Method and reagent for the inhibition of checkpoint kinase-1 (Chk1) enzyme

BACKGROUND OF THE INVENTION

[0001] This patent application claims priority from Jarvis et al., USSN (60/179,983), filed Feb. 3, 2000, entitled “METHOD AND REAGENT FOR THE INHIBITION OF CHECKPOINT KINASE-1 (CHK1) ENZYME”. This application is hereby incorporated by reference herein in its entirety including the drawings.

[0002] The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of kinases which phosphorylate Cdc25 S216, such as Chk1 (checkpoint kinase 1) enzyme.

[0003] The following is a brief description of the current understanding of Chk1. 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] Mammalian cells treated with agents that inhibit DNA replication or cause DNA damage undergo cell cycle arrest due to the presence of multiple checkpoint response mechanisms. Cancer cells frequently lack the p53-induced G1 DNA damage checkpoint response and instead arrest in G2 due to a checkpoint pathway directed towards preventing Cdc2 kinase activation. Inhibition of Cdc2 kinase activity is mediated by Wee1-like kinases, which phosphorylate key residues within the ATP-binding pocket of Cdc2 (accession No. X05360). Maintenance of this arrest also involves repressing Cdc25 function, the phosphatase that removes the Cdc2 inhibitory phosphorylations, by a mechanism involving the binding of 14-3-3 proteins to a phosphorylated serine residue (S216) in Cdc25. Multiple kinases, including Chk1 (accession No. AF016582), Chk2 (Cds1) (accession No. NM—007194), and C-TAK1 (accession No. AL050393), can phosphorylate Cdc25 S216 (accession No. M34065) in-vitro. These kinases may function in the DNA replication and/or DNA damage checkpoint response in vivo.

[0005] Hoekstra et al, International PCT publication No. WO/9955844, describe, in general terms, a method for promoting differentiation of a differentiation-inhibited cell by introducing into a cell a first polynucleotide encoding an antisense polynucleotide that hybridizes to a second polynucleotide encoding a cell cycle checkpoint protein.

SUMMARY OF THE INVENTION

[0006] The invention features novel nucleic acid-based techniques [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 modulate the expression of kinases which phosphorylate Cdc25 S216, such as Chk1 (checkpoint kinase 1) enzyme, Chk2 (Cds1) and C-TAK1.

[0007] The description below of the various aspects and embodiments is provided with reference to the exemplary gene Chk1. However, the various aspects and embodiments are also directed to each of the other genes which phosphorylate Cdc25S216. Those additional genes can be analyzed for target sites as described for Chk1. Further, the nucleic acid-based techniques, molecules, and compositions targeted to those genes can be performed as for Chk1. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

[0008] 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 Chk1. Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of Chk1 gene.

[0009] In another preferred embodiment, the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH, G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of Chk1 gene.

[0010] By “inhibit” it is meant that the activity of Chk1 or level of RNAs or equivalent RNAs encoding one or more protein subunits of Chk1 is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, 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 Chk1 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.

[0011] 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% may 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 may 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).

[0012] 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.

[0013] By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example, see FIGS. 1-5).

[0014] By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid which is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in FIGS. 1-5. That is, these arms contain sequences within a enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention may have binding arms that are contiguous or non-contiguous and may 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 12-100 nucleotides; more preferably 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herrance et al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).

[0015] 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.

[0016] 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.

[0017] 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 may 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.

[0018] 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 may 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.

[0019] 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 may 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.

[0020] By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover.

[0021] 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).

[0022] By “equivalent” RNA to Chk1 is meant to include those naturally occurring RNA molecules having homology (partial or complete) to Chk1 proteins or encoding for proteins with similar function as Chk1 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.

[0023] By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.

[0024] 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 may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol, 40, 1-49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.

[0025] By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; more specifically, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.

[0026] 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).

[0027] 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).

[0028] By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.

[0029] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The enzymatic nucleic acid molecule that cleave the specified sites in Chk1 -specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including cancer.

[0034] 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 may 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 Dreyfuis, 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).

[0035] 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-XIII. 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). 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, Biochemistiy, 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 are 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.

[0036] Preferably, a nucleic acid molecule that down regulates the replication of Chk1 comprises between 12 and 100 bases complementary to a RNA molecule of Chk1. Even more preferably, a nucleic acid molecule that down regulates the replication of Chk1 comprises between 14 and 24 bases complementary to a RNA molecule of Chk1.

[0037] 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 kinases which phosphorylate Cdc25 S216, such as Chk1 proteins (specifically Chk1 gene) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.

[0038] 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 Chk1 gene.

[0039] As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell may be present in an organism which may be a human 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 may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).

[0040] By “Chk1 proteins” is meant, a protein or a mutant protein derivative thereof, comprising phosphorylation activity, preferably to serine residue (S216), or its equivalent, in Cdc25 phosphatase.

[0041] By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

[0042] The nucleic acid-based inhibitors of Chk1 expression are useful for the prevention and/or treatment of diseases and conditions such as cancer, including cancer of the colon, rectum, lung, breast, prostate and any other diseases or conditions that are related to or will respond to the levels of Chk1 in a cell or tissue, alone or in combination with other therapies. In addition, Chk1 inhibition may be used as a therapeutic target for abrogating the G2 DNA damage checkpoint arrest; a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.

[0043] By “related” is meant that the reduction of Chk1 expression (specifically Chk1 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.

[0044] The nucleic acid-based inhibitors of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, 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 VIII. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to VIII. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.

[0045] 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 IX. 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 sequences shown as GeneBloc™ sequences in Table IX. 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 will be complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both.

[0046] 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 may be present which do not interfere with such cleavage. Thus, a core region may, 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 is 5′-GCCGUUAGGC-3′ (SEQ ID NO 3173) 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.

[0047] Sequence X may be a linker of ≧2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides may preferably be internally base-paired to form a stem of preferably ≧2 base pairs. Alternatively or in addition, sequence X may be a non-nucleotide linker. 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.

[0048] In yet another embodiment, the non-nucleotide linker X is as defined herein. The term “non-nucleotide” 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. A “non-nucleotide” further means any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.

[0049] In another aspect of the invention, ribozymes or antisense molecules that cleave target RNA molecules and inhibit Chk1 (specifically Chk1 gene) activity 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 could 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 may 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.

[0050] By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

[0051] 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.

[0052] 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 beincreased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the activity or stability of the nucleic acid molecule can bedecreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.

[0053] 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 Chk1, the patient may be treated, or other appropriate cells may 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.

[0054] 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 could be used in combination with one or more known therapeutic agents to treat cancer, including but not limited to cancer of the colon, rectum, lung, breast and prostate.

[0055] 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., Chk1) capable of progression and/or maintenance of cancer.

[0056] 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 may independently be targeted to the same or different sites.

[0057] 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.

[0058] 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

[0059] First the drawings will be described briefly.

DRAWINGS

[0060]
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 (MIRNA): 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 may 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 may be covalently linked by one or more bases (i.e., r is ≧1 base). Helix 1, 4 or 5 may 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 may 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 may 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 may 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 etal., U.S. Pat. No. 5,631,359).

[0061]
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 may 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.

[0062]
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.

[0063]
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.

[0064]
FIG. 5 shows an example of a DNAzyme motif described by Santoro et al., 1997, PNAS, 94, 4262.

[0065]
FIG. 6 shows a bar graph of a nucleic acid inhibitor (50 to 200 nM GeneBloc™ screen against Chk1 RNA in HeLa cells using 1.25 μg/ml GSV lipid with 24 hour sustained delivery in a 96-well format. Relative amounts of target RNA were measured normalized to actin using real-time PCR monitoring of amplification compared to mismatch nucleic acid and untreated controls. The sequences of GeneBloc™ reagents used in this experiment are shown in Table IX.

[0066]
FIG. 7 shows a bar graph of a lipid optimization study utilizing lead nucleic acid inhibitors (GeneBlocs™) targeting Chk1 RNA in HeLa cells; 96-well plate format, 5000 cells/well, GSV lipid. Six different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitors.

[0067]
FIG. 8 shows a bar graph displaying a time-course inhibition study of a lead nucleic acid inhibitor (GeneBloc™) targeting Chk1 RNA compared to a scrambled nucleic acid control, both at 5 and 100 nM concentrations; 96-well plate format, 5000 cells/well, 1.0 μg/ml GSV lipid.

[0068]
FIG. 9 shows a bar graph representing inhibition of Chk1 RNA via primary lead (GeneBloc™) inhibition as described in FIG. 6, however utilizing a 6-well plate format with a cell density of 150,000 cells per well.

[0069]
FIG. 10 shows a bar graph representing inhibition of Chk1 RNA via primary lead (GeneBloc™) inhibition in conjunction with +/− etoposide and nocodazole treatment; 50 nM GeneBloc™, 1.25 μg/ml GSV lipid, HeLa cells, 6-well plate format, 100,000 cells/well.

[0070]
FIG. 11 shows a bar graph of a lipid optimization study utilizing a lead nucleic acid inhibitor (GeneBloc™) targeting Chk1 RNA in DLD-1 cells; 96-well plate format, 15,000 cells/well, GSV lipid. Four different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitor.

[0071]
FIG. 12 shows a bar graph of a lipid optimization study utilizing a lead nucleic acid inhibitor (GeneBloc™) targeting Chk1 RNA in MCF-7 cells; 96-well plate format, 10,000 cells/well, GSV lipid. Four different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitor.

[0072]
FIG. 13 shows a dose curve of primary and secondary nucleic acid inhibitor (GeneBloc™) leads targeting Chk1 RNA in HeLa cells using 1.25 μg/ml GSV lipid, 24 hr time-point, 96-well plate format with a density of 5000 cells/well.

Mechanism of action of Nucleic Acid Molecules of the Invention

[0073] Antisense: 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, November 1994, BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

[0074] In addition, binding of single stranded DNA to RNA may result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which will 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.

[0075] 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., USSN 60/101,174 which was filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety.

[0076] In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.

[0077] Triplex Forming Olihonucleotides (TFO): 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).

[0078] 2-5A Antisense Chimera: 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). 2-5 A 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.

[0079] (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.

[0080] Enzymatic Nucleic Acid: Seven basic 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.

[0081] Nucleic acid molecules of this invention will block to some extent Chk1 protein expression and can be used to treat disease or diagnose disease associated with the levels of Chk1.

[0082] The enzymatic nature of a ribozyme has significant advantages, such as the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme 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 a ribozyme.

[0083] 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).

[0084] Because of their sequence specificity, trans-cleaving ribozymes can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes 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).

[0085] 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.

[0086] 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 was 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.

Target sites

[0087] 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 Chk1 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 (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence or linker X, the actual sequence is not relevant here). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule. While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted ribozymes may be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.

[0088] Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified. The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.

[0089] 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.

Synthesis of Nucleic acid Molecules

[0090] 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 similarly be synthesized.

[0091] 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 phosphorarnidites 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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).

[0100] 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.

[0101] The sequences of the ribozymes and antisense constructs that are chemically synthesized, useful in this study, are shown in Tables III to IX. 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 IX may 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.

Optimizing Activity of the nucleic acid molecule of the invention.

[0102] 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 at., 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 desired.

[0103] 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., USSN 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.

[0104] 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.

[0105] Nucleic acid molecules having chemical modifications which maintain or enhance activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. Therapeutic nucleic acid molecules delivered exogenously must optimally 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. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

[0106] Use of the nucleic acid-based molecules of the present invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.

[0107] Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously must optimally 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. Clearly, these nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

[0108] 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.

[0109] In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.

[0110] 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 may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or may be present on both termini. In non-limiting examples the 5′-cap is selected from the group comprising inverted abasic residue (moiety), 4′,5′-methylene nucleotide; I-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofaranosyl 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).

[0111] In yet another preferred embodiment, the 3′-cap is selected from a group comprising, 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).

[0112] 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.

[0113] 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 may 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 may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH. The term “alkyl” also includes alkynyl groups 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 may 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.

[0114] Such alkyl groups may 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 may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

[0115] 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-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases may 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.

[0116] 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, -D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases may 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.

[0117] 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.

[0118] 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).

[0119] By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of beta-D-ribo-furanose.

[0120] 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.

[0121] In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′—O—NH2, which may 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.

[0122] Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

[0123] Use of these molecules will 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 may also include combinations of different types of nucleic acid molecules. Therapies may 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.

Administration of Nucleic Acid Molecules

[0124] Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols may be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules 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 may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination is 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 W093/23569, Beigelman et al., PCT W099/05094, and Klimuk et al., PCT W099/04819 all of which have been incorporated by reference herein.

[0125] The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.

[0126] 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. The compositions of the present invention may 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.

[0127] 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.

[0128] 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.

[0129] 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 may 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.

[0130] 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, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies 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.

[0131] 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, Biochim. 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.

[0132] 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 may be used.

[0133] A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

[0134] The nucleic acid molecules of the present invention may 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.

[0135] 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).

[0136] 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 could 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 may be used that provide for transient expression of nucleic acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target MRNA. Delivery of nucleic acid molecule expressing vectors could be systemic, such as by intravenous or intra-muscular 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 (for a review, see Couture et al., 1996, TIG., 12, 510).

[0137] 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.

[0138] 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 may 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).

[0139] 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 will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. 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.

[0140] 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, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. U S A, 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).

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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

[0145] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

[0146] The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver ribozyme molecules and binding/cleavage sites within Chk1 RNA.

Nucleic acid inhibition of Chk1 target RNA

[0147] Control of the cell cycle is one of the most highly orchestrated events in the cell. There is a great deal of interest in discovering the function of genes involved in mitotic checkpoint abrogation, since inhibition of these genes or activities of these gene products could sensitize cells to DNA damaging agents. In these studies, the cell cycle regulatory role of Chk1 (GeneBank Accession #AF016582 is investigated).

[0148] In the fission yeast Schizosaccharomyces pombe, DNA damage by gamma irradiation or a chemical agent such as etoposide leads to activation of Cbk1 by phosphorylation. Chk1, also known as p56chk1, is a Wee 1-like protein kinase, which phosphorylates and inactivates Cdc25. Cdc25 is a phosphatase that acts directly on Cdc2. Chk1 is required for the DNA damage checkpoint, whereas the rad gene products are required for both S—M and DNA damage checkpoints. Wee 1 is also phosphorylated by Chk1 in vitro, also suggesting that Wee 1 is regulated by Chk1 in vivo and the resulting G2 delay is the result of maintaining Y15 phosphorylation on Cdc2. In normal mammalian cells, DNA damage would lead to arrest at G1/S arrest via the p53 pathway, or G2/M arrest via the Cdc2/CyclinB pathway. Thus, p53- cells can remain viable following DNA damage because of the Cdc2/CyclinB arrest pathway. If the Cdc2/CyclinB mediated checkpoint is abrogated via inhibition of Wee1 and Myt1 by small molecule inhibitors in a p53- cell type, then viability is compromised. Chk1 has recently been cloned from mammalian cells. The Chk1 protein is modified in response to DNA damage, and has been shown to bind and phosphorylate Cdc25A, Cdc25B and Cdc25C. The phosphorylation of Cdc25C prevents activation of the Cdc2/CyclinB complex and blocks entry into mitosis, thereby validating the inhibition of Chk1 as a target for nucleic acid based therapeutics.

[0149] To address whether checkpoint kinases function redundantly during DNA replication and/or DNA damage checkpoint responses, applicant undertook an oligonucleotide-based approach to block Chk1 gene function in a human cell line. HeLa cells lacking Chk1 protein failed to maintain a G2 cell cycle arrest after etoposide or gamma radiation-induced DNA damaging treatments. Additionally, Chk1-defeicient cells failed to respond to the DNA replication inhibitor hydroxyurea. Based on these results, applicant concludes that the Chk1 kinase plays an essential role in both the DNA replication and DNA damage checkpoint responses. These results also suggest the neither Chk2 nor C-TAK1 kinases function in these checkpoint responses to a significant level, at least in HeLa cells. Thus, Chk1 is validated as an attractive therapeutic target for abrogating the G2 DNA damage checkpoint arrest; a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.

Example 1

Identification of Potential Target Sites in Human Chk1 RNA

[0150] The sequence of human Chk1 is screened for accessible sites using a computer-folding algorithm. Regions of the RNA are 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-IX.

Example 2

Selection of Enzymatic Nucleic Acid Cleavage Sites in Human Chk1 RNA

[0151] Ribozyme target sites are chosen by analyzing sequences of Human Chk1 (Genbank accession number: AF016582) and prioritizing the sites on the basis of folding. Ribozymes are designed that could 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 ribozyme sequences fold into the appropriate secondary structure. Those ribozymes 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 Ribozymes and Antisense for Efficient Cleavage and/or blocking of Chk1 RNA

[0152] Ribozymes and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The ribozymes and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%.

[0153] Ribozymes and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes 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 ribozymes and antisense constructs used in this study are shown below in Table III-IX.

Example 4

Ribozyme Cleavage of Chk1 RNA Target in vitro

[0154] Ribozymes targeted to the human Chk1 RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the Chk1 RNA are given in Tables III-IX.

[0155] Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme 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 ribozyme in ribozyme 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× ribozyme 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., ribozyme 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 ribozyme 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 Chk1 target RNA in vivo

[0156] Antisense nucleic acid molecules (GeneBlocs™) targeted to the human Chk1 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 Chk1 RNA are given in Tables III-IX.

[0157] Two formats were used to test the efficacy of nucleic acid reagents (GeneBlocs™ targeting Chk1. First, the reagents were tested on asynchronous HeLa cells, to determine the extent of RNA and protein inhibition. To demonstrate whether cells bypass the G2/M checkpoint, HeLa cells (p53−) are treated with etoposide to damage the DNA. Nocodazole and the potential checkpoint inhibitor are added 16 hours later, when all the cells should be arrested in G2. Nocodazole blocks cells from leaving mitosis, so if they have abrogated the checkpoint, the cells will be blocked in mitosis and appear “rounded” in shape. Other surrogate mitotic markers include decreased phosphorylation of cdc-2 at Thr14 and Tyr15, phosphorylation of Myt-1, and phosphorylation of PP1. This study set out to determine whether inhibiting expression of the Chk1 gene would allow the G2/M checkpoint to be bypassed after DNA damage, as well as determining if the presence of p53 influences the DNA-damage checkpoint response.

[0158] Eight GeneBloc™ reagents (e.g.; see Table IX) were selected against the Chk1 cDNA target. RNA inhibition was measured after delivery of these reagents by GSV lipid (Glenn Research) to HeLa cells. Relative amounts of target RNA were measured versus actin using real-time PCR monitoring of amplification (ABI 7700 Taqman®). The results are shown in FIG. 6. The comparison is made to a mixture of 5 oligonucleotide sequences made to unrelated targets (GB-3) or to a randomized oligonucleotide control with the same overall length and chemistry, but randomly substituted at each position (GBC3.2). Primary and secondary lead reagents were chosen for the target and optimization performed. The optimal GSV lipid concentration was chosen after screening for RNA inhibition with oligonucleotides at 5 and 50 nM (FIG. 7). After optimal lipid concentration was chosen, a RNA time-course of inhibition was performed with the lead nucleic acid molecule (GeneBloc™) (FIG. 8). In addition, a cell-plating format was tested for RNA inhibition. The use of a 96-well (5000 cells/well) versus six-well (150,000 cells/well) plating density made no difference in the extent of RNA inhibition (FIG. 9). The phenotypic assays require treatment with etoposide and nocodazole as described above, and RNA inhibition in this assay was also determined (FIG. 10). The various treatments had essentially no effect on RNA levels.

[0159] Optimization of delivery conditions were also performed in DLD-1 (p53−) (FIG. 11) and MCF-7 (FIG. 12) (p53+) cells. Similar levels of inhibition were observed when compared to HeLa cells at the optimal GSV concentration. Dose curves were also generated in HeLa cells with the two best lead nucleic acid molecules (FIG. 13). IC50 values for both leads were in the 1-2 nM range. Similar IC50s were observed in DLD-1 and MCF-7 cells. Protein levels were assessed at 8, 24 and 32 hours after nucleic acid administration, as well as one to five days post delivery. The target protein was significantly reduced (80-90%) by 24 hours after nucleic acid administration and remained low (undetectable by western blot) until at least day 5. Application of nucleic acid inhibitors in the checkpoint abrogation assay resulted in the “rounding up” phenotype for the Chk1 target. Also, there is an increase in Myt 1 phosphorylation and a large increase in PP1 phosphorylation. There also appear to be decreases in phosphorylation of the Y15 and T14 residues on Cdc2, although this is not complete. Most importantly, this evidence demonstrates the role of Chk1 in the G2/M checkpoint and suggests that inhibitors of Chk1 activity can be useful alone or in combination with DNA damaging agents in treatment of certain types of cancer.

Indications

[0160] Particular degenerative and disease states that can be associated with Chk1 expression modulation include but are not limited to cancers of the colon, rectum, lung, breast and prostate

[0161] The present body of knowledge in Chk1 research indicates the need for methods to assay Chk1 activity and for compounds that can regulate Chk1 expression for research, diagnostic, and therapeutic use.

[0162] Radiation and chemotherapeutic treatments are non-limiting examples of 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.

Diagnostic uses

[0163] 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 Chk1 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 will 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 are well known in the art, and include detection of the presence of mRNAs associated with Chk1-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

[0164] 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 is 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 requires two ribozymes, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., Chk1) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

Additional Uses

[0165] Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention might 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 could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.

[0166] 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.

[0167] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] Other embodiments are within the claims that follow.

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 andmaintainance of the active structure.Over 300 known members of this class. Found as an intervening sequencein 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 intron hasbeen 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. The RNAsubunit has been sequenced from bacteria, yeast, rodents, and primates.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 a2′-5′ branch point.Only natural ribozyme with demonstrated participation in DNA cleavage[xx,xxi] in addition to RNA cleavage and ligation.Major structural features largely established through 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 crystal structures[xxvi,xxvii]Minimal ligation activity demonstrated (for engineering through in vitroselection) [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 residuesbegun [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 nosequences 5′ of cleavage site are required. Folded ribozyme contains apseudoknot structure [xl].Reaction mechanism: attack by 2′-OH 5′ to the scissile bond togenerate cleavage products with 2′,3′-cyclic phosphate and 5′-OH ends.Only 2 known members of this class. Found in human HDV.Circular form of HDV is active and shows increased nuclease stability [xli]iMichel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct Biol. (1994), 1(1), 5-7. iiLisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17. iiiHerschlag, 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. ivHerschlag, 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. vKnitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70. viBevilacqua, 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. viiLi, 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. viiiBanerjee, 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. ixZarrinkar, 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. xStrobel, 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. xiStrobel, 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. xiiSullenger, Bruce A.; Cech, Thomas R. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371 (6498), 619-22. xiiiRobertson, H.D.; Altman, S.; Smith, J. D. J. Biol. Chem., 247, 5243-5251 (1972). xivForster, Anthony C.; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D.C., 1883-) (1990), 249(4970), 783-6. xvYuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10. xviHarris, Michael E.; Pace, Norman R. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210-18. xviiPan, 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. xviiiPyle, 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. xixMichels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77. xxZimmerly, 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. xxiGriffin, 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. xxiiMichel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61. xxiiiAbramovitz, 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. xxivDaniels, 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. xxvGuo, 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. xxviScott, 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. xxviiMcKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403. xxviiiLong, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. U.S. Pat. No. 5,633,133. xxixHertel, 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. xxxBeigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708. xxxiHampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Philip. ‘Hairpin’ catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299-304. xxxiiChowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2. xxxiiiBerzal-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. xxxivJoseph, 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. xxxvBerzal-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. xxxviHegg, Lisa A.; Fedor, Martha J. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28. xxxviiGrasby, 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. xxxviiiSchmidt, 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. xxxixPerrotta, 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. xlPerrotta, 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. xliPuttaraju, M.; Perrotta, Anne T.; Been, Michael D. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

[0172]

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
186
233 μL
5 sec
5 sec
5 sec

Imidazole

TCA
176
2.3 μL
21 sec
21 sec
21 sec

Iodine
11.2
1.7 μL
45 sec
45 sec
45 sec

Beaucage
12.9
645 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
6.67 μL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
15
31 μL
45 sec
233 sec
465 sec

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

Acetic Anhydride
655
124 μL
5 sec
5 sec
5 sec

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

Imidazole

TCA
700
732 μL
10 sec
10 sec
10 sec

Iodine
20.6
244 μL
15 sec
15 sec
15 sec

Beaucage
7.7
232 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
2.64 μL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2’-O-

Wait Time* 2’-O-

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

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

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

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

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

Imidazole

TCA
238/475/475
250/500/500μL
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.

