activated for 22 hours (C, D) and stained for the surface activation marker CD69. Unactivated samples are shown in (A, B).

MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

[0077] Antisense:

[0078] Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides which primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 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 Oneogenesis 7, 151-190).

[0079] 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 known to 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.

[0080] 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.

[0081] 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 endogenously in vivo via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.

[0082] Triplex Forming Oligonucleotides (TFO):

[0083] Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra). The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism can result in gene expression or cell death since binding may be irreversible (Mukhopadhyay & Roth, supra).

[0084] 2-5A Antisense Chimera:

[0085] The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2′-5′ oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.

[0086] (2′-5′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.

[0087] Enzymatic Nucleic Acid:

[0088] Several varieties of naturally occurring 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; Nakamaye & 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.

[0089] Nucleic acid molecules of this invention can block to some extent GRID protein expression and can be used to treat disease or diagnose disease associated with levels of GRID.

[0090] The enzymatic nature of an enzymatic nucleic acid has significant advantages, such as the concentration of enzymatic nucleic acid necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid 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 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 completely eliminate catalytic activity of an enzymatic nucleic acid molecule.

[0091] 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).

[0092] Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules show promise 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).

[0093] The nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (e.g., ribozymes, antisense) capable of down-regulating gene expression.

[0094] GeneBlocs are modified oligonucleotides, including ribozymes and modified antisense oligonucleotides, that bind to and target specific mRNA molecules. Because GeneBlocs can be designed to target any specific mRNA, their potential applications are quite broad. Traditional antisense approaches have often relied heavily on the use of phosphorothioate modifications to enhance stability in biological samples, leading to a myriad of specificity problems stemming from non-specific protein binding and general cytotoxicity (Stein, 1995, Nature Medicine, 1, 1119). In contrast, GeneBlocs contain a number of modifications that confer nuclease resistance while making minimal use of phosphorothioate linkages, which reduces toxicity, increases binding affinity, and minimizes non-specific effects compared with traditional antisense oligonucleotides. Similar reagents have recently been utilized successfully in various cell culture systems (Vassar, et al., 1999, Science, 286, 735) and in vivo (Jarvis et al., manuscript in preparation). In addition, novel cationic lipids can be utilized to enhance cellular uptake in the presence of serum. Since ribozymes and antisense oligonucleotides regulate gene expression at the RNA level, the ability to maintain a steady-state dose of GeneBloc over several days is important for target protein and phenotypic analysis. The advances in resistance to nuclease degradation and prolonged activity in vitro have supported the use of GeneBlocs in target validation applications.

[0095] Target Sites

[0096] Targets for useful ribozymes 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. All of these publications are hereby incorporated by reference herein in their 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, all of which are incorporated by reference herein. Rather than repeat the guidance provided in those documents here, specific examples of such methods are provided herein, not limiting to those in the art. Ribozymes 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 GRID 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 ribozyme binding/cleavage sites were identified. These sites are shown in Tables III to VIII and X (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence or linker X as previously defined herein, 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 ribozymes are also useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.

[0097] Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified. The nucleic acid molecules were 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, were eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.

[0098] Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme 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; Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684; and Caruthers et al., 1992, Methods in Enzymology 211,3-19.

[0099] Synthesis of Nucleic Acid Molecules

[0100] Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., 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 be similarly synthesized.

[0101] Oligonucleotides (e.g.; 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-methyl imidazole 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.

[0102] 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:H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

[0103] The method of synthesis used for normal 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; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 and 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-methyl imidazole 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.

[0104] 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:H20/3:1:1, 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.

[0105] 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.

[0106] 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.

[0107] Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides) are 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.

[0108] 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 examples described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.

[0109] 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).

[0110] 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.

[0111] The sequences of the ribozymes and antisense constructs that are chemically synthesized, useful in this study, are shown in Tables III to X. 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 ribozyme and antisense construct sequences listed in Tables III to X can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.

[0112] Optimizing Activity of the Nucleic Acid Molecule of the Invention.

[0113] 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; 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 described herein). All these references are incorporated by reference 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 preferably desired.

[0114] 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 modifications 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 by reference herein in their totalities). 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. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.

[0115] While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications may 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.

[0116] Use of the nucleic acid-based molecules of the invention can lead to improved 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 patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.

[0117] Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously should preferably be 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. The nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents when delivered exogenously. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art (see, e.g., Wincott et al., 1995, Nucleic Acids Res., 23:2677; Carruthers, et al., 1992, Methods in Enzymology, 211:3-19, each 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.

[0118] In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic 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 may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to “maintain” the enzymatic activity of an all RNA ribozyme.

[0119] In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′- cap structure.

[0120] 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 termini. In non-limiting examples, the 5′-cap is selected from the group consisting of 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).

[0121] Suitable 3′-caps include 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).

[0122] 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.

[0123] An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH. The term “alkyl” also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino or SH.

[0124] Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group which has at least one ring having a conjugated π electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

[0125] 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, 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-methylguano sine, N6-methyladeno sine, 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).

[0126] 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.

[0127] 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).

[0128] 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.

[0129] In a preferred embodiment, the invention features modified ribozymes 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.

[0130] By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, (for more details, see Wincott et al., International PCT publication No. WO 97/26270).

[0131] By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.

[0132] 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.

[0133] 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 herein in their entireties.

[0134] Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

[0135] Use of these molecules can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes (including different ribozyme motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules. Therapies can be devised which include a mixture of ribozymes (including different ribozyme motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.

[0136] Administration of Nucleic Acid Molecules

[0137] Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategiesfor Antisense Oligonucleotide Therapeutics, ed.

[0138] 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 may 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 incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, nucleic acid molecules can be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. 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.

[0139] The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (i.e., alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.

[0140] The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient 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 as described in the art. 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 other compositions known in the art.

[0141] 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, including salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

[0142] 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 patient, 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.

[0143] By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes 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 that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

[0144] By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

[0145] The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). All incorporated by reference herein. 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, Biochini. Biophys. Acta, 1238, 86-90). All incorporated by reference herein. 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.

[0146] 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 may be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

[0147] 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.

[0148] 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.

[0149] The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects.

[0150] 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 the references are hereby incorporated in their totality 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 ribozyme (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).

[0151] 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 followed by reintroduction into the patient, or by any other means that allow for introduction into the desired target cell (for a review, see Couture et al., 1996, TIG., 12, 510).

[0152] In one aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules disclosed in the instant invention. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.

[0153] 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).

[0154] 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 po1 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 also can be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 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.

[0155] 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. U S A, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 45 81-9; 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; and 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; and 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).

[0156] In yet another aspect, the invention features an expression vector comprising a 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.

[0157] In another preferred 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.

[0158] 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.

[0159] 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.

EXAMPLES

[0160] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

[0161] The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver enzymatic nucleic acid molecules and binding/cleavage sites within GRID RNA.

[0162] Nucleic Acid Inhibition of GRID Target RNA

[0163] The use of GeneBlocs to modulate the activity of GRID, a putative component of co-stimulatory signaling in T cells, is herein described. An array of GeneBlocs were designed and screened for their ability to reduce GRID mRNA levels whilst leaving transcripts from the closely related genes Grb2 and GRAP unaffected. A series of experiments were conducted to optimize delivery of GeneBlocs to the Jurkat T cell line. Using these conditions, applicant has demonstrated the efficacy of these reagents at both the mRNA and protein level. Anti-CD3/CD28 triggering of Jurkat cells pre-treated with the anti-GRID GeneBloc results in an impairment of CD69 up-regulation consistent with an important role for GRID in transducing the co-stimulatory signal.

Example 1

Identification of Potential Target Sites in Human GRID RNA

[0164] The sequence of human GRID were screened for accessible sites using a computer-folding algorithm. Regions of the RNA were identified that do not form secondary folding structures. These regions contain potential ribozyme and/or antisense binding/cleavage sites. The sequences of these binding/cleavage sites are shown in Tables III-X.

Example 2

Selection of Enzymatic Nucleic Acid Cleavage Sites in Human GRID RNA

[0165] Enzymatic nucleic acid target sites are chosen by analyzing sequences of Human GRID (for example, GenBank accession numbers: AJ011736 and Y18051) and prioritizing the sites on the basis of folding. Enzymatic nucleic acids are designed that bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid sequences fold into the appropriate secondary structure. Those enzymatic nucleic acids with unfavorable intramolecular interactions between the binding arms and the catalytic core are 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.

Example 3

Chemical Synthesis and Purification of Enzymatic Nucleic Acids and Antisense for Efficient Cleavage and/or Blocking of GRID RNA

[0166] Enzymatic nucleic acids and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acids are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The enzymatic nucleic acids and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA or DNA 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 dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%.

[0167] Enzymatic nucleic acids and antisense constructs also can be synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid 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 and antisense constructs used in this study are shown below in Table III-X.

Example 4

Enzymatic Nucleic Acid Cleavage of GRID RNA Target in vitro

[0168] Enzymatic nucleic acids targeted to the human GRID RNA are designed and synthesized as described above. These enzymatic nucleic acids can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.

[0169] Cleavage Reactions:

[0170] Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid 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 in enzymatic nucleic acid 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 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 ribozyme, i.e., enzymatic nucleic acid 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 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.

Example 5

Nucleic Acid Inhibition of GRID in vivo

[0171] Antisense nucleic acid molecules (GeneBlocs) targeted to the human GRID RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.

[0172] GRID shares 60.3% and 57.3% homology at the nucleotide level with the closely related adapter proteins Grb2 and GRAP. In order to discriminate between human GRID and other Grb2 family members, twelve GeneBlocs (see Methods for details) targeting human GRID (GenBank accession number Y18051) were designed, each containing a minimum of six mismatches versus human Grb2 (M96995) and human GRAP (U52518). In order to determine the optimal site for GeneBloc binding and inhibition of the target mRNA, the efficacy of the GeneBlocs was tested on Jurkat cells. A Taqman RNA assay was used to quantify the level of GRID transcript in cells treated continuously for 24hrs. The efficacy of the twelve GeneBlocs, normalized to the levels of a house-keeping gene (β-actin), is shown in FIG. 7. The GeneBloc targeting site 152 (GeneBloc 14540) was the most efficacious, reducing GRID mRNA levels by up to 55% when compared with a randomized control GeneBloc (GBC3.3). To confirm that these effects were target specific, a four base-pair mismatch GeneBloc (GB17477) was synthesized. GRID mRNA expression was unaffected in cells treated with the mismatch control GeneBloc compared to untreated cells.

[0173] Efficacy of the Anti-GRID GeneBloc (GB14540) in Jurkat Cells

[0174] From the primary screen (FIG. 7), the optimal GeneBloc, GB14540, suppressed GRID mRNA levels by up to 55%. However, this represents the inhibition in a bulk population of cells, some of which are refractory to transfection (see FIG. 6D-F). To investigate the correlation between dose and efficacy, GB14540 was spiked with 25% fGB. Based on mixture experiments with active GeneBlocs in other systems, it was not expected that the presence of the fluorescent GeneBloc would interfere with anti-GRID activity of GB14540. Thus, the most highly fluorescent cells represent the population of cells transfected with the highest concentration of active GeneBloc (‘high transfecting’), whilst the cells that appear to be refractory to transfection should contain a significantly lower concentration active GeneBloc (‘low transfecting’).

[0175] Following transfection of a GB14540:fGB mixture, the high transfecting cells (FIG. 8A, Gate M2, the 10% most fluorescent cells) and the low transfecting cells (FIG. 8A, Gate M1, the 10% least fluorescent cells) were purified by FACS sorting. Re-analysis of the sorted cell populations confirmed greater than 95% purity (FIG. 8B-C). Taqman RNA analysis of the treated cells pre- and post-sort (FIG. 8D-F) shows that although GB14540 inhibition of GRID mRNA expression in an unsorted population is variable between experiments (0-30%, FIG. 8D), the level of inhibition is significantly increased to 45-63% in the ‘high transfecting’ fraction (FIG. 8F). In contrast, GRID mRNA levels in the ‘low transfecting’ fraction was similar to that of cells treated with control GBC3.3 (FIG. 8E). These data suggest that the degree of GRID mRNA inhibition is dependent on the dose of GeneBloc delivered to the cells.

[0176] To identify the optimal time-point for inhibition of GRID mRNA levels, samples were sorted as described above at 24 and 72 hours following continuous transfection. Analysis of pre- and post-sort samples at these time-points revealed that in pre-sort samples, inhibition of GRID transcript occurred within 24 hours and did not significantly increase throughout the time-course of the experiment (FIG. 8D). In the ‘high transfecting’ fractions, reduction of GRID transcript was ˜45% at 24 hours and increased only fractionally at the 72 hour time-point (50-65%, FIG. 8F). This suggests that GB14540 reduced GRID mRNA levels rapidly following transfection and that inhibition was sustained in the continued presence of GB14540.

[0177] Analysis of GRID Protein Levels in GB14540 Treated Cells

[0178] To determine whether the reduction in GRID transcript levels was associated with a loss of GRID protein, the level of GRID protein in cells treated continuously with active GeneBloc reagent GB14540 and the mismatch control GB17477 was assessed. When delivered continuously for 72 hours, GB14540 caused a substantial reduction in GRID protein levels as determined by the intensity of the GRID specific band whilst at earlier time-points (24 and 48 hrs) no reduction in protein was observed. Cells treated with the mismatch control GB17477 showed GRID levels comparable to the untreated sample. Cells treated continuously with GB14540 for periods up to 144 hours showed no further reduction in GRID protein levels, suggesting that the effect of the GeneBloc was maximal and sustained from 72 hours onwards. Whilst the effects of the anti-GRID GeneBloc on MRNA levels are seen at 24 hours, the reduction in GRID protein is delayed a further 48 hours indicating that GRID protein may have a relatively long half-life.

[0179] The GeneBlocs were designed to target and discriminate GRID from the closely related adapter proteins Grb2 and GRAP. GB14540 contains 6 and 7 mismatches respectively when aligned with the human Grb2 and GRAP sequences. Due to the presence of these mismatches, GB14540 was not expected to inhibit Grb2 mRNA expression. The Western blots used for the GRID assay were stripped and re-probed using an anti-Grb2 antibody. No difference in Grb2 protein levels was observed between the untreated sample and cells treated with either GB14540 or the mismatch control reagent GB17477, confirming that the GB14540 was specific for GRID.

[0180] Phenotypic Effects of the Anti-GRID GeneBloc on T Cell Activation

[0181] GRID is a novel member of the Grb2 family of adapter proteins. A role for GRID in T cell signaling has been postulated due to its association with known T cell signaling proteins [Law, 1999 #3296][Asada, 1999 #3243][Liu, 1999 #3245] and more recently the T cell co-stimulatory receptor CD28 following activation by cross-linking antibodies (Ellis et al.). To further elucidate the role of GRID in T cell co-stimulatory pathways, applicant studied the expression of early surface activation marker CD69 (Jung et al., 1988, Cellular Immunology, 117, 352, Lanier et al., 1988, J. Exp. Med., 167, 1572) following activation of Jurkat cells treated with GB14540 and GB17477. Jurkat cells were activated by cross-linking anti-CD3 and anti-CD28 monoclonal antibodies using a sub-maximal stimulus to increase the sensitivity of the assay. In cells treated with the mismatch control GeneBloc, GB17477, 5.7% stained CD69 positive following activation compared with 0.7% CD69 positive in unactivated cells (FIG. 9D vs. 9B). In cells treated with the anti-GRID reagent GB14540, there was a marked reduction in the proportion of activated cells, with only 1.3% staining positive for CD69 (FIG. 9C). Expression of CD69 in the unactivated sample remained unaltered at 0.6% (FIG. 9A). As the activation stimulus was increased, the relative difference between the cells treated with GB14540 and GB17477 decreased even though the proportion of cells staining positive for CD69 increased. This can be attributed to the combination of residual GRID protein and supra-maximal activation stimulus. The latter component is particularly relevant to T cell activation since the dependency on co-stimulation is reduced as the strength of the CD3 signal increases (Geppert and Lipsky, 1988, J. Clin. Invest., 81, 1497, Geppert and Lipsky, 1987, Journal of Immunology, 138, 1660).

[0182] Taken together, these data suggest that the phenotypic effects described above can be attributed to GRID and not the closely related adapter protein Grb2. The inhibitory effects of GB14540 on CD69 expression support a role for GRID in T cell co-stimulatory signaling.

Example 6

Delivery of GeneBloc Reagents to Jurkat Cells

[0183] As in many mammalian cell culture systems (Marcusson et al., 1998, Nuc. Acids, Res. 26, 2016), a cationic lipid was found to be necessary to facilitate cellular uptake of oligonucleotide. In preliminary experiments using a fluoresceinated randomized GeneBloc as a marker for uptake, a lipid concentration of 2.5-5.0 μgml−1 was found to be optimal. Although some cells are readily transfected by the GeneBloc, a sub-population of cells remained refractory to transfection (see Gate M2 vs. M1 in FIGS. 6D-6F). In order to minimize the refractory population, the concentration of GeneBloc was varied between 10-200 nM. Transfection frequencies of up to 75% (as determined by fraction of cells in Gate M2) were observed in the 50-100 nM range of GeneBloc concentration. At lower concentrations (10-25nM), the transfection frequency dropped off very steeply whilst at higher concentrations, no further enhancement of transfection was observed. Cationic lipids however are not essential for the use of oligonucleotides in vivo (see McGraw et al., 1997, Anti-Cancer Drug Design, 12, 315-326; Henry et al., 1997, Anti-Cancer Drug Design, 12,409-420).

Example 7

Flow Cytometry

[0184] Cultures were harvested, washed once and re-suspended in PBS containing 2% FCS. Cells were stained with a human anti-CD69 PE-conjugated antibody (Caltag) using an IgG2a PE-conjugate as an isotype control (Becton Dickinson). Cells were analyzed on a Becton Dickinson FACScan using CellQuest software. Cells were sorted on the basis of fluorescence in the FL1 channel using a Becton Dickinson FACStar Plus. In order to compare the efficiency of GeneBloc uptake using different transfection conditions, a coefficient of transfection was calculated by multiplying the proportion of control GeneBloc (as a fraction of total GeneBloc) and the transfection frequency.

Example 8

Protein Studies

[0185] Actively growing Jurkat cells (0.1-1.0×106) were harvested, washed once in PBS and re-suspended in 25 μl PBS. Cells were lysed by the addition of an equal volume of ice-cold 2× RIPA buffer (2% NP40, 1.0% sodium deoxycholate, 0.2% SDS in PBS with 2× protease and phosphatase inhibitors). Following a 30 minute incubation on ice, cell debris was removed by centrifugation and the supernatant denatured at 100° C. for 5 minutes following the addition of an equal volume of 2× SDS protein sample buffer. Prior to separation by SDS-PAGE electrophoresis, protein content was normalized using a Coomassie™ Plus-200 protein assay reagent (Pierce). For Western blotting, SDS-PAGE gels were transferred to PVDF membrane (Millipore). Antisera specific for GRID (rabbit polyclonal courtesy of Claire Ashman, GlaxoWellcome), p85 sub-unit of PI-3-kinase (#06-195, Upstate Biotechnology) and Grb2 (sc-255, Santa Cruz) were used as primary antibodies with an anti-rabbit HRP conjugate as the secondary antibody. Bound antibody was visualized using the SuperSignal® West Dura chemiluminescent reagent. For re-probing, chemiluminescent substrate and bound antibody were removed with TBST (TBS +0.5% Tween-20) and ImmunoPure® IgG Elution Buffer (Pierce) respectively.

Example 9

Cell Culture

[0186] Human Jurkat cell lines E6.1 and J6 were maintained at 37° C. in 5% CO2 in flasks in RPMI 1641 (+25 mM HEPES) supplemented with 10% fetal calf serum and glutamine. Cells were passaged at a density of 1×106 cells ml−1. GeneBlocs were delivered to the cells using a modified centrifugation-based transfection protocol (Verma et al., 1998, BioTechniques, 25, 46). Cells were grown to a density of 1×106 cells ml−1, harvested by centrifugation and re-suspended in fresh media at 0.75×106 cells ml−1. GeneBloc at 10× final concentration and cationic lipid (25 μgml−1) at 10× final concentration were prepared separately in RPMI media (no FCS or glutamine), mixed 1:1 and incubated at 37° C. for 30 minutes. 1.6 ml aliquots of the cell suspension was dispensed into a 6-well tissue-culture treated plate and 0.4 ml of the GeneBloc:lipid mixture added drop-wise. The GeneBloc:lipid solution was evenly distributed by gentle agitation. Following centrifugation at 1000 rpm for 60 minutes at room temperature, the 6-well plates were incubated for 24-72 hours at 37° C.

Example 10

Real-time Quantitative PCR (Taqman)

[0187] Human GRID oligonucleotide Taqman probe 6FAM-(5′-ACTCCAGTTTCCCAAATGGTTTCACGAA-3′) (SEQ ID NO 2237)-TAMRA and human actin Taqman probe JOE-(5′-TCGAGCACGGCATCGTCACCAA-3′) (SEQ ID NO 2238)-TAMRA were purchased from PE Applied Biosystems. GRID primers (forward, 5′-AGGATATGTGCCCAAGAATTTCATA-3′) (SEQ ID NO 2239) and reverse, (5′-TGCCTGGTGTCGAGAGAGG-3′) (SEQ ID NO 2240) and actin primers (forward, 5′-GCATGGGTCAGAAGGATTCCTAT-3′) (SEQ ID NO 2241) and reverse, (5′-TGTAGAAGGTGTGGTGCCAGATT-3′) (SEQ ID NO 2242) were purchased from Life Technologies. The Taqman probes were labeled with a reporter dye (FAM or JOE) at the 5′ termini and a quencher dye (TAMRA) at their 3′ termini. A combination RT-PCR and Taqman PCR was performed for each sample in triplicate on an ABI PRISM 7700 Sequence Detection System using the following program: 48° C. for 30 minutes, 95° C. for 10 minutes and then 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute. The reaction was performed in a total volume of 40 μl with each tube containing 10 U RNase inhibitor (Promega), 1.25 U Amplitaq Gold (PE Biosystems), 100 nM of the GRID and Actin primers, 100 nM GRID FAM Taqman probe, 100 nM Actin JOE Taqman probe and 10 U MuLV reverse transcriptase. PCR Buffer (PE Biosystems #4304441) and dNTPs (PE Biosystems #N808-0261) were added according to the manufacturer's guidelines. A standard curve was generated using serially diluted purified RNA (300, 100, 33 and 11 ng) prepared from untreated Jurkat cells.

Example 11

RNA Isolation

[0188] Total RNA was isolated from Jurkat J6 or Jurkat E6.1 cells using the 96-well RNeasy kit (Qiagen) and a minor modification of their protocol. 90 μl of RLT buffer was added to each sample, followed by an equal volume of 70% ethanol. Samples were mixed and transferred to a RNeasy-96-plate. A vacuum was applied for 15-60 sec until the wells were dry. 80 μl of 1× DNase solution was added (40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM CaCl2, 10 mM NaCl, 1.2 U/μl RNase-free DNase I). Following incubation at room temperature for 15 minutes, 1 ml of Buffer RW1 was added and incubated for a further 5 minutes. The buffer was removed by applying a vacuum. The wells were washed once in 1 ml of RPE. A second 1 ml aliquot of Buffer RPE was added and the RNeasy-96-plate centrifuged at 6000 rpm for 10 minutes. The RNA was eluted by the addition of 100 ml of RNase-free water. Following incubation at room temperature for 1 minute, the RNA was recovered by centrifugation at 6000 rpm for 4 minutes and stored at −70° C.

[0189] Indications

[0190] Particular conditions and disease states that can be associated with GRID expression modulation include, but are not limited to. tissue/graft rejection and cancer, such as leukemia.

[0191] The present body of knowledge in GRID research indicates the need for methods to assay GRID activity and for compounds that can regulate GRID expression for research, diagnostic, and therapeutic use.

[0192] Radiation, chemotherapeutic treatments, and Cyclosporin are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. 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.

[0193] Diagnostic Uses

[0194] The nucleic acid molecules of this invention (e.g., ribozymes) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of GRID RNA in a cell. The close relationship between ribozyme 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 ribozymes 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 ribozymes 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 ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention include detection of the presence of mRNAs associated with GRID-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

[0195] In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme 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 ribozymes to demonstrate the relative ribozyme 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 can require two ribozymes, two substrates and one unknown sample, which are 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., GRID) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and 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.

[0196] Additional Uses

[0197] Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention 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 describes 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.

[0198] 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.

[0199] 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 which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0200] 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.

[0201] The invention illustratively described herein suitably may 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” may 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 may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

[0202] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0203] Other embodiments are within the following claims.