[0173]

3

TABLE III

Human Chk1 Hammerhead Ribozyme and Substrate Sequence

Rz Seq

Pos
Substrate
Seq ID
Ribozyme
ID

12
CGGACAGU C CGCCGAGG
1
CCUCGGCG CUGAUGAG GCCGUUAGGC CGAA ACUGUCCG
1423

25
GAGGUGCU C GGUGGAGU
2
ACUCCACC CUGAUGAG GCCGUUAGGC CGAA AGCACCUC
1424

34
GGUGGAGU C AUGGCAGU
3
ACUGCCAU CUGAUGAG GCCGUUAGGC CGAA ACUCCACC
1425

48
AGUGCCCU U UGUGGAAG
4
CUUCCACA CUGAUGAG GCCGUUAGGC CGAA AGGGCACU
1426

49
GUGCCCUU U GUGGAAGA
5
UCUUCCAC CUGAUGAG GCCGUUAGGC CGAA AAGGGCAC
1427

66
CUGGGACU U GGUGCAAA
6
UUUGCACC CUGAUGAG GCCGUUAGGC CGAA AGUCCCAG
1428

93
AGGUGCCU A UGGAGAAG
7
CUUCUCCA CUGAUGAG GCCGUUAGGC CGAA AGGCACCU
1429

103
GGAGAAGU U CAACUUGC
8
GCAAGUUG CUGAUGAG GCCGUUAGGC CGAA ACUUCUCC
1430

104
GAGAAGUU C AACUUGCU
9
AGCAAGUU CUGAUGAG GCCGUUAGGC CGAA AACUUCUC
1431

109
GUUCAACU U GCUGUGAA
10
UUCACAGC CUGAUGAG GCCGUUAGGC CGAA AGUUGAAC
1432

119
CUGUGAAU A GAGUAACU
11
AGUUACUC CUGAUGAG GCCGUUAGGC CGAA AUUCACAG
1433

123
AAUAGAGU A ACUGAAGA
12
UCUUCAGU CUGAUGAG GCCGUUAGGC CGAA ACUCUAUU
1434

139
GAAGCAGU C GCAGUGAA
13
UUCACUGC CUGAUGAG GCCGUUAGGC CGAA ACUGCUUC
1435

151
GUGAAGAU U GUAGAUAU
14
AUAUCUAC CUGAUGAG GCCGUUAGGC CGAA AUCUUCAC
1436

154
AAGAUUGU A GAUAUGAA
15
UUCAUAUC CUGAUGAG GCCGUUAGGC CGAA ACAAUCUU
1437

158
UUGUAGAU A UGAAGCGU
16
ACGCUUCA CUGAUGAG GCCGUUAGGC CGAA AUCUACAA
1438

172
CGUGCCGU A GACUGUCC
17
GGACAGUC CUGAUGAG GCCGUUAGGC CGAA ACGGCACG
1439

179
UAGACUGU C CAGAAAAU
18
AUUUUCUG CUGAUGAG GCCGUUAGGC CGAA ACAGUCUA
1440

188
CAGAAAAU A UUAAGAAA
19
UUUCUUAA CUGAUGAG GCCGUUAGGC CGAA AUUUUCUG
1441

190
GAAAAUAU U AAGAAAGA
20
UCUUUCUU CUGAUGAG GCCGUUAGGC CGAA AUAUUUUC
1442

191
AAAAUAUU A AGAAAGAG
21
CUCUUUCU CUGAUGAG GCCGUUAGGC CGAA AAUAUUUU
1443

202
AAAGAGAU C UGUAUCAA
22
UUGAUACA CUGAUGAG GCCGUUAGGC CGAA AUCUCUUU
1444

206
AGAUCUGU A UCAAUAAA
23
UUUAUUGA CUGAUGAG GCCGUUAGGC CGAA ACAGAUCU
1445

208
AUCUGUAU C AAUAAAAU
24
AUUUUAUU CUGAUGAG GCCGUUAGGC CGAA AUACAGAU
1446

212
GUAUCAAU A AAAUGCUA
25
UAGCAUUU CUGAUGAG GCCGUUAGGC CGAA AUUGAUAC
1447

220
AAAAUGCU A AAUCAUGA
26
UCAUGAUU CUGAUGAG GCCGUUAGGC CGAA AGCAUUUU
1448

224
UGCUAAAU C AUGAAAAU
27
AUUUUCAU CUGAUGAG GCCGUUAGGC CGAA AUUUAGCA
1449

235
GAAAAUGU A GUAAAAUU
28
AAUUUUAC CUGAUGAG GCCGUUAGGC CGAA ACAUUUUC
1450

238
AAUGUAGU A AAAUUCUA
29
UAGAAUUU CUGAUGAG GCCGUUAGGC CGAA ACUACAUU
1451

243
AGUAAAAU U CUAUGGUC
30
GACCAUAG CUGAUGAG GCCGUUAGGC CGAA AUUUUACU
1452

244
GUAAAAUU C UAUGGUCA
31
UGACCAUA CUGAUGAG GCCGUUAGGC CGAA AAUUUUAC
1453

246
AAAAUUCU A UGGUCACA
32
UGUGACCA CUGAUGAG GCCGUUAGGC CGAA AGAAUUUU
1454

251
UCUAUGGU C ACAGGAGA
33
UCUCCUGU CUGAUGAG GCCGUUAGGC CGAA ACCAUAGA
1455

269
AAGGCAAU A UCCAAUAU
34
AUAUUGGA CUGAUGAG GCCGUUAGGC CGAA AUUGCCUU
1456

271
GGCAAUAU C CAAUAUUU
35
AAAUAUUG CUGAUGAG GCCGUUAGGC CGAA AUAUUGCC
1457

276
UAUCCAAU A UUUAUUUC
36
GAAAUAAA CUGAUGAG GCCGUUAGGC CGAA AUUGGAUA
1458

278
UCCAAUAU U UAUUUCUG
37
CAGAAAUA CUGAUGAG GCCGUUAGGC CGAA AUAUUGGA
1459

279
CCAAUAUU U AUUUCUGG
38
CCAGAAAU CUGAUGAG GCCGUUAGGC CGAA AAUAUUGG
1460

280
CAAUAUUU A UUUCUGGA
39
UCCAGAAA CUGAUGAG GCCGUUAGGC CGAA AAAUAUUG
1461

282
AUAUUUAU U UCUGGAGU
40
ACUCCAGA CUGAUGAG GCCGUUAGGC CGAA AUAAAUAU
1462

283
UAUUUAUU U CUGGAGUA
41
UACUCCAG CUGAUGAG GCCGUUAGGC CGAA AAUAAAUA
1463

284
AUUUAUUU C UGGAGUAC
42
GUACUCCA CUGAUGAG GCCGUUAGGC CGAA AAAUAAAU
1464

291
UCUGGAGU A CUGAUGUG
43
CACUACAG CUGAUGAG GCCGUUAGGC CGAA ACUCCAGA
1465

296
AGUACUGU A GUGGAGGA
44
UCCUCCAC CUGAUGAG GCCGUUAGGC CGAA ACAGUACU
1466

310
GGAGAGCU U UUUGACAG
45
CUGUCAAA CUGAUGAG GCCGUUAGGC CGAA AGCUCUCC
1467

311
GAGAGCUU U UUGACAGA
46
UCUGUCAA CUGAUGAG GCCGUUAGGC CGAA AAGCUCUC
1468

312
AGAGCUUU U UGACAGAA
47
UUCUGUCA CUGAUGAG GCCGUUAGGC CGAA AAAGCUCU
1469

313
GAGCUUUU U GACAGAAU
48
AUUCUGUC CUGAUGAG GCCGUUAGGC CGAA AAAAGCUC
1470

322
GACAGAAU A GAGCCAGA
49
UCUGGCUC CUGAUGAG GCCGUUAGGC CGAA AUUCUGUC
1471

334
CCAGACAU A GGCAUGCC
50
GGCAUGCC CUGAUGAG GCCGUUAGGC CGAA AUGUCUGG
1472

356
CAGAUGCU C AGAGAUUC
51
GAAUCUCU CUGAUGAG GCCGUUAGGC CGAA AGCAUCUG
1473

363
UCAGAGAU U CUUCCAUC
52
GAUGGAAG CUGAUGAG GCCGUUAGGC CGAA AUCUCUGA
1474

364
CAGAGAUU C UUCCAUCA
53
UGAUGGAA CUGAUGAG GCCGUUAGGC CGAA AAUCUCUG
1475

366
GAGAUUCU U CCAUCAAC
54
GUUGAUGG CUGAUGAG GCCGUUAGGC CGAA AGAAUCUC
1476

367
AGAUUCUU C CAUCAACU
55
AGUUGAUG CUGAUGAG GCCGUUAGGC CGAA AAGAAUCU
1477

371
UCUUCCAU C AACUCAUG
56
CAUGAGUU CUGAUGAG GCCGUUAGGC CGAA AUGGAAGA
1478

376
CAUCAACU C AUGGCAGG
57
CCUGCCAU CUGAUGAG GCCGUUAGGC CGAA AGUUGAUG
1479

391
GGGGUGGU U UAUCUGCA
58
UGCAGAUA CUGAUGAG GCCGUUAGGC CGAA ACCACCCC
1480

392
GGGUGGUU U AUCUGCAU
59
AUGCAGAU CUGAUGAG GCCGUUAGGC CGAA AACCACCC
1481

393
GGUGGUUU A UCUGCAUG
60
CAUGCAGA CUGAUGAG GCCGUUAGGC CGAA AAACCACC
1482

395
UGGUUUAU C UGCAUGGU
61
ACCAUGCA CUGAUGAG GCCGUUAGGC CGAA AUAAACCA
1483

404
UGCAUGGU A UUGGAAUA
62
UAUUCCAA CUGAUGAG GCCGUUAGGC CGAA ACCAUGCA
1484

406
CAUGGUAU U GGAAUAAC
63
GUUAUUCC CUGAUGAG GCCGUUAGGC CGAA AUACCAUG
1485

412
AUUGGAAU A ACUCACAG
64
CUGUGAGU CUGAUGAG GCCGUUAGGC CGAA AUUCCAAU
1486

416
GAAUAACU C ACAGGGAU
65
AUCCCUGU CUGAUGAG GCCGUUAGGC CGAA AGUUAUUC
1487

425
ACAGGGAU A UUAAACCA
66
UGGUUUAA CUGAUGAG GCCGUUAGGC CGAA AUCCCUGU
1488

427
AGGGAUAU U AAACCAGA
67
UCUGGUUU CUGAUGAG GCCGUUAGGC CGAA AUAUCCCU
1489

428
GGGAUAUU A AACCAGAA
68
UUCUGGUU CUGAUGAG GCCGUUAGGC CGAA AAUAUCCC
1490

440
CAGAAAAU C UUCUGUUG
69
CAACAGAA CUGAUGAG GCCGUUAGGC CGAA AUUUUCUG
1491

442
GAAAAUCU U CUGUUGGA
70
UCCAACAG CUGAUGAG GCCGUUAGGC CGAA AGAUUUUC
1492

443
AAAAUCUU C UGUUGGAU
71
AUCCAACA CUGAUGAG GCCGUUAGGC CGAA AAGAUUUU
1493

447
UCUUCUGU U GGAUGAAA
72
UUUCAUCC CUGAUGAG GCCGUUAGGC CGAA ACAGAAGA
1494

461
AAAGGGAU A ACCUCAAA
73
UUUGAGGU CUGAUGAG GCCGUUAGGC CGAA AUCCCUUU
1495

466
GAUAACCU C AAAAUCUC
74
GAGAUUUU CUGAUGAG GCCGUUAGGC CGAA AGGUUAUC
1496

472
CUCAAAAU C UCAGACUU
75
AAGUCUGA CUGAUGAG GCCGUUAGGC CGAA AUUUUGAG
1497

474
CAAAAUCU C AGACUUUG
76
CAAAGUCU CUGAUGAG GCCGUUAGGC CGAA AGAUUUUG
1498

480
CUCAGACU U UGGCUUGG
77
CCAAGCCA CUGAUGAG GCCGUUAGGC CGAA AGUCUGAG
1499

481
UCAGACUU U GGCUUGGC
78
GCCAAGCC CUGAUGAG GCCGUUAGGC CGAA AAGUCUGA
1500

486
CUUUGGCU U GGCAACAG
79
CUGUUGCC CUGAUGAG GCCGUUAGGC CGAA AGCCAAAG
1501

496
GCAACAGU A UUUCGGUA
80
UACCGAAA CUGAUGAG GCCGUUAGGC CGAA ACUGUUGC
1502

498
AACAGUAU U UCGGUAUA
81
UAUACCGA CUGAUGAG GCCGUUAGGC CGAA AUACUGUU
1503

499
ACAGUAUU U CGGUAUAA
82
UUAUACCG CUGAUGAG GCCGUUAGGC CGAA AAUACUGU
1504

500
CAGUAUUU C GGUAUAAU
83
AUUAUACC CUGAUGAG GCCGUUAGGC CGAA AAAUACUG
1505

504
AUUUCGGU A UAAUAAUC
84
GAUUAUUA CUGAUGAG GCCGUUAGGC CGAA ACCGAAAU
1506

506
UUCGGUAU A AUAAUCGU
85
ACGAUUAU CUGAUGAG GCCGUUAGGC CGAA AUACCGAA
1507

509
GGUAUAAU A AUCGUGAG
86
CUCACGAU CUGAUGAG GCCGUUAGGC CGAA AUUAUACC
1508

512
AUAAUAAU C GUGAGCGU
87
ACGCUCAC CUGAUGAG GCCGUUAGGC CGAA AUUAUUAU
1509

521
GUGAGCGU U UGUUGAAC
88
GUUCAACA CUGAUGAG GCCGUUAGGC CGAA ACGCUCAC
1510

522
UGAGCGUU U GUUGAACA
89
UGUUCAAC CUGAUGAG GCCGUUAGGC CGAA AACGCUCA
1511

525
GCGUUUGU U GAACAAGA
90
UCUUGUUC CUGAUGAG GCCGUUAGGC CGAA ACAAACGC
1512

542
UGUGUGGU A CUUUACCA
91
UGGUAAAG CUGAUGAG GCCGUUAGGC CGAA ACCACACA
1513

545
GUGGUACU U UACCAUAU
92
AUAUGGUA CUGAUGAG GCCGUUAGGC CGAA AGUACCAC
1514

546
UGGUACUU U ACCAUAUG
93
CAUAUGGU CUGAUGAG GCCGUUAGGC CGAA AAGUACCA
1515

547
GGUACUUU A CCAUAUGU
94
ACAUAUGG CUGAUGAG GCCGUUAGGC CGAA AAAGUACC
1516

552
UUUACCAU A UGUUGCUC
95
GAGCAACA CUGAUGAG GCCGUUAGGC CGAA AUGGUAAA
1517

556
CCAUAUGU U GCUCCAGA
96
UCUGGAGC CUGAUGAG GCCGUUAGGC CGAA ACAUAUGG
1518

560
AUGUUGCU C CAGAACUU
97
AAGUUCUG CUGAUGAG GCCGUUAGGC CGAA AGCAACAU
1519

568
CCAGAACU U CUGAAGAG
98
CUCUUCAG CUGAUGAG GCCGUUAGGC CGAA AGUUCUGG
1520

569
CAGAACUU C UGAAGAGA
99
UCUCUUCA CUGAUGAG GCCGUUAGGC CGAA AAGUUCUG
1521

585
AAGAGAAU U UCAUGCAG
100
CUGCAUGA CUGAUGAG GCCGUUAGGC CGAA AUUCUCUU
1522

586
AGAGAAUU U CAUGCAGA
101
UCUGCAUG CUGAUGAG GCCGUUAGGC CGAA AAUUCUCU
1523

587
GAGAAUUU C AUGCAGAA
102
UUCUGCAU CUGAUGAG GCCGUUAGGC CGAA AAAUUCUC
1524

601
GAACCAGU U GAUGUUUG
103
CAAACAUC CUGAUGAG GCCGUUAGGC CGAA ACUGGUUC
1525

607
GUUGAUGU U UGGUCCUG
104
CAGGACCA CUGAUGAG GCCGUUAGGC CGAA ACAUCAAC
1526

608
UUGAUGUU U GGUCCUGU
105
ACAGGACC CUGAUGAG GCCGUUAGGC CGAA AACAUCAA
1527

612
UGUUUGGU C CUGUGGAA
106
UUCCACAG CUGAUGAG GCCGUUAGGC CGAA ACCAAACA
1528

622
UGUGGAAU A GUACUUAC
107
GUAAGUAC CUGAUGAG GCCGUUAGGC CGAA AUUCCACA
1529

625
GGAAUAGU A CUUACUGC
108
GCAGUAAG CUGAUGAG GCCGUUAGGC CGAA ACUAUUCC
1530

628
AUAGUACU U ACUGCAAU
109
AUUGCAGU CUGAUGAG GCCGUUAGGC CGAA AGUACUAU
1531

629
UAGUACUU A CUGCAAUG
110
CAUUGCAG CUGAUGAG GCCGUUAGGC CGAA AAGUACUA
1532

640
GCAAUGCU C GCUGGAGA
111
UCUCCAGC CUGAUGAG GCCGUUAGGC CGAA AGCAUUGC
1533

651
UGGAGAAU U GCCAUGGG
112
CCCAUGGC CUGAUGAG GCCGUUAGGC CGAA AUUCUCCA
1534

680
ACAGCUGU C AGGAGUAU
113
AUACUCCU CUGAUGAG GCCGUUAGGC CGAA ACAGCUGU
1535

687
UCAGGAGU A UUCUGACU
114
AGUCAGAA CUGAUGAG GCCGUUAGGC CGAA ACUCCUGA
1536

689
AGGAGUAU U CUGACUGG
115
CCAGUCAG CUGAUGAG GCCGUUAGGC CGAA AUACUCCU
1537

690
GGAGUAUU C UGACUGGA
116
UCCAGUCA CUGAUGAG GCCGUUAGGC CGAA AAUACUCC
1538

714
AAAAACAU A CCUCAACC
117
GGUUGAGG CUGAUGAG GCCGUUAGGC CGAA AUGUUUUU
1539

718
ACAUACCU C AACCCUUG
118
CAAGGGUU CUGAUGAG GCCGUUAGGC CGAA AGGUAUGU
1540

725
UCAACCCU U GGAAAAAA
119
UUUUUUCC CUGAUGAG GCCGUUAGGC CGAA AGGGUUGA
1541

736
AAAAAAAU C GAUUCUGC
120
GCAGAAUC CUGAUGAG GCCGUUAGGC CGAA AUUUUUUU
1542

740
AAAUCGAU U CUGCUCCU
121
AGGAGCAG CUGAUGAG GCCGUUAGGC CGAA AUCGAUUU
1543

741
AAUCGAUU C UGCUCCUC
122
GAGGAGCA CUGAUGAG GCCGUUAGGC CGAA AAUCGAUU
1544

746
AUUCUGCU C CUCUAGCU
123
AGCUAGAG CUGAUGAG GCCGUUAGGC CGAA AGCAGAAU
1545

749
CUGCUCCU C AUGCUCUG
124
CAGAGCUA CUGAUGAG GCCGUUAGGC CGAA AGGAGCAG
1546

751
GCUCCUCU A GCUCUGCU
125
AGCAGAGC CUGAUGAG GCCGUUAGGC CGAA AGAGGAGC
1547

755
CUCUAGCU C UGCUGCAU
126
AUGCAGCA CUGAUGAG GCCGUUAGGC CGAA AGCUAGAG
1548

764
UGCUGCAU A AAAUCUUA
127
UAAGAUUU CUGAUGAG GCCGUUAGGC CGAA AUGCAGCA
1549

769
CAUAAAAU C UUAGUUGA
128
UCAACUAA CUGAUGAG GCCGUUAGGC CGAA AUUUUAUG
1550

771
UAAAAUCU U AGUUGAGA
129
UCUCAACU CUGAUGAG GCCGUUAGGC CGAA AGAUUUUA
1551

772
AAAAUCUU A GUUGAGAA
130
UUCUCAAC CUGAUGAG GCCGUUAGGC CGAA AAGAUUUU
1552

775
AUCUUAGU U GAGAAUCC
131
GGAUUCUC CUGAUGAG GCCGUUAGGC CGAA ACUAAGAU
1553

782
UUGAGAAU C CAUCAGCA
132
UGCUGAUG CUGAUGAG GCCGUUAGGC CGAA AUUCUCAA
1554

786
GAAUCCAU C AGCAAGAA
133
UUCUUGCU CUGAUGAG GCCGUUAGGC CGAA AUGGAUUC
1555

796
GCAAGAAU U ACCAUUCC
134
GGAAUGGU CUGAUGAG GCCGUUAGGC CGAA AUUCUUGC
1556

797
CAAGAAUU A CCAUUCCA
135
UGGAAUGG CUGAUGAG GCCGUUAGGC CGAA AAUUCUUG
1557

802
AUUACCAU U CCAGACAU
136
AUGUCUGG CUGAUGAG GCCGUUAGGC CGAA AUGGUAAU
1558

803
UUACCAUU C CAGACAUC
137
GAUGUCUG CUGAUGAG GCCGUUAGGC CGAA AAUGGUAA
1559

811
CCAGACAU C AAAAAAGA
138
UCUUUUUU CUGAUGAG GCCGUUAGGC CGAA AUGUCUGG
1560

821
AAAAAGAU A GAUGGUAC
139
GUACCAUC CUGAUGAG GCCGUUAGGC CGAA AUCUUUUU
1561

828
UAGAUGGU A CAACAAAC
140
GUUUGUUG CUGAUGAG GCCGUUAGGC CGAA ACCAUCUA
1562

841
AAACCCCU C AAGAAAGG
141
CCUUUCUU CUGAUGAG GCCGUUAGGC CGAA AGGGGUUU
1563

868
CCCCGAGU C ACUUCAGG
142
CCUGAAGU CUGAUGAG GCCGUUAGGC CGAA ACUCGGGG
1564

872
GAGUCACU U CAGGUGGU
143
ACCACCUG CUGAUGAG GCCGUUAGGC CGAA AGUGACUC
1565

873
AGUCACUU C AGGUGGUG
144
CACCACCU CUGAUGAG GCCGUUAGGC CGAA AAGUGACU
1566

885
UGGUGUGU C AGAGUCUC
145
GAGACUCU CUGAUGAG GCCGUUAGGC CGAA ACACACCA
1567

891
GUCAGAGU C UCCCAGUG
146
CACUGGGA CUGAUGAG GCCGUUAGGC CGAA ACUCUGAC
1568

893
CAGAGUCU C CCAGUGGA
147
UCCACUGG CUGAUGAG GCCGUUAGGC CGAA AGACUCUG
1569

903
CAGUGGAU U UUCUAAGC
148
GCUUAGAA CUGAUGAG GCCGUUAGGC CGAA AUCCACUG
1570

904
AGUGGAUU U UCUAAGCA
149
UGCUUAGA CUGAUGAG GCCGUUAGGC CGAA AAUCCACU
1571

905
GUGGAUUU U CUAAGCAC
150
GUGCUUAG CUGAUGAG GCCGUUAGGC CGAA AAAUCCAC
1572

906
UGGAUUUU C UAAGCACA
151
UGUGCUUA CUGAUGAG GCCGUUAGGC CGAA AAAAUCCA
1573

908
GAUUUUCU A AGCACAUU
152
AAUGUGCU CUGAUGAG GCCGUUAGGC CGAA AGAAAAUC
1574

916
AAGCACAU U CAAUCCAA
153
UUGGAUUG CUGAUGAG GCCGUUAGGC CGAA AUGUGCUU
1575

917
AGCACAUU C AAUCCAAU
154
AUUGGAUU CUGAUGAG GCCGUUAGGC CGAA AAUGUGCU
1576

921
CAUUCAAU C CAAUUUGG
155
CCAAAUUG CUGAUGAG GCCGUUAGGC CGAA AUUGAAUG
1577

926
AAUCCAAU U UGGACUUC
156
GAAGUCCA CUGAUGAG GCCGUUAGGC CGAA AUUGGAUU
1578

927
AUCCAAUU U GGACUUCU
157
AGAAGUCC CUGAUGAG GCCGUUAGGC CGAA AAUUGGAU
1579

933
UUUGGACU U CUCUCCAG
158
CUGGAGAG CUGAUGAG GCCGUUAGGC CGAA AGUCCAAA
1580

934
UUGGACUU C UCUCCAGU
159
ACUGGAGA CUGAUGAG GCCGUUAGGC CGAA AAGUCCAA
1581

936
GGACUUCU C UCCAGUAA
160
UUACUGGA CUGAUGAG GCCGUUAGGC CGAA AGAAGUCC
1582

938
ACUUCUCU C CAGUAAAC
161
GUUUACUG CUGAUGAG GCCGUUAGGC CGAA AGAGAAGU
1583

943
UCUCCAGU A AACAGUGC
162
GCACUGUU CUGAUGAG GCCGUUAGGC CGAA ACUGGAGA
1584

953
ACAGUGCU U CUAGUGAA
163
UUCACUAG CUGAUGAG GCCGUUAGGC CGAA AGCACUGU
1585

954
CAGUGCUU C UAGUGAAG
164
CUUCACUA CUGAUGAG GCCGUUAGGC CGAA AAGCACUG
1586

956
GUGCUUCU A GUGAAGAA
165
UUCUUCAC CUGAUGAG GCCGUUAGGC CGAA AGAAGCAC
1587

975
UGUGAAGU A CUCCAGUU
166
AACUGGAG CUGAUGAG GCCGUUAGGC CGAA ACUUCACA
1588

978
GAAGUACU C CAGUUCUC
167
GAGAACUG CUGAUGAG GCCGUUAGGC CGAA AGUACUUC
1589

983
ACUCCAGU U CUCAGCCA
168
UGGCUGAG CUGAUGAG GCCGUUAGGC CGAA ACUGGAGU
1590

984
CUCCAGUU C UCAGCCAG
169
CUGGCUGA CUGAUGAG GCCGUUAGGC CGAA AACUGGAG
1591

986
CCAGUUCU C AGCCAGAA
170
UUCUGGCU CUGAUGAG GCCGUUAGGC CGAA AGAACUGG
1592

1007
GCACAGGU C UUUCCUUA
171
UAAGGAAA CUGAUGAG GCCGUUAGGC CGAA ACCUGUGC
1593

1009
ACAGGUCU U UCCUUAUG
172
CAUAAGGA CUGAUGAG GCCGUUAGGC CGAA AGACCUGU
1594

1010
CAGGUCUU U CCUUAUGG
173
CCAUAAGG CUGAUGAG GCCGUUAGGC CGAA AAGACCUG
1595

1011
AGGUCUUU C CUUAUGGG
174
CCCAUAAG CUGAUGAG GCCGUUAGGC CGAA AAAGACCU
1596

1014
UCUUUCCU U AUGGGAUA
175
UAUCCCAU CUGAUGAG GCCGUUAGGC CGAA AGGAAAGA
1597

1015
CUUUCCUU A UGGGAUAC
176
GUAUCCCA CUGAUGAG GCCGUUAGGC CGAA AAGGAAAG
1598

1022
UAUGGGAU A CCAGCCCC
177
GGGGCUGG CUGAUGAG GCCGUUAGGC CGAA AUCCCAUA
1599

1032
CAGCCCCU C AUACAUUG
178
CAAUGUAU CUGAUGAG GCCGUUAGGC CGAA AGGGGCUG
1600

1035
CCCCUCAU A CAUUGAUA
179
UAUCAAUG CUGAUGAG GCCGUUAGGC CGAA AUGAGGGG
1601

1039
UCAUACAU U GAUAAAUU
180
AAUUUAUC CUGAUGAG GCCGUUAGGC CGAA AUGUAUGA
1602

1043
ACAUUGAU A AAUUGGUA
181
UACCAAUU CUGAUGAG GCCGUUAGGC CGAA AUCAAUGU
1603

1047
UGAUAAAU U GGUACAAG
182
CUUGUACC CUGAUGAG GCCGUUAGGC CGAA AUUUAUCA
1604

1051
AAAUUGGU A CAAGGGAU
183
AUCCCUUG CUGAUGAG GCCGUUAGGC CGAA ACCAAUUU
1605

1060
CAAGGGAU C AGCUUUUC
184
GAAAAGCU CUGAUGAG GCCGUUAGGC CGAA AUCCCUUG
1606

1065
GAUCAGCU U UUCCCAGC
185
GCUGGGAA CUGAUGAG GCCGUUAGGC CGAA AGCUGAUC
1607

1066
AUCAGCUU U UCCCAGCC
186
GGCUGGGA CUGAUGAG GCCGUUAGGC CGAA AAGCUGAU
1608

1067
UCAGCUUU U CCCAGCCC
187
GGGCUGGG CUGAUGAG GCCGUUAGGC CGAA AAAGCUGA
1609

1068
CAGCUUUU C CCAGCCCA
188
UGGGCUGG CUGAUGAG GCCGUUAGGC CGAA AAAAGCUG
1610

1082
CCACAUGU C CUGAUCAU
189
AUGAUCAG CUGAUGAG GCCGUUAGGC CGAA ACAUGUGG
1611

1088
GUCCUGAU C AUAUGCUU
190
AAGCAUAU CUGAUGAG GCCGUUAGGC CGAA AUCAGGAC
1612

1091
CUGAUCAU A UGCUUUUG
191
CAAAAGCA CUGAUGAG GCCGUUAGGC CGAA AUGAUCAG
1613

1096
CAUAUGCU U UUGAAUAG
192
CUAUUCAA CUGAUGAG GCCGUUAGGC CGAA AGCAUAUG
1614

1097
AUAUGCUU U UGAAUAGU
193
ACUAUUCA CUGAUGAG GCCGUUAGGC CGAA AAGCAUAU
1615

1098
UAUGCUUU U GAAUAGUC
194
GACUAUUC CUGAUGAG GCCGUUAGGC CGAA AAAGCAUA
1616

1103
UUUUGAAU A GUCAGUUA
195
UAACUGAC CUGAUGAG GCCGUUAGGC CGAA AUUCAAAA
1617

1106
UGAAUAGU C AGUUACUU
196
AAGUAACU CUGAUGAG GCCGUUAGGC CGAA ACUAUUCA
1618

1110
UAGUCAGU U ACUUGGCA
197
UGCCAAGU CUGAUGAG GCCGUUAGGC CGAA ACUGACUA
1619

1111
AGUCAGUU A CUUGGCAC
198
GUGCCAAG CUGAUGAG GCCGUUAGGC CGAA AACUGACU
1620

1114
CAGUUACU U GGCACCCC
199
GGGGUGCC CUGAUGAG GCCGUUAGGC CGAA AGUAACUG
1621

1128
CCCAGGAU C CUCACAGA
200
UCUGUGAG CUGAUGAG GCCGUUAGGC CGAA AUCCUGGG
1622

1131
AGGAUCCU C ACAGAACC
201
GGUUCUGU CUGAUGAG GCCGUUAGGC CGAA AGGAUCCU
1623

1152
GCAGCGGU U GGUCAAAA
202
UUUUGACC CUGAUGAG GCCGUUAGGC CGAA ACCGCUGC
1624

1156
CGGUUGGU C AAAAGAAU
203
AUUCUUUU CUGAUGAG GCCGUUAGGC CGAA ACCAACCG
1625

1173
GACACGAU U CUUUACCA
204
UGGUAAAG CUGAUGAG GCCGUUAGGC CGAA AUCGUGUC
1626

1174
ACACGAUU C UUUACCAA
205
UUGGUAAA CUGAUGAG GCCGUUAGGC CGAA AAUCGUGU
1627

1176
ACGAUUCU U UACCAAAU
206
AUUUGGUA CUGAUGAG GCCGUUAGGC CGAA AGAAUCGU
1628

1177
CGAUUCUU U ACCAAAUU
207
AAUUUGGU CUGAUGAG GCCGUUAGGC CGAA AAGAAUCG
1629

1178
GAUUCUUU A CCAAAUUG
208
CAAUUUGG CUGAUGAG GCCGUUAGGC CGAA AAAGAAUC
1630

1185
UACCAAAU U GGAUGCAG
209
CUGCAUCC CUGAUGAG GCCGUUAGGC CGAA AUUUGGUA
1631

1200
AGACAAAU C UUAUCAAU
210
AUUGAUAA CUGAUGAG GCCGUUAGGC CGAA AUUUGUCU
1632

1202
ACAAAUCU U AUCAAUGC
211
GCAUUGAU CUGAUGAG GCCGUUAGGC CGAA AGAUUUGU
1633

1203
CAAAUCUU A UCAAUGCC
212
GGCAUUGA CUGAUGAG GCCGUUAGGC CGAA AAGAUUUG
1634

1205
AAUCUUAU C AAUGCCUG
213
CAGGCAUU CUGAUGAG GCCGUUAGGC CGAA AUAAGAUU
1635

1223
AAGAGACU U GUGAGAAG
214
CUUCUCAC CUGAUGAG GCCGUUAGGC CGAA AGUCUCUU
1636

1233
UGAGAAGU U GGGCUAUC
215
GAUAGCCC CUGAUGAG GCCGUUAGGC CGAA ACUUCUCA
1637

1239
GUUGGGCU A UCAAUGGA
216
UCCAUUGA CUGAUGAG GCCGUUAGGC CGAA AGCCCAAC
1638

1241
UGGGCUAU C AAUGGAAG
217
CUUCCAUU CUGAUGAG GCCGUUAGGC CGAA AUAGCCCA
1639

1256
AGAAAAGU U GUAUGAAU
218
AUUCAUAC CUGAUGAG GCCGUUAGGC CGAA ACUUUUCU
1640

1259
AAAGUUGU A UGAAUCAG
219
CUGAUUCA CUGAUGAG GCCGUUAGGC CGAA ACAACUUU
1641

1265
GUAUGAAU C AGGUUACU
220
AGUAACCU CUGAUGAG GCCGUUAGGC CGAA AUUCAUAC
1642

1270
AAUCAGGU U ACUAUAUC
221
GAUAUAGU CUGAUGAG GCCGUUAGGC CGAA ACCUGAUU
1643

1271
AUCAGGUU A CUAUAUCA
222
UGAUAUAG CUGAUGAG GCCGUUAGGC CGAA AACCUGAU
1644

1274
AGGUUACU A UAUCAACA
223
UGUUGAUA CUGAUGAG GCCGUUAGGC CGAA AGUAACCU
1645

1276
GUUACUAU A UCAACAAC
224
GUUGUUGA CUGAUGAG GCCGUUAGGC CGAA AUAGUAAC
1646

1278
UACUAUAU C AACAACUG
225
CAGUUGUU CUGAUGAG GCCGUUAGGC CGAA AUAUAGUA
1647

1289
CAACUGAU A GGAGAAAC
226
GUUUCUCC CUGAUGAG GCCGUUAGGC CGAA AUCAGUUG
1648

1301
GAAACAAU A AACUCAUU
227
AAUGAGUU CUGAUGAG GCCGUUAGGC CGAA AUUGUUUC
1649

1306
AAUAAACU C AUUUUCAA
228
UUGAAAAU CUGAUGAG GCCGUUAGGC CGAA AGUUUAUU
1650

1309
AAACUCAU U UUCAAAGU
229
ACUUUGAA CUGAUGAG GCCGUUAGGC CGAA AUGAGUUU
1651

1310
AACUCAUU U UCAAAGUG
230
CACUUUGA CUGAUGAG GCCGUUAGGC CGAA AAUGAGUU
1652

1311
ACUCAUUU U CAAAGUGA
231
UCACUUUG CUGAUGAG GCCGUUAGGC CGAA AAAUGAGU
1653

1312
CUCAUUUU C AAAGUGAA
232
UUCACUUU CUGAUGAG GCCGUUAGGC CGAA AAAAUGAG
1654

1322
AAGUGAAU U UGUUAGAA
233
UUCUAACA CUGAUGAG GCCGUUAGGC CGAA AUUCACUU
1655

1323
AGUGAAUU U GUUAGAAA
234
UUUCUAAC CUGAUGAG GCCGUUAGGC CGAA AAUUCACU
1656

1326
GAAUUUGU U AGAAAUGG
235
CCAUUUCU CUGAUGAG GCCGUUAGGC CGAA ACAAAUUC
1657

1327
AAUUUGUU A GAAAUGGA
236
UCCAUUUC CUGAUGAG GCCGUUAGGC CGAA AACAAAUU
1658

1340
UGGAUGAU A AAAUAUUG
237
CAAUAUUU CUGAUGAG GCCGUUAGGC CGAA AUCAUCCA
1659

1345
GAUAAAAU A UUGGUUGA
238
UCAACCAA CUGAUGAG GCCGUUAGGC CGAA AUUUUAUC
1660

1347
UAAAAUAU U GGUUGACU
239
AGUCAACC CUGAUGAG GCCGUUAGGC CGAA AUAUUUUA
1661

1351
AUAUUGGU U GACUUCCG
240
CGGAAGUC CUGAUGAG GCCGUUAGGC CGAA ACCAAUAU
1662

1356
GGUUGACU U CCGGCUUU
241
AAAGCCGG CUGAUGAG GCCGUUAGGC CGAA AGUCAACC
1663

1357
GUUGACUU C CGGCUUUC
242
GAAAGCCG CUGAUGAG GCCGUUAGGC CGAA AAGUCAAC
1664

1363
UUCCGGCU U UCUAAGGG
243
CCCUUAGA CUGAUGAG GCCGUUAGGC CGAA AGCCGGAA
1665

1364
UCCGGCUU U CUAAGGGU
244
ACCCUUAG CUGAUGAG GCCGUUAGGC CGAA AAGCCGGA
1666

1365
CCGGCUUU C UAAGGGUG
245
CACCCUUA CUGAUGAG GCCGUUAGGC CGAA AAAGCCGG
1667

1367
GGCUUUCU A AGGGUGAU
246
AUCACCCU CUGAUGAG GCCGUUAGGC CGAA AGAAAGCC
1668

1380
UGAUGGAU U GGAGUUCA
247
UGAACUCC CUGAUGAG GCCGUUAGGC CGAA AUCCAUCA
1669

1386
AUUGGAGU U CAAGAGAC
248
GUCUCUUG CUGAUGAG GCCGUUAGGC CGAA ACUCCAAU
1670

1387
UUGGAGUU C AAGAGACA
249
UGUCUCUU CUGAUGAG GCCGUUAGGC CGAA AACUCCAA
1671

1398
GAGACACU U CCUGAAGA
250
UCUUCAGG CUGAUGAG GCCGUUAGGC CGAA AGUGUCUC
1672

1399
AGACACUU C CUGAAGAU
251
AUCUUCAG CUGAUGAG GCCGUUAGGC CGAA AAGUGUCU
1673

1408
CUGAAGAU U AAAGGGAA
252
UUCCCUUU CUGAUGAG GCCGUUAGGC CGAA AUCUUCAG
1674

1409
UGAAGAUU A AAGGGAAG
253
CUUCCCUU CUGAUGAG GCCGUUAGGC CGAA AAUCUUCA
1675

1423
AAGCUGAU U GAUAUUGU
254
ACAAUAUC CUGAUGAG GCCGUUAGGC CGAA AUCAGCUU
1676

1427
UGAUUGAU A UUGUGAGC
255
GCUCACAA CUGAUGAG GCCGUUAGGC CGAA AUCAAUCA
1677

1429
AUUGAUAU U GUGAGCAG
256
CUGCUCAC CUGAUGAG GCCGUUAGGC CGAA AUAUCAAU
1678

1447
CAGAAGGU U UGGCUUCC
257
GGAAGCCA CUGAUGAG GCCGUUAGGC CGAA ACCUUCUG
1679

1448
AGAAGGUU U GGCUUCCU
258
AGGAAGCC CUGAUGAG GCCGUUAGGC CGAA AACCUUCU
1680

1453
GUUUGGCU U CCUGCCAC
259
GUGGCAGG CUGAUGAG GCCGUUAGGC CGAA AGCCAAAC
1681

1454
UUUGGCUU C CUGCCACA
260
UGUGGCAG CUGAUGAG GCCGUUAGGC CGAA AAGCCAAA
1682

1467
CACAUGAU C GGACCAUC
261
GAUGGUCC CUGAUGAG GCCGUUAGGC CGAA AUCAUGUG
1683

1475
CGGACCAU C GGCUCUGG
262
CCAGAGCC CUGAUGAG GCCGUUAGGC CGAA AUGGUCCG
1684

1480
CAUCGGCU C UGGGGAAU
263
AUUCCCCA CUGAUGAG GCCGUUAGGC CGAA AGCCGAUG
1685

1489
UGGGGAAU C CUGGUGAA
264
UUCACCAG CUGAUGAG GCCGUUAGGC CGAA AUUCCCCA
1686

1499
UGGUGAAU A UAGUGCUG
265
CAGCACUA CUGAUGAG GCCGUUAGGC CGAA AUUCACCA
1687

1501
GUGAAUAU A GUGCUGCU
266
AGCAGCAC CUGAUGAG GCCGUUAGGC CGAA AUAUUCAC
1688

1510
GUGCUGCU A UGUUGACA
267
UGUCAACA CUGAUGAG GCCGUUAGGC CGAA AGCAGCAC
1689

1514
UGCUAUGU U GACAUUAU
268
AUAAUGUC CUGAUGAG GCCGUUAGGC CGAA ACAUAGCA
1690

1520
GUUGACAU U AUUCUUCC
269
GGAAGAAU CUGAUGAG GCCGUUAGGC CGAA AUGUCAAC
1691

1521
UUGACAUU A UUCUUCCU
270
AGGAAGAA CUGAUGAG GCCGUUAGGC CGAA AAUGUCAA
1692

1523
GACAUUAU U CUUCCUAG
271
CUAGGAAG CUGAUGAG GCCGUUAGGC CGAA AUAAUGUC
1693

1524
ACAUUAUU C UUCCUAGA
272
UCUAGGAA CUGAUGAG GCCGUUAGGC CGAA AAUAAUGU
1694

1526
AUUAUUCU U CCUAGAGA
273
UCUCUAGG CUGAUGAG GCCGUUAGGC CGAA AGAAUAAU
1695

1527
UUAUUCUU C CUAGAGAA
274
UUCUCUAG CUGAUGAG GCCGUUAGGC CGAA AAGAAUAA
1696

1530
UUCUUCCU A GAGAAGAU
275
AUCUUCUC CUGAUGAG GCCGUUAGGC CGAA AGGAAGAA
1697

1539
GAGAAGAU U AUCCUGUC
276
GACAGGAU CUGAUGAG GCCGUUAGGC CGAA AUCUUCUC
1698

1540
AGAAGAUU A UCCUGUCC
277
GGACAGGA CUGAUGAG GCCGUUAGGC CGAA AAUCUUCU
1699

1542
AAGAUUAU C CUGUCCUG
278
CAGGACAG CUGAUGAG GCCGUUAGGC CGAA AUAAUCUU
1700

1547
UAUCCUGU C CUGCAAAC
279
GUUUGCAG CUGAUGAG GCCGUUAGGC CGAA ACAGGAUA
1701

1563
CUGCAAAU A GUAGUUCC
280
GGAACUAC CUGAUGAG GCCGUUAGGC CGAA AUUUGCAG
1702

1566
CAAAUAGU A GUUCCUGA
281
UCAGGAAC CUGAUGAG GCCGUUAGGC CGAA ACUAUUUG
1703

1569
AUAGUAGU U CCUGAAGU
282
ACUUCAGG CUGAUGAG GCCGUUAGGC CGAA ACUACUAU
1704

1570
UAGUAGUU C CUGAAGUG
283
CACUUCAG CUGAUGAG GCCGUUAGGC CGAA AACUACUA
1705

1580
UGAAGUGU U CACUUCCC
284
GGGAAGUG CUGAUGAG GCCGUUAGGC CGAA ACACUUCA
1706

1581
GAAGUGUU C ACUUCCCU
285
AGGGAAGU CUGAUGAG GCCGUUAGGC CGAA AACACUUC
1707

1585
UGUUCACU U CCCUGUUU
286
AAACAGGG CUGAUGAG GCCGUUAGGC CGAA AGUGAACA
1708

1586
GUUCACUU C CCUGUUUA
287
UAAACAGG CUGAUGAG GCCGUUAGGC CGAA AAGUGAAC
1709

1592
UUCCCUGU U UAUCCAAA
288
UUUGGAUA CUGAUGAG GCCGUUAGGC CGAA ACAGGGAA
1710

1593
UCCCUGUU U AUCCAAAC
289
GUUUGGAU CUGAUGAG GCCGUUAGGC CGAA AACAGGGA
1711

1594
CCCUGUUU A UCCAAACA
290
UGUUUGGA CUGAUGAG GCCGUUAGGC CGAA CUGAUGAG
1712

1596
CUGUUUAU C CAAACAUC
291
GAUGUUUG CUGAUGAG GCCGUUAGGC CGAA AUAAACAG
1713

1604
CCAAACAU C UUCCAAUU
292
AAUUGGAA CUGAUGAG GCCGUUAGGC CGAA AUGUUUGG
1714

1606
AAACAUCU U CCAAUUUA
293
UAAAUUGG CUGAUGAG GCCGUUAGGC CGAA AGAUGUUU
1715

1607
AACAUCUU C CAAUUUAU
294
AUAAAUUG CUGAUGAG GCCGUUAGGC CGAA AAGAUGUU
1716

1612
CUUCCAAU U UAUUUUGU
295
ACAAAAUA CUGAUGAG GCCGUUAGGC CGAA AUUGGAAG
1717

1613
UUCCAAUU U AUUUUGUU
296
AACAAAAU CUGAUGAG GCCGUUAGGC CGAA AAUUGGAA
1718

1614
UCCAAUUU A UUUUGUUU
297
AAACAAAA CUGAUGAG GCCGUUAGGC CGAA AAAUUGGA
1719

1616
CAAUUUAU U UUGUUUGU
298
ACAAACAA CUGAUGAG GCCGUUAGGC CGAA AUAAAUUG
1720

1617
AAUUUAUU U UGUUUGUU
299
AACAAACA CUGAUGAG GCCGUUAGGC CGAA AAUAAAUU
1721

1618
AUUUAUUU U GUUUGUUC
300
GAACAAAC CUGAUGAG GCCGUUAGGC CGAA AAAUAAAU
1722

1621
UAUUUUGU U UGUUCGGC
301
GCCGAACA CUGAUGAG GCCGUUAGGC CGAA ACAAAAUA
1723

1622
AUUUUGUU U GUUCGGCA
302
UGCCGAAC CUGAUGAG GCCGUUAGGC CGAA AACAAAAU
1724

1625
UUGUUUGU U CGGCAUAC
303
GUAUGCCG CUGAUGAG GCCGUUAGGC CGAA ACAAACAA
1725

1626
UGUUUGUU C GGCAUACA
304
UGUAUGCC CUGAUGAG GCCGUUAGGC CGAA AACAAACA
1726

1632
UUCGGCAU A CAAAUAAU
305
AUUAUUUG CUGAUGAG GCCGUUAGGC CGAA AUGCCGAA
1727

1638
AUACAAAU A AUACCUAU
306
AUAGGUAU CUGAUGAG GCCGUUAGGC CGAA AUUUGUAU
1728

1641
CAAAUAAU A CCUAUAUC
307
GAUAUAGG CUGAUGAG GCCGUUAGGC CGAA AUUAUUUG
1729

1645
UAAUACCU A UAUCUUAA
308
UUAAGAUA CUGAUGAG GCCGUUAGGC CGAA AGGUAUUA
1730

1647
AUACCUAU A UCUUAAUU
309
AAUUAAGA CUGAUGAG GCCGUUAGGC CGAA AUAGGUAU
1731

1649
ACCUAUAU C UUAAUUGU
310
ACAAUUAA CUGAUGAG GCCGUUAGGC CGAA AUAUAGGU
1732

1651
CUAUAUCU U AAUUGUAA
311
UUACAAUU CUGAUGAG GCCGUUAGGC CGAA AGAUAUAG
1733

1652
UAUAUCUU A AUUGUAAG
312
CUUACAAU CUGAUGAG GCCGUUAGGC CGAA AAGAUAUA
1734

1655
AUCUUAAU U GUAAGCAA
313
UUGCUUAC CUGAUGAG GCCGUUAGGC CGAA AUUAAGAU
1735

1658
UUAAUUGU A AGCAAAAC
314
GUUUUGCU CUGAUGAG GCCGUUAGGC CGAA ACAAUUAA
1736

1668
GCAAAACU U UGGGGAAA
315
UUUCCCCA CUGAUGAG GCCGUUAGGC CGAA AGUUUUGC
1737

1669
CAAAACUU U GGGGAAAG
316
CUUUCCCC CUGAUGAG GCCGUUAGGC CGAA AAGUUUUG
1738

1685
GGAUGAAU A GAAUUCAU
317
AUGAAUUC CUGAUGAG GCCGUUAGGC CGAA AUUCAUCC
1739

1690
AAUAGAAU U CAUUUGAU
318
AUCAAAUG CUGAUGAG GCCGUUAGGC CGAA AUUCUAUU
1740

1691
AUAGAAUU C AUUUGAUU
319
AAUCAAAU CUGAUGAG GCCGUUAGGC CGAA AAUUCUAU
1741

1694
GAAUUCAU U UGAUUAUU
320
AAUAAUCA CUGAUGAG GCCGUUAGGC CGAA AUGAAUUC
1742

1695
AAUUCAUU U GAUUAUUU
321
AAAUAAUC CUGAUGAG GCCGUUAGGC CGAA AAUGAAUU
1743

1699
CAUUUGAU U AUUUCUUC
322
GAAGAAAU CUGAUGAG GCCGUUAGGC CGAA AUCAAAUG
1744

1700
AUUUGAUU A UUUCUUCA
323
UGAAGAAA CUGAUGAG GCCGUUAGGC CGAA AAUCAAAU
1745

1702
UUGAUUAU U UCUUCAUG
324
CAUGAAGA CUGAUGAG GCCGUUAGGC CGAA AUAAUCAA
1746

1703
UGAUUAUU U CUUCAUGU
325
ACAUGAAG CUGAUGAG GCCGUUAGGC CGAA AAUAAUCA
1747

1704
GAUUAUUU C UUCAUGUG
326
CACAUGAA CUGAUGAG GCCGUUAGGC CGAA AAAUAAUC
1748

1706
UUAUUUCU U CAUGUGUG
327
CACACAUG CUGAUGAG GCCGUUAGGC CGAA AGAAAUAA
1749

1707
UAUUUCUU C AUGUGUGU
328
ACACACAU CUGAUGAG GCCGUUAGGC CGAA AAGAAAUA
1750

1716
AUGUGUGU U AUGUAUCU
329
AGAUACUA CUGAUGAG GCCGUUAGGC CGAA ACACACAU
1751

1717
UGUGUGUU U AGUAUCUG
330
CAGAUACU CUGAUGAG GCCGUUAGGC CGAA AACACACA
1752

1718
GUGUGUUU A GUAUCUGA
331
UCAGAUAC CUGAUGAG GCCGUUAGGC CGAA AAACACAC
1753

1721
UGUUUAGU A UCUGAAUU
332
AAUUCAGA CUGAUGAG GCCGUUAGGC CGAA ACUAAACA
1754

1723
UUUAGUAU C UGAAUUUG
333
CAAAUUCA CUGAUGAG GCCGUUAGGC CGAA AUACUAAA
1755

1729
AUCUGAAU U UGAAACUC
334
GAGUUUCA CUGAUGAG GCCGUUAGGC CGAA AUUCAGAU
1756

1730
UCUGAAUU U GAAACUCA
335
UGAGUUUC CUGAUGAG GCCGUUAGGC CGAA AAUUCAGA
1757

1737
UUGAAACU C AUCUGGUG
336
CACCAGAU CUGAUGAG GCCGUUAGGC CGAA AGUUUCAA
1758

1740
AAACUCAU C UGGUGGAA
337
UUCCACCA CUGAUGAG GCCGUUAGGC CGAA AUGAGUUU
1759

1756
AACCAAGU U UCAGGGGA
338
UCCCCUGA CUGAUGAG GCCGUUAGGC CGAA ACUUGGUU
1760

1757
ACCAAGUU U CAGGGGAC
339
GUCCCCUG CUGAUGAG GCCGUUAGGC CGAA AACUUGGU
1761

1758
CCAAGUUU C AGGGGACA
340
UGUCCCCU CUGAUGAG GCCGUUAGGC CGAA AAACUUGG
1762

1772
ACAUGAGU U UUCCAGCU
341
AGCUGGAA CUGAUGAG GCCGUUAGGC CGAA ACUCAUGU
1763

1773
CAUGAGUU U UCCAGCUU
342
AAGCUGGA CUGAUGAG GCCGUUAGGC CGAA AACUCAUG
1764

1774
AUGAGUUU U CCAGCUUU
343
AAAGCUGG CUGAUGAG GCCGUUAGGC CGAA AAACUCAU
1765

1775
UGAGUUUU C CAGCUUUU
344
AAAAGCUG CUGAUGAG GCCGUUAGGC CGAA AAAACUCA
1766

1781
UUCCAGCU U UUAUACAC
345
GUGUAUAA CUGAUGAG GCCGUUAGGC CGAA AGCUGGAA
1767

1782
UCCAGCUU U UAUACACA
346
UGUGUAUA CUGAUGAG GCCGUUAGGC CGAA AAGCUGGA
1768

1783
CCAGCUUU U AUACACAC
347
GUGUGUAU CUGAUGAG GCCGUUAGGC CGAA AAAGCUGG
1769

1784
CAGCUUUU A UACACACG
348
CGUGUGUA CUGAUGAG GCCGUUAGGC CGAA AAAAGCUG
1770

1786
GCUUUUAU A CACACGUA
349
UACGUGUG CUGAUGAG GCCGUUAGGC CGAA AUAAAAGC
1771

1794
ACACACGU A UCUCAUUU
350
AAAUGAGA CUGAUGAG GCCGUUAGGC CGAA ACGUGUGU
1772

1796
ACACGUAU C UCAUUUUU
351
AAAAAUGA CUGAUGAG GCCGUUAGGC CGAA AUACGUGU
1773

1798
ACGUAUCU C AUUUUUAU
352
AUAAAAAU CUGAUGAG GCCGUUAGGC CGAA AGAUACGU
1774

1801
UAUCUCAU U UUUAUCAA
353
UUGAUAAA CUGAUGAG GCCGUUAGGC CGAA AUGAGAUA
1775

1802
AUCUCAUU U UUAUCAAA
354
UUUGAUAA CUGAUGAG GCCGUUAGGC CGAA AAUGAGAU
1776

1803
UCUCAUUU U UAUCAAAA
355
UUUUGAUA CUGAUGAG GCCGUUAGGC CGAA AAAUGAGA
1777

1804
CUCAUUUU U AUCAAAAC
356
GUUUUGAU CUGAUGAG GCCGUUAGGC CGAA AAAAUGAG
1778

1805
UCAUUUUU A UCAAAACA
357
UGUUUUGA CUGAUGAG GCCGUUAGGC CGAA AAAAAUGA
1779

1807
AUUUUUAU C AAAACAUU
358
AAUGUUUU CUGAUGAG GCCGUUAGGC CGAA AUAAAAAU
1780

Input Sequence = AF016582 Cut Site = UH/.

Stem Length = 8. Core Sequence = CUGAUGAG GCCGUUAGGC CGAA

AF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

[0174] Underlined region can be any X sequence or linker as previously defined herein.