1TABLE ICharacteristics of naturally occurring ribozymesGroup I IntronsSize: ˜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 generatecleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintenance of the active structure.Over 300 known members of this class. Found as an interveningsequence in Tetrahymena thermophila rRNA, fungal mitochondria,chloroplasts, phage T4, blue-green algae, and others.Major structural features largely established through phylogeneticcomparisons, 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 wellestablished [x,xi].The small (4-6 nt) binding site may make this ribozyme too non-specificfor targeted RNA cleavage, however, the Tetrahymena group I intronhas been used to repair a “defective” beta-galactosidase message bythe ligation of new beta-galactosidase sequences onto the defectivemessage[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 cleavageproducts with 3′-OH and 5′-phosphate.RNAse P is found throughout the prokaryotes and eukaryotes. TheRNA subunit has been sequenced from bacteria, yeast, rodents, andprimates.Recruitment of endogenous RNAse P for therapeutic applications ispossible through hybridization of an External Guide Sequence (EGS) tothe target RNA [xiv,xv]Important phosphate and 2′ OH contacts recently identified [xvi,xvii]Group II IntronsSize: >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 cleavageproducts with 3′-OH and a “lariat” RNA containing a 3′-5′ and a 2′-5′branch point.Only natural ribozyme with demonstrated participation in DNAcleavage [xx,xxi] in addition to RNA cleavage and ligation.Major structural features largely established through phylogeneticcomparisons [xxii].Important 2′ OH contacts beginning to be identified [xxiii]Kinetic framework under development [xxiv]Neurospora VS RNASize: ˜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 generatecleavage 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 generatecleavage 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 crystalstructures [xxvi,xxvii]Minimal ligation activity demonstrated (for engineering through invitro selection) [xxviii]Complete kinetic framework established for two or more ribozymes [xxix].Chemical modification investigation of important residues wellestablished [xxx].Hairpin RibozymeSize: ˜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 variablenumber to the 3′-side of the cleavage site.Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generatecleavage products with 2′,3′-cyclic phosphate and 5′-OH ends.3 known members of this class. Found in three plant pathogen (satelliteRNAs of the tobacco ringspot virus, arabis mosaic virus and chicoryyellow 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 ribozymeamenable 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) RibozymeSize: ˜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. Foldedribozyme contains a pseudoknot structure [xl].Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generatecleavage products with 2′,3′-cyclic phosphate and 5′-OH ends.Only 2 known members of this class. Found in human HDV.xliCircular form of HDV isxlii active and shows increased nucleasestability [xliii][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 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 Oigonucleotides: 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] Scoff, W.G., Finch, J.T., Aaron, K. The crystal structure of an all RNA hammerhead ribozyme:A proposed 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 xlii [xliii] Puttaraju, M.; Perrotta, Anne T.; Been, Michael D. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

[0204]

2

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 Imidazole
186
233 μL
5 sec
5 sec
5 sec

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 Imidazole
1245
124 μL
5 sec
5 sec
5 sec

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/

Wait Time*
Wait Time*

Reagent
2′-O-methyl/Ribo
2′-O-methyl/Ribo
Wait Time* DNA
2′-O-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 Imidazole
502/502/502
50/50/50 μL
10 sec
10 sec
10 sec

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.

[0205]

3

TABLE III

Human GRID Hammerhead Ribozyme and Substrate Sequence

Seq

Seq

Pos
Substrate
ID
Ribozyme
ID

13
GGCACAGU U AAUGGAUC
1
GAUCCAUU CUGAUGAG GCCGUUAGGC CGAA ACUGUGCC
906

14
GCACAGUU A AUGGAUCU
2
AGAUCCAU CUGAUGAG GCCGUUAGGC CGAA AACUGUGC
907

21
UAAUGGAU C UGUAAACU
3
AGUUUACA CUGAUGAG GCCGUUAGGC CGAA AUCCAUUA
908

25
GGAUCUGU A AACUUGCA
4
UGCAAGUU CUGAUGAG GCCGUUAGGC CGAA ACAGAUCC
909

30
UGUAAACU U GCACCCUC
5
GAGGGUGC CUGAUGAG GCCGUUAGGC CGAA AGUUUACA
910

38
UGCACCCU C UUUCAGAG
6
CUCUGAAA CUGAUGAG GCCGUUAGGC CGAA AGGGUGCA
911

40
CACCCUCU U UCAGAGUG
7
CACUCUGA CUGAUGAG GCCGUUAGGC CGAA AGAGGGUG
912

41
ACCCUCUU U CAGAGUGG
8
CCACUCUG CUGAUGAG GCCGUUAGGC CGAA AAGAGGGU
913

42
CCCUCUUU C AGAGUGGU
9
ACCACUCU CUGAUGAG GCCGUUAGGC CGAA AAAGAGGG
914

51
AGAGUGGU A CAUGGAAG
10
CUUCCAUG CUGAUGAG GCCGUUAGGC CGAA ACCACUCU
915

76
AAGUGGAU C CAUACUCU
11
AGAGUAUG CUGAUGAG GCCGUUAGGC CGAA AUCCACUU
916

80
GGAUCCAU A CUCUGAAA
12
UUUCAGAG CUGAUGAG GCCGUUAGGC CGAA AUGGAUCC
917

83
UCCAUACU C UGAAAUGC
13
GCAUUUCA CUGAUGAG GCCGUUAGGC CGAA AGUAUGGA
918

95
AAUGCAGU A ACUCUGAU
14
AUCAGAGU CUGAUGAG GCCGUUAGGC CGAA ACUGCAUU
919

99
CAGUAACU C UGAUGCUU
15
AAGCAUCA CUGAUGAG GCCGUUAGGC CGAA AGUUACUG
920

107
CUGAUGCU U GAAUUUGU
16
ACAAAUUC CUGAUGAG GCCGUUAGGC CGAA AGCAUCAG
921

112
GCUUGAAU U UGUUCUCC
17
GGAGAACA CUGAUGAG GCCGUUAGGC CGAA AUUCAAGC
922

113
CUUGAAUU U GUUCUCCC
18
GGGAGAAC CUGAUGAG GCCGUUAGGC CGAA AAUUCAAG
923

116
GAAUUUGU U CUCCCUUC
19
GAAGGGAG CUGAUGAG GCCGUUAGGC CGAA ACAAAUUC
924

117
AAUUUGUU C UCCCUUCU
20
AGAAGGGA CUGAUGAG GCCGUUAGGC CGAA AACAAAUU
925

119
UUUGUUCU C CCUUCUUG
21
CAAGAAGG CUGAUGAG GCCGUUAGGC CGAA AGAACAAA
926

123
UUCUCCCU U CUUGCCAG
22
CUGGCAAG CUGAUGAG GCCGUUAGGC CGAA AGGGAGAA
927

124
UCUCCCUU C UUGCCAGA
23
UCUGGCAA CUGAUGAG GCCGUUAGGC CGAA AAGGGAGA
928

126
UCCCUUCU U GCCAGAAA
24
UUUCUGGC CUGAUGAG GCCGUUAGGC CGAA AGAAGGGA
929

139
GAAAGGAU U CUAAUAAC
25
GUUAUUAG CUGAUGAG GCCGUUAGGC CGAA AUCCUUUC
930

140
AAAGGAUU C UAAUAACU
26
AGUUAUUA CUGAUGAG GCCGUUAGGC CGAA AAUCCUUU
931

142
AGGAUUCU A AUAACUCG
27
CGAGUUAU CUGAUGAG GCCGUUAGGC CGAA AGAAUCCU
932

145
AUUCUAAU A ACUCGGUG
28
CACCGAGU CUGAUGAG GCCGUUAGGC CGAA AUUAGAAU
933

149
UAAUAACU C GGUGUCAA
29
UUGACACC CUGAUGAG GCCGUUAGGC CGAA AGUUAUUA
934

155
CUCGGUGU C AAAGCCAA
30
UUGGCUUU CUGAUGAG GCCGUUAGGC CGAA ACACCGAG
935

169
CAAGACAU A AACUCAAU
31
AUUGAGUU CUGAUGAG GCCGUUAGGC CGAA AUGUCUUG
936

174
CAUAAACU C AAUCUCUU
32
AAGAGAUU CUGAUGAG GCCGUUAGGC CGAA AGUUUAUG
937

178
AACUCAAU C UCUUCUCU
33
AGAGAAGA CUGAUGAG GCCGUUAGGC CGAA AUUGAGUU
938

180
CUCAAUCU C UUCUCUUC
34
GAAGAGAA CUGAUGAG GCCGUUAGGC CGAA AGAUUGAG
939

182
CAAUCUCU U CUCUUCCA
35
UGGAAGAG CUGAUGAG GCCGUUAGGC CGAA AGAGAUUG
940

183
AAUCUCUU C UCUUCCAA
36
UUGGAAGA CUGAUGAG GCCGUUAGGC CGAA AAGAGAUU
941

185
UCUCUUCU C UUCCAAAA
37
UUUUGGAA CUGAUGAG GCCGUUAGGC CGAA AGAAGAGA
942

187
UCUUCUCU U CCAAAAGC
38
GCUUUUGG CUGAUGAG GCCGUUAGGC CGAA AGAGAAGA
943

188
CUUCUCUU C CAAAAGCU
39
AGCUUUUG CUGAUGAG GCCGUUAGGC CGAA AAGAGAAG
944

197
CAAAAGCU U CACGUUAC
40
GUAACGUG CUGAUGAG GCCGUUAGGC CGAA AGCUUUUG
945

198
AAAAGCUU C ACGUUACA
41
UGUAACGU CUGAUGAG GCCGUUAGGC CGAA AAGCUUUU
946

203
CUUCACGU U ACAGCAUG
42
CAUGCUGU CUGAUGAG GCCGUUAGGC CGAA ACGUGAAG
947

204
UUCACGUU A CAGCAUGG
43
CCAUGCUG CUGAUGAG GCCGUUAGGC CGAA AACGUGAA
948

220
GAAGCUGU U GCCAAGUU
44
AACUUGGC CUGAUGAG GCCGUUAGGC CGAA ACAGCUUC
949

228
UGCCAAGU U UGAUUUCA
45
UGAAAUCA CUGAUGAG GCCGUUAGGC CGAA ACUUGGCA
950

229
GCCAAGUU U GAUUUCAC
46
GUGAAAUC CUGAUGAG GCCGUUAGGC CGAA AACUUGGC
951

233
AGUUUGAU U UCACUGCU
47
AGCAGUGA CUGAUGAG GCCGUUAGGC CGAA AUCAAACU
952

234
GUUUGAUU U CACUGCUU
48
AAGCAGUG CUGAUGAG GCCGUUAGGC CGAA AAUCAAAC
953

235
UUUGAUUU C ACUGCUUC
49
GAAGCAGU CUGAUGAG GCCGUUAGGC CGAA AAAUCAAA
954

242
UCACUGCU U CAGGUGAG
50
CUCACCUG CUGAUGAG GCCGUUAGGC CGAA AGCAGUGA
955

243
CACUGCUU C AGGUGAGG
51
CCUCACCU CUGAUGAG GCCGUUAGGC CGAA AAGCAGUG
956

264
ACUGAGCU U UCACACUG
52
CAGUGUGA CUGAUGAG GCCGUUAGGC CGAA AGCUCAGU
957

265
CUGAGCUU U CACACUGG
53
CCAGUGUG CUGAUGAG GCCGUUAGGC CGAA AAGCUCAG
958

266
UGAGCUUU C ACACUGGA
54
UCCAGUGU CUGAUGAG GCCGUUAGGC CGAA AAAGCUCA
959

280
GGAGAUGU U UUGAAGAU
55
AUCUUCAA CUGAUGAG GCCGUUAGGC CGAA ACAUCUCC
960

281
GAGAUGUU U UGAAGAUU
56
AAUCUUCA CUGAUGAG GCCGUUAGGC CGAA AACAUCUC
961

282
AGAUGUUU U GAAGAUUU
57
AAAUCUUC CUGAUGAG GCCGUUAGGC CGAA AAACAUCU
962

289
UUGAAGAU U UUAAGUAA
58
UUACUUAA CUGAUGAG GCCGUUAGGC CGAA AUCUUCAA
963

290
UGAAGAUU U UAAGUAAC
59
GUUACUUA CUGAUGAG GCCGUUAGGC CGAA AAUCUUCA
964

291
GAAGAUUU U AAGUAACC
60
GGUUACUU CUGAUGAG GCCGUUAGGC CGAA AAAUCUUC
965

292
AAGAUUUU A AGUAACCA
61
UGGUUACU CUGAUGAG GCCGUUAGGC CGAA AAAAUCUU
966

296
UUUUAAGU A ACCAAGAG
62
CUCUUGGU CUGAUGAG GCCGUUAGGC CGAA ACUUAAAA
967

312
GGAGUGGU U UAAGGCGG
63
CCGCCUUA CUGAUGAG GCCGUUAGGC CGAA ACCACUCC
968

313
GAGUGGUU U AAGGCGGA
64
UCCGCCUU CUGAUGAG GCCGUUAGGC CGAA AACCACUC
969

314
AGUGGUUU A AGGCGGAG
65
CUCCGCCU CUGAUGAG GCCGUUAGGC CGAA AAACCACU
970

325
GCGGAGCU U GGGAGCCA
66
UGGCUCCC CUGAUGAG GCCGUUAGGC CGAA AGCUCCGC
971

342
GGAAGGAU A UGUGCCCA
67
UGGGCACA CUGAUGAG GCCGUUAGGC CGAA AUCCUUCC
972

356
CCAAGAAU U UCAUAGAC
68
GUCUAUGA CUGAUGAG GCCGUUAGGC CGAA AUUCUUGG
973

357
CAAGAAUU U CAUAGACA
69
UGUCUAUG CUGAUGAG GCCGUUAGGC CGAA AAUUCUUG
974

358
AAGAAUUU C AUAGACAU
70
AUGUCUAU CUGAUGAG GCCGUUAGGC CGAA AAAUUCUU
975

361
AAUUUCAU A GACAUCCA
71
UGGAUGUC CUGAUGAG GCCGUUAGGC CGAA AUGAAAUU
976

367
AUAGACAU C CAGUUUCC
72
GGAAACUG CUGAUGAG GCCGUUAGGC CGAA AUGUCUAU
977

372
CAUCCAGU U UCCCAAAU
73
AUUUGGGA CUGAUGAG GCCGUUAGGC CGAA ACUGGAUG
978

373
AUCCAGUU U CCCAAAUG
74
CAUUUGGG CUGAUGAG GCCGUUAGGC CGAA AACUGGAU
979

374
UCCAGUUU C CCAAAUGG
75
CCAUUUGG CUGAUGAG GCCGUUAGGC CGAA AAACUGGA
980

384
CAAAUGGU U UCACGAAG
76
CUUCGUGA CUGAUGAG GCCGUUAGGC CGAA ACCAUUUG
981

385
AAAUGGUU U CACGAAGG
77
CCUUCGUG CUGAUGAG GCCGUUAGGC CGAA AACCAUUU
982

386
AAUGGUUU C ACGAAGGC
78
GCCUUCGU CUGAUGAG GCCGUUAGGC CGAA AAACCAUU
983

397
GAAGGCCU C UCUCGACA
79
UGUCGAGA CUGAUGAG GCCGUUAGGC CGAA AGGCCUUC
984

399
AGGCCUCU C UCGACACC
80
GGUGUCGA CUGAUGAG GCCGUUAGGC CGAA AGAGGCCU
985

401
GCCUCUCU C GACACCAG
81
CUGGUGUC CUGAUGAG GCCGUUAGGC CGAA AGAGAGGC
986

420
AGAGAACU U ACUCAUGG
82
CCAUGAGU CUGAUGAG GCCGUUAGGC CGAA AGUUCUCU
987

421
GAGAACUU A CUCAUGGG
83
CCCAUGAG CUGAUGAG GCCGUUAGGC CGAA AAGUUCUC
988

424
AACUUACU C AUGGGCAA
84
UUGCCCAU CUGAUGAG GCCGUUAGGC CGAA AGUAAGUU
989

439
AAGGAGGU U GGCUUCUU
85
AAGAAGCC CUGAUGAG GCCGUUAGGC CGAA ACCUCCUU
990

444
GGUUGGCU U CUUCAUCA
86
UGAUGAAG CUGAUGAG GCCGUUAGGC CGAA AGCCAACC
991

445
GUUGGCUU C UUCAUCAU
87
AUGAUGAA CUGAUGAG GCCGUUAGGC CGAA AAGCCAAC
992

447
UGGCUUCU U CAUCAUCC
88
GGAUGAUG CUGAUGAG GCCGUUAGGC CGAA AGAAGCCA
993

448
GGCUUCUU C AUCAUCCG
89
CGGAUGAU CUGAUGAG GCCGUUAGGC CGAA AAGAAGCC
994

451
UUCUUCAU C AUCCGGGC
90
GCCCGGAU CUGAUGAG GCCGUUAGGC CGAA AUGAAGAA
995

454
UUCAUCAU C CGGGCCAG
91
CUGGCCCG CUGAUGAG GCCGUUAGGC CGAA AUGAUGAA
996

471
CCAGAGCU C CCCAGGGG
92
CCCCUGGG CUGAUGAG GCCGUUAGGC CGAA AGCUCUGG
997

483
AGGGGACU U CUCCAUCU
93
AGAUGGAG CUGAUGAG GCCGUUAGGC CGAA AGUCCCCU
998

484
GGGGACUU C UCCAUCUC
94
GAGAUGGA CUGAUGAG GCCGUUAGGC CGAA AAGUCCCC
999

486
GGACUUCU C CAUCUCUG
95
CAGAGAUG CUGAUGAG GCCGUUAGGC CGAA AGAAGUCC
1000

490
UUCUCCAU C UCUGUCAG
96
CUGACAGA CUGAUGAG GCCGUUAGGC CGAA AUGGAGAA
1001

492
CUCCAUCU C UGUCAGGC
97
GCCUGACA CUGAUGAG GCCGUUAGGC CGAA AGAUGGAG
1002

496
AUCUCUGU C AGGCAUGA
98
UCAUGCCU CUGAUGAG GCCGUUAGGC CGAA ACAGAGAU
1003

514
GAUGACGU U CAACACUU
99
AAGUGUUG CUGAUGAG GCCGUUAGGC CGAA ACGUCAUC
1004

515
AUGACGUU C AACACUUC
100
GAAGUGUU CUGAUGAG GCCGUUAGGC CGAA AACGUCAU
1005

522
UCAACACU U CAAGGUCA
101
UGACCUUG CUGAUGAG GCCGUUAGGC CGAA AGUGUUGA
1006

523
CAACACUU C AAGGUCAU
102
AUGACCUU CUGAUGAG GCCGUUAGGC CGAA AAGUGUUG
1007

529
UUCAAGGU C AUGCGAGA
103
UCUCGCAU CUGAUGAG GCCGUUAGGC CGAA ACCUUGAA
1008

548
ACAAGGGU A AUUACUUU
104
AAAGUAAU CUGAUGAG GCCGUUAGGC CGAA ACCCUUGU
1009

551
AGGGUAAU U ACUUUCUG
105
CAGAAAGU CUGAUGAG GCCGUUAGGC CGAA AUUACCCU
1010

552
GGGUAAUU A CUUUCUGU
106
ACAGAAAG CUGAUGAG GCCGUUAGGC CGAA AAUUACCC
1011

555
UAAUUACU U UCUGUGGA
107
UCCACAGA CUGAUGAG GCCGUUAGGC CGAA AGUAAUUA
1012

556
AAUUACUU U CUGUGGAC
108
GUCCACAG CUGAUGAG GCCGUUAGGC CGAA AAGUAAUU
1013

557
AUUACUUU C UGUGGACU
109
AGUCCACA CUGAUGAG GCCGUUAGGC CGAA AAAGUAAU
1014

573
UGAGAAGU U UCCAUCCC
110
GGGAUGGA CUGAUGAG GCCGUUAGGC CGAA ACUUCUCA
1015

574
GAGAAGUU U CCAUCCCU
111
AGGGAUGG CUGAUGAG GCCGUUAGGC CGAA AACUUCUC
1016

575
AGAAGUUU C CAUCCCUA
112
UAGGGAUG CUGAUGAG GCCGUUAGGC CGAA AAACUUCU
1017

579
GUUUCCAU C CCUAAAUA
113
UAUUUAGG CUGAUGAG GCCGUUAGGC CGAA AUGGAAAC
1018

583
CCAUCCCU A AAUAAGCU
114
AGCUUAUU CUGAUGAG GCCGUUAGGC CGAA AGGGAUGG
1019

587
CCCUAAAU A AGCUGGUA
115
UACCAGCU CUGAUGAG GCCGUUAGGC CGAA AUUUAGGG
1020

595
AAGCUGGU A GACUACUA
116
UAGUAGUC CUGAUGAG GCCGUUAGGC CGAA ACCAGCUU
1021

600
GGUAGACU A CUACAGGA
117
UCCUGUAG CUGAUGAG GCCGUUAGGC CGAA AGUCUACC
1022

603
AGACUACU A CAGGACAA
118
UUGUCCUG CUGAUGAG GCCGUUAGGC CGAA AGUAGUCU
1023

614
GGACAAAU U CCAUCUCC
119
GGAGAUGG CUGAUGAG GCCGUUAGGC CGAA AUUUGUCC
1024

615
GACAAAUU C CAUCUCCA
120
UGGAGAUG CUGAUGAG GCCGUUAGGC CGAA AAUUUGUC
1025

619
AAUUCCAU C UCCAGACA
121
UGUCUGGA CUGAUGAG GCCGUUAGGC CGAA AUGGAAUU
1026

621
UUCCAUCU C CAGACAGA
122
UCUGUCUG CUGAUGAG GCCGUUAGGC CGAA AGAUGGAA
1027

637
AAGCAGAU C UUCCUUAG
123
CUAAGGAA CUGAUGAG GCCGUUAGGC CGAA AUCUGCUU
1028

639
GCAGAUCU U CCUUAGAG
124
CUCUAAGG CUGAUGAG GCCGUUAGGC CGAA AGAUCUGC
1029

640
CAGAUCUU C CUUAGAGA
125
UCUCUAAG CUGAUGAG GCCGUUAGGC CGAA AAGAUCUG
1030

643
AUCUUCCU U AGAGACAG
126
CUGUCUCU CUGAUGAG GCCGUUAGGC CGAA AGGAAGAU
1031

644
UCUUCCUU A GAGACAGA
127
UCUGUCUC CUGAUGAG GCCGUUAGGC CGAA AAGGAAGA
1032

671
ACCAGGGU C ACCGGGGC
128
GCCCCGGU CUGAUGAG GCCGUUAGGC CGAA ACCCUGGU
1033

699
CCGGAGGU C CCAGGGAG
129
CUCCCUGG CUGAUGAG GCCGUUAGGC CGAA ACCUCCGG
1034

718
CCACACCU C AGUGGGGC
130
GCCCCACU CUGAUGAG GCCGUUAGGC CGAA AGGUGUGG
1035

742
GAAGAAAU C CGACCUUC
131
GAAGGUCG CUGAUGAG GCCGUUAGGC CGAA AUUUCUUC
1036

749
UCCGACCU U CGAUGAAC
132
GUUCAUCG CUGAUGAG GCCGUUAGGC CGAA AGGUCGGA
1037

750
CCGACCUU C GAUGAACC
133
GGUUCAUC CUGAUGAG GCCGUUAGGC CGAA AAGGUCGG
1038

768
GAAGCUGU C GGAUCACC
134
GGUGAUCC CUGAUGAG GCCGUUAGGC CGAA ACAGCUUC
1039

773
UGUCGGAU C ACCCCCCG
135
CGGGGGGU CUGAUGAG GCCGUUAGGC CGAA AUCCGACA
1040

787
CCGACCCU U CCCCUGCA
136
UGCAGGGG CUGAUGAG GCCGUUAGGC CGAA AGGGUCGG
1041

788
CGACCCUU C CCCUGCAG
137
CUGCAGGG CUGAUGAG GCCGUUAGGC CGAA AAGGGUCG
1042

821
CACAGCCU C CGCAAUAU
138
AUAUUGCG CUGAUGAG GCCGUUAGGC CGAA AGGCUGUG
1043

828
UCCGCAAU A UGCCCCAG
139
CUGGGGCA CUGAUGAG GCCGUUAGGC CGAA AUUGCGGA
1044

873
GCAGCGAU A UCUGCAGC
140
GCUGCAGA CUGAUGAG GCCGUUAGGC CGAA AUCGCUGC
1045

875
AGCGAUAU C UGCAGCAC
141
GUGCUGCA CUGAUGAG GCCGUUAGGC CGAA AUAUCGCU
1046

890
ACCACCAU U UCCACCAG
142
CUGGUGGA CUGAUGAG GCCGUUAGGC CGAA AUGGUGGU
1047

891
CCACCAUU U CCACCAGG
143
CCUGGUGG CUGAUGAG GCCGUUAGGC CGAA AAUGGUGG
1048

892
CACCAUUU C CACCAGGA
144
UCCUGGUG CUGAUGAG GCCGUUAGGC CGAA AAAUGGUG
1049

919
GGCAGCCU U GACAUAAA
145
UUUAUGUC CUGAUGAG GCCGUUAGGC CGAA AGGCUGCC
1050

925
CUUGACAU A AAUGAUGG
146
CCAUCAUU CUGAUGAG GCCGUUAGGC CGAA AUGUCAAG
1051

938
AUGGGCAU U GUGGCACC
147
GGUGCCAC CUGAUGAG GCCGUUAGGC CGAA AUGCCCAU
1052

951
CACCGGCU U GGGCAGUG
148
CACUGCCC CUGAUGAG GCCGUUAGGC CGAA AGCCGGUG
1053

976
GCGGCCCU C AUGCAUCG
149
CGAUGCAU CUGAUGAG GCCGUUAGGC CGAA AGGGCCGC
1054

983
UCAUGCAU C GGAGACAC
150
GUGUCUCC CUGAUGAG GCCGUUAGGC CGAA AUGCAUGA
1055

1009
GUGCAGCU C CAGGCGGC
151
GCCGCCUG CUGAUGAG GCCGUUAGGC CGAA AGCUGCAC
1056

1047
GGCGCUGU A UGACUUUG
152
CAAAGUCA CUGAUGAG GCCGUUAGGC CGAA ACAGCGCC
1057

1053
GUAUGACU U UGAGGCCC
153
GGGCCUCA CUGAUGAG GCCGUUAGGC CGAA AGUCAUAC
1058

1054
UAUGACUU U GAGGCCCU
154
AGGGCCUC CUGAUGAG GCCGUUAGGC CGAA AAGUCAUA
1059

1083
GCUGGGGU U CCACAGCG
155
CGCUGUGG CUGAUGAG GCCGUUAGGC CGAA ACCCCAGC
1060

1084
CUGGGGUU C CACAGCGG
156
CCGCUGUG CUGAUGAG GCCGUUAGGC CGAA AACCCCAG
1061

1108
GUGGAGGU C CUGGAUAG
157
CUAUCCAG CUGAUGAG GCCGUUAGGC CGAA ACCUCCAC
1062

1115
UCCUGGAU A GCUCCAAC
158
GUUGGAGC CUGAUGAG GCCGUUAGGC CGAA AUCCAGGA
1063

1119
GGAUAGCU C CAACCCAU
159
AUGGGUUG CUGAUGAG GCCGUUAGGC CGAA AGCUAUCC
1064

1128
CAACCCAU C CUGGUGGA
160
UCCACCAG CUGAUGAG GCCGUUAGGC CGAA AUGGGUUG
1065

1165
CUGGGCCU C UUCCCUGC
161
GCAGGGAA CUGAUGAG GCCGUUAGGC CGAA AGGCCCAG
1066

1167
GGGCCUCU U CCCUGCCA
162
UGGCAGGG CUGAUGAG GCCGUUAGGC CGAA AGAGGCCC
1067

1168
GGCCUCUU C CCUGCCAA
163
UUGGCAGG CUGAUGAG GCCGUUAGGC CGAA AAGAGGCC
1068

1179
UGCCAACU A CGUGGCAC
164
GUGCCACG CUGAUGAG GCCGUUAGGC CGAA AGUUGGCA
1069

1200
GACCCGAU A AACUCUUC
165
GAAGAGUU CUGAUGAG GCCGUUAGGC CGAA AUCGGGUC
1070

1205
GAUAAACU C UUCAGGGG
166
CCCCUGAA CUGAUGAG GCCGUUAGGC CGAA AGUUUAUC
1071

1207
UAAACUCU U CAGGGGAC
167
GUCCCCUG CUGAUGAG GCCGUUAGGC CGAA AGAGUUUA
1072

1208
AAACUCUU C AGGGGACA
168
UGUCCCCU CUGAUGAG GCCGUUAGGC CGAA AAGAGUUU
1073

1223
CAGAAGCU U UUUGUCUG
169
CAGACAAA CUGAUGAG GCCGUUAGGC CGAA AGCUUCUG
1074

1224
AGAAGCUU U UUGUCUGG
170
CCAGACAA CUGAUGAG GCCGUUAGGC CGAA AAGCUUCU
1075

1225
GAAGCUUU U UGUCUGGA
171
UCCAGACA CUGAUGAG GCCGUUAGGC CGAA AAAGCUUC
1076

1226
AAGCUUUU U GUCUGGAG
172
CUCCAGAC CUGAUGAG GCCGUUAGGC CGAA AAAAGCUU
1077

1229
CUUUUUGU C UGGAGCUG
173
CAGCUCCA CUGAUGAG GCCGUUAGGC CGAA ACAAAAAG
1078

1274
GCUGGACU C CAUGACUA
174
UAGUCAUG CUGAUGAG GCCGUUAGGC CGAA AGUCCAGC
1079

1282
CCAUGACU A UAUAUACA
175
UGUAUAUA CUGAUGAG GCCGUUAGGC CGAA AGUCAUGG
1080

1284
AUGACUAU A UAUACAUA
176
UAUGUAUA CUGAUGAG GCCGUUAGGC CGAA AUAGUCAU
1081

1286
GACUAUAU A UACAUACA
177
UGUAUGUA CUGAUGAG GCCGUUAGGC CGAA AUAUAGUC
1082

1288
CUAUAUAU A CAUACAUC
178
GAUGUAUG CUGAUGAG GCCGUUAGGC CGAA AUAUAUAG
1083

1292
AUAUACAU A CAUCUAUC
179
GAUAGAUG CUGAUGAG GCCGUUAGGC CGAA AUGUAUAU
1084

Input Sequence = HSA011736.

Cut Site = UH/.

Stem Length = 8.

Core Sequence = CUGAUGAG GCCGUUAGGC CGAA

HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp)

[0206] Underlined region can be any X sequence or linker as defined herein.