4TABLE IVHuman Chk1 NCH Ribozyme and Substrate SequenceRz SeqPosSubstrateSeq IDRibozymeID9GGCCGGAC A GUCCGCCG359CGGCGGAC CUGAUGAG GCCGUUAGGC CGAA IUCCGGCC178113GGACAGUC C GCCGAGGU360ACCUCGGC CUGAUGAG GCCGUUAGGC CGAA IACUGUCC178216CAGUCCGC C GAGGUGCU361AGCACCUC CUGAUGAG GCCGUUAGGC CGAA ICGGACUG178324CGAGGUGC U CGGUGGAG362CUCCACCG CUGAUGAG GCCGUUAGGC CGAA ICACCUCG178435GUGGAGUC A UGGCAGUG363CACUGCCA CUGAUGAG GCCGUUAGGC CGAA IACUCCAC178540GUCAUGGC A GUGCCCUU364AAGGGCAC CUGAUGAG GCCGUUAGGC CGAA ICCAUGAC178645GGCAGUGC C CUUUGUGG365CCACAAAG CUGAUGAG GCCGUUAGGC CGAA ICACUGCC178746GCAGUGCC C UUUGUGGA366UCCACAAA CUGAUGAG GCCGUUAGGC CGAA IGCACUGC178847CAGUGCCC U UUGUGGAA367UUCCACAA CUGAUGAG GCCGUUAGGC CGAA IGGCACUG178959UGGAAGAC U GGGACUUG368CAAGUCCC CUGAUGAG GCCGUUAGGC CGAA IUCUUCCA179065ACUGGGAC U UGGUGCAA369UUGCACCA CUGAUGAG GCCGUUAGGC CGAA IUCCCAGU179172CUUGGUGC A AACCCUGG370CCAGGGUU CUGAUGAG GCCGUUAGGC CGAA ICACCAAG179276GUGCAAAC C CUGGGAGA371UCUCCCAG CUGAUGAG GCCGUUAGGC CGAA IUUUGCAC179377UGCAAACC C UGGGAGAA372UUCUCCCA CUGAUGAG GCCGUUAGGC CGAA IGUUUGCA179478GCAAACCC U GGGAGAAG373CUUCUCCC CUGAUGAG GCCGUUAGGC CGAA IGGUUUGC179591GAAGGUGC C UAUGGAGA374UCUCCAUA CUGAUGAG GCCGUUAGGC CGAA ICACCUUC179692AAGGUGCC U AUGGAGAA375UUCUCCAU CUGAUGAG GCCGUUAGGC CGAA IGCACCUU1797105AGAAGUUC A ACUUGCUG376CAGCAAGU CUGAUGAG GCCGUUAGGC CGAA IAACUUCU1798108AGUUCAAC U UGCUGUGA377UCACAGCA CUGAUGAG GCCGUUAGGC CGAA IUUGAACU1799112CAACUUGC U GUGAAUAG378CUAUUCAC CUGAUGAG GCCGUUAGGC CGAA ICAAGUUG1800127AGAGUAAC U GAAGAAGC379GCUUCUUC CUGAUGAG GCCGUUAGGC CGAA IUUACUCU1801136GAAGAAGC A GUCGCAGU380ACUGCGAC CUGAUGAG GCCGUUAGGC CGAA ICUUCUUC1802142GCAGUCGC A GUGAAGAU381AUCUUCAC CUGAUGAG GCCGUUAGGC CGAA ICGACUGC1803169AAGCGUGC C GUAGACUG382CAGUCUAC CUGAUGAG GCCGUUAGGC CGAA ICACGCUU1804176CCGUAGAC U GUCCAGAA383UUCUGGAC CUGAUGAG GCCGUUAGGC CGAA IUCUACGG1805180AGACUGUC C AGAAAAUA384UAUUUUCU CUGAUGAG GCCGUUAGGC CGAA IACAGUCU1806181GACUGUCC A GAAAAUAU385AUAUUUUC CUGAUGAG GCCGUUAGGC CGAA IGACAGUC1807203AAGAGAUC U GUAUCAAU386AUUGAUAC CUGAUGAG GCCGUUAGGC CGAA IAUCUCUU1808209UCUGUAUC A AUAAAAUG387CAUUUUAU CUGAUGAG GCCGUUAGGC CGAA IAUACAGA1809219UAAAAUGC U AAAUCAUG388CAUGAUUU CUGAUGAG GCCGUUAGGC CGAA ICAUUUUA1810225GCUAAAUC A UGAAAAUG389CAUUUUCA CUGAUGAG GCCGUUAGGC CGAA IAUUUAGC1811245UAAAAUUC U AUGGUCAC390GUGACCAU CUGAUGAG GCCGUUAGGC CGAA IAAUUUUA1812252CUAUGGUC A CAGGAGAG391CUCUCCUG CUGAUGAG GCCGUUAGGC CGAA IACCAUAG1813254AUGGUCAC A GGAGAGAA392UUCUCUCC CUGAUGAG GCCGUUAGGC CGAA IUGACCAU1814266GAGAAGGC A AUAUCCAA393UUGGAUAU CUGAUGAG GCCGUUAGGC CGAA ICCUUCUC1815272GCAAUAUC C AAUAUUUA394UAAAUAUU CUGAUGAG GCCGUUAGGC CGAA IAUAUUGC1816273CAAUAUCC A AUAUUUAU395AUAAAUAU CUGAUGAG GCCGUUAGGC CGAA IGAUAUUG1817285UUUAUUUC U GGAGUACU396AGUACUCC CUGAUGAG GCCGUUAGGC CGAA IAAAUAAA1818293UGGAGUAC U GUAGUGGA397UCCACUAC CUGAUGAG GCCGUUAGGC CGAA IUACUCCA1819309AGGAGAGC U UUUUGACA398UGUCAAAA CUGAUGAG GCCGUUAGGC CGAA ICUCUCCU1820317UUUUUGAC A GAAUAGAG399CUCUAUUC CUGAUGAG GCCGUUAGGC CGAA IUCAAAAA1821327AAUAGAGC C AGACAUAG400CUAUGUCU CUGAUGAG GCCGUUAGGC CGAA ICUCUAUU1822328AUAGAGCC A GACAUAGG401CCUAUGUC CUGAUGAG GCCGUUAGGC CGAA IGCUCUAU1823332AGCCAGAC A UAGGCAUG402CAUGCCUA CUGAUGAG GCCGUUAGGC CGAA IUCUGGCU1824338ACAUAGGC A UGCCUGAA403UUCAGGCA CUGAUGAG GCCGUUAGGC CGAA ICCUAUGU1825342AGGCAUGC C UGAACCAG404CUGGUUCA CUGAUGAG GCCGUUAGGC CGAA ICAUGCCU1826343GGCAUGCC U GAACCAGA405UCUGGUUC CUGAUGAG GCCGUUAGGC CGAA IGCAUGCC1827348GCCUGAAC C AGAUGCUC406GAGCAUCU CUGAUGAG GCCGUUAGGC CGAA IUUCAGGC1828349CCUGAACC A GAUGCUCA407UGAGCAUC CUGAUGAG GCCGUUAGGC CGAA IGUUCAGG1829355CCAGAUGC U CAGAGAUU408AAUCUCUG CUGAUGAG GCCGUUAGGC CGAA ICAUCUGG1830357AGAUGCUC A GAGAUUCU409AGAAUCUC CUGAUGAG GCCGUUAGGC CGAA IAGCAUCU1831365AGAGAUUC U UCCAUCAA410UUGAUGGA CUGAUGAG GCCGUUAGGC CGAA IAAUCUCU1832368GAUUCUUC C AUCAACUC411GAGUUGAU CUGAUGAG GCCGUUAGGC CGAA IAAGAAUC1833369AUUCUUCC A UCAACUCA412UGAGUUGA CUGAUGAG GCCGUUAGGC CGAA IGAAGAAU1834372CUUCCAUC A ACUCAUGG413CCAUGAGU CUGAUGAG GCCGUUAGGC CGAA IAUGGAAG1835375CCAUCAAC U CAUGGCAG414CUGCCAUG CUGAUGAG GCCGUUAGGC CGAA IUUGAUGG1836377AUCAACUC A UGGCAGGG415CCCUGCCA CUGAUGAG GCCGUUAGGC CGAA IAGUUGAU1837382CUCAUGGC A GGGGUGGU416ACCACCCC CUGAUGAG GCCGUUAGGC CGAA ICCAUGAG1838396GGUUUAUC U GCAUGGUA417UACCAUGC CUGAUGAG GCCGUUAGGC CGAA IAUAAACC1839399UUAUCUGC A UGGUAUUG418CAAUACCA CUGAUGAG GCCGUUAGGC CGAA ICAGAUAA1840415GGAAUAAC U CACAGGGA419UCCCUGUG CUGAUGAG GCCGUUAGGC CGAA IUUAUUCC1841417AAUAACUC A CAGGGAUA420UAUCCCUG CUGAUGAG GCCGUUAGGC CGAA IAGUUAUU1842419UAACUCAC A GGGAUAUU421AAUAUCCC CUGAUGAG GCCGUUAGGC CGAA IUGAGUUA1843432UAUUAAAC C AGAAAAUC422GAUUUUCU CUGAUGAG GCCGUUAGGC CGAA IUUUAAUA1844433AUUAAACC A GAAAAUCU423AGAUUUUC CUGAUGAG GCCGUUAGGC CGAA IGUUUAAU1845441AGAAAAUC U UCUGUUGG424CCAACAGA CUGAUGAG GCCGUUAGGC CGAA IAUUUUCU1846444AAAUCUUC U GUUGGAUG425CAUCCAAC CUGAUGAG GCCGUUAGGC CGAA IAAGAUUU1847464GGGAUAAC C UCAAAAUC426GAUUUUGA CUGAUGAG GCCGUUAGGC CGAA IUUAUCCC1848465GGAUAACC U CAAAAUCU427AGAUUUUG CUGAUGAG GCCGUUAGGC CGAA IGUUAUCC1849467AUAACCUC A AAAUCUCA428UGAGAUUU CUGAUGAG GCCGUUAGGC CGAA IAGGUUAU1850473UCAAAAUC U CAGACUUU429AAAGUCUG CUGAUGAG GCCGUUAGGC CGAA IAUUUUGA1851475AAAAUCUC A GACUUUGG430CCAAAGUC CUGAUGAG GCCGUUAGGC CGAA IAGAUUUU1852479UCUCAGAC U UUGGCUUG431CAAGCCAA CUGAUGAG GCCGUUAGGC CGAA IUCUGAGA1853485ACUUUGGC U UGGCAACA432UGUUGCCA CUGAUGAG GCCGUUAGGC CGAA ICCAAAGU1854490GGCUUGGC A ACAGUAUU433AAUACUGU CUGAUGAG GCCGUUAGGC CGAA ICCAAGCC1855493UUGGCAAC A GUAUUUCG434CGAAAUAC CUGAUGAG GCCGUUAGGC CGAA IUUGCCAA1856530UGUUGAAC A AGAUGUGU435ACACAUCU CUGAUGAG GCCGUUAGGC CGAA IUUCAACA1857544UGUGGUAC U UUACCAUA436UAUGGUAA CUGAUGAG GCCGUUAGGC CGAA IUACCACA1858549UACUUUAC C AUAUGUUG437CAACAUAU CUGAUGAG GCCGUUAGGC CGAA IUAAAGUA1859550ACUUUACC A UAUGUUGC438GCAACAUA CUGAUGAG GCCGUUAGGC CGAA IGUAAAGU1860559UAUGUUGC U CCAGAACU439AGUUCUGG CUGAUGAG GCCGUUAGGC CGAA ICAACAUA1861561UGUUGCUC C AGAACUUC440GAAGUUCU CUGAUGAG GCCGUUAGGC CGAA IAGCAACA1862562GUUGCUCC A GAACUUCU441AGAAGUUC CUGAUGAG GCCGUUAGGC CGAA IGAGCAAC1863567UCCAGAAC U UCUGAAGA442UCUUCAGA CUGAUGAG GCCGUUAGGC CGAA IUUCUGGA1864570AGAACUUC U GAAGAGAA443UUCUCUUC CUGAUGAG GCCGUUAGGC CGAA IAAGUUCU1865588AGAAUUUC A UGCAGAAC444GUUCUGCA CUGAUGAG GCCGUUAGGC CGAA IAAAUUCU1866592UUUCAUGC A GAACCAGU445ACUGGUUC CUGAUGAG GCCGUUAGGC CGAA ICAUGAAA1867597UGCAGAAC C AGUUGAUG446CAUCAACU CUGAUGAG GCCGUUAGGC CGAA IUUCUGCA1868598GCAGAACC A GUUGAUGU447ACAUCAAC CUGAUGAG GCCGUUAGGC CGAA IGUUCUGC1869613GUUUGGUC C UGUGGAAU448AUUCCACA CUGAUGAG GCCGUUAGGC CGAA IACCAAAC1870614UUUGGUCC U GUGGAAUA449UAUUCCAC CUGAUGAG GCCGUUAGGC CGAA IGACCAAA1871627AAUAGUAC U UACUGCAA450UUGCAGUA CUGAUGAG GCCGUUAGGC CGAA IUACUAUU1872631GUACUUAC U GCAAUGCU451AGCAUUGC CUGAUGAG GCCGUUAGGC CGAA IUAAGUAC1873634CUUACUGC A AUGCUCGC452GCGAGCAU CUGAUGAG GCCGUUAGGC CGAA ICAGUAAG1874639UGCAAUGC U CGCUGGAG453CUCCAGCG CUGAUGAG GCCGUUAGGC CGAA ICAUUGCA1875643AUGCUCGC U GGAGAAUU454AAUUCUCC CUGAUGAG GCCGUUAGGC CGAA ICGAGCAU1876654AGAAUUGC C AUGGGACC455GGUCCCAU CUGAUGAG GCCGUUAGGC CGAA ICAAUUCU1877655GAAUUGCC A UGGGACCA456UGGUCCCA CUGAUGAG GCCGUUAGGC CGAA IGCAAUUC1878662CAUGGGAC C AACCCAGU457ACUGGGUU CUGAUGAG GCCGUUAGGC CGAA IUCCCAUG1879663AUGGGACC A ACCCAGUG458CACUGGGU CUGAUGAG GCCGUUAGGC CGAA IGUCCCAU1880666GGACCAAC C CAGUGACA459UGUCACUG CUGAUGAG GCCGUUAGGC CGAA IUUGGUCC1881667GACCAACC C AGUGACAG460CUGUCACU CUGAUGAG GCCGUUAGGC CGAA IGUUGGUC1882668ACCAACCC A GUGACAGC461GCUGUCAC CUGAUGAG GCCGUUAGGC CGAA IGGUUGGU1883674CCAGUGAC A GCUGUCAG462CUGACAGC CUGAUGAG GCCGUUAGGC CGAA IUCACUGG1884677GUGACAGC U GACAGGAG463CUCCUGAC CUGAUGAG GCCGUUAGGC CGAA ICUGUCAC1885681CAGCUGUC A GGAGUAUU464AAUACUCC CUGAUGAG GCCGUUAGGC CGAA IACAGCUG1886691GAGUAUUC U GACUGGAA465UUCCAGUC CUGAUGAG GCCGUUAGGC CGAA IAAUACUC1887695AUUCUGAC U GGAAAGAA466UUCUUUCC CUGAUGAG GCCGUUAGGC CGAA IUCAGAAU1888712AAAAAAAC A UACCUCAA467UUGAGGUA CUGAUGAG GCCGUUAGGC CGAA IUUUUUUU1889716AAACAUAC C UCAACCCU468AGGGUUGA CUGAUGAG GCCGUUAGGC CGAA IUAUGUUU1890717AACAUACC U CAACCCUU469AAGGGUUG CUGAUGAG GCCGUUAGGC CGAA IGUAUGUU1891719CAUACCUC A ACCCUUGG470CCAAGGGU CUGAUGAG GCCGUUAGGC CGAA IAGGUAUG1892722ACCUCAAC C CUUGGAAA471UUUCCAAG CUGAUGAG GCCGUUAGGC CGAA IUUGAGGU1893723CCUCAACC C UUGGAAAA472UUUUCCAA CUGAUGAG GCCGUUAGGC CGAA IGUUGAGG1894724CUCAACCC U UGGAAAAA473UUUUUCCA CUGAUGAG GCCGUUAGGC CGAA IGGUUGAG1895742AUCGAUUC U GCUCCUCU474AGAGGAGC CUGAUGAG GCCGUUAGGC CGAA IAAUCGAU1896745GAUUCUGC U CCUCUAGC475GCUAGAGG CUGAUGAG GCCGUUAGGC CGAA ICAGAAUC1897747UUCUGCUC C UCUAGCUC476GAGCUAGA CUGAUGAG GCCGUUAGGC CGAA IAGCAGAA1898748UCUGCUCC U CUAGCUCU477AGAGCUAG CUGAUGAG GCCGUUAGGC CGAA IGAGCAGA1899750UGCUCCUC U AGCUCUGC478GCAGAGCU CUGAUGAG GCCGUUAGGC CGAA IAGGAGCA1900754CCUCUAGC U CUGCUGCA479UGCAGCAG CUGAUGAG GCCGUUAGGC CGAA ICUAGAGG1901756UCUAGCUC U GCUGCAUA480UAUGCAGC CUGAUGAG GCCGUUAGGC CGAA IAGCUAGA1902759AGCUCUGC U GCAUAAAA481UUUUAUGC CUGAUGAG GCCGUUAGGC CGAA ICAGAGCU1903762UCUGCUGC A UAAAAUCU482AGAUUUUA CUGAUGAG GCCGUUAGGC CGAA ICAGCAGA1904770AUAAAAUC U UAGUUGAG483CUCAACUA CUGAUGAG GCCGUUAGGC CGAA IAUUUUAU1905783UGAGAAUC C AUCAGCAA484UUGCUGAU CUGAUGAG GCCGUUAGGC CGAA IAUUCUCA1906784GAGAAUCC A UCAGCAAG485CUUGCUGA CUGAUGAG GCCGUUAGGC CGAA IGAUUCUC1907787AAUCCAUC A GCAAGAAU486AUUCUUGC CUGAUGAG GCCGUUAGGC CGAA IAUGGAUU1908790CCAUCAGC A AGAAUUAC487GUAAUUCU CUGAUGAG GCCGUUAGGC CGAA ICUGAUGG1909799AGAAUUAC C AUUCCAGA488UCUGGAAU CUGAUGAG GCCGUUAGGC CGAA IUAAUUCU1910800GAAUUACC A UUCCAGAC489GUCUGGAA CUGAUGAG GCCGUUAGGC CGAA IGUAAUUC1911804UACCAUUC C AGACAUCA490UGAUGUCU CUGAUGAG GCCGUUAGGC CGAA IAAUGGUA1912805ACCAUUCC A GACAUCAA491UUGAUGUC CUGAUGAG GCCGUUAGGC CGAA IGAAUGGU1913809UUCCAGAC A UCAAAAAA492UUUUUUGA CUGAUGAG GCCGUUAGGC CGAA IUCUGGAA1914812CAGACAUC A AAAAAGAU493AUCUUUUU CUGAUGAG GCCGUUAGGC CGAA IAUGUCUG1915830GAUGGUAC A ACAAACCC494GGGUUUGU CUGAUGAG GCCGUUAGGC CGAA IUACCAUC1916833GGUACAAC A AACCCCUC495GAGGGGUU CUGAUGAG GCCGUUAGGC CGAA IUUGUACC1917837CAACAAAC C CCUCAAGA496UCUUGAGG CUGAUGAG GCCGUUAGGC CGAA IUUUGUUG1918838AACAAACC C CUCAAGAA497UUCUUGAG CUGAUGAG GCCGUUAGGC CGAA IGUUUGUU1919839ACAAACCC C UCAAGAAA498UUUCUUGA CUGAUGAG GCCGUUAGGC CGAA IGGUUUGU1920840CAAACCCC U CAAGAAAG499CUUUCUUG CUGAUGAG GCCGUUAGGC CGAA IGGGUUUG1921842AACCCCUC A AGAAAGGG500CCCUUUCU CUGAUGAG GCCGUUAGGC CGAA IAGGGGUU1922853AAAGGGGC A AAAAGGCC501GGCCUUUU CUGAUGAG GCCGUUAGGC CGAA ICCCCUUU1923861AAAAAGGC C CCGAGUCA502UGACUCGG CUGAUGAG GCCGUUAGGC CGAA ICCUUUUU1924862AAAAGGCC C CGAGUCAC503GUGACUCG CUGAUGAG GCCGUUAGGC CGAA IGCCUUUU1925863AAAGGCCC C GAGUCACU504AGUGACUC CUGAUGAG GCCGUUAGGC CGAA IGGCCUUU1926869CCCGAGUC A CUUCAGGU505ACCUGAAG CUGAUGAG GCCGUUAGGC CGAA IACUCGGG1927871CGAGUCAC U UCAGGUGG506CCACCUGA CUGAUGAG GCCGUUAGGC CGAA IUGACUCG1928874GUCACUUC A GGUGGUGU507ACACCACC CUGAUGAG GCCGUUAGGC CGAA IAAGUGAC1929886GGUGUGUC A GAGUCUCC508GGAGACUC CUGAUGAG GCCGUUAGGC CGAA IACACACC1930892UCAGAGUC U CCCAGUGG509CCACUGGG CUGAUGAG GCCGUUAGGC CGAA IACUCUGA1931894AGAGUCUC C CAGUGGAU510AUCCACUG CUGAUGAG GCCGUUAGGC CGAA IAGACUCU1932895GAGUCUCC C AGUGGAUU511AAUCCACU CUGAUGAG GCCGUUAGGC CGAA IGAGACUC1933896AGUCUCCC A GUGGAUUU512AAAUCCAC CUGAUGAG GCCGUUAGGC CGAA IGGAGACU1934907GGAUUUUC U AAGCACAU513AUGUGCUU CUGAUGAG GCCGUUAGGC CGAA IAAAAUCC1935912UUCUAAGC A CAUUCAAU514AUUGAAUG CUGAUGAG GCCGUUAGGC CGAA ICUUAGAA1936914CUAAGCAC A UUCAAUCC515GGAUUGAA CUGAUGAG GCCGUUAGGC CGAA IUGCUUAG1937918GCACAUUC A AUCCAAUU516AAUUGGAU CUGAUGAG GCCGUUAGGC CGAA IAAUGUGC1938922AUUCAAUC C AAUUUGGA517UCCAAAUU CUGAUGAG GCCGUUAGGC CGAA IAUUGAAU1939923UUCAAUCC A AUUUGGAC518GUCCAAAU CUGAUGAG GCCGUUAGGC CGAA IGAUUGAA1940932AUUUGGAC U UCUCUCCA519UGGAGAGA CUGAUGAG GCCGUUAGGC CGAA IUCCAAAU1941935UGGACUUC U CUCCAGUA520UACUGGAG CUGAUGAG GCCGUUAGGC CGAA IAAGUCCA1942937GACUUCUC U CCAGUAAA521UUUACUGG CUGAUGAG GCCGUUAGGC CGAA IAGAAGUC1943939CUUCUCUC C AGUAAACA522UGUUUACU CUGAUGAG GCCGUUAGGC CGAA IAGAGAAG1944940UUCUCUCC A GUAAACAG523CUGUUUAC CUGAUGAG GCCGUUAGGC CGAA IGAGAGAA1945947CAGUAAAC A GUGCUUCU524AGAAGCAC CUGAUGAG GCCGUUAGGC CGAA IUUUACUG1946952AACAGUGC U UCUAGUGA525UCACUAGA CUGAUGAG GCCGUUAGGC CGAA ICACUGUU1947955AGUGCUUC U AGUGAAGA526UCUUCACU CUGAUGAG GCCGUUAGGC CGAA IAAGCACU1948977UGAAGUAC U CCAGUUCU527AGAACUGG CUGAUGAG GCCGUUAGGC CGAA IUACUUCA1949979AAGUACUC C AGUUCUCA528UGAGAACU CUGAUGAG GCCGUUAGGC CGAA IAGUACUU1950980AGUACUCC A GUUCUCAG529CUGAGAAC CUGAUGAG GCCGUUAGGC CGAA IGAGUACU1951985UCCAGUUC U CAGCCAGA530UCUGGCUG CUGAUGAG GCCGUUAGGC CGAA IAACUGGA1952987CAGUUCUC A GCCAGAAC531GUUCUGGC CUGAUGAG GCCGUUAGGC CGAA IAGAACUG1953990UUCUCAGC C AGAACCCC532GGGGUUCU CUGAUGAG GCCGUUAGGC CGAA ICUGAGAA1954991UCUCAGCC A GAACCCCG533CGGGGUUC CUGAUGAG GCCGUUAGGC CGAA IGCUGAGA1955996GCCAGAAC C CCGCACAG534CUGUGCGG CUGAUGAG GCCGUUAGGC CGAA IUUCUGGC1956997CCAGAACC C CGCACAGG535CCUGUGCG CUGAUGAG GCCGUUAGGC CGAA IGUUCUGG1957998CAGAACCC C GCACAGGU536ACCUGUGC CUGAUGAG GCCGUUAGGC CGAA IGGUUCUG19581001AACCCCGC A CAGGUCUU537AAGACCUG CUGAUGAG GCCGUUAGGC CGAA ICGGGGUU19591003CCCCGCAC A GGUCUUUC538GAAAGACC CUGAUGAG GCCGUUAGGC CGAA IUGCGGGG19601008CACAGGUC U UUCCUUAU539AUAAGGAA CUGAUGAG GCCGUUAGGC CGAA IACCUGUG19611012GGUCUUUC C UUAUGGGA540UCCCAUAA CUGAUGAG GCCGUUAGGC CGAA IAAAGACC19621013GUCUUUCC U UAUGGGAU541AUCCCAUA CUGAUGAG GCCGUUAGGC CGAA IGAAAGAC19631024UGGGAUAC C AGCCCCUC542GAGGGGCU CUGAUGAG GCCGUUAGGC CGAA IUAUCCCA19641025GGGAUACC A GCCCCUCA543UGAGGGGC CUGAUGAG GCCGUUAGGC CGAA IGUAUCCC19651028AUACCAGC C CCUCAUAC544GUAUGAGG CUGAUGAG GCCGUUAGGC CGAA ICUGGUAU19661029UACCAGCC C CUCAUACA545UGUAUGAG CUGAUGAG GCCGUUAGGC CGAA IGCUGGUA19671030ACCAGCCC C UCAUACAU546AUGUAUGA CUGAUGAG GCCGUUAGGC CGAA IGGCUGGU19681031CCAGCCCC U CAUACAUU547AAUGUAUG CUGAUGAG GCCGUUAGGC CGAA IGGGCUGG19691033AGCCCCUC A UACAUUGA548UCAAUGUA CUGAUGAG GCCGUUAGGC CGAA IAGGGGCU19701037CCUCAUAC A UUGAUAAA549UUUAUCAA CUGAUGAG GCCGUUAGGC CGAA IUAUGAGG19711053AUUGGUAC A AGGGAUCA550UGAUCCCU CUGAUGAG GCCGUUAGGC CGAA IUACCAAU19721061AAGGGAUC A GCUUUUCC551GGAAAAGC CUGAUGAG GCCGUUAGGC CGAA IAUCCCUU19731064GGAUCAGC U UUUCCCAG552CUGGGAAA CUGAUGAG GCCGUUAGGC CGAA ICUGAUCC19741069AGCUUUUC C CAGCCCAC553GUGGGCUG CUGAUGAG GCCGUUAGGC CGAA IAAAAGCU19751070GCUUUUCC C AGCCCACA554UGUGGGCU CUGAUGAG GCCGUUAGGC CGAA IGAAAAGC19761071CUUUUCCC A GCCCACAU555AUGUGGGC CUGAUGAG GCCGUUAGGC CGAA IGGAAAAG19771074UUCCCAGC C CACAUGUC556GACAUGUG CUGAUGAG GCCGUUAGGC CGAA ICUGGGAA19781075UCCCAGCC C ACAUGUCC557GGACAUGU CUGAUGAG GCCGUUAGGC CGAA IGCUGGGA19791076CCCAGCCC A CAUGUCCU558AGGACAUG CUGAUGAG GCCGUUAGGC CGAA IGGCUGGG19801078CAGCCCAC A UGUCCUGA559UCAGGACA CUGAUGAG GCCGUUAGGC CGAA IUGGGCUG19811083CACAUGUC C UGAUCAUA560UAUGAUCA CUGAUGAG GCCGUUAGGC CGAA IACAUGUG19821084ACAUGUCC U GAUCAUAU561AUAUGAUC CUGAUGAG GCCGUUAGGC CGAA IGACAUGU19831089UCCUGAUC A UAUGCUUU562AAAGCAUA CUGAUGAG GCCGUUAGGC CGAA IAUCAGGA19841095UCAUAUGC U UUUGAAUA563UAUUCAAA CUGAUGAG GCCGUUAGGC CGAA ICAUAUGA19851107GAAUAGUC A GUUACUUG564CAAGUAAC CUGAUGAG GCCGUUAGGC CGAA IACUAUUC19861113UCAGUUAC U UGGCACCC565GGGUGCCA CUGAUGAG GCCGUUAGGC CGAA IUAACUGA19871118UACUUGGC A CCCCAGGA566UCCUGGGG CUGAUGAG GCCGUUAGGC CGAA ICCAAGUA19881120CUUGGCAC C CCAGGAUC567GAUCCUGG CUGAUGAG GCCGUUAGGC CGAA IUGCCAAG19891121UUGGCACC C CAGGAUCC568GGAUCCUG CUGAUGAG GCCGUUAGGC CGAA IGUGCCAA19901122UGGCACCC C AGGAUCCU569AGGAUCCU CUGAUGAG GCCGUUAGGC CGAA IGGUGCCA19911123GGCACCCC A GGAUCCUC570GAGGAUCC CUGAUGAG GCCGUUAGGC CGAA IGGGUGCC19921129CCAGGAUC C UCACAGAA571UUCUGUGA CUGAUGAG GCCGUUAGGC CGAA IAUCCUGG19931130CAGGAUCC U CACAGAAC572GUUCUGUG CUGAUGAG GCCGUUAGGC CGAA IGAUCCUG19941132GGAUCCUC A CAGAACCC573GGGUUCUG CUGAUGAG GCCGUUAGGC CGAA IAGGAUCC19951134AUCCUCAC A GAACCCCU574AGGGGUUC CUGAUGAG GCCGUUAGGC CGAA IUGAGGAU19961139CACAGAAC C CCUGGCAG575CUGCCAGG CUGAUGAG GCCGUUAGGC CGAA IUUCUGUG19971140ACAGAACC C CUGGCAGC576GCUGCCAG CUGAUGAG GCCGUUAGGC CGAA IGUUCUGU19981141CAGAACCC C UGGCAGCG577CGCUGCCA CUGAUGAG GCCGUUAGGC CGAA IGGUUCUG19991142AGAACCCC U GGCAGCGG578CCGCUGCC CUGAUGAG GCCGUUAGGC CGAA IGGGUUCU20001146CCCCUGGC A GCGGUUGG579CCAACCGC CUGAUGAG GCCGUUAGGC CGAA ICCAGGGG20011157GGUUGGUC A AAAGAAUG580CAUUCUUU CUGAUGAG GCCGUUAGGC CGAA IACCAACC20021168AGAAUGAC A CGAUUCUU581AAGAAUCG CUGAUGAG GCCGUUAGGC CGAA IUCAUUCU20031175CACGAUUC U UUACCAAA582UUUGGUAA CUGAUGAG GCCGUUAGGC CGAA IAAUCGUG20041180UUCUUUAC C AAAUUGGA583UCCAAUUU CUGAUGAG GCCGUUAGGC CGAA IUAAAGAA20051181UCUUUACC A AAUUGGAU584AUCCAAUU CUGAUGAG GCCGUUAGGC CGAA IGUAAAGA20061192UUGGAUGC A GACAAAUC585GAUUUGUC CUGAUGAG GCCGUUAGGC CGAA ICAUCCAA20071196AUGCAGAC A AAUCUUAU586AUAAGAUU CUGAUGAG GCCGUUAGGC CGAA IUCUGCAU20081201GACAAAUC U UAUCAAUG587CAUUGAUA CUGAUGAG GCCGUUAGGC CGAA IAUUUGUC20091206AUCUUAUC A AUGCCUGA588UCAGGCAU CUGAUGAG GCCGUUAGGC CGAA IAUAAGAU20101211AUCAAUGC C UGAAAGAG589CUCUUUCA CUGAUGAG GCCGUUAGGC CGAA ICAUUGAU20111212UCAAUGCC U GAAAGAGA590UCUCUUUC CUGAUGAG GCCGUUAGGC CGAA IGCAUUGA20121222AAAGAGAC U UGUGAGAA591UUCUCACA CUGAUGAG GCCGUUAGGC CGAA IUCUCUUU20131238AGUUGGGC U AUCAAUGG592CCAUUGAU CUGAUGAG GCCGUUAGGC CGAA ICCCAACU20141242GGGCUAUC A AUGGAAGA593UCUUCCAU CUGAUGAG GCCGUUAGGC CGAA IAUAGCCC20151266UAUGAAUC A GGUUACUA594UAGUAACC CUGAUGAG GCCGUUAGGC CGAA IAUUCAUA20161273CAGGUUAC U AUAUCAAC595GUUGAUAU CUGAUGAG GCCGUUAGGC CGAA IUAACCUG20171279ACUAUAUC A ACAACUGA596UCAGUUGA CUGAUGAG GCCGUUAGGC CGAA IAUAUAGU20181282AUAUCAAC A ACUGAUAG597CUAUCAGU CUGAUGAG GCCGUUAGGC CGAA IUUGAUAU20191285UCAACAAC U GAUAGGAG598CUCCUAUC CUGAUGAG GCCGUUAGGC CGAA IUUGUUGA20201298GGAGAAAC A AUAAACUC599GAGUUUAU CUGAUGAG GCCGUUAGGC CGAA IUUUCUCC20211305CAAUAAAC U CAUUUUCA600UGAAAAUG CUGAUGAG GCCGUUAGGC CGAA IUUUAUUG20221307AUAAACUC A UUUUCAAA601UUUGAAAA CUGAUGAG GCCGUUAGGC CGAA IAGUUUAU20231313UCAUUUUC A AAGUGAAU602AUUCACUU CUGAUGAG GCCGUUAGGC CGAA IAAAAUGA20241355UGGUUGAC U UCCGGCUU603AAGCCGGA CUGAUGAG GCCGUUAGGC CGAA IUCAACCA20251358UUGACUUC C GGCUUUCU604AGAAAGCC CUGAUGAG GCCGUUAGGC CGAA IAAGUCAA20261362CUUCCGGC U UUCUAAGG605CCUUAGAA CUGAUGAG GCCGUUAGGC CGAA ICCGGAAG20271366CGGCUUUC U AAGGGUGA606UCACCCUU CUGAUGAG GCCGUUAGGC CGAA IAAAGCCG20281388UGGAGUUC A AGAGACAC607GUGUCUCU CUGAUGAG GCCGUUAGGC CGAA IAACUCCA20291395CAAGAGAC A CUUCCUGA608UCAGGAAG CUGAUGAG GCCGUUAGGC CGAA IUCUCUUG20301397AGAGACAC U UCCUGAAG609CUUCAGGA CUGAUGAG GCCGUUAGGC CGAA IUGUCUCU20311400GACACUUC C UGAAGAUU610AAUCUUCA CUGAUGAG GCCGUUAGGC CGAA IAAGUGUC20321401ACACUUCC U GAAGAUUA611UAAUCUUC CUGAUGAG GCCGUUAGGC CGAA IGAAGUGU20331419AGGGAAGC U GAUUGAUA612UAUCAAUC CUGAUGAG GCCGUUAGGC CGAA ICUUCCCU20341436UUGUGAGC A GCCAGAAG613CUUCUGGC CUGAUGAG GCCGUUAGGC CGAA ICUCACAA20351439UGAGCAGC C AGAAGGUU614AACCUUCU CUGAUGAG GCCGUUAGGC CGAA ICUGCUCA20361440GAGCAGCC A GAAGGUUU615AAACCUUC CUGAUGAG GCCGUUAGGC CGAA IGCUGCUC20371452GGUUUGGC U UCCUGCCA616UGGCAGGA CUGAUGAG GCCGUUAGGC CGAA ICCAAACC20381455UUGGCUUC C UGCCACAU617AUGUGGCA CUGAUGAG GCCGUUAGGC CGAA IAAGCCAA20391456UGGCUUCC U GCCACAUG618CAUGUGGC CUGAUGAG GCCGUUAGGC CGAA IGAAGCCA20401459CUUCCUGC C ACAUGAUC619GAUCAUGU CUGAUGAG GCCGUUAGGC CGAA ICAGGAAG20411460UUCCUGCC A CAUGAUCG620CGAUCAUG CUGAUGAG GCCGUUAGGC CGAA IGCAGGAA20421462CCUGCCAC A UGAUCGGA621UCCGAUCA CUGAUGAG GCCGUUAGGC CGAA IUGGCAGG20431472GAUCGGAC C AUCGGCUC622GAGCCGAU CUGAUGAG GCCGUUAGGC CGAA IUCCGAUC20441473AUCGGACC A UCGGCUCU623AGAGCCGA CUGAUGAG GCCGUUAGGC CGAA IGUCCGAU20451479CCAUCGGC U CUGGGGAA624UUCCCCAG CUGAUGAG GCCGUUAGGC CGAA ICCGAUGG20461481AUCGGCUC U GGGGAAUC625GAUUCCCC CUGAUGAG GCCGUUAGGC CGAA IAGCCGAU20471490GGGGAAUC C UGGUGAAU626AUUCACCA CUGAUGAG GCCGUUAGGC CGAA IAUUCCCC20481491GGGAAUCC U GGUGAAUA627UAUUCACC CUGAUGAG GCCGUUAGGC CGAA IGAUUCCC20491506UAUAGUGC U GCUAUGUU628AACAUAGC CUGAUGAG GCCGUUAGGC CGAA ICACUAUA20501509AGUGCUGC U AUGUUGAC629GUCAACAU CUGAUGAG GCCGUUAGGC CGAA ICAGCACU20511518AUGUUGAC A UUAUUCUU630AAGAAUAA CUGAUGAG GCCGUUAGGC CGAA IUCAACAU20521525CAUUAUUC U UCCUAGAG631CUCUAGGA CUGAUGAG GCCGUUAGGC CGAA IAAUAAUG20531528UAUUCUUC C UAGAGAAG632CUUCUCUA CUGAUGAG GCCGUUAGGC CGAA IAAGAAUA20541529AUUCUUCC U AGAGAAGA633UCUUCUCU CUGAUGAG GCCGUUAGGC CGAA IGAAGAAU20551543AGAUUAUC C UGUCCUGC634GCAGGACA CUGAUGAG GCCGUUAGGC CGAA IAUAAUCU20561544GAUUAUCC U GUCCUGCA635UGCAGGAC CUGAUGAG GCCGUUAGGC CGAA IGAUAAUC20571548AUCCUGUC C UGCAAACU636AGUUUGCA CUGAUGAG GCCGUUAGGC CGAA IACAGGAU20581549UCCUGUCC U GCAAACUG637CAGUUUGC CUGAUGAG GCCGUUAGGC CGAA IGACAGGA20591552UGUCCUGC A AACUGCAA638UUGCAGUU CUGAUGAG GCCGUUAGGC CGAA ICAGGACA20601556CUGCAAAC U GCAAAUAG639CUAUUUGC CUGAUGAG GCCGUUAGGC CGAA IUUUGCAG20611559CAAACUGC A AAUAGUAG640CUACUAUU CUGAUGAG GCCGUUAGGC CGAA ICAGUUUG20621571AGUAGUUC C UGAAGUGU641ACACUUCA CUGAUGAG GCCGUUAGGC CGAA IAACUACU20631572GUAGUUCC U GAAGUGUU642AACACUUC CUGAUGAG GCCGUUAGGC CGAA IGAACUAC20641582AAGUGUUC A CUUCCCUG643CAGGGAAG CUGAUGAG GCCGUUAGGC CGAA IAACACUU20651584GUGUUCAC U UCCCUGUU644AACAGGGA CUGAUGAG GCCGUUAGGC CGAA IUGAACAC20661587UUCACUUC C CUGUUUAU645AUAAACAG CUGAUGAG GCCGUUAGGC CGAA IAAGUGAA20671588UCACUUCC C UGUUUAUC646GAUAAACA CUGAUGAG GCCGUUAGGC CGAA IGAAGUGA20681589CACUUCCC U GUUUAUCC647GGAUAAAC CUGAUGAG GCCGUUAGGC CGAA IGGAAGUG20691597UGUUUAUC C AAACAUCU648AGAUGUUU CUGAUGAG GCCGUUAGGC CGAA IAUAAACA20701598GUUUAUCC A AACAUCUU649AAGAUGUU CUGAUGAG GCCGUUAGGC CGAA IGAUAAAC20711602AUCCAAAC A UCUUCCAA650UUGGAAGA CUGAUGAG GCCGUUAGGC CGAA IUUUGGAU20721605CAAACAUC U UCCAAUUU651AAAUUGGA CUGAUGAG GCCGUUAGGC CGAA IAUGUUUG20731608ACAUCUUC C AAUUUAUU652AAUAAAUU CUGAUGAG GCCGUUAGGC CGAA IAAGAUGU20741609CAUCUUCC A AUUUAUUU653AAAUAAAU CUGAUGAG GCCGUUAGGC CGAA IGAAGAUG20751630UGUUCGGC A UACAAAUA654UAUUUGUA CUGAUGAG GCCGUUAGGC CGAA ICCGAACA20761634CGGCAUAC A AAUAAUAC655GUAUUAUU CUGAUGAG GCCGUUAGGC CGAA IUAUGCCG20771643AAUAAUAC C UAUAUCUU656AAGAUAUA CUGAUGAG GCCGUUAGGC CGAA IUAUUAUU20781644AUAAUACC U AUAUCUUA657UAAGAUAU CUGAUGAG GCCGUUAGGC CGAA IGUAUUAU20791650CCUAUAUC U UAAUUGUA658UACAAUUA CUGAUGAG GCCGUUAGGC CGAA IAUAUAGG20801662UUGUAAGC A AAACUUUG659CAAAGUUU CUGAUGAG GCCGUUAGGC CGAA ICUUACAA20811667AGCAAAAC U UUGGGGAA660UUCCCCAA CUGAUGAG GCCGUUAGGC CGAA IUUUUGCU20821692UAGAAUUC A UUUGAUUA661UAAUCAAA CUGAUGAG GCCGUUAGGC CGAA IAAUUCUA20831705AUUAUUUC U UCAUGUGU662ACACAUGA CUGAUGAG GCCGUUAGGC CGAA IAAAUAAU20841708AUUUCUUC A UGUGUGUU663AACACACA CUGAUGAG GCCGUUAGGC CGAA IAAGAAAU20851724UUAGUAUC U GAAUUUGA664UCAAAUUC CUGAUGAG GCCGUUAGGC CGAA IAUACUAA20861736UUUGAAAC U CAUCUGGU665ACCAGAUG CUGAUGAG GCCGUUAGGC CGAA IUUUCAAA20871738UGAAACUC A UCUGGUGG666CCACCAGA CUGAUGAG GCCGUUAGGC CGAA IAGUUUCA20881741AACUCAUC U GGUGGAAA667UUUCCACC CUGAUGAG GCCGUUAGGC CGAA IAUGAGUU20891751GUGGAAAC C AAGUUUCA668UGAAACUU CUGAUGAG GCCGUUAGGC CGAA IUUUCCAC20901752UGGAAACC A AGUUUCAG669CUGAAACU CUGAUGAG GCCGUUAGGC CGAA IGUUUCCA20911759CAAGUUUC A GGGGACAU670AUGUCCCC CUGAUGAG GCCGUUAGGC CGAA IAAACUUG20921766CAGGGGAC A UGAGUUUU671AAAACUCA CUGAUGAG GCCGUUAGGC CGAA IUCCCCUG20931776GAGUUUUC C AGCUUUUA672UAAAAGCU CUGAUGAG GCCGUUAGGC CGAA IAAAACUC20941777AGUUUUCC A GCUUUUAU673AUAAAAGC CUGAUGAG GCCGUUAGGC CGAA IGAAAACU20951780UUUCCAGC U UUUAUACA674UGUAUAAA CUGAUGAG GCCGUUAGGC CGAA ICUGGAAA20961788UUUUAUAC A CACGUAUC675GAUACGUG CUGAUGAG GCCGUUAGGC CGAA IUAUAAAA20971790UUAUACAC A CGUAUCUC676GAGAUACG CUGAUGAG GCCGUUAGGC CGAA IUGUAUAA20981797CACGUAUC U CAUUUUUA677UAAAAAUG CUGAUGAG GCCGUUAGGC CGAA IAUACGUG20991799CGUAUCUC A UUUUUAUC678GAUAAAAA CUGAUGAG GCCGUUAGGC CGAA IAGAUACG21001808UUUUUAUC A AAACAUUU679AAAUGUUU CUGAUGAG GCCGUUAGGC CGAA IAUAAAAA21011813AUCAAAAC A UUUUGUUU680AAACAAAA CUGAUGAG GCCGUUAGGC CGAA IUUUUGAU2102Input Sequence = AF016582 Cut Site = CH/. Stem Length = 8. Core Sequence = CUGAUGAG GCCGUUAGGC CGAA AF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