4TABLE IVHumam GRID NCH Ribozyme and Substrate SequenceSeqSeqPosSubstrateIDRibozymeID10GGAGGCAC A GUUAAUGG180CCAUUAAC CUGAUGAG GCCGUUAGGC CGAA IUGCCUCC108522AAUGGAUC U GUAAACUU181AAGUUUAC CUGAUGAG GCCGUUAGGC CGAA IAUCCAUU108629CUGUAAAC U UGCACCCU182AGGGUGCA CUGAUGAG GCCGUUAGGC CGAA IUUUACAG108733AAACUUGC A CCCUCUUU183AAAGAGGG CUGAUGAG GCCGUUAGGC CGAA ICAAGUUU108835ACUUGCAC C CUCUUUCA184UGAAAGAG CUGAUGAG GCCGUUAGGC CGAA IUGCAAGU108936CUUGCACC C UCUUUCAG185CUGAAAGA CUGAUGAG GCCGUUAGGC CGAA IGUGCAAG109037UUGCACCC U CUUUCAGA186UCUGAAAG CUGAUGAG GCCGUUAGGC CGAA IGGUGCAA109139GCACCCUC U UUCAGAGU187ACUCUGAA CUGAUGAG GCCGUUAGGC CGAA IAGGGUGC109243CCUCUCUC A GAGUGGUA188UACCACUC CUGAUGAG GCCGUUAGGC CGAA IAAAGAGG109353AGUGGUAC A UGGAAGAC189GUCUUCCA CUGAUGAG GCCGUUAGGC CGAA IUACCACU109462UGGAAGAC A GCACAAAG190CUUUGUGC CUGAUGAG GCCGUUAGGC CGAA IUCUUCCA109565AAGACAGC A CAAAGUGG191CCACUUUG CUGAUGAG GCCGUUAGGC CGAA ICUGUCUU109667GACAGCAC A AAGUGGAU192AUCCACUU CUGAUGAG GCCGUUAGGC CGAA IUGCUGUC109777AGUGGAUC C AUACUCUG193CAGAGUAU CUGAUGAG GCCGUUAGGC CGAA IAUCCACU109878GUGGAUCC A UACUCUGA194UCAGAGUA CUGAUGAG GCCGUUAGGC CGAA IGAUCCAC109982AUCCAUAC U CUGAAAUG195CAUUUCAG CUGAUGAG GCCGUUAGGC CGAA IUAUGGAU110084CCAUACUC U GAAAUGCA196UGCAUUUC CUGAUGAG GCCGUUAGGC CGAA IAGUAUGG110192UGAAAUGC A GUAACUCU197AGAGUUAC CUGAUGAG GCCGUUAGGC CGAA ICAUUUCA110298GCAGUAAC U CUGAUGCU198AGCAUCAG CUGAUGAG GCCGUUAGGC CGAA IUUACUGC1103100AGUAACUC U GAUGCUUG199CAAGCAUC CUGAUGAG GCCGUUAGGC CGAA IAGUUACU1104106UCUGAUGC U UGAAUUUG200CAAAUUCA CUGAUGAG GCCGUUAGGC CGAA ICAUCAGA1105118AUUUGUUC U CCCUUCUU201AAGAAGGG CUGAUGAG GCCGUUAGGC CGAA IAACAAAU1106120UUGUUCUC C CUUCUUGC202GCAAGAAG CUGAUGAG GCCGUUAGGC CGAA IAGAACAA1107121UGUUCUCC C UUCUUGCC203GGCAAGAA CUGAUGAG GCCGUUAGGC CGAA IGAGAACA1108122GUUCUCCC U UCUUGCCA204UGGCAAGA CUGAUGAG GCCGUUAGGC CGAA IGGAGAAC1109125CUCCCUUC U UGCCAGAA205UUCUGGCA CUGAUGAG GCCGUUAGGC CGAA IAAGGGAG1110129CUUCUUGC C AGAAAGGA206UCCUUUCU CUGAUGAG GCCGUUAGGC CGAA ICAAGAAG1111130UUCUUGCC A GAAAGGAU207AUCCUUUC CUGAUGAG GCCGUUAGGC CGAA IGCAAGAA1112141AAGGAUUC U AAUAACUC208GAGUUAUU CUGAUGAG GCCGUUAGGC CGAA IAAUCCUU1113148CUAAUAAC U CGGUGUCA209UGACACCG CUGAUGAG GCCGUUAGGC CGAA IUUAUUAG1114156UCGGUGUC A AAGCCAAG210CUUGGCUU CUGAUGAG GCCGUUAGGC CGAA IACACCGA1115161GUCAAAGC C AAGACAUA211UAUGUCUU CUGAUGAG GCCGUUAGGC CGAA ICUUUGAC1116162UCAAAGCC A AGACAUAA212UUAUGUCU CUGAUGAG GCCGUUAGGC CGAA IGCUUUGA1117167GCCAAGAC A UAAACUCA213UGAGUUUA CUGAUGAG GCCGUUAGGC CGAA IUCUUGGC1118173ACAUAAAC U CAAUCUCU214AGAGAUUG CUGAUGAG GCCGUUAGGC CGAA IUUUAUGU1119175AUAAACUC A AUCUCUUC215GAAGAGAU CUGAUGAG GCCGUUAGGC CGAA IAGUUUAU1120179ACUCAAUC U CUUCUCUU216AAGAGAAG CUGAUGAG GCCGUUAGGC CGAA IAUUGAGU1121181UCAAUCUC U UCUCUUCC217GGAAGAGA CUGAUGAG GCCGUUAGGC CGAA IAGAUUGA1122184AUCUCUUC U CUUCCAAA218UUUGGAAG CUGAUGAG GCCGUUAGGC CGAA IAAGAGAU1123186CUCUUCUC U UCCAAAAG219CUUUUGGA CUGAUGAG GCCGUUAGGC CGAA IAGAAGAG1124189UUCUCUUC C AAAAGCUU220AAGCUUUU CUGAUGAG GCCGUUAGGC CGAA IAAGAGAA1125190UCUCUUCC A AAAGCUUC221GAAGCUUU CUGAUGAG GCCGUUAGGC CGAA IGAAGAGA1126196CCAAAAGC U UCACGUUA222UAACGUGA CUGAUGAG GCCGUUAGGC CGAA ICUUUUGG1127199AAAGCUUC A CGUUACAG223CUGUAACG CUGAUGAG GCCGUUAGGC CGAA IAAGCUUU1128206CACGUUAC A GCAUGGAA224UUCCAUGC CUGAUGAG GCCGUUAGGC CGAA IUAACGUG1129209GUUACAGC A UGGAAGCU225AGCUUCCA CUGAUGAG GCCGUUAGGC CGAA ICUGUAAC1130217AUGGAAGC U GUUGCCAA226UUGGCAAC CUGAUGAG GCCGUUAGGC CGAA ICUUCCAU1131223GCUGUUGC C AAGUUUCA227UCAAACUU CUGAUGAG GCCGUUAGGC CGAA ICAACAGC1132224CUGUUGCC A AGUUUGAU228AUCAAACU CUGAUGAG GCCGUUAGGC CGAA IGCAACAG1133236UUGAUUUC A CUCCUUCA229UGAAGCAG CUGAUGAG GCCGUUAGGC CGAA IAAAUCAA1134238GAUUUCAC U GCUUCAGG230CCUGAAGC CUGAUGAG GCCGUUAGGC CGAA IUGAAAUC1135241UUCACUGC U UCAGGUGA231UCACCUGA CUGAUGAG GCCGUUAGGC CGAA ICAGUGAA1136244ACUGCUCC A GGUGAGGA232UCCUCACC CUGAUGAG GCCGUUAGGC CGAA IAAGCAGU1137258GGAUGAAC U GAGCUUUC233GAAAGCUC CUGAUGAG GCCGUUAGGC CGAA IUUCAUCC1138263AACUGAGC U UUCACACU234AGUGUGAA CUGAUGAG GCCGUUAGGC CGAA ICUCAGUU1139267GAGCUUUC A CACUGGAG235CUCCAGUG CUGAUGAG GCCGUUAGGC CGAA IAAAGCUC1140269GCUUUCAC A CUGGAGAU236AUCUCCAG CUGAUGAG GCCGUUAGGC CGAA IUGAAAGC1141271UUUCACAC U GGAGAUGU237ACAUCUCC CUGAUGAG GCCGUUAGGC CGAA IUGUGAAA1142299UAAGUAAC C AAGAGGAG238CUCCUCUU CUGAUGAG GCCGUUAGGC CGAA IUUACUUA1143300AAGUAACC A AGAGGAGU239ACUCCUCU CUGAUGAG GCCGUUAGGC CGAA IGUUACUU1144324GGCGGAGC U UGGGAGCC240GGCUCCCA CUGAUGAG GCCGUUAGGC CGAA ICUCCGCC1145332UUGGGAGC C AGGAAGGA241UCCUUCCU CUGAUGAG GCCGUUAGGC CGAA ICUCCCAA1146333UGGGAGCC A GGAAGGAU242AUCCUUCC CUGAUGAG GCCGUUAGGC CGAA IGCUCCCA1147348AUAUGUGC C CAAGAAUU243AAUUCUUG CUGAUGAG GCCGUUAGGC CGAA ICACAUAU1148349UAUCUGCC C AAGAAUUU244AAAUUCUU CUGAUGAG GCCGUUAGGC CGAA IGCACAUA1149350AUGUGCCC A AGAAUUUC245GAAAUUCU CUGAUGAG GCCGUUAGGC CGAA IGGCACAU1150359AGAAUUUC A UAGACAUC246GAUGUCUA CUGAUGAG GCCGUUAGGC CGAA IAAAUUCU1151365UCAUAGAC A UCCAGUUU247AAACUGGA CUGAUGAG GCCGUUAGGC CGAA IUCUAUGA1152368UAGACAUC C AGUUUCCC248AGGAAACU CUGAUGAG GCCGUUAGGC CGAA IAUGUCUA1153369AGACAUCC A GUUUCCCA249AGGGAAAC CUGAUGAG GCCGUUAGGC CGAA IGAUGUCU1154375CCAGUUCC C CAAAUGGU250ACCAUUUG CUGAUGAG GCCGUUAGGC CGAA IAAACUGG1155376CAGUUUCC C AAAUGGUU251AACCAUUU CUGAUGAG GCCGUUAGGC CGAA IGAAACUG1156377AGUUUCCC A AAUGGUUU252AAACCAUU CUGAUGAG GCCGUUAGGC CGAA IGGAAACU1157387AUGGUUUC A CGAAGGCC253GGCCUUCG CUGAUGAG GCCGUUAGGC CGAA IAAACCAU1158395ACGAAGGC C UCUCUCGA254UCGAGAGA CUGAUGAG GCCGUUAGGC CGAA ICCUUCGU1159396CGAAGGCC U CUCUCGAC255GUCGAGAG CUGAUGAG GCCGUUAGGC CGAA IGCCUUCG1160398AAGGCCUC U CUCGACAC256GUGUCGAG CUGAUGAG GCCGUUAGGC CGAA IAGGCCUU1161400GGCCUCUC U CGACACCA257UGGUGUCG CUGAUGAG GCCGUUAGGC CGAA IAGAGGCC1162405CUCUCGAC A CCAGGCAG258CUGCCUGG CUGAUGAG GCCGUUAGGC CGAA IUCGAGAG1163407CUCGACAC C AGGCAGAG259CUCUGCCU CUGAUGAG GCCGUUAGGC CGAA IUGUCGAG1164408UCGACACC A GGCAGAGA260UCUCUGCC CUGAUGAG GCCGUUAGGC CGAA IGUGUCGA1165412CACCAGGC A GAGAACUU261AAGUUCUC CUGAUGAG GCCGUUAGGC CGAA ICCUGGUG1166419CAGAGAGC U UACUCAUG262CAUGAGUA CUGAUGAG GCCGUUAGGC CGAA IUUCUCUG1167423GAACUUAC U CAUGGGCA263UGCCCAUG CUGAUGAG GCCGUUAGGC CGAA IUAAGUUC1168425ACUUACUC A UGGGCAAG264CUUGCCCA CUGAUGAG GCCGUUAGGC CGAA IAGUAAGU1169431UCAUGGGC A AGGAGGUU265AACCUCCU CUGAUGAG GCCGUUAGGC CGAA ICCCAUGA1170443AGGUUGGC U UCUUCAUC266GAUGAAGA CUGAUGAG GCCGUUAGGC CGAA ICCAACCU1171446UUGGCUUC U UCAUCAUC267GAUGAUGA CUGAUGAG GCCGUUAGGC CGAA IAAGCCAA1172449GCUUCUUC A UCAUCCGG268CCGGAUGA CUGAUGAG GCCGUUAGGC CGAA IAAGAAGC1173452UCUUCAUC A UCCGGGCC269GGCCCGGA CUGAUGAG GCCGUUAGGC CGAA IAUGAAGA1174455UCAUCAUC C GGGCCAGC270GCUGGCCC CUGAUGAG GCCGUUAGGC CGAA IAUGAUGA1175460AUCCGGGC C AGCCAGAG271CUCUGGCU CUGAUGAG GCCGUUAGGC CGAA ICCCGGAU1176461UCCGGGCC A GCCAGAGC272GCUCUGGC CUGAUGAG GCCGUUAGGC CGAA IGCCCGGA1177464GGGCCAGC C AGAGCUCC273GGAGCUCU CUGAUGAG GCCGUUAGGC CGAA ICUGGCCC1178465GGCCAGCC A GAGCUCCC274GGGAGCUC CUGAUGAG GCCGUUAGGC CGAA IGCUGGCC1179470GCCAGAUC U CCCCAGGG275CCCUGGGG CUGAUGAG GCCGUUAGGC CGAA ICUCUGGC1180472CAGACCUC C CCAGGGGA276UCCCCUGG CUGAUGAG GCCGUUAGGC CGAA IAGCUCUG1181473AGAGCUCC C CAGGGGAC277GUCCCCUG CUGAUGAG GCCGUUAGGC CGAA IGAGCUCU1182474GAGCUCCC C AGGGGACU278AGUCCCCU CUGAUGAG GCCGUUAGGC CGAA IGGAGGUC1183475AGCUCCCC A GGGGACUU279AAGUCCCC CUGAUGAG GCCGUUAGGC CGAA IGGGAGCU1184482CAGGGGAC U UCUCCAUC280GAUGGAGA CUGAUGAG GCCGUUAGGC CGAA IUCCCCUG1185485GGGACUUC U CCAUCUCU281AGAGAUGG CUGAUGAG GCCGUUAGGC CGAA IAAGUCCC1186487GACCUCUC C AUCUCUGU282ACAGAGAU CUGAUGAG GCCGUUAGGC CGAA IAGAAGUC1187488ACUUCUCC A UCUCUGUC283GACAGAGA CUGAUGAG GCCGUUAGGC CGAA IGAGAAGU1188491UCUCCAUC U CUGUCAGG284CCUGACAG CUGAUGAG GCCGUUAGGC CGAA IAUGGAGA1189493UCCAUCUC U GUCAGGCA285UGCCUGAC CUGAUGAG GCCGUUAGGC CGAA IAGAUGGA1190497UCUCUGUC A GGCAUGAG286CUCAUGCC CUGAUGAG GCCGUUAGGC CGAA IACAGAGA1191501UGUCAGGC A UGAGGAUG287CAUCCUCA CUGAUGAG GCCGUUAGGC CGAA ICCUGACA1192516UGACGUUC A ACACUUCA288UGAAGUGU CUGAUGAG GCCGUUAGGC CGAA IAACGUCA1193519CGUUCAAC A CUUCAAGG289ACUUGAAG CUGAUGAG GCCGUUAGGC CGAA IUUGAACG1194521UUCAACAC U UCAAGGUC290GACCUUGA CUGAUGAG GCCGUUAGGC CGAA IUGUUGAA1195524AACACUUC A AGGUCAUG291CAUGACCU CUGAUGAG GCCGUUAGGC CGAA IAAGUGUU1196530UCAAGGUC A UGCGAGAC292GUCUCGCA CUGAUGAG GCCGUUAGGC CGAA IACCUUGA1197539UGCGAGAC A ACAAGGGU293ACCCUUGU CUGAUGAG GCCGUUAGGC CGAA IUCUCGCA1198542GAGACAAC A AGGGUAAU294AUUACCCU CUGAUGAG GCCGUUAGGC CGAA IUUGUCUC1199554GUAAUUAC U UUCUGUGG295CCACAGAA CUGAUGAG GCCGUUAGGC CGAA IUAAUUAC1200558UUACUUUC U GUGGACUG296CAGUCCAC CUGAUGAG GCCGUUAGGC CGAA IAAAGUAA1201565CUGUGGAC U GAGAAGUU297AACUUCUC CUGAUGAG GCCGUUAGGC CGAA IUCCACAG1202576GAAGUUUC C AUCCCUAA298UUAGGGAU CUGAUGAG GCCGUUAGGC CGAA IAAACUUC1203577AAGUUUCC A UCCCUAAA299UUUAGGGA CUGAUGAG GCCGUUAGGC CGAA IGAAACUU1204580UUUCCAUC C CUAAAUAA300UUAUUUAG CUGAUGAG GCCGUUAGGC CGAA IAUGGAAA1205581UUCCAUCC C UAAAUAAG301CUUAUUUA CUGAUGAG GCCGUUAGGC CGAA IGAUGGAA1206582UCCAUCCC U AAAUAAGC302GCUUAUUU CUGAUGAG GCCGUUAGGC CGAA IGGAUGGA1207591AAAUAAGC U GGUAGACU303AGUCUACC CUGAUGAG GCCGUUAGGC CGAA ICUUAUUU1208599UGGUAGAC U ACUACAGG304CCUGUAGU CUGAUGAG GCCGUUAGGC CGAA IUCUACCA1209602UAGACUAC U ACAGGACA305UGUCCUGU CUGAUGAG GCCGUUAGGC CGAA IUAGUCUA1210605ACUACUAC A GGACAAAU306AUUUGUCC CUGAUGAG GCCGUUAGGC CGAA IUAGUAGU1211610UACAGGAC A AAUUCCAU307AUGGAAUU CUGAUGAG GCCGUUAGGC CGAA IUCCUGUA1212616ACAAAUUC C AUCUCCAG308CUGGAGAC CUGAUGAG GCCGUUAGGC CGAA IAAUUUGU1213617CAAAUUCC A UCUCCAGA309UCUGGAGA CUGAUGAG GCCGUUAGGC CGAA IGAAUUUG1214620AUUCCAUC U CCAGACAG310CUGUCUGG CUGAUGAG GCCGUUAGGC CGAA IAUGGAAU1215622UCCAUCUC C AGACAGAA311UUCUGUCU CUGAUGAG GCCGUUAGGC CGAA IAGAUGGA1216623CCAUCUCC A GACAGAAG312CUUCUGUC CUGAUGAG GCCGUUAGGC CGAA IGAGAUGG1217627CUCCAGAC A GAAGCAGA313UCUGCUUC CUGAUGAG GCCGUUAGGC CGAA IUCUGGAG1218633ACAGAAGC A GAUCUUCC314GGAAGAUC CUGAUGAG GCCGUUAGGC CGAA ICUUCUGU1219638AGCAGAUC U UCCUUAGA315UCUAAGGA CUGAUGAG GCCGUUAGGC CGAA IAUCUGCU1220641AGAUCUUC C UUAGAGAC316GUCUCUAA CUGAUGAG GCCGUUAGGC CGAA IAAGAUCU1221642GAUCUUCC U UAGAGACA317UGUCUCUA CUGAUGAG GCCGUUAGGC CGAA IGAAGAUC1222650UUAGAGAC A GAACCCGA318UCGGGUUC CUGAUGAG GCCGUUAGGC CGAA IUCUCUAA1223655GACAGAAC C CGAGAAGA319UCUUCUCG CUGAUGAG GCCGUUAGGC CGAA IUUCUGUC1224656ACAGAACC C GAGAAGAC320GUCUUCUC CUGAUGAG GCCGUUAGGC CGAA IGUUCUGU1225665GAGAAGAC C AGGGUCAC321GUGACCCU CUGAUGAG GCCGUUAGGC CGAA IUCUUCUC1226666AGAAGACC A GGGUCACC322GGUGACCC CUGAUGAG GCCGUUAGGC CGAA IGUCUUCU1227672CCAGGGUC A CCGGGGCA323UGCCCCGG CUGAUGAG GCCGUUAGGC CGAA IACCCUGG1228674AGGGUCAC C GGGGCAAC324GUUGCCCC CUGAUGAG GCCGUUAGGC CGAA IUGACCCU1229680ACCGGGGC A ACAGCCUG325CAGGCUGU CUGAUGAG GCCGUUAGGC CGAA ICCCCGGU1230683GGGGCAAC A GCCUGGAC326GUCCAGGC CUGAUGAG GCCGUUAGGC CGAA IUUGCCCC1231686GCAACAGC C UGGACCGG327CCGGUCCA CUGAUGAG GCCGUUAGGC CGAA ICUGUUGC1232687CAACAGCC U GGACCGGA328UCCGGUCC CUGAUGAG GCCGUUAGGC CGAA IGCUGUUG1233692GCCUGGAC C GGAGGUCC329GGACCUCC CUGAUGAG GCCGUUAGGC CGAA IUCCAGGC1234700CGGAGGUC C CAGGGAGG330CCUCCCUG CUGAUGAG GCCGUUAGGC CGAA IACCUCCG1235701GGAGGUCC C AGGGAGGC331GCCUCCCU CUGAUGAG GCCGUUAGGC CGAA IGACCUCC1236702GAGGUCCC A GGGAGGCC332GGCCUCCC CUGAUGAG GCCGUUAGGC CGAA IGGACCUC1237710AGGGAGGC C CACACCUC333GAGGUGUG CUGAUGAG GCCGUUAGGC CGAA ICCUCCCU1238711GGGAGGCC C ACACCUCA334UGAGGUGU CUGAUGAG GCCGUUAGGC CGAA IGCCUCCC1239712GGAGGCCC A CACCUCAG335CUGAGGUG CUGAUGAG GCCGUUAGGC CGAA IGGCCUCC1240714AGGCCCAC A CCUCAGUG336CACUGAGG CUGAUGAG GCCGUUAGGC CGAA IUGGGCCU1241716GCCCACAC C UCAGUGGG337CCCACUGA CUGAUGAG GCCGUUAGGC CGAA IUGUGGGC1242717CCCACACC U CAGUGGGG338CCCCACUG CUGAUGAG GCCGUUAGGC CGAA IGUGUGGG1243719CACACCUC A GUGGGGCU339AGCCCCAC CUGAUGAG GCCGUUAGGC CGAA IAGGUGUG1244727AGUGGGGC U GUGGGAGA340UCUCCCAC CUGAUGAG GCCGUUAGGC CGAA ICCCCACU1245743AAGAAAUC C GACCUUCG341CGAAGGUC CUGAUGAG GCCGUUAGGC CGAA IAUUUCUU1246747AAUCCGAC C UUCGAUGA342UCAUCGAA CUGAUGAG GCCGUUAGGC CGAA IUCGGAUU1247748AUCCGACC U UCGAUGAA343UUCAUCGA CUGAUGAG GCCGUUAGGC CGAA IGUCGGAU1248758CGAUGAAC C GGAAGCUG344CAGCUUCC CUGAUGAG GCCGUUAGGC CGAA IUUCAUCG1249765CCGGAAGC U GUCGGAUC345GAUCCGAC CUGAUGAG GCCGUUAGGC CGAA ICUUCCGG1250774GUCGGAUC A CCCCCCGA346UCGGGGGG CUGAUGAG GCCGUUAGGC CGAA IAUCCGAC1251776CGGAUCAC C CCCCGACC347GGUCGGGG CUGAUGAG GCCGUUAGGC CGAA IUGAUCCG1252777GGAUCACC C CCCGACCC348GGGUCGGG CUGAUGAG GCCGUUAGGC CGAA IGUGAUCC1253778GAUCACCC C CCGACCCU349AGGGUCGG CUGAUGAG GCCGUUAGGC CGAA IGGUGAUC1254779AUCACCCC C CGACCCUU350AAGGGUCG CUGAUGAG GCCGUUAGGC CGAA IGGGUGAU1255780UCACCCCC C GACCCUUC351GAAGGGUC CUGAUGAG GCCGUUAGGC CGAA IGGGGUGA1256784CCCCCGAC C CUUCCCCU352AGGGGAAG CUGAUGAG GCCGUUAGGC CGAA IUCGGGGG1257785CCCCGACC C UUCCCCUG353CAGGGGAA CUGAUGAG GCCGUUAGGC CGAA IGUCGGGG1258786CCCGACCC U UCCCCUGC354GCAGGGGA CUGAUGAG GCCGUUAGGC CGAA IGGUCGGG1259789GACCCUUC C CCUGCAGC355GCUGCAGG CUGAUGAG GCCGUUAGGC CGAA IAAGGGUC1260790ACCCUUCC C CUGCAGCA356UGCUGCAG CUGAUGAG GCCGUUAGGC CGAA IGAAGGGU1261791CCCUUCCC C UGCAGCAG357CUGCUGCA CUGAUGAG GCCGUUAGGC CGAA IGGAAGGG1262792CCUUCCCC U GCAGCAGC358GCUGCUGC CUGAUGAG GCCGUUAGGC CGAA IGGGAAGG1263795UCCCCUGC A GCAGCACC359GGUGCUGC CUGAUGAG GCCGUUAGGC CGAA ICAGGGGA1264798CCUGCAGC A GCACCAGC360GCUGGUGC CUGAUGAG GCCGUUAGGC CGAA ICUGCAGG1265801GCAGCAGC A CCAGCACC361GGUGCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCUGC1266803AGCAGCAC C AGCACCAG362CUGGUGCU CUGAUGAG GCCGUUAGGC CGAA IUGCUGCU1267804GCACCACC A GCACCAGC363GCUGGUGC CUGAUGAG GCCGUUAGGC CGAA IGUGCUGC1268807GCACCAGC A CCAGCCAC364GUGGCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGGUGC1269809ACCACCAC C AGCCACAG365CUGUGGCU CUGAUGAG GCCGUUAGGC CGAA IUGCUGGU1270810CCAGCACC A GCCACAGC366GCUGUGGC CUGAUGAG GCCGUUAGGC CGAA IGUGCUGG1271813GCACCAGC C ACAGCCUC367GAGGCUGU CUGAUGAG GCCGUUAGGC CGAA ICUGGUGC1272814CACCAGCC A CAGCCUCC368GGAGGCUG CUGAUGAG GCCGUUAGGC CGAA IGCUGGUG1273816CCAGCCAC A GCCUCCGC369GCGGAGGC CUGAUGAG GCCGUUAGGC CGAA IUGGCUGG1274819GCCACAGC C UCCGCAAU370AUUGCGGA CUGAUGAG GCCGUUAGGC CGAA ICUGUGGC1275820CCACAGCC U CCGCAAUA371UAUUGCGG CUGAUGAG GCCGUUAGGC CGAA IGCUGUGG1276822ACAGCCUC C GCAAUAUG372CAUAUUGC CUGAUGAG GCCGUUAGGC CGAA IAGGCUGU1277825GCCUCCGC A AUAUGCCC373GGGCAUAU CUGAUGAG GCCGUUAGGC CGAA ICGGAGGC1278832CAAUAUGC C CCAGCGCC374GGCGCUGG CUGAUGAG GCCGUUAGGC CGAA ICAGACUG1279833AAUAUGCC C CAGCGCCC375GGGCGCUG CUGAUGAG GCCGUUAGGC CGAA IGCAUAUU1280834AUAUGCCC C AGCGCCCC376GGGGCGCU CUGAUGAG GCCGUUAGGC CGAA IGGCAUAU1281835UAUGCCCC A GCGCCCCA377UGGGGCGC CUGAUGAG GCCGUUAGGC CGAA IGGGCAUA1282840CCCAGCGC C CCAGCAGC378GCUGCUGG CUGAUGAG GCCGUUAGGC CGAA ICGCUGGG1283841CCAGCGCC C CAGCAGCU379AGCUGCUG CUGAUGAG GCCGUUAGGC CGAA IGCGCUGG1284842CAGCGCCC C AGCAGCUG380CAGCUGCU CUGAUGAG GCCGUUAGGC CGAA IGGCGCUG1285843AGCGCCCC A GCAGCUGC381GCAGCUGC CUGAUGAG GCCGUUAGGC CGAA IGGGCGCU1286846GCCCCAGC A GCUGCAGC382GCUGCAGC CUGAUGAG GCCGUUAGGC CGAA ICUGGGGC1287849CCAGCAGC U GCAGCAGC383GCUGCUGC CUGAUGAG GCCGUUAGGC CGAA ICUGCUGG1288852GCAGCUGC A GCAGCCCC384GGGGCUGC CUGAUGAG GCCGUUAGGC CGAA ICAGCUGC1289855GCUGCAGC A GCCCCCAC385GUGGGGGC CUGAUGAG GCCGUUAGGC CGAA ICUGCAGC1290858GCAGCAGC C CCCACAGC386GCUGUGGG CUGAUGAG GCCGUUAGGC CGAA ICUGCUGC1291859CAGCAGCC C CCACAGCA387UGCUGUGG CUGAUGAG GCCGUUAGGC CGAA IGCUGCUG1292860AGCAGCCC C CACAGCAG388CUGCUGUG CUGAUGAG GCCGUUAGGC CGAA IGGCUGCU1293861GCAGCCCC C ACAGCAGC389GCUGCUGU CUGAUGAG GCCGUUAGGC CGAA IGGGCUGC1294862CAGCCCCC A CAGCAGCG390CGCUGCUG CUGAUGAG GCCGUUAGGC CGAA IGGGGCUG1295864GCCCCCAC A GCAGCGAU391AUCGCUGC CUGAUGAG GCCGUUAGGC CGAA IUGGGGGC1296867CCCACAGC A GCGAUAUC392GAUAUCGC CUGAUGAG GCCGUUAGGC CGAA ICUGUGGG1297876GCGAUAUC U GCAGCACC393GGUGCUGC CUGAUGAG GCCGUUAGGC CGAA IAUAUCGC1298879AUAUCUGC A GCACCACC394GGUGGUGC CUGAUGAG GCCGUUAGGC CGAA ICAGAUAU1299882UCUGCAGC A CCACCAUU395AAUGGUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCAGA1300884UGCAGCAC C ACCAUCUC396GAAAUGGU CUGAUGAG GCCGUUAGGC CGAA IUGCUGCA1301885GCAGCACC A CCAUUUCC397GGAAAUGG CUGAUGAG GCCGUUAGGC CGAA IGUGCUGC1302887AGCACCAC C AUCUCCAC398GUGGAAAU CUGAUGAG GCCGUUAGGC CGAA IUGGUGCU1303888GCACCACC A UUUCCACC399GGUGGAAA CUGAUGAG GCCGUUAGGC CGAA IGUGGUGC1304893ACCAUCUC C ACCAGGAA400UUCCUGGU CUGAUGAG GCCGUUAGGC CGAA IAAAUGGU1305894CCAUUUCC A CCAGGAAC401GUUCCUGG CUGAUGAG GCCGUUAGGC CGAA IGAAAUGG1306896AUCUCCAC C AGGAACGC402GCGUUCCU CUGAUGAG GCCGUUAGGC CGAA IUGGAAAU1307897UUUCCACC A GGAACGCC403GGCGUUCC CUGAUGAG GCCGUUAGGC CGAA IGUGGAAA1308905AGGAACGC C GAGGAGGC404GCCUCCUC CUGAUGAG GCCGUUAGGC CGAA ICGUUCCU1309914GAGGAGUC A GCCUUGAC405GUCAAGGC CUGAUGAG GCCGUUAGGC CGAA ICCUCCUC1310917GAGGCAGC C UUGACAUA406UAUGUCAA CUGAUGAG GCCGUUAGGC CGAA ICUGCCUC1311918AGGCAGCC U UGACAUAA407UCAUGUCA CUGAUGAG GCCGUUAGGC CGAA IGCUGCCU1312923GCCUUGAC A UAAAUGAU408AUCAUUUA CUGAUGAG GCCGUUAGGC CGAA IUCAAGGC1313936UGAUGGGC A UUGUGGCA409UGCCACAA CUGAUGAG GCCGUUAGGC CGAA ICCCAUCA1314944AUUGUGGC A CCGGCUUG410CAAGCCGG CUGAUGAG GCCGUUAGGC CGAA ICCACAAU1315946UGUGGCAC C GGCUUGGG411CCCAAGCC CUGAUGAG GCCGUUAGGC CGAA IUGCCACA1316950GCACCGGC U UGGGCAGU412ACUGCCCA CUGAUGAG GCCGUUAGGC CGAA ICCGGUGC1317956GCUUGGGC A GUGAAAUG413CACUCCAC CUGAUGAG GCCGUUAGGC CGAA ICCCAAGC1318973AAUGCGGC C CUCAUGCA414UGCAUGAG CUGAUGAG GCCGUUAGGC CGAA ICCUCAUC1319974AUGCGGCC C UCAUGCAU415AUGCAUGA CUGAUGAG GCCGUUAGGC CGAA IGCCGCAU1320975UGCGGCCC U CAUGCAUC416GAUGCAUG CUGAUGAG GCCGUUAGGC CGAA IGGCCGCA1321977CGGCCCUC A UGCAUCGG417CCGAUGCA CUGAUGAG GCCGUUAGGC CGAA IAGGGCCG1322981CCUCAUGC A UCGGAGAC418GUCUCCGA CUGAUGAG GCCGUUAGGC CGAA ICAUGAGG1323990UCGGAGAC A CACAGACC419GGUCUGUG CUGAUGAG GCCGUUAGGC CGAA IUCUCCGA1324992GGAGACAC A CAGACCCA420UGGGUCUG CUGAUGAG GCCGUUAGGC CGAA IUGUCUCC1325994AGACACAC A GACCCAGU421ACUGGGUC CUGAUGAG GCCGUUAGGC CGAA IUGUGUCU1326998ACACAGAC C CAGUCCAG422CUGCACUG CUGAUGAG GCCGUUAGGC CGAA IUCUGUGU1327999CACAGAUC C AGUGCAGC423GCUGCACU CUGAUGAG GCCGUUAGGC CGAA IGUCUGUG13281000ACAGACCC A GUGCAGCU424AGCUGCAC CUGAUGAG GCCGUUAGGC CGAA IGGUCUGU13291005CCCAGUGC A GCUCCAGG425CCUGGAGC CUGAUGAG GCCGUUAGGC CGAA ICACUGGG13301008AGUGCAGC U CCAGGCGG426CCGCCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCACU13311010UGCAGCUC C AGGCGGCA427UGCCGCCU CUGAUGAG GCCGUUAGGC CGAA IAGCUGCA13321011GCAGCUCC A GGCGGCAG428CUGCCGCC CUGAUGAG GCCGUUAGGC CGAA IGAGCUGC13331018CAGGUGGC A GGGCGAGU429ACUCGCCC CUGAUGAG GCCGUUAGGC CGAA ICCGCCUG13341036CUGUGGUC C CGGGCGCU430AGCGCCCG CUGAUGAG GCCGUUAGGC CGAA ICCCACCG13351037GGUGGGCC C GGGCGCUG431CAGCGCCC CUGAUGAG GCCGUUAGGC CGAA IGCCCACC13361044CCGGGCGC U GUAUGACU432AGUCAUAC CUGAUGAG GCCGUUAGGC CGAA ICGCCCGG13371052UGUAUGAC U UUGAGGCC433GGCCUCAA CUGAUGAG GCCGUUAGGC CGAA IUCAUACA13381060UUUGAGGC C CUGGAGGA434UCCUCCAG CUGAUGAG GCCGUUAGGC CGAA ICCUCAAA13391061UUGAGGCC C UGGAGGAU435AUCCUCCA CUGAUGAG GCCGUUAGGC CGAA IGCCUCAA13401062UGAGGCCC U GGAGGAUG436CAUCCUCC CUGAUGAG GCCGUUAGGC CGAA IGGCCUCA13411077UGACGAGC U GGGGUUCC437GGAACCCC CUGAUGAG GCCGUUAGGC CGAA ICUCGUCA13421085UGGGGUUC C ACAGCGGG438CCCGCUGU CUGAUGAG GCCGUUAGGC CGAA IAACCCCA13431086GGGGUUCC A CAGCGGGG439CCCCGCUG CUGAUGAG GCCGUUAGGC CGAA IGAACCCC13441088GGUUCCAC A GCGGGGAG440CUCCCCGC CUGAUGAG GCCGUUAGGC CGAA IUGGAACC13451109UGGAGGUC C UGGAUAGC441GCUAUCCA CUGAUGAG GCCGUUAGGC CGAA IACCUCCA13461110GGAGGUCC U GGAUAGCU442AGCUAUCC CUGAUGAG GCCGUUAGGC CGAA IGACCUCC13471118UGGAUAGC U CCAACCCA443UGGGUUGG CUGAUGAG GCCGUUAGGC CGAA ICUAUCCA13481120GAUAGCUC C AACCCAUC444GAUGGGUU CUGAUGAG GCCGUUAGGC CGAA IAGCUAUC13491121AUAGCUCC A ACCCAUCC445GGAUGGGU CUGAUGAG GCCGUUAGGC CGAA IGAGCUAU13501124GCUCCAAC C CAUCCUGG446CCAGGAUG CUGAUGAG GCCGUUAGGC CGAA IUUGGAGC13511125CUCCAACC C AUCCUGGU447ACCAGGAU CUGAUGAG GCCGUUAGGC CGAA IGUUGGAG13521126UCCAACCC A UCCUGGUG448CACCAGGA CUGAUGAG GCCGUUAGGC CGAA IGGUUGGA13531129AACCCAUC C UGGUGGAC449GUCCACCA CUGAUGAG GCCGUUAGGC CGAA IAUGGGUU13541130ACCCAUCC U GGUGGACC450GGUCCACC CUGAUGAG GCCGUUAGGC CGAA IGAUGGGU13551138UGGUGGAC C GGCCGCCU451AGGCGGCC CUGAUGAG GCCGUUAGGC CGAA IUCCACCA13561142GGACCGGC C GCCUGCAC452GUGCAGGC CUGAUGAG GCCGUUAGGC CGAA ICCGGUCC13571145CCGGCCGC C UGCACAAC453GUUGUGCA CUGAUGAG GCCGUUAGGC CGAA ICGGCCGG13581146CGGCCGCC U GCACAACA454UGUUGUGC CUGAUGAG GCCGUUAGGC CGAA IGCGGCCG13591149CCGCCUGC A CAACAAGC455GCUUGUUG CUGAUGAG GCCGUUAGGC CGAA ICAGGCGG13601151GCCUGCAC A ACAAGCUG456CAGCUUGU CUGAUGAG GCCGUUAGGC CGAA IUGCAGGC13611154UGCACAAC A AGCUGGGC457GCCCAGCU CUGAUGAG GCCGUUAGGC CGAA IUUGUGCA13621158CAACAAGC U GGGCCUCU458AGAGGCCC CUGAUGAG GCCGUUAGGC CGAA ICUUGUUG13631163AGCUGGGC C UCUUCCCU459AGGGAAGA CUGAUGAG GCCGUUAGGC CGAA ICCCAGCU13641164GCUGGGCC U CUUCCCUG460CAGGGAAG CUGAUGAG GCCGUUAGGC CGAA IGCCCAGC13651166UGGGCCUC U UCCCUGCC461GGCAGGGA CUGAUGAG GCCGUUAGGC CGAA IAGGCCCA13661169GCCUCUUC C CUGCCAAC462GUUGGCAG CUGAUGAG GCCGUUAGGC CGAA IAAGAGGC13671170CCUCUUCC C UGCCAACU463AGUUGGCA CUGAUGAG GCCGUUAGGC CGAA IGAAGAGG13681171CUCUUCCC U GCCAACUA464UAGUUGGC CUGAUGAG GCCGUUAGGC CGAA IGGAAGAG13691174UUCCCUGC C AACUACGU465ACGUAGUU CUGAUGAG GCCGUUAGGC CGAA ICAGGGAA13701175UCCCUGCC A ACUACGUG466CACGUAGU CUGAUGAG GCCGUUAGGC CGAA IGCAGGGA13711178CUGCCAAC U ACGUGGCA467UGCCACGU CUGAUGAG GCCGUUAGGC CGAA IUUGGCAG13721186UACGUGGC A CCCAUGAC468GUCAUGGG CUGAUGAG GCCGUUAGGC CGAA ICCACGUA13731188CGUGGCAC C CAUGACCC469GGGUCAUG CUGAUGAG GCCGUUAGGC CGAA IUGCCACG13741189GUGGCACC C AUGACCCG470CGGGUCAU CUGAUGAG GCCGUUAGGC CGAA IGUGCCAC13751190UGGCACCC A UGACCCGA471UCGGGUCA CUGAUGAG GCCGUUAGGC CGAA IGGUGCCA13761195CCCAUGAC C CGAUAAAC472GUUUAUCG CUGAUGAG GCCGUUAGGC CGAA IUCAUGGG13771196CCAUGACC C GAUAAACU473AGUUUAUC CUGAUGAG GCCGUUAGGC CGAA IGUCAUGG13781204CGAUAAAC U CUUCAGGG474CCCUGAAG CUGAUGAG GCCGUUAGGC CGAA IUUUAUCG13791206AUAAACUC U UCAGGGGA475UCCCCUGA CUGAUGAG GCCGUUAGGC CGAA IAGUUUAU13801209AACUCUUC A GGGGACAG476CUGUCCCC CUGAUGAG GCCGUUAGGC CGAA IAAGAGUU13811216CAGGGGAC A GAAGCUUU477AAAGCUUC CUGAUGAG GCCGUUAGGC CGAA IUCCCCUG13821222ACAGAAGC U UUUUGUCU478AGACAAAA CUGAUGAG GCCGUUAGGC CGAA ICUUCUGU13831230UUUUUGUC U GGAGCUGC479GCAGCUCC CUGAUGAG GCCGUUAGGC CGAA IACAAAAA13841236UCUGGAGC U GCCCACAA490UUGUGGGC CUGAUGAG GCCGUUAGGC CGAA ICUCCAGA13851239GGAGCUGC C CACAAGAA481UUCUUGUG CUGAUGAG GCCGUUAGGC CGAA ICAGCUCC13861240GAGCUGCC C ACAAGAAA482UUUCUUGU CUGAUGAG GCCGUUAGGC CGAA IGCAGCUC13871241AGCUGCCC A CAAGAAAG483CUUUCUUG CUGAUGAG GCCGUUAGGC CGAA IGGCAGCU13881243CUGCCCAC A AGAAAGAG484CUCUUUCU CUGAUGAG GCCGUUAGGC CGAA IUGGGCAG13891255AAGAGGGC A AGGAAAAA485UUUUUCCU CUGAUGAG GCCGUUAGGC CGAA ICCCUCUU13901268AAAAAGGC U GGACUCCA486UGGAGUCC CUGAUGAG GCCGUUAGGC CGAA ICCUUUUU13911273GGCUGGAC U CCAUGACU487AGUCAUGG CUGAUGAG GCCGUUAGGC CGAA IUCCAGCC13921275CUGGACUC C AUGACUAU488AUAGUCAU CUGAUGAG GCCGUUAGGC CGAA IAGUCCAG13931276UGGACUCC A UGACUAUA489UAUAGUCA CUGAUGAG GCCGUUAGGC CGAA IGAGUCCA13941281UCCAUGAC U AUAUAUAC490GUAUAUAU CUGAUGAG GCCGUUAGGC CSAA IUCAUGGA13951290AUAUAUAC A UACAUCUA491UAGAUGUA CUGAUGAG GCCGUUAGGC CGAA IUAUAUAU13961294AUACAUAC A UCUAUCUA492UAGAUAGA CUGAUGAG GCCGUUAGGC CGAA IUAUGUAU1397Input Sequence = HSA011736. Cut Site = CH/. Stem Length = 8. Core Sequence = CUGAUGAG GCCGUUAGGC CGAA HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp) Underlined region can be any X sequence or linker as defined herein. I = Inosine

[0207]