[0175] Underlined region can be any X sequence or linker as previously defined herein.

[0176] I=Inosine

[0177] 5

5TABLE VHuman Chk1 G-Cleaver Ribozyme and Substrate SequencePosSubstrateSeq IDRibozymeRz Seq ID14GACAGUCC G CCGAGGUG681CACCUCGG UGAUGGCAUGCACUAUGCGCG GGACUGUC210317AGUCCGCC G AGGUGCUC682GAGCACCU UGAUGGCAUGCACUAUGCGCG GGCGGACU210422GCCGAGGU G CUCGGUGG683CCACCGAG UGAUGGCAUGCACUAUGCGCG ACCUCGGC210543AUGGCAGU G CCCUUUGU684ACAAAGGG UGAUGGCAUGCACUAUGCGCG ACUGCCAU210650UGCCCUUU G UGGAAGAC685GUCUUCCA UGAUGGCAUGCACUAUGCGCG AAAGGGCA210770GACUUGGU G CAAACCCU686AGGGUUUG UGAUGGCAUGCACUAUGCGCG ACCAAGUC210889GAGAAGGU G CCUAUGGA687UCCAUAGG UGAUGGCAUGCACUAUGCGCG ACCUUCUC2109110UUCAACUU G CUGUGAAU688AUUCACAG UGAUGGCAUGCACUAUGCGCG AAGUUGAA2110113AACUUGCU G UGAAUAGA689UCUAUUCA UGAUGGCAUGCACUAUGCGCG AGCAAGUU2111115CUUGCUGU G AAUAGAGU690ACUCUAUU UGAUGGCAUGCACUAUGCGCG ACAGCAAG2112128GAGUAACU G AAGAAGCA691UGCUUCUU UGAUGGCAUGCACUAUGCGCG AGUUACUC2113140AAGCAGUC G CAGUGAAG692CUUCACUG UGAUGGCAUGCACUAUGCGCG GACUGCUU2114145GUCGCAGU G AAGAUUGU693ACAAUCUU UGAUGGCAUGCACUAUGCGCG ACUGCGAC2115152UGAAGAUU G UAGAUAUG694CAUAUCUA UGAUGGCAUGCACUAUGCGCG AAUCUUCA2116160GUAGAUAU G AAGCGUGC695GCACGCUU UGAUGGCAUGCACUAUGCGCG AUAUCUAC2117167UGAAGCGU G CCGUAGAC696GUCUACGG UGAUGGCAUGCACUAUGCGCG ACGCUUCA2118177CGUAGACU G UCCAGAAA697UUUCUGGA UGAUGGCAUGCACUAUGCGCG AGUCUACG2119204AGAGAUCU G UAUCAAUA698UAUUGAUA UGAUGGCAUGCACUAUGCGCG AGAUCUCU2120217AAUAAAAU G CUAAAUCA699UGAUUUAG UGAUGGCAUGCACUAUGCGCG AUUUUAUU2121227UAAAUCAU G AAAAUGUA700UACAUUUU UGAUGGCAUGCACUAUGCGCG AUGAUUUA2122233AUGAAAAU G AUGUAAAA701UUUUACUA UGAUGGCAUGCACUAUGCGCG AUUUUCAU2123294GGAGUACU G UAGUGGAG702CUCCACUA UGAUGGCAUGCACUAUGCGCG AGUACUCC2124314AGCUUUUU G ACAGAAUA703UAUUCUGU UGAUGGCAUGCACUAUGCGCG AAAAAGCU2125340AUAGGCAU G CCUGAACC704GGUUCAGG UGAUGGCAUGCACUAUGCGCG AUGCCUAU2126344GCAUGCCU G AACCAGAU705AUCUGGUU UGAUGGCAUGCACUAUGCGCG AGGCAUGC2127353AACCAGAU G CUCAGAGA706UCUCUGAG UGAUGGCAUGCACUAUGCGCG AUCUGGUU2128397GUUUAUCU G CAUGGUAU707AUACCAUG UGAUGGCAUGCACUAUGCGCG AGAUAAAC2129445AAUCUUCU G UUGGAUGA708UCAUCCAA UGAUGGCAUGCACUAUGCGCG AGAAGAUU2130452UGUUGGAU G AAAGGGAU709AUCCCUUU UGAUGGCAUGCACUAUGCGCG AUCCAACA2131515AUAAUCGU G AGCGUUUG710CAAACGCU UGAUGGCAUGCACUAUGCGCG ACGAUUAU2132523GAGCGUUU G UUGAACAA711UUGUUCAA UGAUGGCAUGCACUAUGCGCG AAACGCUC2133526CGUUUGUU G AACAAGAU712AUCUUGUU UGAUGGCAUGCACUAUGCGCG AACAAACG2134535AACAAGAU G UGUGGUAC713GUACCACA UGAUGGCAUGCACUAUGCGCG AUCUUGUU2135537CAAGAUGU G UGGUACUU714AAGUACCA UGAUGGCAUGCACUAUGCGCG ACAUCUUG2136554UACCAUAU G UUGCUCCA715UGGAGCAA UGAUGGCAUGCACUAUGCGCG AUAUGGUA2137557CAUAUGUU G CUCCAGAA716UUCUGGAG UGAUGGCAUGCACUAUGCGCG AACAUAUG2138571GAACUUCU G AAGAGAAG717CUUCUCUU UGAUGGCAUGCACUAUGCGCG AGAAGUUC2139590AAUUUCAU G CAGAACCA718UGGUUCUG UGAUGGCAUGCACUAUGCGCG AUGAAAUU2140602AACCAGUU G AUGUUUGG719CCAAACAU UGAUGGCAUGCACUAUGCGCG AACUGGUU2141605CAGUUGAU G UUUGGUCC720GGACCAAA UGAUGGCAUGCACUAUGCGCG AUCAACUG2142615UUGGUCCU G UGGAAUAG721CUAUUCCA UGAUGGCAUGCACUAUGCGCG AGGACCAA2143632UACUUACU G CAAUGCUC722GAGCAUUG UGAUGGCAUGCACUAUGCGCG AGUAAGUA2144637ACUGCAAU G CUCGCUGG723CCAGCGAG UGAUGGCAUGCACUAUGCGCG AUUGCAGU2145641CAAUGCUC G CUGGAGAA724UUCUCCAG UGAUGGCAUGCACUAUGCGCG GAGCAUUG2146652GGAGAAUU G CCAUGGGA725UCCCAUGG UGAUGGCAUGCACUAUGCGCG AAUUCUCC2147671AACCCAGU G ACAGCUGU726ACAGCUGU UGAUGGCAUGCACUAUGCGCG ACUGGGUU2148678UGACAGCU G UCAGGAGU727ACUCCUGA UGAUGGCAUGCACUAUGCGCG AGCUGUCA2149692AGUAUUCU G ACUGGAAA728UUUCCAGU UGAUGGCAUGCACUAUGCGCG AGAAUACU2150737AAAAAAUC G AUUCUGCU729AGCAGAAU UGAUGGCAUGCACUAUGCGCG GAUUUUUU2151732UCGAUUCU G CUCCUCUA730UAGAGGAG UGAUGGCAUGCACUAUGCGCG AGAAUCGA2152757CUAGCUCU G CUGCAUAA731UUAUGCAG UGAUGGCAUGCACUAUGCGCG AGAGCUAG2153760GCUCUGCU G CAUAAAAU732AUUUUAUG UGAUGGCAUGCACUAUGCGCG AGCAGAGC2154776UCUUAGUU G AGAAUCCA733UGGAUUCU UGAUGGCAUGCACUAUGCGCG AACUAAGA2155864AAGGCCCC G AGUCACUU734AAGUGACU UGAUGGCAUGCACUAUGCGCG GGGGCCUU2156881CAGGUGGU G UGUCAGAG735CUCUGACA UGAUGGCAUGCACUAUGCGCG ACCACCUG2157883GGUGGUGU G UCAGAGUC736GACUCUGA UGAUGGCAUGCACUAUGCGCG ACACCACC2158950UAAACAGU G CUUCUAGU737ACUAGAAG UGAUGGCAUGCACUAUGCGCG ACUGUUUA2159959CUUCUAGU G AAGAAAAU738AUUUUCUU UGAUGGCAUGCACUAUGCGCG ACUAGAAG2160968AAGAAAAU G UGAAGUAC739GUACUUCA UGAUGGCAUGCACUAUGCGCG AUUUUCUU2161970GAAAAUGU G AAGUACUC740GAGUACUU UGAUGGCAUGCACUAUGCGCG ACAUUUUC2162999AGAACCCC G CACAGGUC741GACCUGUG UGAUGGCAUGCACUAUGCGCG GGGGUUCU21631040CAUACAUU G AUAAAUUG742CAAUUUAU UGAUGGCAUGCACUAUGCGCG AAUGUAUG21641080GCCCACAU G UCCUGAUC743GAUCAGGA UGAUGGCAUGCACUAUGCGCG AUGUGGGC21651085CAUGUCCU G AUCAUAUG744CAUAUGAU UGAUGGCAUGCACUAUGCGCG AGGACAUG21661093GAUCAUAU G CUUUUGAA745UUCAAAAG UGAUGGCAUGCACUAUGCGCG AUAUGAUC21671099AUGCUUUU G AAUAGUCA746UGACUAUU UGAUGGCAUGCACUAUGCGCG AAAAGCAU21681165AAAAGAAU G ACACGAUU747AAUCGUGU UGAUGGCAUGCACUAUGCGCG AUUCUUUU21691170AAUGACAC G AUUCUUUA748UAAAGAAU UGAUGGCAUGCACUAUGCGCG GUGUCAUU21701190AAUUGGAU G CAGACAAA749UUUGUCUG UGAUGGCAUGCACUAUGCGCG AUCCAAUU21711209UUAUCAAU G CCUGAAAG750CUUUCAGG UGAUGGCAUGCACUAUGCGCG AUUGAUAA21721213CAAUGCCU G AAAGAGAC751GUCUCUUU UGAUGGCAUGCACUAUGCGCG AGGCAUUG21731224AGAGACUU G UGAGAAGU752ACUUCUCA UGAUGGCAUGCACUAUGCGCG AAGUCUCU21741226AGACUUGU G AGAAGUUG753CAACUUCU UGAUGGCAUGCACUAUGCGCG ACAAGUCU21751257GAAAAGUU G UAUGAAUC754GAUUCAUA UGAUGGCAUGCACUAUGCGCG AACUUUUC21761261AGUUGUAU G AAUCAGGU755ACCUGAUU UGAUGGCAUGCACUAUGCGCG AUACAACU21771286CAACAACU G AUAGGAGA756UCUCCUAU UGAUGGCAUGCACUAUGCGCG AGUUGUUG21781318UUCAAAGU G AAUUUGUU757AACAAAUU UGAUGGCAUGCACUAUGCGCG ACUUUGAA21791324GUGAAUUU G UUAGAAAU758AUUUCUAA UGAUGGCAUGCACUAUGCGCG AAAUUCAC21801337AAAUGGAU G AUAAAAUA759UAUUUUAU UGAUGGCAUGCACUAUGCGCG AUCCAUUU21811352UAUUGGUU G ACUUCCGG760CCGGAAGU UGAUGGCAUGCACUAUGCGCG AACCAAUA21821373CUAAGGGU G AUGGAUUG761CAAUCCAU UGAUGGCAUGCACUAUGCGCG ACCCUUAG21831402CACUUCCU G AAGAUUAA762UUAAUCUU UGAUGGCAUGCACUAUGCGCG AGGAAGUG21841420GGGAAGCU G AUUGAUAU763AUAUCAAU UGAUGGCAUGCACUAUGCGCG AGCUUCCC21851424AGCUGAUU G AUAUUGUG764CACAAUAU UGAUGGCAUGCACUAUGCGCG AAUCAGCU21861430UUGAUAUU G UGAGCAGC765GCUGCUCA UGAUGGCAUGCACUAUGCGCG AAUAUCAA21871432GAUAUUGU G AGCAGCCA766UGGCUGCU UGAUGGCAUGCACUAUGCGCG ACAAUAUC21881457GGCUUCCU G CCACAUGA767UCAUGUGG UGAUGGCAUGCACUAUGCGCG AGGAAGCC21891464UGCCACAU G AUCGGACC768GGUCCGAU UGAUGGCAUGCACUAUGCGCG AUGUGGCA21901495AUCCUGGU G AAUAUAGU769ACUAUAUU UGAUGGCAUGCACUAUGCGCG ACCAGGAU21911504AAUAUAGU G CUGCUAUG770CAUAGCAG UGAUGGCAUGCACUAUGCGCG ACUAUAUU21921507AUAGUGCU G CUAUGUUG771CAACAUAG UGAUGGCAUGCACUAUGCGCG AGCACUAU21931512GCUGCUAU G UUGACAUU772AAUGUCAA UGAUGGCAUGCACUAUGCGCG AUAGCAGC21941515GCUAUGUU G ACAUUAUU773AAUAAUGU UGAUGGCAUGCACUAUGCGCG AACAUAGC21951545AUUAUCCU G UCCUGCAA774UUGCAGGA UGAUGGCAUGCACUAUGCGCG AGGAUAAU21961550CCUGUCCU G CAAACUGC775GCAGUUUG UGAUGGCAUGCACUAUGCGCG AGGACAGG21971557UGCAAACU G CAAAUAGU776ACUAUUUG UGAUGGCAUGCACUAUGCGCG AGUUUGCA21981573UAGUUCCU G AAGUGUUC777GAACACUU UGAUGGCAUGCACUAUGCGCG AGGAACUA21991578CCUGAAGU G UUCACUUC778GAAGUGAA UGAUGGCAUGCACUAUGCGCG ACUUCAGG22001590ACUUCCCU G UUUAUCCA779UGGAUAAA UGAUGGCAUGCACUAUGCGCG AGGGAAGU22011619UUUAUUUU G UUUGUUCG780CGAACAAA UGAUGGCAUGCACUAUGCGCG AAAAUAAA22021623UUUUGUUU G UUCGGCAU781AUGCCGAA UGAUGGCAUGCACUAUGCGCG AAACAAAA22031656UCUUAAUU G UAAGCAAA782UUUGCUUA UGAUGGCAUGCACUAUGCGCG AAUUAAGA22041681GAAAGGAU G AAUAGAAU783AUUCUAUU UGAUGGCAUGCACUAUGCGCG AUCCUUUC22051696AUUCAUUU G AUUAUUUC784GAAAUAAU UGAUGGCAUGCACUAUGCGCG AAAUGAAU22061710UUCUUCAU G UGUGUUUA785UAAACACA UGAUGGCAUGCACUAUGCGCG AUGAAGAA22071712CUUCAUGU G UGUUUAGU786ACUAAACA UGAUGGCAUGCACUAUGCGCG ACAUGAAG22081714UCAUGUGU G UUUAGUAU787AUACUAAA UGAUGGCAUGCACUAUGCGCG ACACAUGA22091725UAGUAUCU G AAUUUGAA788UUCAAAUU UGAUGGCAUGCACUAUGCGCG AGAUACUA22101731CUGAAUUU G AAACUCAU789AUGAGUUU UGAUGGCAUGCACUAUGCGCG AAAUUCAG22111768GGGGACAU G AGUUUUCC790GGAAAACU UGAUGGCAUGCACUAUGCGCG AUGUCCCC2212Input Sequence = AF016582. Cut Site = YG/M or UG/U. Stem Length = 8. Core Sequence = UGAUG GCAUGCACUAUGC GCGAF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

[0178]