5

TABLE V

Human GRID G-cleaver Ribozyme and Substrate Sequence

Pos
Substrate
Seq ID
Ribozyme
Seq ID

31
GUAAACUU G CACCCUCU
493
AGAGGGUG UGAUG GCAUGCACUAUGC GCG AAGUUUAC
1398

85
CAUACUCU G AAAUGCAG
494
CUGCAUUU UGAUG GCAUGCACUAUGC GCG AGAGUAUG
1399

90
UCUGAAAU G CAGUAACU
495
AGUUACUG UGAUG GCAUGCACUAUGC GCG AUUUCAGA
1400

101
GUAACUCU G AUGCUUGA
496
UCAAGCAU UGAUG GCAUGCACUAUGC GCG AGAGGUAC
1401

104
ACUCUGAU G CUUGAAUU
497
AAUUCAAG UGAUG GCAUGCACUAUGC GCG AUCAGAGU
1402

108
UGAUGCUU G AAUUUGUU
498
AACAAAUU UGAUG GCAUGCACUAUGC GCG AAGCAUCA
1403

127
CCCUUCUU G CCAGAAAG
499
CUUUCUGG UGAUG GCAUGCACUAUGC GCG AAGAAGGG
1404

221
AAGCUGUU G CCAAGUUU
500
AAACUUGG UGAUG GCAUGCACUAUGC GCG AACAGCUU
1405

230
CCAAGUUU G AUUUCACU
501
AGUGAAAU UGAUG GCAUGCACUAUGC GCG AAACUUGG
1406

239
AUUUCACU G CUUCAGGU
502
ACCUGAAG UGAUG GCAUGCACUAUGC GCG AGUGAAAU
1407

248
CUUCAGGU G AGGAUGAA
503
UUCAUCCU UCAUG GCAUGCACUAUGC GCG ACCUGAAG
1408

254
GUGAGGAU G AACUGAGC
504
GCUCAGUU UGAUG GCAUGCACUAUGC GCG AUCCUCAC
1409

259
GAUGAACU G AGCUUUCA
505
UGAAAGCU UGAUG GCAUGCACUAUGC GCG AGUUCAUC
1410

283
GAUGUUUU G AAGAUUUU
506
AAAAUCUU UGAUG GCAUGCACUAUGC GCG AAAACAUC
1411

346
GGAUAUGU G CCCAAGAA
507
UUCUUGGG UGAUG GCAUGCACUAUGC GCG ACAUAUCC
1412

389
GGUUUCAC G AAGGCCUC
508
CAGGCCUU UGAUG GCAUGCACUAUGC GCG GUGAAACC
1413

402
CCUCUCUC G ACACCAGG
509
CCUGGUGU UGAUG GCAUGCACUAUGC GCG GAGAGAGG
1414

503
UCAGGCAU G AGGAUGAC
510
GUCAUCCU UGAUG GCAUGCACUAUGC GCG AUGUCUGA
1415

509
AUGAGGAU G ACGUUCAA
511
UUGAACGU UGAUG GCAUGCACUAUGC GCG AUCCUCAU
1416

532
AAGGUCAU G CGAGACAA
512
UUGUCUCG UCAUG GCAUGCACUAUGC GCC AUGACCUU
1417

534
GGUCAUGC G AGACAACA
513
UGUUGUCU UCAUG GCAUGCACUAUGC GCG GCAUGACC
1418

566
UGUGGACU G AGAAGUUU
514
AAACUUCU UGAUG GCAUGCACUAUGC GCG AGUCCACA
1419

657
CAGAACCC G AGAAGACC
515
GGUCUUCU UGAUG GCAUGCACUAUGC GCG GGGUUCUG
1420

744
AGAAAUCC G ACCUUCGA
516
UCGAAGGU UGAUG GCAUGCACUAUGC GCG GGAUUUCU
1421

751
CGACCUUC G AUGAACCG
517
CGGUUCAU UGAUG GCAUGCACUAUGC GCG GAAGGUCG
1422

754
CCUUCGAU G AACCGGAA
518
UUCCGGUU UGAUG GCAUGCACUAUGC GCG AUCGAAGG
1423

781
CACCCCCC G ACCCUUCC
519
GGAAGGGU UGAUG GCAUGCACUAUGC GCG GGGGGGUG
1424

793
CUUCCCCU G CAGCAGCA
520
UGCUGCUG UGAUG GCAUGCACUAUGC GCG AGGGGAAG
1425

823
CAGCCUCC G CAAUAUGC
521
GCAUAUUG UCAUG GCAUGCACUAUGC GCG GGAGGCUG
1426

830
CGCAAUAU G CCCCAGCG
522
CGCUGGGG UGAUG GCAUGCACUAUGC GCG AUAUUGCG
1427

838
GCCCCAGC G CCCCAGCA
523
UGCUGGGG UCAUG GCAUGCACUAUGC GCG GCUGGGGC
1428

850
CAGCAGUC G CAGCAGCC
524
GGCUGCUG UGAUG GCAUGCACUAUGC GCG AGCUGCUG
1429

870
ACAGCAGC G AUAUCUGC
525
GCAGAUAU UCAUG GCAUGCACUAUGC GCG GCUGCUGU
1430

877
CGAUAUCU G CAGCACCA
526
UGGUGCUG UGAUG GCAUGCACUAUGC GCG AGAUAUCG
1431

903
CCAGGAAC G CCGAGGAG
527
CUCCUCGG UGAUG GCAUGCACUAUGC GCG GUUCCUGG
1432

906
GGAACGCC G AGGAGGCA
528
UGCCUCCU UGAUG GCAUGCACUAUGC GCG GGCGUUCC
1433

920
GCAGCCUU G ACAUAAAU
529
AUUUAUGU UCAUG GCAUGCACUAUGC GCG AAGGCUGC
1434

929
ACAUAAAU G AUGGGCAU
530
AUGCCCAU UGAUG GCAUGCACUAUGC GCG AUUUAUGU
1435

959
UGGGCAGU G AAAUGAAU
531
AUUCAUUU UGAUG GCAUGCACUAUGC GCG ACUGCCCA
1436

964
AGUGAAAU G AAUGCGGC
532
GCCGCAUU UGAUG GCAUGCACUAUGC GCG AUUUCACU
1437

968
AAAUGAAU G CGGCCCUC
533
GAGGGCCG UCAUG GCAUGCACUAUGC GCG AUUCAUUU
1438

979
GCCCUCAU G CAUCGGAG
534
CUCCGAUG UGAUG GCAUGCACUAUGC GCG AUGAGGGC
1439

1003
GACCCAGU G CAGCUCCA
535
UGGAGCUG UGAUG GCAUGCACUAUGC GCG ACUGGGUC
1440

1023
GGCAGGGC G AGUGCGGU
536
ACCGCACU UCAUG GCAUGCACUAUGC GCG GCCCUGCC
1441

1027
GGGCGAGU G CGGUGGGC
537
GCCCACCG UGAUG GCAUGCACUAUGC GCG ACUCGCCC
1442

1042
GCCCGGGC G CUGUAUGA
538
UCAUACAG UGAUG GCAUGCACUAUGC GCG GCCCGGGC
1443

1049
CGCUGUAU G ACUUUGAG
539
CUCAAAGU UGAUG GCAUGCACUAUGC GCG AUACAGCG
1444

1055
AUGACUUU G AGGCCCUG
540
CAGGGCCU UGAUG GCAUGCACUAUGC GCG AAAGUCAU
1445

1070
UGGAGGAG G ACGAGCUG
541
CAGCUCGU UGAUG GCAUGCACUAUGC GCG AUCCUCCA
1446

1073
AGGAUGAC G AGCUGGGG
542
CCCCAGCU UGAUG GCAUGCACUAUGC GCG GUCAUCCU
1447

1143
GACCCGCC G CCUGCACA
543
UGUGCAGG UGAUG GCAUGCACUAUGC GCG GGCCGGUC
1448

1147
GGCCGCCU G CACAACAA
544
UUGUUGUG UCAUG GCAUGCACUAUGC GCG AGGCGGCC
1449

1172
UCUUCCCU G CCAACUAC
545
GUAGUUGG UGAUG GCAUGCACUAUGC GCG AGGGAAGA
1450

1192
CCACCCAU G ACCCGAUA
546
UAUCGGGU UGAUG GCAUGCACUAUGC GCG AUGGGUGC
1451

1197
CAUGACCC G AUAAACUC
547
GAGUUUAU UCAUG GCAUGCACUAUGC GCG GGGUCAUG
1452

1237
CUCGACCU G CCCACAAG
548
CUUGUGGG UGAUG GCAUGCACUAUGC GCG AGCUCCAG
1453

1278
GACUCCAU G ACUAUAUA
549
UAUAUAGU UGAUG GCAUGCACUAUGC GCG AUGGAGUC
1454

Input Sequence = HSA011736.

Cut Site = YG/M or UG/U.

Stem Length = 8.

Core Sequence = UCAUG GCAUGCACUAUGC GCG

HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp)

[0208]

6

TABLE VI

Human GRID Zinzyme and Substrate Sequence

Pos
Substrate
Seq ID
Zinzyme
Seq ID

11
GAGGCACA G UUAAUGGA
550
UCCAUUAA GCCGAAAGGCGAGUCAAGGUCU UGUGCCUC
1455

23
AUGGAUCU G UAAACUUG
551
CAAGUUUA GCCGAAAGGCGAGUCAAGGUCU AGAUCCAU
1456

31
GUAAACUU G CACCCUCU
493
AGAGGGUG GCCGAAAGGCGAGUCAAGGUCU AAGUUUAC
1457

46
CUUUCAGA G UGGUACAU
552
AUGUACCA GCCGAAAGGCGAGUCAAGGUCU UCUGAAAG
1458

49
UCAGAGUG G UACAUGGA
553
UCCAUGUA GCCGAAAGGCGAGUCAAGGUCU CACUCUGA
1459

63
GGAAGACA G CACAAAGU
554
ACUUUGUG GCCGAAAGGCGAGUCAAGGUCU UGUCUUCC
1460

70
AGCACAAA G UGGAUCCA
555
UGGAUCCA GCCGAAAGGCGAGUCAAGGUCU UUUGUGCU
1461

90
UCUGAAAU G CAGUAACU
495
AGUUACUG GCCGAAAGGCGAGUCAAGGUCU AUGUCAGA
1462

93
GAAAUGCA G UAACUCUG
556
CAGAGUUA GCCGAAAGGCGAGUCAAGGUCU UGCAUUUC
1463

104
ACUCUGAU G CUUGAAUU
497
AAUUCAAG GCCGAAAGGCGAGUCAAGGUCU AUCAGAGU
1464

114
UUGAAUUU G UUCUCCCU
557
AGGGAGAA GCCGAAAGGCGAGUCAAGGUCU AAAUUCAA
1465

127
CCCUUCUU G CCAGAAAG
499
CUUUCUGG GCCGAAAGGCGAGUCAAGGUCU AAGAAGGG
1466

151
AUAACUCG G UGUCAAAG
558
CUCUGACA GCCGAAAGGCGAGUCAAGGUCU CGAGUUAU
1467

153
AACUCGGU G UCAAAGCC
559
GGCUUUGA GCCGAAAGGCGAGUCAAGGUCU ACCGAGUU
1468

159
GUGUCAAA G CCAAGACA
560
UGUCUUGG GCCGAAAGGCGAGUCAAGGUCU UUUGACAC
1469

194
UUCCAAAA G CUUCACGU
561
ACGUGAAG GCCGAAAGGCGAGUCAAGGUCU UUUUGGAA
1470

201
AGCUUCAC G UUACAGCA
562
UGCUGUAA GCCGAAAGGCGAGUCAAGGUCU GUGAAGCU
1471

207
ACGUUACA G CAUGGAAG
563
CUUCCAUG GCCGAAAGGCGAGUCAAGGUCU UGUAACGU
1472

215
GCAUGGAA G CUGUUGCC
564
GGCAACAG GCCGAAAGGCGAGUCAAGGUCU UUCCAUGC
1473

218
UGGAAGCU G UUGCCAAG
565
CUUGGCAA GCCGAAAGGCGAGUCAAGGUCU AGCUUCCA
1474

221
AAGCUGUU G CCAAGUUU
500
AAACUUGG GCCGAAAGGCGAGUCAAGGUCU AACAGCUU
1475

226
GUUGCCAA G UUUGAUUU
566
AAAUCAAA GCCGAAAGGCGAGUCAAGGUCU UUGGCAAC
1476

239
AUUUCACU G CUUCAGGU
502
ACCUGAAG GCCGAAAGGCGAGUCAAGGUCU AGUGAAAU
1477

246
UGCUUCAG G UGAGGAUG
567
CAUCCUCA GCCGAAAGGCGAGUCAAGGUCU CUGAAGCA
1478

261
UGAACUGA G CUCUCACA
568
UGUGAAAG GCCGAAAGGCGAGUCAAGGUCU UCAGUUCA
1479

278
CUGGAGAU G UUUUGAAG
569
CUUCAAAA GCCGAAAGGCGAGUCAAGGUCU AUCUCCAG
1480

294
GAUUUUAA G UAACCAAG
570
CUUGGUUA GCCGAAAGGCGAGUCAAGGUCU UUAAAAUC
1481

307
CAAGAGGA G UGGUUUAA
571
UUAAACCA GCCGAAAGGCGAGUCAAGGUCU UCCUCUUG
1482

310
GAGGAGUG G UUUAAGGC
572
GCCUUAAA GCCGAAAGGCGAGUCAAGGUCU CACUCCUC
1483

317
GGUUUAAG G CGGAGCUU
573
AAGCUCCG GCCGAAAGGCGAGUCAAGGUCU CUCCUUGC
1494

322
AAGGCGGA G CUUGGGAG
574
CUCCCAAG GCCGAAAGGCGAGUCAAGGUCU UCCGCCUU
1485

330
GCUUGGGA G CCAGGAAG
575
CUUCCUGG GCCGAAAGGCGAGUCAAGGUCU UCCCAAGC
1486

344
AAGGAUAU G UGCCCAAG
576
CUUGGGCA GCCGAAAGGCGAGUCAAGGUCU AUAUCCUU
1487

346
GGAUAUGU G CCCAAGAA
507
UUCUUGGG GCCGAAAGGCGAGUCAAGGUCU ACAUAUCC
1488

370
GACAUCCA G UUUCCCAA
577
UUGGGAAA GCCGAAAGGCGAGUCAAGGUCU UGGAUGUC
1489

392
CCCAAAUG G UCUCACGA
578
UCGUGAAA GCCGAAAGGCGAGUCAAGGUCU CAUUUGGG
1490

393
UCACGAAG G CCUCUCUC
579
GAGAGAGG GCCGAAAGGCGAGUCAAGGUCU CUUCGUGA
1491

410
GACACCAG G CAGAGAAC
580
GUUCUCUG GCCGAAAGGCGAGUCAAGGUCU CUGGUGUC
1492

429
ACUCAUGG G CAAGGAGG
581
CCUCCUUG GCCGAAAGGCGAGUCAAGGUCU CCAUGAGU
1493

437
GCAAGGAG G UUGGCUUC
582
GAAGCCAA GCCGAAAGGCGAGUCAAGGUCU CUCCUUGC
1494

441
GGAGGUUG G CUUCUUCA
583
UGAAGAAG GCCGAAAGGCGAGUCAAGGUCU CAACCUCC
1495

458
UCAUCCGG G CCAGCCAG
584
CUGGCUGG GCCGAAAGGCGAGUCAAGGUCU CCGGAUGA
1496

462
CCGGGCCA G CCAGAGCU
585
AGCUCUGG GCCGAAAGGCGAGUCAAGGUCU UGGCCCGG
1497

468
CAGCCAGA G CUCCCCAG
586
CUGGGGAG GCCGAAAGGCGAGUCAAGGUCU UCUGGCUG
1498

494
CCAUCUCU G UCAGGCAU
587
AUGCCUGA GCCGAAAGGCGAGUCAAGGUCU AGAGAUGG
1499

499
UCUGUCAG G CAUGAGGA
588
UCCUCAUG GCCGAAAGGCGAGUCAAGGUCU CUGACAGA
1500

512
AGGAUGAC G UUCAACAC
589
GUGUUGAA GCCGAAAGGCGAGUCAAGGUCU GUCAUCCU
1501

527
ACUUCAAG G UCAUGCGA
590
UCGCAUGA GCCGAAAGGCGAGUCAAGGUCU CUUGAAGU
1502

532
AAGGUCAU G CGAGACAA
512
UUGUCUCG GCCGAAAGGCGAGUCAAGGUCU AUGACCUU
1503

546
CAACAAGG G UAAUUACU
591
AGUAAUUA GCCGAAAGGCGAGUCAAGGUCU CCUUGUUG
1504

559
UACUUUCU G UGGACUGA
592
UCAGUCCA GCCGAAAGGCGAGUCAAGGUCU AGAAAGUA
1505

571
ACUGAGAA G UUUCCAUC
593
GAUGGAAA GCCGAAAGGCGAGUCAAGGUCU UUCUCAGU
1506

589
CUAAAUAA G CUGGUAGA
594
UCUACCAG GCCGAAAGGCGAGUCAAGGUCU UUAUUUAG
1507

593
AUAAGCUG G UAGACUAC
595
GUAGUCUA GCCGAAAGGCGAGUCAAGGUCU CAGCUUAU
1508

631
AGACAGAA G CAGAUCUU
596
AAGAUCUG GCCGAAAGGCGAGUCAAGGUCU UUCUGUCU
1509

669
AGACCAGG G UCACCGGG
597
CCCGGUGA GCCGAAAGGCGAGUCAAGGUCU CCUGGUCU
1510

678
UCACCGGG G CAACAGCC
598
GGCUGUUG GCCGAAAGGCGAGUCAAGGUCU CCCGGUGA
1511

684
GGGCAACA G CCUGGACC
599
GGUCCAGG GCCGAAAGGCGAGUCAAGGUCU UGUUGCCC
1512

697
GACCGGAG G UCCCAGGG
600
CCCUGGGA GCCGAAAGGCGAGUCAAGGUCU CUCCGGUC
1513

708
CCAGGGAG G CCCACACC
601
GGUGUGGG GCCGAAAGGCGAGUCAAGGUCU CUCCCUGG
1514

720
ACACCUCA G UGGGGCUG
602
CAGCCCCA GCCGAAAGGCGAGUCAAGGUCU UGAGGUGU
1515

725
UCAGUGGG G CUGUGGGA
603
UCCCACAG GCCGAAAGGCGAGUCAAGGUCU CCCACUGA
1516

728
GUGGGGCU G UGGGAGAA
604
UUCUCCCA GCCGAAAGGCGAGUCAAGGUCU AGCCCCAC
1517

763
AACCGGAA G CUGUCGGA
605
UCCGACAG GCCGAAAGGCGAGUCAAGGUCU UUCCGGUU
1518

766
CGGAAGCU G UCGGAUCA
606
UGAUCCGA GCCGAAAGGCGAGUCAAGGUCU AGCUUCCG
1519

793
CUUCCCCU G CAGCAGCA
520
UGCUGCUG GCCGAAAGGCGAGUCAAGGUCU AGGGGAAG
1520

796
CCCCUGCA G CAGCACCA
607
UGGUGCUG GCCGAAAGGCGAGUCAAGGUCU UGCAGGGG
1521

799
CUGCAGCA G CACCAGCA
608
UGCUGGUG GCCGAAAGGCGAGUCAAGGUCU UGCUGCAG
1522

805
CAGCACCA G CACCAGCC
609
GGCUGGUG GCCGAAAGGCGAGUCAAGGUCU UGGUGCUG
1523

811
CAGCACCA G CCACAGCC
610
GGCUGUGG GCCGAAAGGCGAGUCAAGGUCU UGGUGCUG
1524

817
CAGCCACA G CCUCCGCA
611
UGCGGAGG GCCGAAAGGCGAGUCAAGGUCU UGUGGCUG
1525

823
CAGCCUCC G CAAUAUGC
521
GCAUAUUG GCCGAAAGGCGAGUCAAGGUCU GGAGGCUG
1526

830
CGCAAUAU G CCCCAGCG
522
CGCUGGGG GCCGAAAGGCGAGUCAAGGUCU AUAUUGCG
1527

836
AUGCCCCA G CGCCCCAG
612
CUGGGGCG GCCGAAAGGCGAGUCAAGGUCU UGGGGCAU
1528

838
GCCCCAGC G CCCCAGCA
523
UGCUGGGG GCCGAAAGGCGAGUCAAGGUCU GCUGGGGC
1529

844
GCGCCCCA G CAGCUGCA
613
UGCAGCUG GCCGAAAGGCGAGUCAAGGUCU UGGGGCGC
1530

847
CCCCAGCA G CUGCAGCA
614
UGCUGCAG GCCGAAAGGCGAGUCAAGGUCU UGCUGGGG
1531

850
CAGCAGCU G CAGCAGCC
524
GGCUGCUG GCCGAAAGGCGAGUCAAGGUCU AUCUGCUG
1532

853
CAGCUGCA G CAUCCCCC
615
GGGGGCUG GCCGAAAGGCGAGUCAAGGUCU UGCAGCUG
1533

856
CUGCAGCA G CCCCCACA
616
UGUGGGGG GCCGAAAGGCGAGUCAAGGUCU UGCUGCAG
1534

865
CCCCCACA G CAGCGAUA
617
UAUCGCUG GCCGAAAGGCGAGUCAAGGUCU UGUGGGGG
1535

868
CCACAGCA G CGAUAUCU
618
AGAUAUCG GCCGAAAGGCGAGUCAAGGUCU UGCUGUGG
1536

877
CGAUAUCU G CAGCACCA
526
UGGUGCUG GCCGAAAGGCGAGUCAAGGUCU AGAUAUCG
1537

880
UAUCUGCA G CACCACCA
619
UGGUGGUG GCCGAAAGGCGAGUCAAGGUCU UGCAGAUA
1538

903
CCAGGAAC G CCGAGGAG
527
CUCCUCGG GCCGAAAGGCGAGUCAAGGUCU GUUCCUGG
1539

912
CCGAGGAG G CAGCCUUG
620
CAAGGCUG GCCGAAAGGCGAGUCAAGGUCU CUCCUCGG
1540

915
AGGAGGCA G CCUUGACA
621
UGUCAAGG GCCGAAAGGCGAGUCAAGGUCU UGCCUCCU
1541

934
AAUGAUGG G CAUUGUGG
622
CCACAAUG GCCGAAAGGCGAGUCAAGGUCU CCAUCAUU
1542

939
UGGGCAUU G UGGCACCG
623
CGGUGCCA GCCGAAAGGCGAGUCAAGGUCU AAUGCCCA
1543

942
GCAUUGUG G CACCGGCU
624
AGCCGGUG GCCGAAAGGCGAGUCAAGGUCU CACAAUGC
1544

948
UGGCACCG G CUUGGGCA
625
UGCCCAAG GCCGAAAGGCGAGUCAAGGUCU CGGUGCCA
1545

954
CGGCUUGG G CAGUGAAA
626
UUUCACUG GCCGAAAGGCGAGUCAAGGUCU CCAAGCCG
1546

957
CUCGGGCA G UGAAAUGA
627
UCAUUUCA GCCGAAAGGCGAGUCAAGGUCU UGCCCAAG
1547

968
AAAUGAAU G CGGCCCUC
533
GAGGGCCG GCCGAAAGGCGAGUCAAGGUCU AUUCAUUU
1548

971
UGAAUGCG G CCCUCAUG
628
CAUGAGGG GCCGAAAGGCGAGUCAAGGUCU CGCAUUCA
1549

979
GCCCUCAU G CAUCGGAG
534
CUCCGAUG GCCGAAAGGCGAGUCAAGGUCU AUGAGGGC
1550

1001
CAGACCCA G UGCAGCUC
629
GAGCUGCA GCCGAAAGGCGAGUCAAGGUCU UGGGUCUG
1551

1003
GACCCAGU G CAGCUCCA
535
UGGAGCUG GCCGAAAGGCGAGUCAAGGUCU ACUGGGUC
1552

1006
CCAGUGCA G CUCCAGGC
630
GCCUGGAG GCCGAAAGGCGAGUCAAGGUCU UGCACUGG
1553

1013
AGCUCCAG G CGGCAGGG
631
CCCUGCCG GCCGAAAGGCGAGUCAAGGUCU CUGGAGCU
1554

1016
UCCAGGGG G CAGGGCGA
632
UCGUCCUG GCCGAAAGGCGAGUCAAGGUCU CGCCUGGA
1555

1021
GCGGCAGG G CGAGUGCG
633
CGCACUCG GCCGAAAGGCGAGUCAAGGUCU CCUGCCGC
1556

1025
CAGGGCGA G UGCGGUGG
634
CCACCGCA GCCGAAAGGCGAGUCAAGGUCU UCGCCCUG
1557

1027
GGGCGACU G CGGUGGGC
537
GCCCACCG GCCGAAAGGCGAGUCAAGGUCU ACUCGCCC
1558

1030
CGAGUGCG G UGGGCCCG
635
CGGGCCCA GCCGAAAGGCGAGUCAAGGUCU CGCACUCG
1559

1034
UGCGGUGG G CCCGGGCG
636
CGCCCGGG GCCGAAAGGCGAGUCAAGGUCU CCACCGCA
1560

1040
GGGCCCGG G CGCUGUAU
637
AUACAGCG GCCGAAAGGCGAGUCAAGGUCU CCGGGCCC
1561

1042
GCCCGGGC G CUGUACGA
538
UCAGACAG GCCGAAAGGCGAGUCAAGGUCU GCCCGGGC
1562

1045
CGGGCGCU G UAUGACUU
638
AAGUCAUA GCCGAAAGGCGAGUCAAGGUCU AGCGCCCG
1563

1058
ACUCUGAG G CCCUGGAG
639
CUCCAGGG GCCGAAAGGCGAGUCAAGGUCU CUCAAAGU
1564

1075
GAUGACGA G CUGGGGUU
640
AACCCCAG GCCGAAAGGCGAGUCAAGGUCU UCGUCAUC
1565

1081
GAGCUGGG G UUCCACAG
641
CUGUGGAA GCCGAAAGGCGAGUCAAGGUCU CCCAGCUC
1566

1089
GUUCCACA G CGGGGACC
642
CCUCCCCG GCCGAAAGGCGAGUCAAGGUCU UGUGGAAC
1567

1097
GCGGGGAG G UGGUGGAG
643
CUCCACCA GCCGAAAGGCGAGUCAAGGUCU CUCCCCGC
1568

1100
GGGAGGUG G UGGAGGUC
644
GACCUCCA GCCGAAAGGCGAGUCAAGGUCU CACCUCCC
1569

1106
UGGUGGAG G UCCUGGAU
645
AUCCAGGA GCCGAAAGGCGAGUCAAGGUCU CUCCACCA
1570

1116
CCUGGAUA G CUCCAACC
646
GGUUGGAG GCCGAAAGGCGAGUCAAGGUCU UAUCCAGG
1571

1132
CCAUCCUG G UGGACCGG
647
CCGGUCCA GCCGAAAGGCGAGUCAAGGUCU CAGGAUGG
1572

1140
GUGGACCG G CCGCCUGC
648
GCAGGCGG GCCGAAAGGCGAGUCAAGGUCU CGGUCCAC
1573

1143
GACCGGCC G CCUGCACA
543
UGUGCAGG GCCGAAAGGCGAGUCAAGGUCU GGCCGGUC
1574

1147
GGCCGCCU G CACAACAA
544
UUGUUGUG GCCGAAAGGCGAGUCAAGGUCU AGGCGGCC
1575

1156
CACAACAA G CUGGGCCU
649
AGGCCCAG GCCGAAAGGCGAGUCAAGGUCU UUGUUGUG
1576

1161
CAAGCUGG G CCUCUUCC
650
GGAAGAGG GCCGAAAGGCGAGUCAAGGUCU CCAGCUUG
1577

1172
UCUUCCCU G CCAACUAC
545
GUAGUUGG GCCGAAAGGCGAGUCAAGGUCU AGGGAAGA
1578

1181
CCAACUAC G UGGCACCC
651
GGGUGCCA GCCGAAAGGCGAGUCAAGGUCU GUAGUUGG
1579

1184
ACUACGUG G CACCCAUG
652
CAUGGGUG GCCGAAAGGCGAGUCAAGGUCU CACGUAGU
1580

1220
GGACAGAA G CUUUUUGU
653
ACAAAAAG GCCGAAAGGCGAGUCAAGGUCU UUCUGUCC
1581

1227
AGCUUUUU G UCUGGAGC
654
GCUCCAGA GCCGAAAGGCGAGUCAAGGUCU AAAAAGCU
1582

1234
UGUCUGGA G CUGCCCAC
655
GUGGGCAG GCCGAAAGGCGAGUCAAGGUCU UCCAGACA
1583

1237
CUGGAGCU G CCCACAAG
548
CUUGUGGG GCCGAAAGGCGAGUCAAGGUCU AGCUCCAG
1584

1253
GAAAGAGG G CAAGGAAA
656
UUUCCUUG GCCGAAAGGCGAGUCAAGGUCU CCUCUUUC
1585

1266
GAAAAAAG G CUGGACUC
657
GAGUCCAG GCCGAAAGGCGAGUCAAGGUCU CUUUUUUC
1586

Input Sequence = HSA011736.