6

TABLE VI

Human Chk1 Zinzyme Ribozyme and Substrate Sequence

Rz Seq

Pos
Substrate
Seq ID
Ribozyme
ID

10
GCCGGACA G UCCGCCGA
791
UCGGCGGA GCCGAAAGGCGAGUCAAGGUCU UGUCCGGC
2213

14
GACAGUCC G CCGAGGUG
792
CACCUCGG GCCGAAAGGCGAGUCAAGGUCU GGACUGUC
2214

20
CCGCCGAG G UGCUCGGU
793
ACCGAGCA GCCGAAAGGCGAGUCAAGGUCU CUCGGCGG
2215

22
GCCGAGGU G CUCGGUGG
794
CCACCGAG GCCGAAAGGCGAGUCAAGGUCU ACCUCGGC
2216

27
GGUGCUCG G UGGAGUCA
795
UGACUCCA GCCGAAAGGCGAGUCAAGGUCU CGAGCACC
2217

32
UCGGUGGA G UCAUGGCA
796
UGCCAUGA GCCGAAAGGCGAGUCAAGGUCU UCCACCGA
2218

38
GAGUCAUG G CAGUGCCC
797
GGGCACUG GCCGAAAGGCGAGUCAAGGUCU CAUGACUC
2219

41
UCAUGGCA G UGCCCUUU
798
AAAGGGCA GCCGAAAGGCGAGUCAAGGUCU UGCCAUGA
2220

43
AUGGCAGU G CCCUUUGU
799
ACAAAGGG GCCGAAAGGCGAGUCAAGGUCU ACUGCCAU
2221

50
UGCCCUUU G UGGAAGAC
800
GUCUUCCA GCCGAAAGGCGAGUCAAGGUCU AAAGGGCA
2222

68
GGGACUUG G UGCAAACC
801
GGUUUGCA GCCGAAAGGCGAGUCAAGGUCU CAAGUCCC
2223

70
GACUUGGU G CAAACCCU
802
AGGGUUUG GCCGAAAGGCGAGUCAAGGUCU ACCAAGUC
2224

87
GGGAGAAG G UGCCUAUG
803
CAUAGGCA GCCGAAAGGCGAGUCAAGGUCU CUUCUCCC
2225

89
GAGAAGGU G CCUAUGGA
804
UCCAUAGG GCCGAAAGGCGAGUCAAGGUCU ACCUUCUC
2226

101
AUGGAGAA G UUCAACUU
805
AAGUUGAA GCCGAAAGGCGAGUCAAGGUCU UUCUCCAU
2227

110
UUCAACUU G CUGUGAAU
806
AUUCACAG GCCGAAAGGCGAGUCAAGGUCU AAGUUGAA
2228

113
AACUUGCU G UGAAUAGA
807
UCUAAUCA GCCGAAAGGCGAGUCAAGGUCU AGCAAGUU
2229

122
UGAAUAGA G UAACUGAA
808
UUCAGUUA GCCGAAAGGCGAGUCAAGGUCU UCUAUUCA
2230

134
CUGAAGAA G CAGUCGCA
809
UGCGACUG GCCGAAAGGCGAGUCAAGGUCU UUCUUCAG
2231

137
AAGAAGCA G UCGCAGUG
810
CACUGCGA GCCGAAAGGCGAGUCAAGGUCU UGCUUCUU
2232

140
AAGCAGUC G CAGUGAAG
811
CUUCACUG GCCGAAAGGCGAGUCAAGGUCU GACUGCUU
2233

143
CAGUCGCA G UGAAGAUU
812
AAUCUUCA GCCGAAAGGCGAGUCAAGGUCU UGCGACUG
2234

152
UGAAGAUU G UAGAUAUG
813
CAUAUCUA GCCGAAAGGCGAGUCAAGGUCU AAUCUUCA
2235

163
GAUAUGAA G CGUGCCGU
814
ACGGCACG GCCGAAAGGCGAGUCAAGGUCU UUCAUAUC
2236

165
UAUGAAGC G UGCCGUAG
815
CUACGGCA GCCGAAAGGCGAGUCAAGGUCU GCUUCAUA
2237

167
UGAAGCGU G CCGUAGAC
816
GUCUACGG GCCGAAAGGCGAGUCAAGGUCU ACGCUUCA
2238

170
AGCGUGCC G UAGACUGU
817
ACAGUCUA GCCGAAAGGCGAGUCAAGGUCU GGCACGCU
2239

177
CGUAGACU G UCCAGAAA
818
UUUCUGGA GCCGAAAGGCGAGUCAAGGUCU AGUCUACG
2240

204
AGAGAUCU G UAUCAAUA
819
UAUUGAUA GCCGAAAGGCGAGUCAAGGUCU AGAUCUCU
2241

217
AAUAAAAU G CUAAAUCA
820
UGAUUUAG GCCGAAAGGCGAGUCAAGGUCU AUUUUAUU
2242

233
AUGAAAAU G UAGUAAAA
821
UUUUACUA GCCGAAAGGCGAGUCAAGGUCU AUUUUCAU
2243

236
AAAAUGUA G UAAAAUUC
822
GAAUUUUA GCCGAAAGGCGAGUCAAGGUCU UACAUUUU
2244

249
AUUCUAUG G UCACAGGA
823
UCCUGUGA GCCGAAAGGCGAGUCAAGGUCU CAUAGAAU
2245

264
GAGAGAAG G CAAUAUCC
824
GGAUAUUG GCCGAAAGGCGAGUCAAGGUCU CUUCUCUC
2246

289
UUUCUGGA G UACUGUAG
825
CUACAGUA GCCGAAAGGCGAGUCAAGGUCU UCCAGAAA
2247

294
GGAGUACU G UAGUGGAG
826
CUCCACUA GCCGAAAGGCGAGUCAAGGUCU AGUACUCC
2248

297
GUACUGUA G UGGAGGAG
827
CUCCUCCA GCCGAAAGGCGAGUCAAGGUCU UACAGUAC
2249

307
GGAGGAGA G CUUUUUGA
828
UCAAAAAG GCCGAAAGGCGAGUCAAGGUCU UCUCCUCC
2250

325
AGAAUAGA G CCAGACAU
829
AUGUCUGG GCCGAAAGGCGAGUCAAGGUCU UCUAUUCU
2251

336
AGACAUAG G CAUGCCUG
830
CAGGCAUG GCCGAAAGGCGAGUCAAGGUCU CUAUGUCU
2252

340
AUAGGCAU G CCUGAACC
831
GGUUCAGG GCCGAAAGGCGAGUCAAGGUCU AUGCCUAU
2253

353
AACCAGAU G CUCAGAGA
832
UCUCUGAG GCCGAAAGGCGAGUCAAGGUCU AUCUGGUU
2254

380
AACUCAUG G CAGGGGUG
833
CACCCCUG GCCGAAAGGCGAGUCAAGGUCU CAUGAGUU
2255

386
UGGCAGGG G UGGUUUAU
834
AUAAACCA GCCGAAAGGCGAGUCAAGGUCU CCCUGCCA
2256

389
CAGGGGUG G UUUAUCUG
835
CAGAUAAA GCCGAAAGGCGAGUCAAGGUCU CACCCCUG
2257

397
GUUUAUCU G CAUGGUAU
836
AUACCAUG GCCGAAAGGCGAGUCAAGGUCU AGAUAAAC
2258

402
UCUGCAUG G UAUUGGAA
837
UUCCAAUA GCCGAAAGGCGAGUCAAGGUCU CAUGCAGA
2259

445
AAUCUUCU G UUGGAUGA
838
UCAUCCAA GCCGAAAGGCGAGUCAAGGUCU AGAAGAUU
2260

483
AGACUUUG G CUUGGCAA
839
UUGCCAAG GCCGAAAGGCGAGUCAAGGUCU CAAAGUCU
2261

488
UUGGCUUG G CAACAGUA
840
UACUGUUG GCCGAAAGGCGAGUCAAGGUCU CAAGCCAA
2262

494
UGGCAACA G UAUUUCGG
841
CCGAAAUA GCCGAAAGGCGAGUCAAGGUCU UGUUGCCA
2263

502
GUAUUUCG G UAUAAUAA
842
UUAUUAUA GCCGAAAGGCGAGUCAAGGUCU CGAAAUAC
2264

513
UAAUAAUC G UGAGCGUU
843
AACGCUCA GCCGAAAGGCGAGUCAAGGUCU GAUUAUUA
2265

517
AAUCGUGA G CGUUUGUU
844
AACAAACG GCCGAAAGGCGAGUCAAGGUCU UCACGAUU
2266

519
UCGUGAGC G UUUGUUGA
845
UCAACAAA GCCGAAAGGCGAGUCAAGGUCU GCUCACGA
2267

523
GAGCGUUU G UUGAACAA
846
UUGUUCAA GCCGAAAGGCGAGUCAAGGUCU AAACGCUC
2268

535
AACAAGAU G UGUGGUAC
847
GUACCACA GCCGAAAGGCGAGUCAAGGUCU AUCUUGUU
2269

537
CAAGAUGU G UGGUACUU
848
AAGUACCA GCCGAAAGGCGAGUCAAGGUCU ACAUCUUG
2270

540
GAUGUGUG G UACUUUAC
849
GUAAAGUA GCCGAAAGGCGAGUCAAGGUCU CACACAUC
2271

554
UACCAUAU G UUGCUCCA
850
UGGAGCAA GCCGAAAGGCGAGUCAAGGUCU AUAUGGUA
2272

557
CAUAUGUU G CUCCAGAA
851
UUCUGGAG GCCGAAAGGCGAGUCAAGGUCU AACAUAUG
2273

590
AAUUUCAU G CAGAACCA
852
UGGUUCUG GCCGAAAGGCGAGUCAAGGUCU AUGAAAUU
2274

599
CAGAACCA G UUGAUGUU
853
AACAUCAA GCCGAAAGGCGAGUCAAGGUCU UGGUUCUG
2275

605
CAGUUGAU G UUUGGUCC
854
GGACCAAA GCCGAAAGGCGAGUCAAGGUCU AUCAACUG
2276

610
GAUGUUUG G UCCUGUGG
855
CCACAGGA GCCGAAAGGCGAGUCAAGGUCU CAAACAUC
2277

615
UUGGUCCU G UGGAAUAG
856
CUAUUCCA GCCGAAAGGCGAGUCAAGGUCU AGGACCAA
2278

623
GUGGAAUA G UACUUACU
857
AGUAAGUA GCCGAAAGGCGAGUCAAGGUCU UAUUCCAC
2279

632
UACUUACU G CAAUGCUC
858
GAGCAUUG GCCGAAAGGCGAGUCAAGGUCU AGUAAGUA
2280

637
ACUGCAAU G CUCGCUGG
859
CCAGCGAG GCCGAAAGGCGAGUCAAGGUCU AUUGCAGU
2281

641
CAAUGCUC G CUGGAGAA
860
UUCUCCAG GCCGAAAGGCGAGUCAAGGUCU GAGCAUUG
2282

652
GGAGAAUU G CCAUGGGA
861
UCCCAUGG GCCGAAAGGCGAGUCAAGGUCU AAUUCUCC
2283

669
CCAACCCA G UGACAGCU
862
AGCUGUCA GCCGAAAGGCGAGUCAAGGUCU UGGGUUGG
2284

675
CAGUGACA G CUGUCAGG
863
CCUGACAG GCCGAAAGGCGAGUCAAGGUCU UGUCACUG
2285

678
UGACAGCU G UCAGGAGU
864
ACUCCUGA GCCGAAAGGCGAGUCAAGGUCU AGCUGUCA
2286

685
UGUCAGGA G UAUUCUGA
865
UCAGAAUA GCCGAAAGGCGAGUCAAGGUCU UCCUGACA
2287

743
UCGAUUCU G CUCCUCUA
866
UAGAGGAG GCCGAAAGGCGAGUCAAGGUCU AGAAUCGA
2288

752
CUCCUCUA G CUCUGCUG
867
CAGCAGAG GCCGAAAGGCGAGUCAAGGUCU UAGAGGAG
2289

757
CUAGCUCU G CUGCAUAA
868
UUAUGCAG GCCGAAAGGCGAGUCAAGGUCU AGAGCUAG
2290

760
GCUCUGCU G CAUAAAAU
869
AUUUUAUG GCCGAAAGGCGAGUCAAGGUCU AGCAGAGC
2291

773
AAAUCUUA G UUGAGAAU
870
AUUCUCAA GCCGAAAGGCGAGUCAAGGUCU UAAGAUUU
2292

788
AUCCAUCA G CAAGAAUU
871
AAUUCUUG GCCGAAAGGCGAGUCAAGGUCU UGAUGGAU
2293

826
GAUAGAUG G UACAACAA
872
UUGUUGUA GCCGAAAGGCGAGUCAAGGUCU CAUCUAUC
2294

851
AGAAAGGG G CAAAAAGG
873
CCUUUUUG GCCGAAAGGCGAGUCAAGGUCU CCCUUUCU
2295

859
GCAAAAAG G CCCCGAGU
874
ACUCGGGG GCCGAAAGGCGAGUCAAGGUCU CUUUUUGC
2296

866
GGCCCCGA G UCACUUCA
875
UGAAGUGA GCCGAAAGGCGAGUCAAGGUCU UCGGGGCC
2297

876
CACUUCAG G UGGUGUGU
876
ACACACCA GCCGAAAGGCGAGUCAAGGUCU CUGAAGUG
2298

879
UUCAGGUG G UGUGUCAG
877
CUGACACA GCCGAAAGGCGAGUCAAGGUCU CACCUGAA
2299

881
CAGGUGGU G UGUCAGAG
878
CUCUGACA GCCGAAAGGCGAGUCAAGGUCU ACCACCUG
2300

883
GGUGGUGU G UCAGAGUC
879
GACUCUGA GCCGAAAGGCGAGUCAAGGUCU ACACCACC
2301

889
GUGUCAGA G UCUCCCAG
880
CUGGGAGA GCCGAAAGGCGAGUCAAGGUCU UCUGACAC
2302

897
GUCUCCCA G UGGAUUUU
881
AAAAUCCA GCCGAAAGGCGAGUCAAGGUCU UGGGAGAC
2303

910
UUUUCUAA G CACAUUCA
882
UGAAUGUG GCCGAAAGGCGAGUCAAGGUCU UUAGAAAA
2304

941
UCUCUCCA G UAAACAGU
883
ACUGUUUA GCCGAAAGGCGAGUCAAGGUCU UGGAGAGA
2305

948
AGUAAACA G UGCUUCUA
884
UAGAAGCA GCCGAAAGGCGAGUCAAGGUCU UGUUUACU
2306

950
UAAACAGU G CUUCUAGU
885
ACUAGAAG GCCGAAAGGCGAGUCAAGGUCU ACUGUUUA
2307

957
UGCUUCUA G UGAAGAAA
886
UUUCUUCA GCCGAAAGGCGAGUCAAGGUCU UAGAAGCA
2308

968
AAGAAAAU G UGAAGUAC
887
GUACUUCA GCCGAAAGGCGAGUCAAGGUCU AUUUUCUU
2309

973
AAUGUGAA G UACUCCAG
888
CUGGAGUA GCCGAAAGGCGAGUCAAGGUCU UUCACAUU
2310

981
GUACUCCA G UUCUCAGC
889
GCUGAGAA GCCGAAAGGCGAGUCAAGGUCU UGGAGUAC
2311

988
AGUUCUCA G CCAGAACC
890
GGUUCUGG GCCGAAAGGCGAGUCAAGGUCU UGAGAACU
2312

999
AGAACCCC G CACAGGUC
891
GACCUGUG GCCGAAAGGCGAGUCAAGGUCU GGGGUUCU
2313

1005
CCGCACAG G UCUUUCCU
892
AGGAAAGA GCCGAAAGGCGAGUCAAGGUCU CUGUGCGG
2314

1026
GGAUACCA G CCCCUCAU
893
AUGAGGGG GCCGAAAGGCGAGUCAAGGUCU UGGUAUCC
2315

1049
AUAAAUUG G UACAAGGG
894
CCCUUGUA GCCGAAAGGCGAGUCAAGGUCU CAAUUUAU
2316

1062
AGGGAUCA G CUUUUCCC
895
GGGAAAAG GCCGAAAGGCGAGUCAAGGUCU UGAUCCCU
2317

1072
UUUUCCCA G CCCACAUG
896
CAUGUGGG GCCGAAAGGCGAGUCAAGGUCU UGGGAAAA
2318

1080
GCCCACAU G UCCUGAUC
897
GAUCAGGA GCCGAAAGGCGAGUCAAGGUCU AUGUGGGC
2319

1093
GAUCAUAU G CUUUUGAA
898
UUCAAAAG GCCGAAAGGCGAGUCAAGGUCU AUAUGAUC
2320

1104
UUUGAAUA G UCAGUUAC
899
GUAACUGA GCCGAAAGGCGAGUCAAGGUCU UAUUCAAA
2321

1108
AAUAGUCA G UUACUUGG
900
CCAAGUAA GCCGAAAGGCGAGUCAAGGUCU UGACUAUU
2322

1116
GUUACUUG G CACCCCAG
901
CUGGGGUG GCCGAAAGGCGAGUCAAGGUCU CAAGUAAC
2323

1144
AACCCCUG G CAGCGGUU
902
AACCGCUG GCCGAAAGGCGAGUCAAGGUCU CAGGGGUU
2324

1147
CCCUGGCA G CGGUUGGU
903
ACCAACCG GCCGAAAGGCGAGUCAAGGUCU UGCCAGGG
2325

1150
UGGCAGCG G UUGGUCAA
904
UUGACCAA GCCGAAAGGCGAGUCAAGGUCU CGCUGCCA
2326

1154
AGCGGUUG G UCAAAAGA
905
UCUUUUGA GCCGAAAGGCGAGUCAAGGUCU CAACCGCU
2327

1190
AAUUGGAU G CAGACAAA
906
UUUGUCUG GCCGAAAGGCGAGUCAAGGUCU AUCCAAUU
2328

1209
UUAUCAAU G CCUGAAAG
907
CUUUCAGG GCCGAAAGGCGAGUCAAGGUCU AUUGAUAA
2329

1224
AGAGACUU G UGAGAAGU
908
ACUUCUCA GCCGAAAGGCGAGUCAAGGUCU AAGUCUCU
2330

1231
UGUGAGAA G UUGGGCUA
909
UAGCCCAA GCCGAAAGGCGAGUCAAGGUCU UUCUCACA
2331

1236
GAAGUUGG G CUAUCAAU
910
AUUGAUAG GCCGAAAGGCGAGUCAAGGUCU CCAACUUC
2332

1254
GAAGAAAA G UUGUAUGA
911
UCAUACAA GCCGAAAGGCGAGUCAAGGUCU UUUUCUUC
2333

1257
GAAAAGUU G UAUGAAUC
912
GAUUCAUA GCCGAAAGGCGAGUCAAGGUCU AACUUUUC
2334

1268
UGAAUCAG G UUACUAUA
913
UAUAGUAA GCCGAAAGGCGAGUCAAGGUCU CUGAUUCA
2335

1316
UUUUCAAA G UGAAUUUG
914
CAAAUUCA GCCGAAAGGCGAGUCAAGGUCU UUUGAAAA
2336

1324
GUGAAUUU G UUAGAAAU
915
AUUUCUAA GCCGAAAGGCGAGUCAAGGUCU AAAUUCAC
2337

1349
AAAUAUUG G UUGACUUC
916
GAAGUCAA GCCGAAAGGCGAGUCAAGGUCU CAAUAUUU
2338

1360
GACUUCCG G CUUUCUAA
917
UUAGAAAG GCCGAAAGGCGAGUCAAGGUCU CGGAAGUC
2339

1371
UUCUAAGG G UGAUGGAU
918
AUCCAUCA GCCGAAAGGCGAGUCAAGGUCU CCUUAGAA
2340

1384
GGAUUGGA G UUCAAGAG
919
CUCUUGAA GCCGAAAGGCGAGUCAAGGUCU UCCAAUCC
2341

1417
AAAGGGAA G CUGAUUGA
920
UCAAUCAG GCCGAAAGGCGAGUCAAGGUCU UUCCCUUU
2342

1430
UUGAUAUU G UGAGCAGC
921
GCUGCUCA GCCGAAAGGCGAGUCAAGGUCU AAUAUCAA
2343

1434
UAUUGUGA G CAGCCAGA
922
UCUGGCUG GCCGAAAGGCGAGUCAAGGUCU UCACAAUA
2344

1437
UGUGAGCA G CCAGAAGG
923
CCUUCUGG GCCGAAAGGCGAGUCAAGGUCU UGCUCACA
2345

1445
GCCAGAAG G UUUGGCUU
924
AAGCCAAA GCCGAAAGGCGAGUCAAGGUCU CUUCUGGC
2346

1450
AAGGUUUG G CUUCCUGC
925
GCAGGAAG GCCGAAAGGCGAGUCAAGGUCU CAAACCUU
2347

1457
GGCUUCCU G CCACAUGA
926
UCAUGUGG GCCGAAAGGCGAGUCAAGGUCU AGGAAGCC
2348

1477
GACCAUCG G CUCUGGGG
927
CCCCAGAG GCCGAAAGGCGAGUCAAGGUCU CGAUGGUC
2349

1493
GAAUCCUG G UGAAUAUA
928
UAUAUUCA GCCGAAAGGCGAGUCAAGGUCU CAGGAUUC
2350

1502
UGAAUAUA G UGCUGCUA
929
UAGCAGCA GCCGAAAGGCGAGUCAAGGUCU UAUAUUCA
2351

1504
AAUAUAGU G CUGCUAUG
930
CAUAGCAG GCCGAAAGGCGAGUCAAGGUCU ACUAUAUU
2352

1507
AUAGUGCU G CUAUGUUG
931
CAACAUAG GCCGAAAGGCGAGUCAAGGUCU AGCACUAU
2353

1512
GCUGCUAU G UUGACAUU
932
AAUGUCAA GCCGAAAGGCGAGUCAAGGUCU AUAGCAGC
2354

1545
AUUAUCCU G UCCUGCAA
933
UUGCAGGA GCCGAAAGGCGAGUCAAGGUCU AGGAUAAU
2355

1550
CCUGUCCU G CAAACUGC
934
GCAGUUUG GCCGAAAGGCGAGUCAAGGUCU AGGACAGG
2356

1557
UGCAAACU G CAAAUAGU
935
ACUAUUUG GCCGAAAGGCGAGUCAAGGUCU AGUUUGCA
2357

1564
UGCAAAUA G UAGUUCCU
936
AGGAACUA GCCGAAAGGCGAGUCAAGGUCU UAUUUGCA
2358

1567
AAAUAGUA G UUCCUGAA
937
UUCAGGAA GCCGAAAGGCGAGUCAAGGUCU UACUAUUU
2359

1576
UUCCUGAA G UGUUCACU
938
AGUGAACA GCCGAAAGGCGAGUCAAGGUCU UUCAGGAA
2360

1578
CCUGAAGU G UUCACUUC
939
GAAGUGAA GCCGAAAGGCGAGUCAAGGUCU ACUUCAGG
2361

1590
ACUUCCCU G UUUAUCCA
940
UGGAUAAA GCCGAAAGGCGAGUCAAGGUCU AGGGAAGU
2362

1619
UUUAUUUU G UUUGUUCG
941
CGAACAAA GCCGAAAGGCGAGUCAAGGUCU AAAAUAAA
2363

1623
UUUUGUUU G UUCGGCAU
942
AUGCCGAA GCCGAAAGGCGAGUCAAGGUCU AAACAAAA
2364

1628
UUUGUUCG G CAUACAAA
943
UUUGUAUG GCCGAAAGGCGAGUCAAGGUCU CGAACAAA
2365

1656
UCUUAAUU G UAAGCAAA
944
UUUGCUUA GCCGAAAGGCGAGUCAAGGUCU AAUUAAGA
2366

1660
AAUUGUAA G CAAAACUU
945
AAGUUUUG GCCGAAAGGCGAGUCAAGGUCU UUACAAUU
2367

1710
UUCUUCAU G UGUGUUUA
946
UAAACACA GCCGAAAGGCGAGUCAAGGUCU AUGAAGAA
2368

1712
CUUCAUGU G UGUUUAGU
947
ACUAAACA GCCGAAAGGCGAGUCAAGGUCU ACAUGAAG
2369

1714
UCAUGUGU G UUUAGUAU
948
AUACUAAA GCCGAAAGGCGAGUCAAGGUCU ACACAUGA
2370

1719
UGUGUUUA G UAUCUGAA
949
UUCAGAUA GCCGAAAGGCGAGUCAAGGUCU UAAACACA
2371

1743
CUCAUCUG G UGGAAACC
950
GGUUUCCA GCCGAAAGGCGAGUCAAGGUCU CAGAUGAG
2372

1754
GAAACCAA G UUUCAGGG
951
CCCUGAAA GCCGAAAGGCGAGUCAAGGUCU UUGGUUUC
2373

1770
GGACAUGA G UUUUCCAG
952
CUGGAAAA GCCGAAAGGCGAGUCAAGGUCU UCAUGUCC
2374

1778
GUUUUCCA G CUUUUAUA
953
UAUAAAAG GCCGAAAGGCGAGUCAAGGUCU UGGAAAAC
2375

1792
AUACACAC G UAUCUCAU
954
AUGAGAUA GCCGAAAGGCGAGUCAAGGUCU GUGUGUAU
2376

Input Sequence = AF016582. Cut Site = G/Y

Stem Length = 8. Core Sequence = GCcgaaagGCGaGuCaaGGuCu

AF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

[0179]

7

TABLE VII

Human Chk1 DNAzyme and Substrate Sequence

Pos
Substrate
Seq ID
DNAzyme
Seq ID

10
GCCGGACA G UCCGCCGA
791
TCGGCGGA GGCTAGCTACAACGA TGTCCGGC
2377

14
GACAGUCC G CCGAGGUG
792
CACCTCGG GGCTAGCTACAACGA GGACTGTC
2378

20
CCGCCGAG G UGCUCGGU
793
ACCGAGCA GGCTAGCTACAACGA CTCGGCGG
2379

22
GCCGAGGU G CUCGGUGG
794
CCACCGAG GGCTAGCTACAACGA ACCTCGGC
2380

27
GGUGCUCG G UGGAGUCA
795
TGACTCCA GGCTAGCTACAACGA CGAGCACC
2381

32
UCGGUGGA G UCAUGGCA
796
TGCCATGA GGCTAGCTACAACGA TCCACCGA
2382

38
GAGUCAUG G CAGUGCCC
797
GGGCACTG GGCTAGCTACAACGA CATGACTC
2383

41
UCAUGGCA G UGCCCUUU
798
AAAGGGCA GGCTAGCTACAACGA TGCCATGA
2384

43
AUGGCAGU G CCCUUUGU
799
ACAAAGGG GGCTAGCTACAACGA ACTGCCAT
2385

50
UGCCCUUU G UGGAAGAC
800
GTCTTCCA GGCTAGCTACAACGA AAAGGGCA
2386

68
GGGACUUG G UGCAAACC
801
GGTTTGCA GGCTAGCTACAACGA CAAGTCCC
2387

70
GACUUGGU G CAAACCCU
802
AGGGTTTG GGCTAGCTACAACGA ACCAAGTC
2388

87
GGGAGAAG G UGCCUAUG
803
CATAGGCA GGCTAGCTACAACGA CTTCTCCC
2389

89
GAGAAGGU G CCUAUGGA
804
TCCATAGG GGCTAGCTACAACGA ACCTTCTC
2390

101
AUGGAGAA G UUCAACUU
805
AAGTTGAA GGCTAGCTACAACGA TTCTCCAT
2391

110
UUCAACUU G CUGUGAAU
806
ATTCACAG GGCTAGCTACAACGA AAGTTGAA
2392

113
AACUUGCU G UGAAUAGA
807
TCTATTCA GGCTAGCTACAACGA AGCAAGTT
2393

122
UGAAUAGA G UAACUGAA
808
TTCAGTTA GGCTAGCTACAACGA TCTATTCA
2394

134
CUGAAGAA G CAGUCGCA
809
TGCGACTG GGCTAGCTACAACGA TTCTTCAG
2395

137
AAGAAGCA G UCGCAGUG
810
CACTGCGA GGCTAGCTACAACGA TGCTTCTT
2396

140
AAGCAGUC G CAGUGAAG
811
CTTCACTG GGCTAGCTACAACGA GACTGCTT
2397

143
CAGUCGCA G UGAAGAUU
812
AATCTTCA GGCTAGCTACAACGA TGCGACTG
2398

152
UGAAGAUU G UAGAUAUG
813
CATATCTA GGCTAGCTACAACGA AATCTTCA
2399

163
GAUAUGAA G CGUGCCGU
814
ACGGCACG GGCTAGCTACAACGA TTCATATC
2400

165
UAUGAAGC G UGCCGUAG
815
CTACGGCA GGCTAGCTACAACGA GCTTCATA
2401

167
UGAAGCGU G CCGUAGAC
816
GTCTACGG GGCTAGCTACAACGA ACGCTTCA
2402

170
AGCGUGCC G UAGACUGU
817
ACAGTCTA GGCTAGCTACAACGA GGCACGCT
2403

177
CGUAGACU G UCCAGAAA
818
TTTCTGGA GGCTAGCTACAACGA AGTCTACG
2404

204
AGAGAUCU G UAUCAAUA
819
TATTGATA GGCTAGCTACAACGA AGATCTCT
2405

217
AAUAAAAU G CUAAAUCA
820
TGATTTAG GGCTAGCTACAACGA ATTTTATT
2406

233
AUGAAAAU G UAGUAAAA
821
TTTTACTA GGCTAGCTACAACGA ATTTTCAT
2407

236
AAAAUGUA G UAAAAUUC
822
GAATTTTA GGCTAGCTACAACGA TACATTTT
2408

249
AUUCUAUG G UCACAGGA
823
TCCTGTGA GGCTAGCTACAACGA CATAGAAT
2409

264
GAGAGAAG G CAAUAUCC
824
GGATATTG GGCTAGCTACAACGA CTTCTCTC
2410

289
UUUCUGGA G UACUGUAG
825
CTACAGTA GGCTAGCTACAACGA TCCAGAAA
2411

294
GGAGUACU G UAGUGGAG
826
CTCCACTA GGCTAGCTACAACGA AGTACTCC
2412

297
GUACUGUA G UGGAGGAG
827
CTCCTCCA GGCTAGCTACAACGA TACAGTAC
2413

307
GGAGGAGA G CUUUUUGA
828
TCAAAAAG GGCTAGCTACAACGA TCTCCTCC
2414

325
AGAAUAGA G CCAGACAU
829
ATGTCTGG GGCTAGCTACAACGA TCTATTCT
2415

336
AGACAUAG G CAUGCCUG
830
CAGGCATG GGCTAGCTACAACGA CTATGTCT
2416

340
AUAGGCAU G CCUGAACC
831
GGTTCAGG GGCTAGCTACAACGA ATGCCTAT
2417

353
AACCAGAU G CUCAGAGA
832
TCTCTGAG GGCTAGCTACAACGA ATCTGGTT
2418

380
AACUCAUG G CAGGGGUG
833
CACCCCTG GGCTAGCTACAACGA CATGAGTT
2419

386
UGGCAGGG G UGGUUUAU
834
ATAAACCA GGCTAGCTACAACGA CCCTGCCA
2420

389
CAGGGGUG G UUUAUCUG
835
CAGATAAA GGCTAGCTACAACGA CACCCCTG
2421

397
GUUUAUCU G CAUGGUAU
836
ATACCATG GGCTAGCTACAACGA AGATAAAC
2422

402
UCUGCAUG G UAUUGGAA
837
TTCCAATA GGCTAGCTACAACGA CATGCAGA
2423

445
AAUCUUCU G UUGGAUGA
838
TCATCCAA GGCTAGCTACAACGA AGAAGATT
2424

483
AGACUUUG G CUUGGCAA
839
TTGCCAAG GGCTAGCTACAACGA CAAAGTCT
2425

488
UUGGCUUG G CAACAGUA
840
TACTGTTG GGCTAGCTACAACGA CAAGCCAA
2426

494
UGGCAACA G UAUUUCGG
841
CCGAAATA GGCTAGCTACAACGA TGTTGCCA
2427

502
GUAUUUCG G UAUAAUAA
842
TTATTATA GGCTAGCTACAACGA CGAAATAC
2428

513
UAAUAAUC G UGAGCGUU
843
AACGCTCA GGCTAGCTACAACGA GATTATTA
2429

517
AAUCGUGA G CGUUUGUU
844
AACAAACG GGCTAGCTACAACGA TCACGATT
2430

519
UCGUGAGC G UUUGUUGA
845
TCAACAAA GGCTAGCTACAACGA GCTCACGA
2431

523
GAGCGUUU G UUGAACAA
846
TTGTTCAA GGCTAGCTACAACGA AAACGCTC
2432

535
AACAAGAU G UGUGGUAC
847
GTACCACA GGCTAGCTACAACGA ATCTTGTT
2433

537
CAAGAUGU G UGGUACUU
848
AAGTACCA GGCTAGCTACAACGA ACATCTTG
2434

540
GAUGUGUG G UACUUUAC
849
GTAAAGTA GGCTAGCTACAACGA CACACATC
2435

554
UACCAUAU G UUGCUCCA
850
TGGAGCAA GGCTAGCTACAACGA ATATGGTA
2436

557
CAUAUGUU G CUCCAGAA
851
TTCTGGAG GGCTAGCTACAACGA AACATATG
2437

590
AAUUUCAU G CAGAACCA
852
TGGTTCTG GGCTAGCTACAACGA ATGAAATT
2438

599
CAGAACCA G UUGAUGUU
853
AACATCAA GGCTAGCTACAACGA TGGTTCTG
2439

605
CAGUUGAU G UUUGGUCC
854
GGACCAAA GGCTAGCTACAACGA ATCAACTG
2440

610
GAUGUUUG G UCCUGUGG
855
CCACAGGA GGCTAGCTACAACGA CAAACATC
2441

615
UUGGUCCU G UGGAAUAG
856
CTATTCCA GGCTAGCTACAACGA AGGACCAA
2442

623
GUGGAAUA G UACUUACU
857
AGTAAGTA GGCTAGCTACAACGA TATTCCAC
2443

632
UACUUACU G CAAUGCUC
858
GAGCATTG GGCTAGCTACAACGA AGTAAGTA
2444

637
ACUGCAAU G CUCGCUGG
859
CCAGCGAG GGCTAGCTACAACGA ATTGCAGT
2445

641
CAAUGCUC G CUGGAGAA
860
TTCTCCAG GGCTAGCTACAACGA GAGCATTG
2446

652
GGAGAAUU G CCAUGGGA
861
TCCCATGG GGCTAGCTACAACGA AATTCTCC
2447

669
CCAACCCA G UGACAGCU
862
AGCTGTCA GGCTAGCTACAACGA TGGGTTGG
2448

675
CAGUGACA G CUGUCAGG
863
CCTGACAG GGCTAGCTACAACGA TGTCACTG
2449

678
UGACAGCU G UCAGGAGU
864
ACTCCTGA GGCTAGCTACAACGA AGCTGTCA
2450

685
UGUCAGGA G UAUUCUGA
865
TCAGAATA GGCTAGCTACAACGA TCCTGACA
2451

743
UCGAUUCU G CUCCUCUA
866
TAGAGGAG GGCTAGCTACAACGA AGAATCGA
2452

752
CUCCUCUA G CUCUGCUG
867
CAGCAGAG GGCTAGCTACAACGA TAGAGGAG
2453

757
CUAGCUCU G CUGCAUAA
868
TTATGCAG GGCTAGCTACAACGA AGAGCTAG
2454

760
GCUCUGCU G CAUAAAAU
869
ATTTTATG GGCTAGCTACAACGA AGCAGAGC
2455

773
AAAUCUUA G UUGAGAAU
870
ATTCTCAA GGCTAGCTACAACGA TAAGATTT
2456

788
AUCCAUCA G CAAGAAUU
871
AATTCTTG GGCTAGCTACAACGA TGATGGAT
2457

826
GAUAGAUG G UACAACAA
872
TTGTTGTA GGCTAGCTACAACGA CATCTATC
2458

851
AGAAAGGG G CAAAAAGG
873
CCTTTTTG GGCTAGCTACAACGA CCCTTTCT
2459

859
GCAAAAAG G CCCCGAGU
874
ACTCGGGG GGCTAGCTACAACGA CTTTTTGC
2460

866
GGCCCCGA G UCACUUCA
875
TGAAGTGA GGCTAGCTACAACGA TCGGGGCC
2461

876
CACUUCAG G UGGUGUGU
876
ACACACCA GGCTAGCTACAACGA CTGAAGTG
2462

879
UUCAGGUG G UGUGUCAG
877
CTGACACA GGCTAGCTACAACGA CACCTGAA
2463

881
CAGGUGGU G UGUCAGAG
878
CTCTGACA GGCTAGCTACAACGA ACCACCTG
2464

883
GGUGGUGU G UCAGAGUC
879
GACTCTGA GGCTAGCTACAACGA ACACCACC
2465

889
GUGUCAGA G UCUCCCAG
880
CTGGGAGA GGCTAGCTACAACGA TCTGACAC
2466

897
GUCUCCCA G UGGAUUUU
881
AAAATCCA GGCTAGCTACAACGA TGGGAGAC
2467

910
UUUUCUAA G CACAUUCA
882
TGAATGTG GGCTAGCTACAACGA TTAGAAAA
2468

941
UCUCUCCA G UAAACAGU
883
ACTGTTTA GGCTAGCTACAACGA TGGAGAGA
2469

948
AGUAAACA G UGCUUCUA
884
TAGAAGCA GGCTAGCTACAACGA TGTTTACT
2470

950
UAAACAGU G CUUCUAGU
885
ACTAGAAG GGCTAGCTACAACGA ACTGTTTA
2471

957
UGCUUCUA G UGAAGAAA
886
TTTCTTCA GGCTAGCTACAACGA TAGAAGCA
2472

968
AAGAAAAU G UGAAGUAC
887
GTACTTCA GGCTAGCTACAACGA ATTTTCTT
2473

973
AAUGUGAA G UACUCCAG
888
CTGGAGTA GGCTAGCTACAACGA TTCACATT
2474

981
GUACUCCA G UUCUCAGC
889
GCTGAGAA GGCTAGCTACAACGA TGGAGTAC
2475

988
AGUUCUCA G CCAGAACC
890
GGTTCTGG GGCTAGCTACAACGA TGAGAACT
2476

999
AGAACCCC G CACAGGUC
891
GACCTGTG GGCTAGCTACAACGA GGGGTTCT
2477

1005
CCGCACAG G UCUUUCCU
892
AGGAAAGA GGCTAGCTACAACGA CTGTGCGG
2478

1026
GGAUACCA G CCCCUCAU
893
ATGAGGGG GGCTAGCTACAACGA TGGTATCC
2479

1049
AUAAAUUG G UACAAGGG
894
CCCTTGTA GGCTAGCTACAACGA CAATTTAT
2480

1062
AGGGAUCA G CUUUUCCC
895
GGGAAAAG GGCTAGCTACAACGA TGATCCCT
2481

1072
UUUUCCCA G CCCACAUG
896
CATGTGGG GGCTAGCTACAACGA TGGGAAAA
2482

1080
GCCCACAU G UCCUGAUC
897
GATCAGGA GGCTAGCTACAACGA ATGTGGGC
2483

1093
GAUCAUAU G CUUUUGAA
898
TTCAAAAG GGCTAGCTACAACGA ATATGATC
2484

1104
UUUGAAUA G UCAGUUAC
899
GTAACTGA GGCTAGCTACAACGA TATTCAAA
2485

1108
AAUAGUCA G UUACUUGG
900
CCAAGTAA GGCTAGCTACAACGA TGACTATT
2486

1116
GUUACUUG G CACCCCAG
901
CTGGGGTG GGCTAGCTACAACGA CAAGTAAC
2487

1144
AACCCCUG G CAGCGGUU
902
AACCGCTG GGCTAGCTACAACGA CAGGGGTT
2488

1147
CCCUGGCA G CGGUUGGU
903
ACCAACCG GGCTAGCTACAACGA TGCCAGGG
2489

1150
UGGCAGCG G UUGGUCAA
904
TTGACCAA GGCTAGCTACAACGA CGCTGCCA
2490

1154
AGCGGUUG G UCAAAAGA
905
TCTTTTGA GGCTAGCTACAACGA CAACCGCT
2491

1190
AAUUGGAU G CAGACAAA
906
TTTGTCTG GGCTAGCTACAACGA ATCCAATT
2492

1209
UUAUCAAU G CCUGAAAG
907
CTTTCAGG GGCTAGCTACAACGA ATTGATAA
2493

1224
AGAGACUU G UGAGAAGU
908
ACTTCTCA GGCTAGCTACAACGA AAGTCTCT
2494

1231
UGUGAGAA G UUGGGCUA
909
TAGCCCAA GGCTAGCTACAACGA TTCTCACA
2495

1236
GAAGUUGG G CUAUCAAU
910
ATTGATAG GGCTAGCTACAACGA CCAACTTC
2496

1254
GAAGAAAA G UUGUAUGA
911
TCATACAA GGCTAGCTACAACGA TTTTCTTC
2497

1257
GAAAAGUU G UAUGAAUC
912
GATTCATA GGCTAGCTACAACGA AACTTTTC
2498

1268
UGAAUCAG G UUACUAUA
913
TATAGTAA GGCTAGCTACAACGA CTGATTCA
2499

1316
UUUUCAAA G UGAAUUUG
914
CAAATTCA GGCTAGCTACAACGA TTTGAAAA
2500

1324
GUGAAUUU G UUAGAAAU
915
ATTTCTAA GGCTAGCTACAACGA AAATTCAC
2501

1349
AAAUAUUG G UUGACUUC
916
GAAGTCAA GGCTAGCTACAACGA CAATATTT
2502

1360
GACUUCCG G CUUUCUAA
917
TTAGAAAG GGCTAGCTACAACGA CGGAAGTC
2503

1371
UUCUAAGG G UGAUGGAU
918
ATCCATCA GGCTAGCTACAACGA CCTTAGAA
2504

1384
GGAUUGGA G UUCAAGAG
919
CTCTTGAA GGCTAGCTACAACGA TCCAATCC
2505

1417
AAAGGGAA G CUGAUUGA
920
TCAATCAG GGCTAGCTACAACGA TTCCCTTT
2506

1430
UUGAUAUU G UGAGCAGC
921
GCTGCTCA GGCTAGCTACAACGA AATATCAA
2507

1434
UAUUGUGA G CAGCCAGA
922
TCTGGCTG GGCTAGCTACAACGA TCACAATA
2508

1437
UGUGAGCA G CCAGAAGG
923
CCTTCTGG GGCTAGCTACAACGA TGCTCACA
2509

1445
GCCAGAAG G UUUGGCUU
924
AAGCCAAA GGCTAGCTACAACGA CTTCTGGC
2510

1450
AAGGUUUG G CUUCCUGC
925
GCAGGAAG GGCTAGCTACAACGA CAAACCTT
2511

1457
GGCUUCCU G CCACAUGA
926
TCATGTGG GGCTAGCTACAACGA AGGAAGCC
2512

1477
GACCAUCG G CUCUGGGG
927
CCCCAGAG GGCTAGCTACAACGA CGATGGTC
2513

1493
GAAUCCUG G UGAAUAUA
928
TATATTCA GGCTAGCTACAACGA CAGGATTC
2514

1502
UGAAUAUA G UGCUGCUA
929
TAGCAGCA GGCTAGCTACAACGA TATATTCA
2515

1504
AAUAUAGU G CUGCUAUG
930
CATAGCAG GGCTAGCTACAACGA ACTATATT
2516

1507
AUAGUGCU G CUAUGUUG
931
CAACATAG GGCTAGCTACAACGA AGCACTAT
2517

1512
GCUGCUAU G UUGACAUU
932
AATGTCAA GGCTAGCTACAACGA ATAGCAGC
2518

1545
AUUAUCCU G UCCUGCAA
933
TTGCAGGA GGCTAGCTACAACGA AGGATAAT
2519

1550
CCUGUCCU G CAAACUGC
934
GCAGTTTG GGCTAGCTACAACGA AGGACAGG
2520

1557
UGCAAACU G CAAAUAGU
935
ACTATTTG GGCTAGCTACAACGA AGTTTGCA
2521

1564
UGCAAAUA G UAGUUCCU
936
AGGAACTA GGCTAGCTACAACGA TATTTGCA
2522

1567
AAAUAGUA G UUCCUGAA
937
TTCAGGAA GGCTAGCTACAACGA TACTATTT
2523

1576
UUCCUGAA G UGUUCACU
938
AGTGAACA GGCTAGCTACAACGA TTCAGGAA
2524

1578
CCUGAAGU G UUCACUUC
939
GAAGTGAA GGCTAGCTACAACGA ACTTCAGG
2525

1590
ACUUCCCU G UUUAUCCA
940
TGGATAAA GGCTAGCTACAACGA AGGGAAGT
2526

1619
UUUAUUUU G UUUGUUCG
941
CGAACAAA GGCTAGCTACAACGA AAAATAAA
2527

1623
UUUUGUUU G UUCGGCAU
942
ATGCCGAA GGCTAGCTACAACGA AAACAAAA
2528

1628
UUUGUUCG G CAUACAAA
943
TTTGTATG GGCTAGCTACAACGA CGAACAAA
2529

1656
UCUUAAUU G UAAGCAAA
944
TTTGCTTA GGCTAGCTACAACGA AATTAAGA
2530

1660
AAUUGUAA G CAAAACUU
945
AAGTTTTG GGCTAGCTACAACGA TTACAATT
2531

1710
UUCUUCAU G UGUGUUUA
946
TAAACACA GGCTAGCTACAACGA ATGAAGAA
2532

1712
CUUCAUGU G UGUUUAGU
947
ACTAAACA GGCTAGCTACAACGA ACATGAAG
2533

1714
UCAUGUGU G UUUAGUAU
948
ATACTAAA GGCTAGCTACAACGA ACACATGA
2534

1719
UGUGUUUA G UAUCUGAA
949
TTCAGATA GGCTAGCTACAACGA TAAACACA
2535

1743
CUCAUCUG G UGGAAACC
950
GGTTTCCA GGCTAGCTACAACGA CAGATGAG
2536

1754
GAAACCAA G UUUCAGGG
951
CCCTGAAA GGCTAGCTACAACGA TTGGTTTC
2537

1770
GGACAUGA G UUUUCCAG
952
CTGGAAAA GGCTAGCTACAACGA TCATGTCC
2538

1778
GUUUUCCA G CUUUUAUA
953
TATAAAAG GGCTAGCTACAACGA TGGAAAAC
2539

1792
AUACACAC G UAUCUCAU
954
ATGAGATA GGCTAGCTACAACGA GTGTGTAT
2540

35
GUGGAGUC A UGGCAGUG
955
CACTGCCA GGCTAGCTACAACGA GACTCCAC
2541

57
UGUGGAAG A CUGGGACU
956
AGTCCCAG GGCTAGCTACAACGA CTTCCACA
2542

63
AGACUGGG A CUUGGUGC
957
GCACCAAG GGCTAGCTACAACGA CCCAGTCT
2543

74
UGGUGCAA A CCCUGGGA
958
TCCCAGGG GGCTAGCTACAACGA TTGCACCA
2544

93
AGGUGCCU A UGGAGAAG
959
CTTCTCCA GGCTAGCTACAACGA AGGCACCT
2545

106
GAAGUUCA A CUUGCUGU
960
ACAGCAAG GGCTAGCTACAACGA TGAACTTC
2546

117
UGCUGUGA A UAGAGUAA
961
TTACTCTA GGCTAGCTACAACGA TCACAGCA
2547

125
AUAGAGUA A CUGAAGAA
962
TTCTTCAG GGCTAGCTACAACGA TACTCTAT
2548

149
CAGUGAAG A UUGUAGAU
963
ATCTACAA GGCTAGCTACAACGA CTTCACTG
2549

156
GAUUGUAG A UAUGAAGC
964
GCTTCATA GGCTAGCTACAACGA CTACAATC
2550

158
UUGUAGAU A UGAAGCGU
965
ACGCTTCA GGCTAGCTACAACGA ATCTACAA
2551

174
UGCCGUAG A CUGUCCAG
966
CTGGACAG GGCTAGCTACAACGA CTACGGCA
2552

186
UCCAGAAA A UAUUAAGA
967
TCTTAATA GGCTAGCTACAACGA TTTCTGGA
2553

188
CAGAAAAU A UUAAGAAA
968
TTTCTTAA GGCTAGCTACAACGA ATTTTCTG
2554

200
AGAAAGAG A UCUGUAUC
969
GATACAGA GGCTAGCTACAACGA CTCTTTCT
2555

206
AGAUCUGU A UCAAUAAA
970
TTTATTGA GGCTAGCTACAACGA ACAGATCT
2556

210
CUGUAUCA A UAAAAUGC
971
GCATTTTA GGCTAGCTACAACGA TGATACAG
2557

215
UCAAUAAA A UGCUAAAU
972
ATTTAGCA GGCTAGCTACAACGA TTTATTGA
2558

222
AAUGCUAA A UCAUGAAA
973
TTTCATGA GGCTAGCTACAACGA TTAGCATT
2559

225
GCUAAAUC A UGAAAAUG
974
CATTTTCA GGCTAGCTACAACGA GATTTAGC
2560

231
UCAUGAAA A UGUAGUAA
975
TTACTACA GGCTAGCTACAACGA TTTCATGA
2561

241
GUAGUAAA A UUCUAUGG
976
CCATAGAA GGCTAGCTACAACGA TTTACTAC
2562

246
AAAAUUCU A UGGUCACA
977
TGTGACCA GGCTAGCTACAACGA AGAATTTT
2563

252
CUAUGGUC A CAGGAGAG
978
CTCTCCTG GGCTAGCTACAACGA GACCATAG
2564

267
AGAAGGCA A UAUCCAAU
979
ATTGGATA GGCTAGCTACAACGA TGCCTTCT
2565

269
AAGGCAAU A UCCAAUAU
980
ATATTGGA GGCTAGCTACAACGA ATTGCCTT
2566

274
AAUAUCCA A UAUUUAUU
981
AATAAATA GGCTAGCTACAACGA TGGATATT
2567

276
UAUCCAAU A UUUAUUUC
982
GAAATAAA GGCTAGCTACAACGA ATTGGATA
2568

280
CAAUAUUU A UUUCUGGA
983
TCCAGAAA GGCTAGCTACAACGA AAATATTG
2569

291
UCUGGAGU A CUGUAGUG
984
CACTACAG GGCTAGCTACAACGA ACTCCAGA
2570

315
GCUUUUUG A CAGAAUAG
985
CTATTCTG GGCTAGCTACAACGA CAAAAAGC
2571

320
UUGACAGA A UAGAGCCA
986
TGGCTCTA GGCTAGCTACAACGA TCTGTCAA
2572

330
AGAGCCAG A CAUAGGCA
987
TGCCTATG GGCTAGCTACAACGA CTGGCTCT
2573

332
AGCCAGAC A UAGGCAUG
988
CATGCCTA GGCTAGCTACAACGA GTCTGGCT
2574

338
ACAUAGGC A UGCCUGAA
989
TTCAGGCA GGCTAGCTACAACGA GCCTATGT
2575

346
AUGCCUGA A CCAGAUGC
990
GCATCTGG GGCTAGCTACAACGA TCAGGCAT
2576

351
UGAACCAG A UGCUCAGA
991
TCTGAGCA GGCTAGCTACAACGA CTGGTTCA
2577

361
GCUCAGAG A UUCUUCCA
992
TGGAAGAA GGCTAGCTACAACGA CTCTGAGC
2578

369
AUUCUUCC A UCAACUCA
993
TGAGTTGA GGCTAGCTACAACGA GGAAGAAT
2579

373
UUCCAUCA A CUCAUGGC
994
GCCATGAG GGCTAGCTACAACGA TGATGGAA
2580

377
AUCAACUC A UGGCAGGG
995
CCCTGCCA GGCTAGCTACAACGA GAGTTGAT
2581

393
GGUGGUUU A UCUGCAUG
996
CATGCAGA GGCTAGCTACAACGA AAACCACC
2582

399
UUAUCUGC A UGGUAUUG
997
CAATACCA GGCTAGCTACAACGA GCAGATAA
2583

404
UGCAUGGU A UUGGAAUA
998
TATTCCAA GGCTAGCTACAACGA ACCATGCA
2584

410
GUAUUGGA A UAACUCAC
999
GTGAGTTA GGCTAGCTACAACGA TCCAATAC
2585

413
UUGGAAUA A CUCACAGG
1000
CCTGTGAG GGCTAGCTACAACGA TATTCCAA
2586

417
AAUAACUC A CAGGGAUA
1001
TATCCCTG GGCTAGCTACAACGA GAGTTATT
2587

423
UCACAGGG A UAUUAAAC
1002
GTTTAATA GGCTAGCTACAACGA CCCTGTGA
2588

425
ACAGGGAU A UUAAACCA
1003
TGGTTTAA GGCTAGCTACAACGA ATCCCTGT
2589

430
GAUAUUAA A CCAGAAAA
1004
TTTTCTGG GGCTAGCTACAACGA TTAATATC
2590

438
ACCAGAAA A UCUUCUGU
1005
ACAGAAGA GGCTAGCTACAACGA TTTCTGGT
2591

450
UCUGUUGG A UGAAAGGG
1006
CCCTTTCA GGCTAGCTACAACGA CCAACAGA
2592

459
UGAAAGGG A UAACCUCA
1007
TGAGGTTA GGCTAGCTACAACGA CCCTTTCA
2593

462
AAGGGAUA A CCUCAAAA
1008
TTTTGAGG GGCTAGCTACAACGA TATCCCTT
2594

470
ACCUCAAA A UCUCAGAC
1009
GTCTGAGA GGCTAGCTACAACGA TTTGAGGT
2595

477
AAUCUCAG A CUUUGGCU
1010
AGCCAAAG GGCTAGCTACAACGA CTGAGATT
2596

491
GCUUGGCA A CAGUAUUU
1011
AAATACTG GGCTAGCTACAACGA TGCCAAGC
2597

496
GCAACAGU A UUUCGGUA
1012
TACCGAAA GGCTAGCTACAACGA ACTGTTGC
2598

504
AUUUCGGU A UAAUAAUC
1013
GATTATTA GGCTAGCTACAACGA ACCGAAAT
2599

507
UCGGUAUA A UAAUCGUG
1014
CACGATTA GGCTAGCTACAACGA TATACCGA
2600

510
GUAUAAUA A UCGUGAGC
1015
GCTCACGA GGCTAGCTACAACGA TATTATAC
2601

528
UUUGUUGA A CAAGAUGU
1016
ACATCTTG GGCTAGCTACAACGA TCAACAAA
2602

533
UGAACAAG A UGUGUGGU
1017
ACCACACA GGCTAGCTACAACGA CTTGTTCA
2603

542
UGUGUGGU A CUUUACCA
1018
TGGTAAAG GGCTAGCTACAACGA ACCACACA
2604

547
GGUACUUU A CCAUAUGU
1019
ACATATGG GGCTAGCTACAACGA AAAGTACC
2605

550
ACUUUACC A UAUGUUGC
1020
GCAACATA GGCTAGCTACAACGA GGTAAAGT
2606

552
UUUACCAU A UGUUGCUC
1021
GAGCAACA GGCTAGCTACAACGA ATGGTAAA
2607

565
GCUCCAGA A CUUCUGAA
1022
TTCAGAAG GGCTAGCTACAACGA TCTGGAGC
2608

583
AGAAGAGA A UUUCAUGC
1023
GCATGAAA GGCTAGCTACAACGA TCTCTTCT
2609

588
AGAAUUUC A UGCAGAAC
1024
GTTCTGCA GGCTAGCTACAACGA GAAATTCT
2610

595
CAUGCAGA A CCAGUUGA
1025
TCAACTGG GGCTAGCTACAACGA TCTGCATG
2611

603
ACCAGUUG A UGUUUGGU
1026
ACCAAACA GGCTAGCTACAACGA CAACTGGT
2612

620
CCUGUGGA A UAGUACUU
1027
AAGTACTA GGCTAGCTACAACGA TCCACAGG
2613

625
GGAAUAGU A CUUACUGC
1028
GCAGTAAG GGCTAGCTACAACGA ACTATTCC
2614

629
UAGUACUU A CUGCAAUG
1029
CATTGCAG GGCTAGCTACAACGA AAGTACTA
2615

635
UUACUGCA A UGCUCGCU
1030
AGCGAGCA GGCTAGCTACAACGA TGCAGTAA
2616

649
GCUGGAGA A UUGCCAUG
1031
CATGGCAA GGCTAGCTACAACGA TCTCCAGC
2617

655
GAAUUGCC A UGGGACCA
1032
TGGTCCCA GGCTAGCTACAACGA GGCAATTC
2618

660
GCCAUGGG A CCAACCCA
1033
TGGGTTGG GGCTAGCTACAACGA CCCATGGC
2619

664
UGGGACCA A CCCAGUGA
1034
TCACTGGG GGCTAGCTACAACGA TGGTCCCA
2620

672
ACCCAGUG A CAGCUGUC
1035
GACAGCTG GGCTAGCTACAACGA CACTGGGT
2621

687
UCAGGAGU A UUCUGACU
1036
AGTCAGAA GGCTAGCTACAACGA ACTCCTGA
2622

693
GUAUUCUG A CUGGAAAG
1037
CTTTCCAG GGCTAGCTACAACGA CAGAATAC
2623

710
AAAAAAAA A CAUACCUC
1038
GAGGTATG GGCTAGCTACAACGA TTTTTTTT
2624

712
AAAAAAAC A UACCUCAA
1039
TTGAGGTA GGCTAGCTACAACGA GTTTTTTT
2625

714
AAAAACAU A CCUCAACC
1040
GGTTGAGG GGCTAGCTACAACGA ATGTTTTT
2626

720
AUACCUCA A CCCUUGGA
1041
TCCAAGGG GGCTAGCTACAACGA TGAGGTAT
2627

734
GGAAAAAA A UCGAUUCU
1042
AGAATCGA GGCTAGCTACAACGA TTTTTTCC
2628

738
AAAAAUCG A UUCUGCUC
1043
GAGCAGAA GGCTAGCTACAACGA CGATTTTT
2629

762
UCUGCUGC A UAAAAUCU
1044
AGATTTTA GGCTAGCTACAACGA GCAGCAGA
2630

767
UGCAUAAA A UCUUAGUU
1045
AACTAAGA GGCTAGCTACAACGA TTTATGCA
2631

780
AGUUGAGA A UCCAUCAG
1046
CTGATGGA GGCTAGCTACAACGA TCTCAACT
2632

784
GAGAAUCC A UCAGCAAG
1047
CTTGCTGA GGCTAGCTACAACGA GGATTCTC
2633

794
CAGCAAGA A UUACCAUU
1048
AATGGTAA GGCTAGCTACAACGA TCTTGCTG
2634

797
CAAGAAUU A CCAUUCCA
1049
TGGAATGG GGCTAGCTACAACGA AATTCTTG
2635

800
GAAUUACC A UUCCAGAC
1050
GTCTGGAA GGCTAGCTACAACGA GGTAATTC
2636

807
CAUUCCAG A CAUCAAAA
1051
TTTTGATG GGCTAGCTACAACGA CTGGAATG
2637

809
UUCCAGAC A UCAAAAAA
1052
TTTTTTGA GGCTAGCTACAACGA GTCTGGAA
2638

819
CAAAAAAG A UAGAUGGU
1053
ACCATCTA GGCTAGCTACAACGA CTTTTTTG
2639

823
AAAGAUAG A UGGUACAA
1054
TTGTACCA GGCTAGCTACAACGA CTATCTTT
2640

828
UAGAUGGU A CAACAAAC
1055
GTTTGTTG GGCTAGCTACAACGA ACCATCTA
2641

831
AUGGUACA A CAAACCCC
1056
GGGGTTTG GGCTAGCTACAACGA TGTACCAT
2642

835
UACAACAA A CCCCUCAA
1057
TTGAGGGG GGCTAGCTACAACGA TTGTTGTA
2643

869
CCCGAGUC A CUUCAGGU
1058
ACCTGAAG GGCTAGCTACAACGA GACTCGGG
2644

901
CCCAGUGG A UUUUCUAA
1059
TTAGAAAA GGCTAGCTACAACGA CCACTGGG
2645

912
UUCUAAGC A CAUUCAAU
1060
ATTGAATG GGCTAGCTACAACGA GCTTAGAA
2646

914
CUAAGCAC A UUCAAUCC
1061
GGATTGAA GGCTAGCTACAACGA GTGCTTAG
2647

919
CACAUUCA A UCCAAUUU
1062
AAATTGGA GGCTAGCTACAACGA TGAATGTG
2648

924
UCAAUCCA A UUUGGACU
1063
AGTCCAAA GGCTAGCTACAACGA TGGATTGA
2649

930
CAAUUUGG A CUUCUCUC
1064
GAGAGAAG GGCTAGCTACAACGA CCAAATTG
2650

945
UCCAGUAA A CAGUGCUU
1065
AAGCACTG GGCTAGCTACAACGA TTACTGGA
2651

966
UGAAGAAA A UGUGAAGU
1066
ACTTCACA GGCTAGCTACAACGA TTTCTTCA
2652

975
UGUGAAGU A CUCCAGUU
1067
AACTGGAG GGCTAGCTACAACGA ACTTCACA
2653

994
CAGCCAGA A CCCCGCAC
1068
GTGCGGGG GGCTAGCTACAACGA TCTGGCTG
2654

1001
AACCCCGC A CAGGUCUU
1069
AAGACCTG GGCTAGCTACAACGA GCGGGGTT
2655

1015
CUUUCCUU A UGGGAUAC
1070
GTATCCCA GGCTAGCTACAACGA AAGGAAAG
2656

1020
CUUAUGGG A UACCAGCC
1071
GGCTGGTA GGCTAGCTACAACGA CCCATAAG
2657

1022
UAUGGGAU A CCAGCCCC
1072
GGGGCTGG GGCTAGCTACAACGA ATCCCATA
2658

1033
AGCCCCUC A UACAUUGA
1073
TCAATGTA GGCTAGCTACAACGA GAGGGGCT
2659

1035
CCCCUCAU A CAUUGAUA
1074
TATCAATG GGCTAGCTACAACGA ATGAGGGG
2660

1037
CCUCAUAC A UUGAUAAA
1075
TTTATCAA GGCTAGCTACAACGA GTATGAGG
2661

1041
AUACAUUG A UAAAUUGG
1076
CCAATTTA GGCTAGCTACAACGA CAATGTAT
2662

1045
AUUGAUAA A UUGGUACA
1077
TGTACCAA GGCTAGCTACAACGA TTATCAAT
2663

1051
AAAUUGGU A CAAGGGAU
1078
ATCCCTTG GGCTAGCTACAACGA ACCAATTT
2664

1058
UACAAGGG A UCAGCUUU
1079
AAAGCTGA GGCTAGCTACAACGA CCCTTGTA
2665

1076
CCCAGCCC A CAUGUCCU
1080
AGGACATG GGCTAGCTACAACGA GGGCTGGG
2666

1078
CAGCCCAC A UGUCCUGA
1081
TCAGGACA GGCTAGCTACAACGA GTGGGCTG
2667

1086
AUGUCCUG A UCAUAUGC
1082
GCATATGA GGCTAGCTACAACGA CAGGACAT
2668

1089
UCCUGAUC A UAUGCUUU
1083
AAAGCATA GGCTAGCTACAACGA GATCAGGA
2669

1091
CUGAUCAU A UGCUUUUG
1084
CAAAAGCA GGCTAGCTACAACGA ATGATCAG
2670

1101
GCUUUUGA A UAGUCAGU
1085