Cut Site = G/Y

Stem Length = 8.

Core Sequence = GCcgaaagGCGaGuCaaGGuCu

HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp)

[0209]

7

TABLE VII

Human GRID DNAzyme and Substrate Sequence

Seq

Seq

Pos
Substrate
ID
DNAzyme
ID

11
GAGGCACA G UUAAUGGA
550
TCCATTAA GGCTAGCTACAACGA TGTGCCTC
1587

15
CACAGUUA A UGGAUCUG
658
CAGATCCA GGCTAGCTACAACGA TAACTGTG
1588

19
GUUAAUGG A UCUGUAAA
659
TTTACAGA GGCTAGCTACAACGA CCATTAC
1589

23
AUGGAUCU G UAAACUUG
551
CAAGTTTA GGCTAGCTACAACGA AGATCCAT
1590

27
AUCUGUAA A CUUGCACC
660
GGTGCAAG GGCTAGCTACAACGA TTACAGAT
1591

31
GUAAACUU G CACCCUCU
493
AGAGGUTO GGCTAGCTACAACGA AAGTTTAC
1592

33
AAACUUGC A CCCUCUUU
183
AAAGAGGG GGCTAGCTACAACGA GCAAGTTT
1593

46
CUUUCAGA G UGGUACAU
552
ATGTAOCA GGCTAGCTACAACGA TCTGAAAG
1594

49
UCAGAGUG G UACAUGGA
553
TCCATGTA GGCTAGCTACAACGA CACTCTGA
1595

51
AGAGUGGU A CAUGGAAG
10
CTTCCATG GGCTAGCTACAACGA ACCACTOT
1596

53
AGUGGUAC A UGGAAGAC
189
GTCTTCCA GGCTAGCTACAACGA UTACCACT
1597

60
CAUGGAAG A CAGCACAA
661
TTGTGCTG GGCTAGCTACAACGA CTTCCATG
1599

63
GGAAGACA G CACAAAGU
554
ACTTTGTG GGCTAGCTACAACGA TGTCTTCC
1599

65
AAGACAGC A CAAAGUGG
191
CCACTTTG GGCTAGCTACAACGA GCTGTCTT
1600

70
AGCACAAA G UGGAUCCA
555
TGGATCCA GGCTAGCTACAACGA TTTGTGCT
1601

74
CAAAGUGG A UCCAUACU
662
AUTATOGA GGCTAGCTACAACGA CCACTTTG
1602

78
GUGGAUCC A UACUCUGA
194
TCAGAGTA GGCTAGCTACAACGA GGATCCAC
1603

80
GGAUCCAU A CUCUGAAA
12
TTTCAGAG GGCTAGCTACAACGA ATGGATCC
1604

88
ACUCUGAA A UGCAGUAA
663
TTACTGCA GGCTAGCTACAACGA TTCAGAGT
1605

90
UCUGAAAU G CAGUAACU
495
AGTTACTG GGCTAGCTACAACGA ATTTCAGA
1606

93
GAAAUGCA G UAACUCUG
556
CAGAGTTA GGCTAGCTACAACGA TGCATTTC
1607

96
AUGCAGUA A CUCUGAUG
664
CATOAGAG GGCTAGCTACAACGA TACTUCAT
1608

102
UAACUCUG A UGCUUGAA
665
TTCAAGCA GGCTAGCTACAACGA CAGAGTTA
1609

104
ACUCUGAU G CUUGAAUU
497
AATTCAAG GUCTAGCTACAACGA ATCAGAUT
1610

110
AUGCUUGA A UUUGUUCU
666
AGAACAAA GGCTAGCTACAACGA TCAAGCAT
1611

114
UUGAAUUU G UUCUCCCU
557
AGGGAUAA GGCTAGCTACAACGA AAATTCAA
1612

127
CCCUUCUU G CCAGAAAG
499
CTTTCTGG GGCTAGCTACAACGA AAGAAGGU
1613

137
CAGAAAGG A UUCUAAUA
667
TATTAGAA GGCTAGCTACAACGA CCTTTCTG
1614

143
GGAUUCUA A UAACUCGG
668
CCGAGTTA GGCTAGCTACAACGA TAGAATCC
1615

146
UUCUAACA A CUCGGUGC
669
ACACCUAG GGCTAGCTACAACGA TATTAGAA
1616

151
AUAACUCG G UGUCAAAG
558
CTTTGACA GGCTAUCTACAACGA CGAUTTAT
1617

153
AACUCGGU G UCAAAGCC
559
UGCTTTGA GGCTAGCTACAACGA ACCUAGTT
1618

159
GUGUCAAA G CCAAGACA
560
TUTOTTUG GGCTAGCTACAACGA TTTUACAC
1619

165
AAGCCAAG A CAUAAACU
670
AUTTTATG UGCTAGCTACAACGA CTTGGCTT
1620

167
GCCAAGAC A UAAACUCA
213
TGAGTTTA GGCTAGCTACAACGA GTCTTGUC
1621

171
AGACAUAA A CUCAAUCU
671
AGATTUAG GGCTAGCTACAACGA TTATUTCT
1622

176
UAAACUCA A UCUCUUCU
672
AGAAGAGA GGCTAGCTACAACGA TGAGTTTA
1623

194
UUCCAAAA G CUUCACGU
561
ACGTGAAG GGCTAGCTACAACGA TTTTGGAA
1624

199
AAAGCUUC A CGUUACAG
223
CTGTAACG GGCTAGCTACAACGA GAAGCTTT
1625

201
AGCUUCAC G UUACAGCA
562
TGCTGTAA GGCTAGCTACAACGA GTGAAGCT
1626

204
UUCACGUU A CAGCAUGG
43
CCATGCTG GUCTAGCTACAACGA AACGTGAA
1627

207
ACGUUACA G CAUGGAAG
563
CTTCCATU GGCTAGCTACAACGA TGTAACGT
1628

209
GUUACAGC A UGGAAGCU
225
AGCTTCCA GGCTAGCTACAACGA GCTGTAAC
1629

215
GCAUGGAA G CUGUUGCC
564
GGCAACAG GGCTAGCTACAACGA TTCCATUC
1630

218
UGGAAGCU G UUGCCAAG
565
CTTGGCAA GUCTAUCTACAACGA AUCTTCCA
1631

221
AAGCUGUU G CCAAGUUU
500
AAACTTGG GGCTAGCTACAACGA AACAUCTT
1632

226
GUUGCCAA G UUUGAUUU
566
AAATCAAA GGCTAGCTACAACGA TTGGCAAC
1633

231
CAAGUUUG A UUUCACUG
673
CAGTGAAA GGCTAGCTACAACGA CAAACTTG
1634

236
UUGAUUUC A CUGCUUCA
229
TGAAGCAC GGCTAGCTACAACGA GAAATCAA
1635

239
AUUUCACU G CUUCAGGU
502
ACCTGAAC GCCTAGCTACAACGA ACTGAAAT
1636

246
UGCUUCAG G UGAGGAUG
567
CATCCTCA GGCTAGCTACAACGA CTGAAGCA
1637

252
AGGUGAGG A UGAACUGA
674
TCAGTTCA GGCTAGCTACAACGA CCTCACCT
1638

256
GAGGAUGA A CUGAGCUU
675
AAGCTCAG GGCTAGCTACAACGA TCATCCTC
1639

261
UGAACUGA G CUUUCACA
568
TGTGAAAG GGCTAGCTACAACGA TCAGTTCA
1640

267
GAGCUUUC A CACUGGAG
235
CTCCAGTG GGCTAGCTACAACGA GAAAGCTC
1641

269
GCUUUCAC A CUGGAGAU
236
ATCTCCAG GGCTAGCTACAACGA GTGAAAGC
1642

276
CACUGGAG A UGUUUUGA
676
TCAAAACA GGCTAGCTACAACGA CTCCAGTG
1643

278
CUGGAGAU G UUUUGAAG
569
CTTCAAAA GGCTAGCTACAACGA ATUTOCAG
1644

287
UUUUGAAG A UUUUAAGU
677
ACTTAAAA GGCTAGCTACAACGA CTTCAAAA
1645

294
GAUUUUAA G UAACCAAG
570
CTTGGTTA GGCTAGCTACAACGA TTAAAATC
1646

297
UUUAAGUA A CCAAGAGG
678
CCTCTTGG GGCTAGCTACAACGA TACTTAAA
1647

307
CAAGAGGA G UGGUUUAA
571
TTAAACCA GGCTAGCTACAACGA TCCTCTTG
1648

310
GAGGAGUG G UUUAAGGC
572
GCCTTAAA GGCTAGCTACAACGA CACTCCTC
1649

317
GGUUUAAG C CGGAGCUU
573
AAGCTCCG GGCTAGCTACAACGA CTTAAACC
1650

322
AAGGCGGA G CUUGGGAG
574
CTCCCAAG GGCTAGCTACAACGA TCCGCCTT
1651

330
GUUUGGGA U CCAGGAAG
575
CTTCCTGG GGCTAGCTACAACGA TCCCAAGC
1652

340
CAGGAAGG A UAUGUGCC
679
GGCACATA GGCTAGCTACAAUGA CCTTCCTG
1653

342
GGAAGGAU A UGUGCCCA
67
TGGGCACA GGCTAGCTACAACGA ATCCTTCC
1654

344
AAGGAUAU G UGCCCAAG
576
CTTGGGCA GGCTAGCTACAACGA ATATCCTT
1655

346
GGAUAUGU G CCCAAGAA
507
TTCTTGGG GGCTAGCTACAACGA ACATATCC
1656

354
GCCCAAGA A UUUCAUAG
680
CTATGAAA GGCTAGCTACAACGA TCTTGGGC
1657

359
AGAAUUUC A UAGACAUC
246
GATGTCTA GGCTAGCTACAACGA GAAATTCT
1658

363
UCUCAGAG A CAUCCAGU
681
ACTGGATG GGCTAGCTACAACGA CTATGAAA
1659

365
UCACAGAC A UCCAGUUU
247
AAACTGGA GGCTAGCTACAACGA GTCTATGA
1660

370
GACAUCCA C UUUCCCAA
577
TTGGGAAA GGCTAGCTACAACGA TGGATGTC
1661

379
UUUCCCAA A UGGUCUCA
682
TGAAACCA GGCTAGCTACAACGA TTGGGAAA
1662

382
CCCAAAUG U UUUCACGA
578
TCGTGAAA GGCTAGCTACAACGA CATTTGGG
1663

387
AUGGUCUC A CGAAGGCU
253
GGCCTTCG GGCTAGCTACAACGA GAAACCAT
1664

393
UCACGAAG C CCUCUCUC
579
GAGAGAGG GGCTAGCTACAACGA CTTCGTGA
1665

403
CUCUCUCO A CACCAGGC
683
GCCTGGTG GGCTAGCTACAAUGA CGAGAGAG
1666

405
CUCUCGAC A CCAGGCAG
258
CTGCCTGG GGCTAGCTACAACGA GTCGAGAG
1667

410
GACACCAG C CAGAGAAC
580
GTTCTCTG GGCTAGCTACAACGA CTGGTGTC
1668

417
GGCAGAGA A CUUACUCA
684
CGAGTAAG GGCTAGCTACAACGA TCTCTGCC
1669

421
GAGAACUU A CUCAUGGG
83
CCCATGAG GGCTAGCTACAACGA AAGTTCTC
1670

425
ACUWACUC A UGGGCAAG
264
CTTGCCCA GGCTAGCTACAACGA GAGTAAGT
1671

429
ACUCAUGO U CAAGGAGG
581
CCTCCTTG GGCTAGCTACAACGA CCATGAGT
1672

437
GCAAGGAG G UUGGCUUC
582
GAAGCCAA GGCTAGCTACAACGA CCATGAGT
1673

441
GGAGGUUG C CUUCUUCA
583
TGAAGAAG GGCTAGCTACAACCA CAACCTCC
1674

449
GCUUCUUC A UCAUCCUG
268
CCGGATGA GGCTAGCTACAACGA GAAGAAGC
1675

452
UCUOCAUC A UCCGGGCC
269
GGCCCGGA GGCTAGCTACAACGA GATGAAGA
1676

458
UCAUCCUG U CCAGCCAG
584
CTGGCTGG GGCTAGCTACAACGA CCGGATGA
1677

462
UCOGUCCA C CCAGAGCU
585
AGCTCTCG GCCTACCTACAACCA TGGCCCGG
1678

468
CAUCCAGA C CUCCOCAG
586
CTGGGGAG GGCTAGCTACAACGA TCTGCCTG
1679

480
UCCAGGUG A CUUCUCCA
685
TGGAGAAG CGCTAGCTACAACGA CCCCTGGG
1680

488
ACUUCUCC A UCUCUGUC
283
CACAGAGA GGCTAGCTACAACGA GGACAAGT
1681

494
CCAUCUCU G UCAGGCAU
587
ATGCCTGA GGCTAGCTACAACGA AGAGATGG
1682

499
UCUGUCAG C CAUCACCA
588
TCCTCATG CGCTAGCTACAACGA CTCACACA
1682

501
UGUCAGUC A UGAGGAUG
287
CATCCTCA GGCLAGCTACAACGA GCCTGACA
1684

507
GCAUGACG A UGACGUUC
686
GAACGTCA GGCTAGCTACAACGA CCTCATGC
1685

510
UGAGGAUG A CGUUCAAC
687
GTTGAACG GGCTAGCTACAACGA CATCCTCA
1686

512
AGGAUGAC C UUCAACAC
589
GTGTTGAA GGCTAGCTACAACGA GTCATCCT
1687

517
GACUCUCA A CACUUCAA
688
TTGAAGTG GGCTAGCTACAACGA TGAACGTC
1688

519
CGUUCAAC A CUUCAAGG
289
CCTTGAAG GGCTAGCTACAACGA GTTGAACG
1689

527
ACUUCAAG C UCAUGCGA
590
TCGCATGA GGCTAGCTACAACGA CTTGAAGT
1690

530
UCAAGGUC A UGGUAGAC
292
GTCTCGCA GGCTAGCTACAACGA GACCTTGA
1691

532
AAGGUCAU G CGAGACAA
512
TTGTCTCG GGCTAGCTACAACGA ATGACCTT
1692

537
CAUGCGAG A CAACAAGG
689
CCTTGTTG GGCTAGCTACAACGA CTCGCATG
1693

540
GCGAGACA A CAAGGGUA
690
TACCCTTG GGCTAGCTACAACGA TGTCTCGC
1694

546
CAACAAGG C UAAUUACU
591
ACTAATTA GGCTAGCTACAACGA CCTTGTTG
1695

549
CAAGGGUA A UUACUUUC
691
GAAAGTAA GGCTAGCTACAACGA TACCCTTG
1696

552
GGGUAAUU A CUUUCUGU
106
ACAGAAAG GGCTAGCTACAACGA AATTACCC
1697

559
UACGUUCU G UGGACUGA
592
TCAGTCCA GGCTAGCTACAACGA AGAAAGTA
1698

563
UUCUGUGG A CUGAGAAG
692
CTTCTCAG GGCTAGCTACAACGA CCACAGAA
1699

571
ACUGAGAA G UUUCCAUC
593
GATGGAAA GGCTAGCTACAACGA TTCTCAGT
1700

577
AAGUUUCC A UCCCUAAA
299
TTTAGGGA GGCTAGCTACAACGA GGAAACTT
1701

585
AUCCCUAA A UAAGCUGG
693
CCAGCTTA GGCTAGCTACAACGA TTAGGGAT
1702

589
CUAAAUAA G CUGGUAGA
594
TCTACCAG GGCTAGCTACAACGA TTATTTAG
1703

593
AUAAGCUG G UAGACUAC
595
GTAGTCTA GGCTAGCTACAACGA CAGCTTAT
1704

597
UCUGGUAG A CUACUACA
694
TGTAGTAG GGCTAGCTACAACGA CTACCAGC
1705

600
GGUAGACU A CUACAGGA
117
TCCTGTAG GGCTAGCTACAACGA AGTCTACC
1706

603
AGACUACU A CAGGACAA
118
TTGTCCTG GGCTAGCTACAACGA AGTAGTCT
1707

608
ACUACAGG A CAAAUUCC
695
GGAATTTG GGCTAGCTACAACGA CCTGTAGT
1708

612
CAGGACAA A UUCCAUCU
696
AGATGGAA GGCTAGCTACAACGA TTGTCCTG
1709

617
CAAAUUCC A UCUCCAGA
309
TCTGGAGA GGCTAGCTACAACGA GGAATTTG
1710

625
AUCUCCAG A CAGAAGCA
697
TGCTTCTG GGCTAGCTACAACGA CTGGAGAT
1711

631
AGACAGAA G CAGAUCUU
596
AAGATCTG GGCTAGCTACAACGA TTCTGTCP
1712

635
AGAAGCAG A UCUUCCUU
698
AAGGAAGA GGCTAGCTACAACGA CTGCTTCT
1713

648
CCUUAGAG A CAGAACCC
699
GGGTTCTG GGCTAGCTACAACGA CTCTAAGG
1714

653
GAGACAGA A CCCGAGAA
700
TTCTCGGG GGCTAGCTACAACGA TCTGTCTC
1715

663
CCGAGAAG A CCAGGGUC
701
GACCCTGG GGCTAGCTACAACGA CTTCTCGG
1716

669
AGACCAGG C UCACCGGG
597
CCCGGTGA GGCTAGCTACAACGA CCTGGTCT
1717

672
CCAGGGUC A CCGGGGCA
323
TGCCCCGG GGCTAGCTACAACGA GACCCTGG
1718

678
UCACCGGG C CAACAGCC
598
GGCTGTTG GCCTAGCTACAACCA CCCGGTGA
1719

681
CCGGGGCA A CAGCCUGG
702
CCAGGCTG GGCTAGCTACAACGA TGCCCCGG
1720

684
GGGCAACA C CCUCGACC
599
GGTCCAGG GGCTAGCTACAACGA TGTTGCCC
1721

690
CAGCCUGG A CCGGACGU
703
ACCTCCGG GGCTAGCTACAACGA CCAGGCTG
1722

697
GACCGGAG C UCCCAGGG
600
CCCTGGGA GGCTAGCTACAACGA CTCCGCTC
1723

708
CCAGGGAG C CCCACACC
601
GGTGTGCG GGCTAGCTACAACGA CTCCCTGG
1724

712
GGAGGCCC A CACCUCAG
335
CTGAGGTC GGCTAGCTACAACGA GGCCCTCC
1725

714
AGGCCCAC A CCUCAGUG
336
CACTGAGG GGCTAGCTACAACGA GTCGGCCT
1726

720
ACACCUCA C UGGGGCUG
602
CAGCCCCA GGCTAGCTACAACGA TGAGGTGT
1727

725
UCAGUGGG C CUCUCGCA
603
TCCCACAG GGCTAGCTACAACGA CCCACTGA
1728

728
GUGGGGCU C UGGGAGAA
604
TTCTCCCA GGCTAGCTACAACGA AGCCCCAC
1729

740
GAGAAGAA A UCCGACCU
704
AGCTCGGA GGCTAGCTACAACGA TTCTTCTC
1730

745
GAAAUCCG A CCUUCGAU
705
ATCGAAGG GGCTAGCTACAACGA CGGATTTC
1731

752
GACCUCG A UGAACCGG
706
CCGGTTCA GGCTAGCTACAACGA CGAAGGTC
1732

756
UUCGAUGA A CCGGAAGC
707
GCTTCCGG GGCTAGCTACAACGA TCATCGAA
1733

763
AACCGGAA G CUGUCGGA
605
TCCGACAG GGCTAGCTACAACGA TTCCGGTT
1734

766
CCGAAGCU G UCGGAUCA
606
TGATCCGA GGCTAGCTACAACGA AGCTTCCG
1735

771
GCUGUCGG A UCACCCCC
708
GGGGGTGA GGCTAGCTACAAOGA CCGACAGC
1736

774
CUCGGAC A CCCCCCGA
346
TCGGGGGG GGCTAGCTACAACGA GATCCGAC
1737

782
ACCCCCCG A CCCUUCCC
709
GGGAAGGG GGCTAGCTACAACGA CGGGGGGT
1738

793
CUUCCCCU G CAGCAGCA
520
TGCTGCTG GGCTAGCTACAACGA AGGGGAAG
1739

796
CCCCUGCA G CAGCACCA
607
TGGTGCTG GGCTAGCTACAACGA TGCAGGGG
1740

799
CUGCAGCA G CACCAGCA
608
TGCTGGTG GGCTAGCTACAACGA TGCTGCAG
1741

801
GCAGCAGC A CCAGCACC
361
GGTGCTGG GGCTAGCTACAACGA GCTGCTGC
1742

805
CAGCACCA G CACCAGCC
609
GGCTGGTG GGCTAGCTACAACGA TGGTGCTG
1743

807
GCACCAGC A CCAGCCAC
364
GTGGCTGG GGCTAGCTACAACGA GCTGGTGC
1744

811
CAGCACCA G CCACAGCC
610
GGCTGTGG GGCTAGCTACAACGA TGGTGCTG
1745

814
CACCAGCC A CAGCCUCC
368
GGAGGCTG GGCTAGCTACAACGA GGCTGGTG
1746

817
CAGCCACA G CCUCCGCA
611
TGCGGAGG GGCTAGCTACAACGA TGTGGCTG
1747

823
CAGCCUCC G CAAUAUGC
521
GCATATTG GGCTAGCTACAACGA GGAGGCTG
1748

826
CCUCCGCA A UAUGCCCC
710
GGGGCATA GCCTAGCTACAACGA TGCGGAGG
1749

828
UCCGCAAU A UGCCCCAG
139
CTGGGGCA GGCTAGCTACAACGA ATTGCGGA
1750

830
CGCAAAU C CCCCAGCG
522
CGCTGCGG GGCTAGCTACAACGA ATATTGCG
1751

836
AUCCUCCA C CGCCCCAG
612
CTGGGCCG GGCTAGCTACAACGA IGUGGUAT
1752

838
GCCCCAUC C CCCCAGCA
523
TOCTUGGO GGCTAGCTACAACGA GOTUGGOC
1753

844
CUGOCOCA C CACCUCCA
613
TCCACCTG GCCTACCTACAACCA TGGGGCGC
1754

847
CCCCAGCA G CUGCAGCA
614
TUCTOCAC GGCTAGCTACAACCA TGCTCGCG
1755

850
CACCAGUD G CAGCAGCC
524
GGCTGCTG GGCTAGCTACAACGA AGCTGCTG
1756

853
CACCUGCA C CACCCCCC
615
GGGGGCTG GGCTAGCTACAACGA TGCACCTC
1757

856
CUGCAGCA C CCCCCACA
616
TGTGGGGG GCCTACCTACAACCA TGCTCCAG
1758

862
CACUCCUC A CACCACUC
390
CGCTGCTG CGCTAGCTACAACGA CCGGCCTG
1759

865
OCUCCACA G CAGCCAUA
617
TATCCCTC CGCTAGCTACAACCA TGTCGGGC
1760

868
OCACACCA C CGAUAUCU
618
ACATATCC CGCTACCTACAACCA TCCTGTGC
1761

871
CAGUACOC A UAUOUCCA
711
TOCACATA GCCTACCTACAACCA CCCTGCTC
1762

873
GCAGCCAU A UCUGCAGC
140
GCTCCAGA GGCTAGCTACAACGA ATCCCTCC
1763

877
CCAUAUCU G CACCACCA
526
TGGTGCTC CGCTAGCTACAACCA AGATATCC
1764

880
UAUCUGCA C CACCACCA
619
TGGTGGTC GGCTAGCTACAACGA TGCAGATA
1765

882
UCUGCAGC A CCACCAUU
395
AATCGTCG CCCTACCTACAACCA CCTGCACA
1766

885
GCAGCACC A CCAUUUCC
397
GCAAATCG GGCTAGCTACAACGA GGTGCTGC
1767

888
GCACCACC A UUUCCACC
399
CGTCCAAA CGCTAGCTACAACGA GCTGCTGC
1768

894
CCAUUUCC A CCAGGAAC
401
CTTCCTGC GGCTACCTACAACGA GGAAATGG
1769

901
CACCAGGA A CGCCGAGG
712
CCTCGGCG GGCTAGCTACAACGA TCCTGGTG
1770

903
CCAGGAAC G CCGAGCAG
527
CTCCTCGC CCCTACCTACAACCA GTTCCTCC
1771

912
CCGACCAG C CACCCUUG
620
CAAGCCTG GGCTAGCTACAACGA CTCCTCGG
1772

915
ACGACGCA C CCUUCACA
621
TGTCAACG GCCTACCTACAACGA TCCCTCCT
1773

921
CACCC3UC A CAUAAAUG
713
CATTTATG GGCTACCTACAACGA CAAGGCTG
1774

923
GCCUUGAC A UAAAUGAU
408
ATCATTTA GGCTAGCTACAACGA GTCAACGC
1775

927
UGACAUAA A UGAUCGGC
714
CCCCATCA GGCTAGCTACAACCA TTATGTCA
1776

930
CAUAAAUG A UGGGCAUU
715
AATCCCCA GGCTAGCTACAACCA CATTTATG
1777

934
AAUCAUGG G CAUUGUCC
622
CCACAATC CGCTAGCTACAACCA CCATCATT
1778

936
UCAUGGCC A UUCUCCCA
409
TGCCACAA CCCTAGCTACAACCA GCCCATCA
1779

939
UGCAGCA3 C UGGCACCG
623
CGGTGCCA CGCTAGCTACAACGA AATGCCCA
1780

942
CCAUUGUG C CACCCCCU
624
AGCCCGTC CCCTACCTACAACGA CACAATGC
1781

944
AUUGUCGC A CCGCCUUC
410
CAAGCCGG GGCTAGCTACAACGA GCCACAAT
1782

949
UCGCACCG G CUUGGGCA
625
TGCCCAAC GGCTAGCTACAACGA CCCTCCCA
1783

954
CGGCUUCG G CAGUGAAA
626
TTTCACTC CGCTACCTACAACGA COAACCCG
1784

957
CUUGGGCA C UGAAAUCA
627
TCATTTCA GGCTACCTACAACGA TGCCCAAC
1785

962
GCAGUCAA A UGAAUGCG
716
CCCATTCA GGCTACCPACAACCA TTCAOTCC
1786

966
UGAAAUGA A UCCCGCCC
717
CCGCCGCA GGCTAGCTACAACGA TCATTTCA
1797

968
AAAUGAAU G CGGCCCUC
533
GAGCGCCC CGCTAGCTACAACGA ATTCATTT
1788

971
UCAAUGCC G CCCUCAUG
628
CATGAGGG GCCTAGCTACAACGA CCCATTCA
1789

977
CGCCCCUC A UGCAUCGC
417
CCCATCCA CCCTAGCTACAACCA CAGCGCCC
1790

979
GCCCUCAU G CAUCGCAC
534
CTCCGATG GGCTACCTACAACGA ATCACCCC
1791

981
CCUCAUGC A UCGGAGAC
418
GTCTCCGA CGCTAGCTACAACGA CCATGACG
1792

988
CAUCGCAG A CACACAGA
718
TCTGTGTG CGCTAGCTACAACCA CTCCGATC
1793

990
UCGGAGAC A CACAGACC
419
GGTCTGTC GGCTACCTACAACGA CTCTCCGA
1794

992
GGAGACAC A CAGACCCA
420
TGGCTCTG GGCTACCTACAACCA GTGTCTCC
1795

996
ACACACAC A CCCAGUGC
719
CCACTCGC GGCTAGCTACAACGA CTGTGTGT
1796

1001
CAGACCCA G UGCAGCUC
629
CACCTCCA CGCTAGCTACAACGA TCCGTCTC
1797

1003
GACCCAGU C CAGCUCCA
535
TCCACCTC GCCTACCTACAACCA ACTCCCTC
1798

1006
CCAGUGCA G CUCCAGUC
630
CCCTCCAG GCCTACCTACAACGA TGCACTGG
1799

1013
AGCUCCAG G CGGCAGCC
631
CCCTCCCG GGCTAGCTACAACGA CTGGACCT
1800

1016
UCCAGGCG G CAGGUCCA
632
TCGCCCTG GGCTAGCTACAACGA CGCCTCGA
1801

1021
CCGGCAGC G CGAGUGCG
633
CGCACTCG GCCTAGCTACAACCA CCTGCCGC
1802

1025
CACGGCGA G UGCGGUGG
634
CCACCGCA GGCTAGCTACAACGA TCGCCCTG
1803

1027
GGCCGACU C CGCUCCGC
537
CCCCACCC GCCTACCTACAACCA ACTCCCCC
1804

1030
CGAGUCCG C UGGGCCCG
635
CGGGCCCA CCCTACCTACAACGA CCCACTCC
1805

1034
UCCCCUCC C CCCCGCCC
636
CGCCCGGG CCCTACCTACAACCA CCACCCCA
1806

1040
CCCCCCCC C CCCUCUAU
637
ATACAGCG CCCTACCTACAACCA CCCCCCCC
1807

1042
CCCCCGCC C CUCUAUCA
538
TCATACAG CCCTACCTACAACCA CCCCCCCC
1808

1045
CGGGCCCU C UAUCACUU
638
AACTCATA CCCTACCTACAACCA ACCCCCCC
1809

1047
CCCCCUCU A UCACUUUC
152
CAAACTCA CCCTACCTACAACCA ACACCCCC
1810

1050
CCUCUAUC A CUUUCACC
720
CCTCAAAC CCCTACCTACAACCA CATACAGC
1811

1058
ACUUUGAG C CCCUGGAG
639
CTCCAGGG CCCTACCTACAACCA CTCAAACT
1812

1068
CCUGCAGC A UGACGAGC
721
CCTCGTCA CCCTACCTACAACCA CCTCCAGC
1813

1071
GGAGGAUG A CGAGCUGG
722
CCACCTCC CCCTACCTACAACCA CATCCTCC
1814

1075
GAUCACCA C CUCCUCUC
640
AACCCCAC CCCTACCTACAACCA TCCTCATC
1815

1081
GAGCUGGG C UUCCACAG
641
CTCTCCAA CCCTACCTACAACCA CCCACCTC
1816

1086
GGGGUUCC A CACCCCCG
439
CCCCCCTC CCCTACCTACAACCA CCAACCCC
1817

1089
GUUCCACA C CCCCCAGC
642
CCTCCCCC CCCTACCTACAACCA TCTCCAAC
1818

1097
GCGCCCAC C UCCUGGAC
643
CTCCACCA CCCTACCTACAACCA CTCCCCCC
1819

1100
GGGGGCUG C UGGAGGUC
644
CACCTCCA CCCTACCTACAACCA CACCTCCC
1820

1106
UCCUCCAG C UCCUGGAU
645
ATCCACCA CCCTACCTACAACCA CTCCACCA
1821

1113
GGUGGUCC A UACCUGGA
723
TUCACOTA CCCTACCTACAACCA CCAGCACC
1822

1116
CCUCCAUA C CUCCAACC
646
CCTTCCAC CCCTACCTACAACCA TATCCACC
1823

1122
UAGCUCCA A CCCAUCCU
724
ACCATCCC CCCTACCTACAACCA TGCACCTA
1824

1126
UCCAACCC A UCCUGGUG
448
CACCACCA CGCTAGCTACAACGA CCGTTCCA
1825

1132
CCAUCCUG G UGGACCGG
647
CCCCTCCA CCCTACCTACAACCA CACCATUG
1826

1136
CCUGGUGG A CCGGCCGC
725
CCCGCCCG CCCTACCTACAACCA CCACCACC
1827

1140
GUCCACCG C CCGCCUGC
648
CCACCCCC CCCTACCTACAACCA CCCTCCAC
1828

1143
GACCGGCC G CCUGCACA
543
TGTGCAGG GGCTAGCTACAACGA GGCCGGTC
1829

1147
GGCCCCCU C CACAACAA
544
TTCTTCTC CGCTAGCTACAACGA ACCCCCCC
1830

1149
CCGCCCGC A CAACAAGC
455
CCTTCTTG GGCTAGCTACAACCA GCAGGCGG
1831

1152
CCUGCACA A CAACCUGG
726
CCAGCTTG GGCTAGCTACAACGA TGTGCAGG
1832

1156
CACAACAA C CUGGGCCU
649
AGGCCCAG GGCTAGCTACAACGA TTCTTGTG
1833

1161
CAACCUCC C CCJCUUCC
650
GGAACACG GCCTAGCTACAACGA CCACCTTC
1834

1172
UCUUCCCU C CCAACUAC
545
GTAGTTGG GCCTACCTACAACCA ACCCAACA
1835

1176
CCCUCCCA A CUACGUGC
727
CCACCTAG CGCTAGCTACAACGA TGCCAGGG
1836

1179
UCCCAACU A CCUCCCAC
164
GTCCCACC CCCTAGCTACAACGA AGTTCCCA
1837

1181
CCAACUAC G UCCCACCC
651
CGCTCCCA CCCTACCTACAACCA CTAGTTGG
1838

1184
ACLACGDC G CACCCAUG
652
CATGCGTG CGCTACCTACAACCA CACCTAGT
1839

1186
UACCUGCC A CCCAUCAC
468
GTCATGGG GCCTAGCTACAACGA CCCACGTA
1840

1190
UCGCACCC A UCACCCCA
471
TCGCGTCA GGCTAGCTACAACCA CGCTCCCA
1841

1193
CACCCAUG A CCCGAUAA
728
TTATCGCC CGCTACCTACAACGA CATCCCTC
1842

1198
AUCACCCC A UAAACUCU
729
AGACTTTA GGCTAGCTACAACCA CGGGTCAT
1843

1202
CCCGAUAA A CUCUUCAC
730
CTGAAGAC GCCTAGCTACAACGA TTATCGGC
1844

1214
UUCAGGCC A CAGAAGCU
731
AGCTTCTG CCCTAGCTACAACGA CCCCTGAA
1845

1220
GGACAGAA G CUUUUUGU
653
ACAAAAAC GGCTAGCTACAACCA TTCTCTCC
1846

1227
AGCUUUUU C UCUGGAGC
654
COTOCAGA GGCTAGCTACAACCA AAAAACCT
1847

1234
UGUCUGGA G CUGCCCAC
655
CTCGGCAG GGCTAGCTACAACCA TCCAGACA
1848

1237
CUGCAGCU G CCCACAAG
548
CTTGTCGC GGCTAGCTACAACGA AGCTCCAG
1849

1241
AGCUGCCC A CAAGAAAG
483
CTTTCTTG GGCTAGCTACAACGA GGGCAGCT
1850

1253
CAAAGAGG G CAAGGAAA
656
TTTCCTTG GGCTAGCTACAACCA CCTCTTTC
1851

1266
GAAAAAAG C CUGGACUC
657
CAGTCCAG GCCTAGCTACAACGA CTTTTTTC
1852

1271
AAGGCUCG A CUCCAUGA
732
TCATCCAC CCCTACCTACAACCA CCAGCCTT
1853

1276
UGGACUCC A UGACUAUA
489
TATACTCA CCCTACCTACAACCA CCACTCCA
1854

1279
ACUCCAUG A CUAUAUAU
733
ATATATAG CGCTACCTACAACCA CATGCAGT
1855

1282
CCAUGACU A UAUAUACA
175
TCTATATA CCCTACCTACAACGA AGTCATCG
1856

1284
AUGACUAU A UAUACAUA
176
TATCTATA CCCTACCTACAACGA ATACTCAT
1857

1286
GACUAUAU A UACAUACA
177
TCTATGTA GCCTAGCTACAACCA ATATACTC
1858

1288
CUAUAUAU A CAUACAUC
178
CATGTATG CCCTACCTACAACCA ATATATAC
1859

1290
AUAUAUAC A UACAUCUA
491
TACATCTA CGCTACCTACAACCA CTATATAT
1860

1292
AUAUACAU A CAUCUAUC
179
CATACATC CCCTACCTACAACCA ATCTATAT
1861

1294
AUACAUAC A UCUAUCUA
492
TACATACA CCCTACCTACAACCA CTATCTAT
1862

Input Sequence = HSA011736.

Cut Site = R/Y

Stem Length = 8.

Core Sequence = GGCTAGCTACAACGA

HSA011736 (Home sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp)

[0210]

8

TABLE VIII

Human GRID Amberzyme and Substrate Sequence

Pos
Substrate
Seq ID
Amberzyme
Seq ID

11
GAGGGACA G UUAAUGGA
550
UCCAUUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGCCUC
1863

17
CAGUUAAU G GAUCCUGA
734
GACAGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUAACUG
1864

18
AGUUAAUG G AUCUGUAA
735
UUACAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUAACU
1865

23
AUGGAUCU G UAAACUUG
551
CAAGUUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUCCAU
1866

31
GUAAACUU G CACCCUCU
493
AGAGGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUUUAC
1867

44
CUCUUUCA G AGUGGUAC
736
GUACCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAAGAG
1868

46
CUUUCAGA G UGGUACAU
552
AUGUACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGAAAG
1869

48
UUCAGAGU G GUACAUGG
737
CCAUGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCUGAA
1870

49
UCAGAGUG G UACAUGGA
553
UCCAUGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUCUCA
1871

55
UGGUACAG G GAAGACAG
738
CUGUCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUACCA
1872

56
GGUACAUG G AAGACAGC
739
GCUGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGUACC
1873

59
ACAUGGAA G ACAGCACA
740
UGUGCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCAUGU
1874

63
GCAAGACA G CACAAACU
554
ACUGUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUUCC
1875

70
ACCACASA G UGGAUCCA
555
UGGAUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUGUGCU
1876

72
CACAAACU G CAUCCAUA
741
GAUGGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUUGUG
1877

73
ACAAACUC G AUCCAUAC
742
GUAUGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUUUGU
1878

85
CAUACUCU G AAAUGCAG
494
CUGCAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGUAUG
1879

90
UCUGAAAU G CAGUAACU
495
AGGUACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUCAGA
1880

93
CAAAUCCA G UAACUCUG
556
CAGACUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAUUUC
1861

101
GUAACUCU G AUGCUUGA
496
UCAACCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUUAC
1882

104
ACUCUGAU G CUUGAAUU
497
AAUUCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAGAGU
1883

108
UGAUGCUU G AAUUUCUU
498
AACAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCAUCA
1884

114
UUGAAUUU G UUCUCCCU
557
AGCGACAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUUCAA
1885

127
CCCUUCUU G CCAGAAAG
499
CUUUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGAAGGG
1886

131
UCUUGCCA G AAAGGAUU
743
AAUCCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCAAGA
1887

135
GCCAGAAA G CAUUCUAA
744
UUACAAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUCCC
1888

136
CCACAAAG G AUUCUAAU
745
AUUAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUCUCC
1889

150
AAUAACUC G CUCUCAAA
746
UUUGACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUUAUU
1890

151
AUAACUCC G UCUCAAAC
558
CUUUGACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGUUAU
1891

153
AACUCCCU G UCAAACCC
559
CCCUUUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCACUU
1892

159
GUGUCAAA G CCAAGACA
560
UCUCUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCACAC
1893

164
AAAGCCAA G ACAUAAAC
747
GUUUAUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCCUUU
1894

194
UUCCAAAA G CUUCACGU
561
ACGUGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUGGAA
1895

201
AGCCUCAC G UUACAGCA
562
UGCUGUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGCU
1896

207
ACGUUACA G CAUGGAAG
563
CUUCCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUAACGU
1897

211
UACAGCAU G GAAGCUGU
748
ACAGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCUGUA
1898

212
ACAGCAUG G AAGCUGUU
749
AACAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGCUGU
1899

215
GCAUGGAA G CUGUUGCC
564
GGCAACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCAUGC
1900

218
UGGAAGCU G UUGCCAAG
565
CUUGGCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUCCA
1901

221
AACCUGUU G CCAAGUUU
500
AAACUUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAGCUU
1902

226
GUUGCCAA G UUUGAUUU
586
AAAUCAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGCAAC
1903

230
CCAACUUU G AUUUCACU
501
ACUGAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACUUGC
1904

239
AUUUCACU G CUUCAGGU
502
ACCUGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUGAAAU
1905

245
CUGCUUCA G GUGAGGAU
750
AUCCUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAACCAG
1906

246
UCCUUCAC G UCAGGAUG
567
CAUCCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGCA
1907

248
CUUCAGCU G AGGAUGAA
503
UUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCAAG
1908

250
UCAGGUCA G CAUCAACU
751
ACUUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACCUCA
1909

251
CAGGUCAC G AUCAACUC
752
CACUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCACCUG
1910

254
GUGACCAU G AACUCACC
504
CCUCACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCAC
1911

259
GAUCAACU G ACCUUUCA
505
UCAAACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUCAUC
1912

261
UGAACUCA G CUUUCACA
568
UGUGAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACUUCA
1913

272
UUCACACU G GACAUCUU
753
AACAUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCUGAA
1914

273
UCACACUC G ACAUCUUU
754
AAACAUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUGUGA
1915

275
ACACUCCA G AUCUUUUG
755
CAAAACAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGUGU
1916

278
CUCCACAU G UUUUCAAG
569
CUUCAAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUCCAG
1917

283
CAUCUUUU G AACAUUUU
506
AAAAUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAACAUC
1918

286
GUUUUCAA G AUUUUAAC
756
CUUAAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAAAAC
1919

294
GAUUUUAA G UAACCAAC
570
CUUCGUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAAAUC
1920

302
CUAACCAA G AGGACUCC
757
CCACUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCGUUAC
1921

304
AACCAACA G CACUGCUU
758
AACCACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUCCUU
1922

305
ACCAAGAC G ACUCGUUU
759
AAACCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCUUCCU
1923

307
CAACACGA G UCCUUUAA
571
UUAAACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCUUC
1924

309
ACACCACU G CUUUAAGG
760
CCUUAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCCUCU
1925

310
GACCACUC G UUUAACCC
572
GCCUUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUCCUC
1926

316
UGGUUUAA G GCGCACCU
761
ACCUCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAACCA
1927

317
CCUUUAAC G CCCACCUU
573
AAGCUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUAAACC
1928

319
UUUAAGGC G GAGCUUGG
762
CCAAGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCUUAAA
1929

320
UUAAGGCG G AGCUUGCG
763
CCCAAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCCUUAA
1930

322
AACCCGGA G CUUCCGAG
574
CUCCCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGCCUU
1931

326
CUGACCUC G GGAGCCAG
764
CUGGCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACCUCCG
1932

327
GGAGCUUG G GAGCCAGG
765
CCUCGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGCUCC
1933

328
GAGCUUGC G AGCCAGGA
766
UCCUGGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAAGCUC
1934

330
GCUUGCGA G CCACGAAG
575
CUUCCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCAAGC
1935

334
GGGACCCA G GAAGGAUA
767
UAUCCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUCOC
1936

335
GGACCCAC G AAGCAUAU
768
AUAUCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGCUCC
1937

338
GCCACGAA G GAUAUCUC
769
CACAUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCUCCC
1938

339
CCAGGAAG G AUAUGUGC
770
GCACAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCCUCG
1939

344
AAGGAUAU G UGCCCAAC
576
CUUCGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUCCUU
1940

346
GGAUAUGU G CCCAACAA
507
UUCUUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAUAUCC
1941

352
GUGCCCAA G AAUUUCAU
771
AUCASAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGCCAC
1942

362
AUUUCAUA G ACAUCCAG
772
CUCGAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUGAAAU
1943

370
GACAUCCA G UUUCCCAA
577
UUGCGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAUCUC
1944

391
UCCCAAAU G GUUUCACC
773
CGUCAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUGGCA
1945

382
CCCAAAUG G UUUCACGA
578
UCGUGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUUCCC
1946

389
GGUUUCAC G AAGGCCUC
508
GAGGCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGAAACC
1947

392
UUCACGAA G GCCUCUCU
774
AGAGACGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCUCAA
1948

393
UCACGAAG G CCUCUCUC
579
GAGAGACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCCUCA
1949

402
CCUCUCUC G ACACCAGG
509
CCUCCUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACACACC
1950

409
CGACACCA G GCAGAGAA
775
UUCUCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCUCC
1951

410
GACACCAG G GAGAGAAC
580
COUCUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUCUC
1952

413
ACCAGGCA G AGAACUUA
776
UAACUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCUCCU
1953

415
CAGGCAGA G AACUUACU
777
ACUAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUCCCUC
1954

427
UUACUCAU G GGCAAGGA
778
UCCUUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCACUAA
1955

428
UACUCAUG G GCAAGGAG
779
CUCCUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCACUA
1956

429
ACUCAUGG G CAAGGAGG
581
CCUCCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUCACU
1957

433
AUGGGCAA G GAGGUUGG
780
CCAACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCCCAU
1958

434
UGGGCAAG G AGGUUGGC
781
GCCAACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGCCCA
1959

436
GGCAAGGA G GUUGGCUU
782
AACCCAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUUCCC
1960

437
GCAAGGAG G UUGGCUUC
582
CAACCCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUUGC
1961

440
AGGAGCUU G GCUUCUUC
783
CAACAACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACCUCCU
1962

441
GGAGCUUG G CUUCUUCA
593
UGAAGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACCUCC
1963

456
CAUCAUCC G GGCCAGCC
784
GGCUGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAUGAUG
1964

457
AUCAUCCG G GCCAGCCA
785
UGGCUGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGAUGAU
1965

458
UCAUCCGC G CCAGCCAG
584
CUCGCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGGAUGA
1966

462
CCGGGCCA G CCACAGCU
565
AGCUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCCCGG
1967

466
GCCAGCCA G AGCUCCCC
786
GGGGAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUGGC
1968

468
CAGCCACA G CUCOCCAG
586
CUGGGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGGCUC
1969

476
GCUCCCCA G GGGACUUC
787
GAAGUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGGACC
1970

477
CUCCCCAG G GGACUUCU
788
ACAAGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGGCAG
1971

478
UCCCCAGG G GACUUCUC
789
GAGAAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGCGCA
1972

479
CCCCAGGC G ACUUCUCC
790
GGAGAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGCGG
1973

494
CCAUCUCU G UCAGGCAU
587
AUGCCUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACAUCG
1974

498
CUCUGUCA G GCAUGACG
791
CCUCAUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACAGAG
1975

499
UCUGUCAG G CAUCAGGA
598
UCCUCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCACAGA
1976

503
UCAGCCAU G AGGAUGAC
510
GUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCCUGA
1977

505
AGOCAUCA G GAUGACGU
792
ACCUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAUGCCU
1979

506
CCCAUGAC G AUCACGUU
793
AACGGUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCAUGCC
1979

509
AUGAGGAU G ACGUUCAA
511
UUCAACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCAU
1988

512
ACCAUGAC G UUCAACAC
589
GUGUUCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCAUCCU
1981

526
CACUUCAA G GUCAUGCG
794
CGCAUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAACUC
1982

527
ACUUCAAC G UCAUGCGA
598
UCCCAUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCAACU
1983

532
AAGGUCAU G CGACACAA
512
UUCUCUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCACCUU
1984

534
GGUCAUGC G AGACAACA
513
UCUUCUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUCACC
1985

536
UCAUGCGA G ACAACAAG
795
CUUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCAUCA
1986

544
GACAACAA G GGUAAUUA
796
UAAUUACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUUCUC
1987

545
ACAACAAG G GUAAUUAC
797
CUAAUUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCUUCU
1988

546
CAACAACG G UAAUUACU
591
AGUAUUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUCUUG
1989

559
UACUUUCU G UGGACUCA
592
UCACUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAACUA
1990

561
CUUUCUGU G GACUGAGA
798
UCUCACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACAAAC
1991

562
UUUCUGUG G ACUGAGAA
799
UUCUCAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACACAAA
1992

566
UGUGGACU G ACAACUUU
514
AAACUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCCACA
1993

568
UCCACUGA G AAGUUUCC
800
CCAAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACUCCA
1994

571
ACUGAGAA G UUUCCAUC
593
CAUCCAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCACU
1995

589
CUAAAUAA G CUCCUAGA
594
UCUACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAUUUAC
1996

592
AAUAAGCU G GUAGACUA
801
UAGUCUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUAUU
1997

593
AUAAGCUG G UAGACUAC
595
GUAGUCUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUUAU
1998

596
AGCUGGUA G ACUACUAC
802
GUAGUAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACCAGCU
1999

606
CUACUACA G GACAAAUU
803
AAUUUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUAGUAG
2000

607
UACUACAG G ACAAAUUC
804
GAAUUUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUAGUA
2001

624
CAUCUCCA G ACAGAAGC
805
GCUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGAUG
2002

628
UCCAGACA G AAGCAGAU
806
AUCUGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUGGA
2003

631
AGACAGAA G CAGAUCUU
596
AAGAUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGUCU
2004

634
CAGAAGCA G AUCUUCCU
807
AGGAAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUUCUG
2005

645
CUUCCUUA G AGACAGAA
808
UUCUGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAGGAAG
2006

647
UCCUUAGA G ACAGAACC
809
CGUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUAAGGA
2007

651
UAGAGACA G AACCCGAG
810
CUCCGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUCUA
2008

657
CAGAACCC G AGAAGACC
515
GGUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGUUCUG
2009

659
GAACCCCA G AAGACCAG
911
CUGGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGGUUC
2010

662
CCCGAGAA G ACCAGGGU
812
ACCCUGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCGGG
2011

667
GAAGACCA G GGUCACCG
813
CGGUGACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUCUUC
2012

668
AAGACCAG G GUCACCGG
814
CCCCUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGUCUU
2013

668
AGACCAGG G UCACCCGG
597
CCCCGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCGUCU
2014

675
GGGUCACC G GCGCAACA
815
UGUUGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUCACCC
2015

676
GGUCACCG G GCCAACAG
816
CUGUUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUCACC
2016

677
GUCACCGG G GCAACAGC
817
GCUGUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCCUCAC
2017

678
UCACCGGG G CAACAGCC
598
GGCUCUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCCCUGA
2018

684
GGGCAACA G CCUGGACC
599
GGUCCACG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUUCCCC
2019

688
AACAGCCU G CACCCGAG
818
CUCCCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCUCUU
2020

689
ACAGCCUG G ACCCCAGG
819
CCUCCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCCUCU
2021

693
CCUGGACC G CAGGUCCC
820
CCCACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGUCCAGG
2022

694
CUGGACCC G AGGUCCCA
821
UCCCACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUCCAG
2023

696
GGACCGCA G CUCCCAGG
822
CCUGGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGGUCC
2024

697
CACCGGAG G UCCCAGCG
600
CCCUGGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCGGUC
2025

703
ACCUCCCA G GGAGCCCC
823
GGGCCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGACCU
2026

704
CCUCCCAG G GACGCCCA
924
UGGGCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGGACC
2027

705
GUCCCAGG G AGCCCCAC
825
GUGGGCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCCCAC
2028

707
CCCAGGCA G GCCCACAC
826
GUCUCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCUGGC
2029

708
CCAGGGAG G CCCACACC
601
CCUCUGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCCUGC
2030

720
ACACCUCA G UGGGGCUG
602
CAGCCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGGUGU
2031

722
ACCUCAUC G GGGCUGUC
827
CACAGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGAGGU
2032

723
CCUCAGUG G GUCUGUGG
828
CCACAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUGAGG
2033

724
CUCAGUGG G GCUGUGGG
829
CCCACAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACUGAG
2034

725
UCAGUGGG G CUGUGGGA
603
UCCCACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCACUGA
2035

728
GUGGGGCU G UGGGACAA
604
UUCUCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCCCAC
2036

730
GGGUCUGU G GGAGAAGA
830
UCUCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGCCCC
2037

731
GUGCUGUG G GAGAAGAA
831
UUCUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGCCC
2038

732
GGCUGUGG G AGAAGAAA
832
UUUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACAGCC
2038

734
CUGUGGGA G AAGAAAUC
833
GAUUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCACAG
2040

737
UGGGAGAA G AAAUCCGA
834
UCGGAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCCCA
2041

744
AGAAAUCC G ACCUUCGA
516
UCGAAGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAUUUCU
2042

751
CGACCUUC G AUGAACCG
517
CGGUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGGUCG
2043

754
CCUUCGAU G AACCGGAA
518
UUCCGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCGAAGG
2044

759
GAUGAACC G GAAGCUGU
835
ACAGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUUCAUC
2045

760
AUGAACCG G AAGCUGUC
836
GACAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUUCAU
2046

763
AACCGGAA G CUGUCGGA
605
UCCUACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCGGUU
2047

766
CGGAAGCU G UCGGAUCA
606
UGAUCCGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUCCG
2048

769
AAGCUGUC G GAUCACCC
837
GGGUGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GACAGCUU
2048

770
AGCUGUCG G AUCACCCC
838
GGGGUGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGACAGCU
2050

781
CACCCCCC G ACCCUUCC
519
GGAAGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGCGGUG
2051

793
CUUCCCCU G CACCACCA
520
UCCUCCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGGAAG
2052

796
CCCCUGCA G CACCACCA
607
UCCUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCACCCC
2053

799
CUCCACCA G CACCACCA
608
UCCUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCCAC
2054

805
CACCACCA G CACCACCC
609
CCCUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCCUC
2055

811
CACCACCA G CCACACCC
610
CCCUCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCCUC
2056

817
CAGCCACA G CCUCCCCA
611
UCCCCACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUCCCUC
2057

823
CACCCUCC G CAAUAUGC
521
CCAUAUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACCCUC
2058

830
CGCAAUAU G CCCCAGCC
522
CCCUCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUUCCC
2059

836
AUCCCCCA G CCCCCCAC
612
CUCCCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCCCAU
2060

838
CCCCCACC G CCCCACCA
523
UCCUCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCCCCC
2061

844
GCCCCCCA G CAGCUGCA
613
UCCACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCCCCC
2062

847
CCCCACCA G CUGCAGCA
614
UCCUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCCCC
2063

850
CACCACCU G CAGCAGCC
524
CCCUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCCUC
2064

853
CAGCUGCA G CAGCCCCC
615
GGGGGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAGCUG
2065

856
CUGCAGCA G CCCCCACA
616
UGUOGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGCAG
2066

865
CCCCCACA G CAGCGAUA
617
UAUCGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGGUGG
2067

868
CCACAGCA G CGAUAUCU
618
AGAUAUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGUGG
2068

870
ACAGCAGC G AUAUCUGC
525
GCAGAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGCUGU
2069

877
CGAUAUCU G CAGCACCA
526
UGGUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUAUCG
2070

880
UAUCUGCA G CACCACCA
619
UGGUGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGAUA
2071

898
UUCCACCA G GAACGCCG
839
CGGCGUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGGAA
2072

899
UCCACCAC G AACGCCCA
840
UCGGCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGUGGA
2073

903
CCAGGAAC G COGAGGAG
527
CUCCUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUUCCUGC
2074

906
GGAACGCC G AGGACGCA
528
UCCCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCGUUCC
2075

908
AACGCCGA G GAGGCAGC
841
GCUGCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGCGUU
2076

909
ACGCCGAG G AGGCAGCC
842
GCCUGCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGGCGU
2077

911
CCCGAGGA G GCAGCCUU
843
AAGGCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCGGC
2078

912
CCGAGGAG G CAGOCCUG
620
CAAGGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUCGG
2079

915
AGGAGGCA G CCUUGACA
621
UGUCAAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCUCCU
2080

920
GCAGCCUU G ACAUAAAU
529
AUUUAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCCUCC
2081

929
ACAUAAAU G AUGCGCAU
530
AUGCCCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUAUGU
2082

932
UAAAUGAU G CGCAUUGU
844
ACAAUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAUUUA
2083

933
AAAUCAUG G GCAUUGUG
845
CACAAUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCAUUU
2084

934
AAUGAUGG G CAUUCUGG
622
CCACAAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUCAUU
2085

939
UGGGCAUU G UGGCACCG
623
CGGUGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUCCCCA
2086

941
GGCAUUGU G CCACCGGC
846
GCCCGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAUGCC
2087

942
GCAUUCUC G CACCCGCU
624
AGCCGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAAUGC
2088

947
GUGOCACO G GCUUGGGC
847
GCCCAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUGCCAC
2089

948
UGGCACCG G CUUGGGCA
625
UGCCCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUGCCA
2090

952
ACCCGCUU G GGCAGUGA
848
UCACUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCCGGU
2091

953
CCCCCUUC G CCAGUGAA
849
UUCACUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACCCGG
2092

954
CCCCUUGC G CAGUGAAA
626
UUUCACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAACCCG
2093

957
CUUGGGCA G UGAAAUGA
627
UCAUUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCCAAG
2094

959
UCGGCAGU G AAAUCAAU
531
AUUCAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGOCCA
2095

964
AGUGAAAU G AAUCCCCC
532
GCCGCAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUCACU
2096

968
AAAUCAAU G CCCCCCUC
533
GAGCCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUCAUUU
2097

970
AUCAAUGC G GCCCUCAU
850
AUGAGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUUCAU
2098

971
UGAAUGCG G CCCUCAUC
628
CAUGAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCAUCCA
2099

979
GCCCUCAU G CAUCGGAG
534
CUCCGAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAGGGC
2100

984
CAUCCAUC G GAGACACA
851
UGUGUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUGGAUG
2101

985
AUGCAUCG G AGACACAC
852
GUGUGUOU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAUGCAU
2102

987
GCAUCGGA G ACACACAG
853
CUGUGUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGAUGC
2103

995
GACACACA G ACCOAGUG
854
CACUGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGUGUC
2104

1001
CAGACCCA G UGCAGCUC
629
GAGCUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGUCUG
2105

1003
GACCCAGU G CAGCUCCA
535
UGGACCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGGGUC
2106

1006
CCAGUGGA G CUCCAGGC
630
GCCUGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCACUGG
2107

1012
CAGCUCCA G GCGGCAGG
855
CCUGCCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGCUG
2108

1013
AGCUCCAG G CGGCAGGG
631
CCCUGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGAGCU
2109

1015
CUCCAGGC G GCAGGGCG
856
CGCCCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCUGGAG
2110

1016
UCCAGGCG G CAGGGCGA
632
UCGCCCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCCUGGA
2111

1019
AGGCGGCA G GGCGAGUG
857
CACUCGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCGCCU
2112

1020
GGCGGCAG G GCGAGUGC
858
GCACUCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGCCGCC
2113

1021
GCGGCAGG G CGAGUGCG
633
CGCACUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGCCGC
2114

1023
GGCAGGGC G AGUGCGGU
536
ACCGCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCUGCC
2115

1025
CAGGGCGA G UGCCGUGG
634
CCACCGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGCCCUG
2116

1027
GGGCCAGU G CGGUGGGC
537
GCCCACCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCGCCC
2117

1029
GCGAGUGC G GUGGGCCC
859
GGGCCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCACUCGC
2118

1030
CGACUCCG G UGGCCCCG
635
CGGCCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCACUCG
2119

1032
AGUGCGCU G GCCCCGGG
860
CCCGGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCCACU
2120

1033
GUGCCCUG G GGCCCCGC
861
CCCCCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCGCAC
2121

1034
UGCCCUCG G CCCGGGCG
636
CCCCCGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACCGCA
2122

1038
GUGGCCCC G CGCCCUGU
862
ACACCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCCCAC
2123

1039
UGCCCCCC G GCCCUGUA
863
UACACCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGGCCCA
2124

1040
GGCCCCGG G CGCUGUAU
637
AUACACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGGGCCC
2125

1042
GCCCGGGC G CUGUAUGA
538
UCAUACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCGGGC
2126

1045
CGGGCGCU G UAUGACUU
638
AACUCAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGCCCG
2127

1049
CGCUGUAU G ACUUUGAG
539
CUCAAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUACAGCG
2128

1055
AUGACUUU G AGGCCCUG
540
CACCCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAGUCAU
2129

1057
GACUUUGA G GCCCUGCA
864
UCCACCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAAACUC
2130

1058
ACUUUGAG G CCCUGGAG
639
CUCCACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCAAAGU
2131

1063
GAGGCCCU G GAGGAUGA
865
UCAUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGCCUC
2132

1064
AGGCCCUG G AGGAUGAC
866
GUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGCCU
2133

1066
GCCCUGGA G GAUGACGA
867
UCCUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGGGC
2134

1067
CCCUGGAG G AUGACCAG
666
CUCGUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCAGGG
2135

1070
UGGAGGAU G ACGAGCUG
541
CAGCUCGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCCA
2136

1073
AGGAUGAC G AGCUGGGG
542
CCCCAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCAUCCU
2137

1075
GAUGACGA G CUGGGGUU
640
AACCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGUCAUC
2138

1078
GACGAGCU G GGGUUCCA
869
UGGAACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUCGUC
2139

1079
ACGAGCUG G GGUUCCAC
870
GUGGAACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUCGU
2140

1080
CGAGCUGG G GOUCCACA
871
UGUGGAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCUCG
2141

1081
GACCUGGG G UUCCACAG
641
CUGUGGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAGCUC
2142

1069
GUUCCACA G CGGGGAGG
642
CCUCCCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGGAAC
2143

1091
UCCACAGC G GCGAGGUG
872
CACCUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUCUGGA
2144

1092
CCACAGCC G GGAGGUGG
873
CCACCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCUGUCC
2145

1093
CACAGCGG G GAGGUGGU
874
ACCACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGCUGUC
2146

1094
ACACCGGG G ACCUGGUG
875
CACCACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCGCUCU
2147

1096
ACCGGGGA G GUGGUGGA
876
UCCACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCCGCU
2148

1097
GCCCCCAG G UGGUGGAG
643
CUCCACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCCCGC
2149

1099
GGGCAGGU G GUGGAGGU
877
ACCUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCCCC
2150

1100
GGGAGGUG G UGGAGGUC
644
GACCUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCUCCC
2151

1102
GAGGUGGU G GAGGUCCU
878
AGGACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCUC
2152

1103
AGGUGGUC G AGGUCCUG
879
CAGGACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCACCU
2153

1105
GUGGUGGA G GUCCUGGA
880
UCCAGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCACCAC
2154

1106
UGGUGGAG G UCCUGGAU
645
AUCCAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCACCA
2155

1111
GAGGUCCU G GAUAGCUC
881
CAGCUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCUC
2156

1112
AGGUCCUC G AUAGCUCC
882
CGACCUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGACCU
2157

1116
CCUGGAUA G CUCCAACC
646
CGUUCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUCCACC
2158

1131
CCCAUCCU G GUGGACCG
883
CCCUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAUGGC
2159

1132
CCAUCCUG G UCGACCCG
647
CCCCUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCAUCC
2160

1134
AUCCUCGU G GACCGGCC
884
CCCCCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCAU
2161

1135
UCCUGGUG G ACCCCCCG
885
CGGCCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCACCA
2162

1139
GCUGCACC G CCCCCCUC
886
CACCCCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCCACC
2163

1140
GUGGACCC G CCGCCUGC
648
CCACGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUCCAC
2164

1143
GACCGGCC G CCUGCACA
543
UCUCCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCCCCUC
2165

1147
GCCCGCCU G CACAACAA
544
UUCUUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCGCCC
2166

1156
CACAACAA G CUGGGCCU
649
AGGCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUUGUG
2167

1159
AACAAGCU G GGCCUCUU
887
AAGAGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUGUU
2168

1160
ACAAGCUG G GCCUCUUC
888
GAAGAGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUUGU
2169

1161
CAAGCUGG G ocucuwec
650
GGAAGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCUUG
2170

1172
UCUUCCCU G CCAACUAC
545
GUAGUUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGAAGA
2171

1181
CCAACUAC G UGGCACCC
651
GGGUGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUAGUUGG
2172

1183
AACUACGU G GCACCCAU
889
AUGGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGUAGUU
2173

1184
ACUACGUG G CACCCAUG
652
CAUGGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACGUAGU
2174

1192
GCACCCAU G ACCCGAUA
546
UAUCGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGGUGC
2175

1197
CAUGACCC G AUAAACUC
547
GAGUUUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GOGUCAUG
2176

1210
ACUCUUCA G GOGACAGA
890
UCUGUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAGAGU
2177

1211
CUCUUCAG G GGACAGAA
891
UUCUGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGAG
2178

1212
UCUUCAGG G GACAGAAG
892
CUUCUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGAAGA
2178

1213
CUUCAGGG G ACAGAAGC
893
GCUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGAAG
2180

1217
AGGGGACA G AAGCUUUU
894
AAAAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCCCCU
2181

1220
GGACAGAA G CUUUUUGU
653
ACAAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGUCC
2182

1227
AGCUUUUU G UCUGGAGC
654
GCUCCAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAAAGCU
2183

1231
UUUUGUCU G GAGCUGCC
895
GGCAGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACAAAA
2184

1232
UUUGUCUG G AGCUGCCC
896
GGGCAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGACAAA
2185

1234
UGUCUGGA G CUGCCCAC
655
GUGGGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGACA
2186

1237
CUGGAGCU G CCCACAAG
548
CUUGUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUCCAG
2187

1245
GCCCACAA G AAAGAGGG
897
CCCUCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUGGGC
2188

1249
ACAAGAAA G AGGGCAAG
898
CUUGCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUUGU
2189

1251
AAGAAAGA G GGCAAGGA
899
UCCUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUUCUU
2190

1252
AGAAAGAG G GCAAGGAA
900
UUCCUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCUUUCU
2191

1253
GAAAGAGG G CAAGGAAA
656
UUUCCUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCUUUC
2192

1257
GAGGGCAA G GAAAAAAG
901
CUUUUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCCCUC
2193

1258
AGGGCAAC G AAAAAAGG
902
CCUUUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCCCCU
2194

1265
GGAAAAAA G GCUGGACU
903
AGUCCAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUUUCC
2195

1266
GAAAAAAG G CUGGACUC
657
GAGUCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUUUUC
2196

1269
AAAAGCCU G GACUCCAU
904
AUGGAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCUUUU
2197

1270
AAAGGCUG G ACUCCAUC
905
CAUGCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCCUUU
2198

1278
CACUCCAU G ACUAUAUA
549
UAUAUACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGACUC
2199

Input Sequence = HSA011736.

Cut Site = G/.

Stem Length = 8.

Core Sequence = GGAGGAAACCCC CU UCAAGGACAUCGUCCGGG

HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRGLG); 1303 bp)

TABLE IX

Human GRID GeneBloc and Substrate Sequence

Pos
Substrate
Seq ID
GeneBloc
GB Seq ID
GB RPI#

324
GAGGAGUGGUUUAAGGCGGAGCU
2201
asgscsuccgCsCsTsTsAsAsAsCsCsacucscsusc B
2212
14006

445
UCAUGGGCAAGGAGGUUGGCUUC
2201
gsasasgccaAsCsCsTsCsCsTsTsGscccasusgsa B
2213
14007

456
GAGGUUGGCUUCUUCAUCAUCCG
2202
csgsgsaugaTsGsAsAsGsAsAsGsCscaacscsusc B
2214
14008

736
ACCUCAGUGGGGCUGUGGGAGAA
2203
ususcsucccAsCsAsGsCsCsCsCsAscugasgsgsu B
2215
14009

819
CAGCACCAGCACCAGCCACAGCC
2204
gsgscsugugGsCsTsGsGsTsGsCsTsggugscsusg B
2216
14010

825
CAGCACCAGCCACAGCCUCCGCA
2205
usgscsggagGsCsTsGsTsGsGsCsTsggugscsusg B
2217
14011

951
GAUGGGCAUUGUGGCACCGGCUU
2206
asasgsccggTsGsCsCsAsCsAsAsTsgcccsasusc B
2218
14012

1108
ACAGCGGGGAGGUGGUGGAGGUC
2207
gscsusauccAsCsCsAsCsCsTsCsCsccgcsusgsu B
2219
14013

1117
AGGUGGUGGAGGUCCUGGAUAGC
2208
gsascscuccAsGsGsAsCsCsTsCsCsaccascscsu B
2220
14014

1162
GCCGCCUGCACAACAAGCUGGGC
2209
gscscscagcTsTsGsTsTsGsTsGsCsaggcsgsgsc B
2221
14015

1166
CCUGCACAACAAGCUGGGCCUCU
2210
asgsasggccCsCsCsAsGsCsTsTsGsuugusgscsa B
2222
14016

1168
UGCACAACAAGCUGGGCCUCUUC
2211
gsasasgaggCsCsCsAsGsCsTsTsGsuugusgscsa B
2223
14017

324
GAGGAGUGGUUUAAGGCGGAGCU
2200

B
agcuccgCsCsTsTsAsAsAsCsCsacuccuc B
2224
14540

445
UCAUGGGCAAGGAGGUUGGCUUC
2201

B
gaagccaAsCsCsTsCsCsTsTsGscccauga B
2225
14541

456
GAGGUUGGCUUCUUCAUCAUCCG
2202

B
cggaugaTsGsAsAsGsAsAsGsCscaaccuc B
2226
14542

736
ACCUCAGUGGGGCUGUGGGAGAA
2203

B
uucucccAsCsAsGsCsCsCsCsAscugaggu B
2227
14543

819
CAGCACCAGCACCAGCCACAGCC
2204

B
ggcugugGsCsTsGsGsTsGsCsTsggugcug B
2228
14544

825
CAGCACCAGCCACAGCCUCCGCA
2205

B
ugcggagGsCsTsGsTsGsGsCsTsggugcug B
2229
14545

951
GAUGGGGAUUGUGGCACCGGCUU
2206

B
aagccggTsGsCsCsAsCsAsAsTsgcccauc B
2230
14546

11O8
ACAGCGGGGAGGUGGUGGAGGUC
2207

B
gaccuccAsCsCsAsCsCsTsCsCsccgcugu B
2231
14547

1117
AGGUGGUGGAGGUCCUGGAUAGC
2208

B
gcuauccAsGsGsAsCsCsTsCsCsaccaccu B
2232
14548

1162
GCCGCCUGCACAACAAGGUGGGC
2209

B
gcccagcTsTsGsTsTsGsTsGsCsaggcggc B
2233
14549

1166
CCUGCACAACAAGCUGGGCCUCU
2210

B
agaggccCsAsGsCsTsTsGsTsTsgugcagg B
2234
14550

1168
UGCACAACAAGCUGGGCCUCUUC
2211

B
gaagaggCsCsCsAsGsCsTsTsGsuugugca B
2235
14551

Upper Case = Ribo

Lower Case = 2′-O-Methyl

s = phosphorothioate linkage

B
= inverted deoxyabasic ribonucleotide

Input Sequence = HSA011736

GB Length = 23

HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp)

[0211]

9

TABLE X

Human Grid Enzymatic nucleic acid and target sequence

ref

Seq

pos
Target
Seq ID
RPI#
Enzymatic Nucleic Acid
ID
Motif

13
GCACAGU U AAUGGAU
2256
23891
auccauu CUGAUGAggccguuaggccGAA Acugugc B
2280
Hammerhead

178
ACUCAAU C UCUUCUC
2257
23892
Gagaaga CUGAUGAggccguuaggccCAA Auugagu B
2281
Hammerhead

183
AUCUCUU C UCUUCCA
2258
23901
uggaaga CUGAUGAggccguuaggccGAA Aagagau B
2282
Hammerhead

317
AGUGGUU U AAGGCGG
2259
23902
ccgccuu CUGAUGAggccguuaggccGAA Aaccacu B
2283
Hammerhead

401
CCUCUCU C GACACCA
2260
23910
ugguguc CUGAUGAggccguuaggccGAA Agagagg B
2284
Hammerhead

179
CUCAAUC U CUUCUCU
2261
23911
agagaag CUGAUGAggccguuaggccGAA Iauugag B
2285
Inozyme

395
CGAAGGC C UCUCUCG
2262
23920
cgagaga CUGAUGAggccguuaggcccAA Iccuucg B
2286
Inozyme

412
ACCAGGC A GAGAACU
2263
23921
aguucuc CUGAUGAggccguuaggccGAA Iccuggu B
2287
Inozyme

638
GCAGAUC U UCCUUAG
2284
23929
cuaagga CUGAUGAggccguuaggcccAA Iaucugc B
2288
Inozyme

1268
AAAAGGC U GGACUCC
2265
23930
ggagucc CUGAUGAggccguuaggccGAA Iccuuuu B
2289
Inozyme

11
AGGCACA G UUAAUGG
2288
23890
ccauuaa gccgaaaggCgaqugaGGuCu ugugccu B
2290
Zinzyme

153
ACUCGGU G UCAAAGC
2267
23900
gcuuuga gccgaaaggCgagugaGGuCu accgagu B
2291
Zinzyme

370
ACAUCCA G UUUCCCA
2260
23909
ugggaaa gccgaaaggCgagugaGGuCu uggaugu B
2292
Zinzyme

817
AGCCACA G CCUCCGC
2269
23919
gcggagg gccgaaaggCgagugaGGuCu uguggcu B
2293
Zinzyme

880
AUCUGCA G CACCACC
2270
23928
gguggug gccgaaaggCgagugaGGuCu ugcagau B
2294
Zinzyme

409
GACACCA G GCAGAGA
2271
23893
ucucugc CgaggaaacucC CUUCaaggacaucgucCGGG ugguguc B
2295
Amberzyme

413
CCAGGCA G AGAACUU
2272
23903
aaguucu GgaggaaacucC CUUCaaggacaucgucCGGG ugccugg B
2296
Amherzyme

628
CCAGACA G AAGCAGA
2273
23912
ucugcuu GgaggaaacucC CUUCaaggacaucgucCGGG ugucugg B
2297
Amberzyme

1070
GGAGGAU G ACGAGCU
2274
23922
agcucgu GgaggaaacucC CUUCaaggacaucgucCGGG auccucc B
2298
Amberzyme

110
AGGUGGU G GAGGUCC
2275
23931
ggaccuc GgaggaaacucC CUUCaaggacaucgucCGGG accaccu B
2299
Amberzyme

8
AGGAGGU A CAGUUAA
2276
23889
uuaacug GGCTAGCTACAACGA gccuccu B
2300
DNAzyme

102
AACUCUG A UGCUUGA
2277
23899
ucaagca GGCTAGCTACAACGA cagaguu B
2301
DNAzyme

176
AAACUCA A UCUCUUC
2278
23908
gaagaga GGCTAGCTACAACGA ugaguuu B
2302
DNAzyme

370
ACAUCCA G UUUCCCA
2268
23918
ugggaaa GGCTAGCTACAACGA uggaugu B
2303
DNAzyme

403
UCUCUCG A CACCAGG
2279
23927
cauggug GGCTAGCTACAACGA cgagaga B
2304
DNAzyme

Lowercase = 2′-O-methyl nucleotide

Uppercase = ribonucleotide

B = inverted deoxyabasic moiety

Method and reagent for the inhibition of grid

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Parent Case Info

Provisional Applications (1)