ACTGACTA GGCTAGCTACAACGA TCAAAAGC
2671

1111
AGUCAGUU A CUUGGCAC
1086
GTGCCAAG GGCTAGCTACAACGA AACTGACT
2672

1118
UACUUGGC A CCCCAGGA
1087
TCCTGGGG GGCTAGCTACAACGA GCCAAGTA
2673

1126
ACCCCAGG A UCCUCACA
1088
TGTGAGGA GGCTAGCTACAACGA CCTGGGGT
2674

1132
GGAUCCUC A CAGAACCC
1089
GGGTTCTG GGCTAGCTACAACGA GAGGATCC
2675

1137
CUCACAGA A CCCCUGGC
1090
GCCAGGGG GGCTAGCTACAACGA TCTGTGAG
2676

1163
UCAAAAGA A UGACACGA
1091
TCGTGTCA GGCTAGCTACAACGA TCTTTTGA
2677

1166
AAAGAAUG A CACGAUUC
1092
GAATCGTG GGCTAGCTACAACGA CATTCTTT
2678

1168
AGAAUGAC A CGAUUCUU
1093
AAGAATCG GGCTAGCTACAACGA GTCATTCT
2679

1171
AUGACACG A UUCUUUAC
1094
GTAAAGAA GGCTAGCTACAACGA CGTGTCAT
2680

1178
GAUUCUUU A CCAAAUUG
1095
CAATTTGG GGCTAGCTACAACGA AAAGAATC
2681

1183
UUUACCAA A UUGGAUGC
1096
GCATCCAA GGCTAGCTACAACGA TTGGTAAA
2682

1188
CAAAUUGG A UGCAGACA
1097
TGTCTGCA GGCTAGCTACAACGA CCAATTTG
2683

1194
GGAUGCAG A CAAAUCUU
1098
AAGATTTG GGCTAGCTACAACGA CTGCATCC
2684

1198
GCAGACAA A UCUUAUCA
1099
TGATAAGA GGCTAGCTACAACGA TTGTCTGC
2685

1203
CAAAUCUU A UCAAUGCC
1100
GGCATTGA GGCTAGCTACAACGA AAGATTTG
2686

1207
UCUUAUCA A UGCCUGAA
1101
TTCAGGCA GGCTAGCTACAACGA TGATAAGA
2687

1220
UGAAAGAG A CUUGUGAG
1102
CTCACAAG GGCTAGCTACAACGA CTCTTTCA
2688

1239
GUUGGGCU A UCAAUGGA
1103
TCCATTGA GGCTAGCTACAACGA AGCCCAAC
2689

1243
GGCUAUCA A UGGAAGAA
1104
TTCTTCCA GGCTAGCTACAACGA TGATAGCC
2690

1259
AAAGUUGU A UGAAUCAG
1105
CTGATTCA GGCTAGCTACAACGA ACAACTTT
2691

1263
UUGUAUGA A UCAGGUUA
1106
TAACCTGA GGCTAGCTACAACGA TCATACAA
2692

1271
AUCAGGUU A CUAUAUCA
1107
TGATATAG GGCTAGCTACAACGA AACCTGAT
2693

1274
AGGUUACU A UAUCAACA
1108
TGTTGATA GGCTAGCTACAACGA AGTAACCT
2694

1276
GUUACUAU A UCAACAAC
1109
GTTGTTGA GGCTAGCTACAACGA ATAGTAAC
2695

1280
CUAUAUCA A CAACUGAU
1110
ATCAGTTG GGCTAGCTACAACGA TGATATAG
2696

1283
UAUCAACA A CUGAUAGG
1111
CCTATCAG GGCTAGCTACAACGA TGTTGATA
2697

1287
AACAACUG A UAGGAGAA
1112
TTCTCCTA GGCTAGCTACAACGA CAGTTGTT
2698

1296
UAGGAGAA A CAAUAAAC
1113
GTTTATTG GGCTAGCTACAACGA TTCTCCTA
2699

1299
GAGAAACA A UAAACUCA
1114
TGAGTTTA GGCTAGCTACAACGA TGTTTCTC
2700

1303
AACAAUAA A CUCAUUUU
1115
AAAATGAG GGCTAGCTACAACGA TTATTGTT
2701

1307
AUAAACUC A UUUUCAAA
1116
TTTGAAAA GGCTAGCTACAACGA GAGTTTAT
2702

1320
CAAAGUGA A UUUGUUAG
1117
CTAACAAA GGCTAGCTACAACGA TCACTTTG
2703

1331
UGUUAGAA A UGGAUGAU
1118
ATCATCCA GGCTAGCTACAACGA TTCTAACA
2704

1335
AGAAAUGG A UGAUAAAA
1119
TTTTATCA GGCTAGCTACAACGA CCATTTCT
2705

1338
AAUGGAUG A UAAAAUAU
1120
ATATTTTA GGCTAGCTACAACGA CATCCATT
2706

1343
AUGAUAAA A UAUUGGUU
1121
AACCAATA GGCTAGCTACAACGA TTTATCAT
2707

1345
GAUAAAAU A UUGGUUGA
1122
TCAACCAA GGCTAGCTACAACGA ATTTTATC
2708

1353
AUUGGUUG A CUUCCGGC
1123
GCCGGAAG GGCTAGCTACAACGA CAACCAAT
2709

1374
UAAGGGUG A UGGAUUGG
1124
CCAATCCA GGCTAGCTACAACGA CACCCTTA
2710

1378
GGUGAUGG A UUGGAGUU
1125
AACTCCAA GGCTAGCTACAACGA CCATCACC
2711

1393
UUCAAGAG A CACUUCCU
1126
AGGAAGTG GGCTAGCTACAACGA CTCTTGAA
2712

1395
CAAGAGAC A CUUCCUGA
1127
TCAGGAAG GGCTAGCTACAACGA GTCTCTTG
2713

1406
UCCUGAAG A UUAAAGGG
1128
CCCTTTAA GGCTAGCTACAACGA CTTCAGGA
2714

1421
GGAAGCUG A UUGAUAUU
1129
AATATCAA GGCTAGCTACAACGA CAGCTTCC
2715

1425
GCUGAUUG A UAUUGUGA
1130
TCACAATA GGCTAGCTACAACGA CAATCAGC
2716

1427
UGAUUGAU A UUGUGAGC
1131
GCTCACAA GGCTAGCTACAACGA ATCAATCA
2717

1460
UUCCUGCC A CAUGAUCG
1132
CGATCATG GGCTAGCTACAACGA GGCAGGAA
2718

1462
CCUGCCAC A UGAUCGGA
1133
TCCGATCA GGCTAGCTACAACGA GTGGCAGG
2719

1465
GCCACAUG A UCGGACCA
1134
TGGTCCGA GGCTAGCTACAACGA CATGTGGC
2720

1470
AUGAUCGG A CCAUCGGC
1135
GCCGATGG GGCTAGCTACAACGA CCGATCAT
2721

1473
AUCGGACC A UCGGCUCU
1136
AGAGCCGA GGCTAGCTACAACGA GGTCCGAT
2722

1487
UCUGGGGA A UCCUGGUG
1137
CACCAGGA GGCTAGCTACAACGA TCCCCAGA
2723

1497
CCUGGUGA A UAUAGUGC
1138
GCACTATA GGCTAGCTACAACGA TCACCAGG
2724

1499
UGGUGAAU A UAGUGCUG
1139
CAGCACTA GGCTAGCTACAACGA ATTCACCA
2725

1510
GUGCUGCU A UGUUGACA
1140
TGTCAACA GGCTAGCTACAACGA AGCAGCAC
2726

1516
CUAUGUUG A CAUUAUUC
1141
GAATAATG GGCTAGCTACAACGA CAACATAG
2727

1518
AUGUUGAC A UUAUUCUU
1142
AAGAATAA GGCTAGCTACAACGA GTCAACAT
2728

1521
UUGACAUU A UUCUUCCU
1143
AGGAAGAA GGCTAGCTACAACGA AATGTCAA
2729

1537
UAGAGAAG A UUAUCCUG
1144
CAGGATAA GGCTAGCTACAACGA CTTCTCTA
2730

1540
AGAAGAUU A UCCUGUCC
1145
GGACAGGA GGCTAGCTACAACGA AATCTTCT
2731

1554
UCCUGCAA A CUGCAAAU
1146
ATTTGCAG GGCTAGCTACAACGA TTGCAGGA
2732

1561
AACUGCAA A UAGUAGUU
1147
AACTACTA GGCTAGCTACAACGA TTGCAGTT
2733

1582
AAGUGUUC A CUUCCCUG
1148
CAGGGAAG GGCTAGCTACAACGA GAACACTT
2734

1594
CCCUGUUU A UCCAAACA
1149
TGTTTGGA GGCTAGCTACAACGA AAACAGGG
2735

1600
UUAUCCAA A CAUCUUCC
1150
GGAAGATG GGCTAGCTACAACGA TTGGATAA
2736

1602
AUCCAAAC A UCUUCCAA
1151
TTGGAAGA GGCTAGCTACAACGA GTTTGGAT
2737

1610
AUCUUCCA A UUUAUUUU
1152
AAAATAAA GGCTAGCTACAACGA TGGAAGAT
2738

1614
UCCAAUUU A UUUUGUUU
1153
AAACAAAA GGCTAGCTACAACGA AAATTGGA
2739

1630
UGUUCGGC A UACAAAUA
1154
TATTTGTA GGCTAGCTACAACGA GCCGAACA
2740

1632
UUCGGCAU A CAAAUAAU
1155
ATTATTTG GGCTAGCTACAACGA ATGCCGAA
2741

1636
GCAUACAA A UAAUACCU
1156
AGGTATTA GGCTAGCTACAACGA TTGTATGC
2742

1639
UACAAAUA A UACCUAUA
1157
TATAGGTA GGCTAGCTACAACGA TATTTGTA
2743

1641
CAAAUAAU A CCUAUAUC
1158
GATATAGG GGCTAGCTACAACGA ATTATTTG
2744

1645
UAAUACCU A UAUCUUAA
1159
TTAAGATA GGCTAGCTACAACGA AGGTATTA
2745

1647
AUACCUAU A UCUUAAUU
1160
AATTAAGA GGCTAGCTACAACGA ATAGGTAT
2746

1653
AUAUCUUA A UUGUAAGC
1161
GCTTACAA GGCTAGCTACAACGA TAAGATAT
2747

1665
UAAGCAAA A CUUUGGGG
1162
CCCCAAAG GGCTAGCTACAACGA TTTGCTTA
2748

1679
GGGAAAGG A UGAAUAGA
1163
TCTATTCA GGCTAGCTACAACGA CCTTTCCC
2749

1683
AAGGAUGA A UAGAAUUC
1164
GAATTCTA GGCTAGCTACAACGA TCATCCTT
2750

1688
UGAAUAGA A UUCAUUUG
1165
CAAATGAA GGCTAGCTACAACGA TCTATTCA
2751

1692
UAGAAUUC A UUUGAUUA
1166
TAATCAAA GGCTAGCTACAACGA GAATTCTA
2752

1697
UUCAUUUG A UUAUUUCU
1167
AGAAATAA GGCTAGCTACAACGA CAAATGAA
2753

1700
AUUUGAUU A UUUCUUCA
1168
TGAAGAAA GGCTAGCTACAACGA AATCAAAT
2754

1708
AUUUCUUC A UGUGUGUU
1169
AACACACA GGCTAGCTACAACGA GAAGAAAT
2755

1721
UGUUUAGU A UCUGAAUU
1170
AATTCAGA GGCTAGCTACAACGA ACTAAACA
2756

1727
GUAUCUGA A UUUGAAAC
1171
GTTTCAAA GGCTAGCTACAACGA TCAGATAC
2757

1734
AAUUUGAA A CUCAUCUG
1172
CAGATGAG GGCTAGCTACAACGA TTCAAATT
2758

1738
UGAAACUC A UCUGGUGG
1173
CCACCAGA GGCTAGCTACAACGA GAGTTTCA
2759

1749
UGGUGGAA A CCAAGUUU
1174
AAACTTGG GGCTAGCTACAACGA TTCCACCA
2760

1764
UUCAGGGG A CAUGAGUU
1175
AACTCATG GGCTAGCTACAACGA CCCCTGAA
2761

1766
CAGGGGAC A UGAGUUUU
1176
AAAACTCA GGCTAGCTACAACGA GTCCCCTG
2762

1784
CAGCUUUU A UACACACG
1177
CGTGTGTA GGCTAGCTACAACGA AAAAGCTG
2763

1786
GCUUUUAU A CACACGUA
1178
TACGTGTG GGCTAGCTACAACGA ATAAAAGC
2764

1788
UUUUAUAC A CACGUAUC
1179
GATACGTG GGCTAGCTACAACGA GTATAAAA
2765

1790
UUAUACAC A CGUAUCUC
1180
GAGATACG GGCTAGCTACAACGA GTGTATAA
2766

1794
ACACACGU A UCUCAUUU
1181
AAATGAGA GGCTAGCTACAACGA ACGTGTGT
2767

1799
CGUAUCUC A UUUUUAUC
1182
GATAAAAA GGCTAGCTACAACGA GAGATACG
2768

1805
UCAUUUUU A UCAAAACA
1183
TGTTTTGA GGCTAGCTACAACGA AAAAATGA
2769

1811
UUAUCAAA A CAUUUUGU
1184
ACAAAATG GGCTAGCTACAACGA TTTGATAA
2770

1813
AUCAAAAC A UUUUGUUU
1185
AAACAAAA GGCTAGCTACAACGA GTTTTGAT
2771

Input Sequence = AF016582 Cut Site = R/Y

Stem Length = 8. Core Sequence = GGCTAGCTACAACGA

AF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

[0180]

8

TABLE VIII

Human Chk1 Amberzyme Ribozyme and Substrate Sequence

Pos
Substrate
Seq ID
Ribozyme
Rz Seq ID

10
GCCGGACA G UCCGCCGA
791
UCGGCGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCCGGC
2772

14
GACAGUCC G CCGAGGUG
792
CACCUCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGACUGUC
2773

20
CCGCCGAG G UGCUCGGU
793
ACCGAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGGCGG
2774

22
GCCGAGGU G CUCGGUGG
794
CCACCGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCGGC
2775

27
GGUGCUCG G UGGAGUCA
795
UGACUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGCACC
2776

32
UCGGUGGA G UCAUGGCA
796
UGCCAUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCACCGA
2777

38
GAGUCAUG G CAGUGCCC
797
GGGCACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGACUC
2778

41
UCAUGGCA G UGCCCUUU
798
AAAGGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCAUGA
2779

43
AUGGCAGU G CCCUUUGU
799
ACAAAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGCCAU
2780

50
UGCCCUUU G UGGAAGAC
800
GUCUUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAGGGCA
2781

68
GGGACUUG G UGCAAACC
801
GGUUUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGUCCC
2782

70
GACUUGGU G CAAACCCU
802
AGGGUUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAAGUC
2783

87
GGGAGAAG G UGCCUAUG
803
CAUAGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCUCCC
2784

89
GAGAAGGU G CCUAUGGA
804
UCCAUAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUUCUC
2785

101
AUGGAGAA G UUCAACUU
805
AAGUUGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCCAU
2786

110
UUCAACUU G CUGUGAAU
806
AUUCACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUUGAA
2787

113
AACUUGCU G UGAAUAGA
807
UCUAUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAAGUU
2788

122
UGAAUAGA G UAACUGAA
808
UUCAGUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUAUUCA
2789

134
CUGAAGAA G CAGUCGCA
809
UGCGACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUUCAG
2790

137
AAGAAGCA G UCGCAGUG
810
CACUGCGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUUCUU
2791

140
AAGCAGUC G CAGUGAAG
811
CUUCACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GACUGCUU
2792

143
CAGUCGCA G UGAAGAUU
812
AAUCUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCGACUG
2793

152
UGAAGAUU G UAGAUAUG
813
CAUAUCUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUCUUCA
2794

163
GAUAUGAA G CGUGCCGU
814
ACGGCACG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAUAUC
2795

165
UAUGAAGC G UGCCGUAG
815
CUACGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAUAUC
2796

167
UGAAGCGU G CCGUAGAC
816
GUCUACGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGCUUCA
2797

170
AGCGUGCC G UAGACUGU
817
ACAGUCUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCACGCU
2798

177
CGUAGACU G UCCAGAAA
818
UUUCUGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUCUACG
2799

204
AGAGAUCU G UAUCAAUA
819
UAUUGAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUCUCU
2800

217
AAUAAAAU G CUAAAUCA
820
UGAUUUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUUAUU
2801

233
AUGAAAAU G UAGUAAAA
821
UUUUACUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUUCAU
2802

236
AAAAUGUA G UAAAAUUC
822
GAAUUUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACAUUUU
2803

249
AUUCUAUG G UCACAGGA
823
UCCUGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUAGAAU
2804

264
GAGAGAAG G CAAUAUCC
824
GGAUAUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCUCUC
2805

289
UUUCUGGA G UACUGUAG
825
CUACAGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGAAA
2806

294
GGAGUACU G UAGUGGAG
826
CUCCACUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUACUCC
2807

297
GUACUGUA G UGGAGGAG
827
CUCCUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACAGUAC
2808

307
GGAGGAGA G CUUUUUGA
828
UCAAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUCCUCC
2809

325
AGAAUAGA G CCAGACAU
829
AUGUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUAUUCU
2810

336
AGACAUAG G CAUGCCUG
830
CAGGCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUAUGUCU
2811

340
AUAGGCAU G CCUGAACC
831
GGUUCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCCUAU
2812

353
AACCAGAU G CUCAGAGA
832
UCUCUGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUGGUU
2813

380
AACUCAUG G CAGGGGUG
833
CACCCCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGAGUU
2814

386
UGGCAGGG G UGGUUUAU
834
AUAAACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGCCA
2815

389
CAGGGGUG G UUUAUCUG
835
CAGAUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCCCUG
2816

397
GUUUAUCU G CAUGGUAU
836
AUACCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUAAAC
2817

402
UCUGCAUG G UAUUGGAA
837
UUCCAAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGCAGA
2818

445
AAUCUUCU G UUGGAUGA
838
UCAUCCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAGAUU
2819

483
AGACUUUG G CUUGGCAA
839
UUGCCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAAGUCU
2820

488
UUGGCUUG G CAACAGUA
840
UACUGUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGCCAA
2821

494
UGGCAACA G UAUUUCGG
841
CCGAAAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUUGCCA
2822

502
GUAUUUCG G UAUAAUAA
842
UUAUUAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAAAUAC
2823

513
UAAUAAUC G UGAGCGUU
843
AACGCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUUAUUA
2824

517
AAUCGUGA G CGUUUGUU
844
AACAAACG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGU UCACGAUU
2825

519
UCGUGAGC G UUUGUUGA
845
UCAACAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUCACGA
2826

523
GAGCGUUU G UUGAACAA
846
UUGUUCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACGCUC
2827

535
AACAAGAU G UGUGGUAC
847
GUACCACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUUGUU
2828

537
CAAGAUGU G UGGUACUU
848
AAGUACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAUCUUG
2829

540
GAUGUGUG G UACUUUAC
849
GUAAAGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACACAUC
2830

554
UACCAUAU G UUGCUCCA
850
UGGAGCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUGGUA
2831

557
CAUAUGUU G CUCCAGAA
851
UUCUGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAUAUG
2832

590
AAUUUCAU G CAGAACCA
852
UGGUUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAAAUU
2833

599
CAGAACCA G UUGAUGUU
853
AACAUCAA GGAGGAAACUCC CU UCAACGACAUCGUCCGGG UGGUUCUG
2834

605
CAGUUGAU G UUUGGUCC
854
GGACCAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAACUG
2835

610
GAUGUUUG G UCCUGUGG
855
CCACAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAACAUC
2836

615
UUGGUCCU G UGGAAUAG
856
CUAUUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGACCAA
2837

623
GUGGAAUA G UACUUACU
857
AGUAAGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUUCCAC
2838

632
UACUUACU G CAAUGCUC
858
GAGCAUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUAAGUA
2839

637
ACUGCAAU G CUCGCUGG
859
CCAGCGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUGCAGU
2840

641
CAAUGCUC G CUGGAGAA
860
UUCUCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGCAUUG
2841

652
GGAGAAUU G CCAUGGGA
861
UCCCAUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUUCUCC
2842

669
CCAACCCA G UGACAGCU
862
AGCUGUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGUUGG
2843

675
CAGUGACA G CUGUCAGG
863
CCUGACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCACUG
2844

676
UGACAGCU G UCAGGAGU
864
ACUCCUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUGUCA
2845

685
UGUCAGGA G UAUUCUGA
865
UCAGAAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUGACA
2846

743
UCGAUUCU G CUCCUCCA
866
UAGAGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAUCGA
2847

752
CUCCUCUA G CUCUGCUG
867
CAGCAGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAGAGGAG
2848

757
CUAGCUCU G CUGCAUAA
868
UUAUGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUAGC
2849

760
GCUCUGCU G CAUAAAAU
869
AUUUUAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAGAGC
2850

773
AAAUCUUA G UUGAGAAU
870
AUUCUCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAGAUUU
2851

788
AUCCAUCA G CAAGAAUU
871
AAUUCUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAUGGAU
2852

826
GAUAGAUG G UACAACAA
872
UUGUUGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCUAUC
2853

851
AGAAAGGG G CAAAAAGG
873
CCUUUUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUUUCU
2854

859
GCAAAAAG G CCCCGAGU
874
ACUCGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUUUGC
2855

866
GGCCCCGA G UCACUUCA
875
UGAAGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGGGCC
2856

876
CACUUCAG G UGGUGUGU
876
ACACACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGUG
2857

879
UUCAGGUG G UGUGUCAG
877
CUGACACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCUGAA
2858

881
CAGGUGGU G UGUCAGAG
878
CUCUGACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCUG
2859

883
GGUGGUGU G UCAGAGUC
879
GACUCUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACCACC
2860

889
GUGUCAGA G UCUCCCAG
880
CUGGGAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGACAC
2861

897
GUCUCCCA G UGGAUUUU
881
AAAAUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGAGAC
2862

910
UUUUCUAA G CACAUUCA
882
UGAAUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAGAAAA
2863

941
UCUCUCCA G UAAACAGU
883
ACUGUUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGAGA
2864

948
AGUAAACA G UGCUUCUA
884
UAGAAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUUUACU
2865

950
UAAACAGU G CUUCUAGU
885
ACUAGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGUUUA
2866

957
UGCUUCUA G UGAAGAAA
886
UUUCUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAGAAGCA
2867

968
AAGAAAAU G UGAAGUAC
887
GUACUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUUCUU
2868

973
AAUGUGAA G UACUCCAG
888
CUGGAGUA GGAGGAAACUCC CU UCAACGACAUCGUCCGGG UUCACAUU
2869

981
GUACUCCA G UUCUCAGC
889
GCUGAGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGUAC
2870

988
AGUUCUCA G CCAGAACC
890
GGUUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGAACU
2871

999
AGAACCCC G CACAGGUC
891
GACCUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGGUUCU
2872

1005
CCGCACAG G UCUUUCCU
892
AGGAAAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUGCGG
2873

1026
GGAUACCA G CCCCUCAU
893
AUGAGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUAUCC
2874

1049
AUAAAUUG G UACAAGGG
894
CCCUUGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUUUAU
2875

1062
AGGGAUCA G CUUUUCCC
895
GGGAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAUCCCU
2876

1072
UUUUCCCA G CCCACAUG
896
CAUGUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGAAAA
2877

1080
GCCCACAU G UCCUGAUC
897
GAUCAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUGGGC
2878

1093
GAUCAUAU G CUUUUGAA
898
UUCAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUGAUC
2879

1104
UUUGAAUA G UCAGUUAC
899
GUAACUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUUCAAA
2880

1108
AAUAGUCA G UUACUUGG
900
CCAAGUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGACUAUU
2881

1116
GUUACUUG G CACCCCAG
901
CUGGGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGUAAC
2882

1144
AACCCCUG G CAGCGGUU
902
AACCGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGGUU
2883

1147
CCCUGGCA G CGGUUGGU
903
ACCAACCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCAGGG
2884

1150
UGGCAGCG G UUGGUCAA
904
UUGACCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCUGCCA
2885

1154
AGCGGUUG G UCAAAAGA
905
UCUUUUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACCGCU
2886

1190
AAUUGGAU G CAGACAAA
906
UUUGUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCAAUU
2887

1209
UUAUCAAU G CCUGAAAG
907
CUUUCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUGAUAA
2888

1224
AGAGACUU G UGAGAAGU
908
ACUUCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUCUCU
2889

1231
UGUGAGAA G UUGGGCUA
909
UAGCCCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCACA
2890

1236
GAAGUUGG G CUAUCAAU
910
AUUGAUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAACUUC
2891

1254
GAAGAAAA G UUGUAUGA
911
UCAUACAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUCUUC
2892

1257
GAAAAGUU G UAUGAAUC
912
GAUUCAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACUUUUC
2893

1268
UGAAUCAG G UUACUAUA
913
UAUAGUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAUUCA
2894

1316
UUUUCAAA G UGAAUUUG
914
CAAAUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUGAAAA
2895

1324
GUGAAUUU G UUAGAAAU
915
AUUUCUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUUCAC
2896

1349
AAAUAUUG G UUGACUUC
916
GAAGUCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUAUUU
2897

1360
GACUUCCG G CUUUCUAA
917
UUAGAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGAAGUC
2898

1371
UUCUAAGG G UGAUGGAU
918
AUCCAUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUAGAA
2899

1384
GGAUUGGA G UUCAAGAG
919
CUCUUGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAAUCC
2900

1417
AAAGGGAA G CUGAUUGA
920
UCAAUCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCCUUU
2901

1430
UUGAUAUU G UGAGCAGC
921
GCUGCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUAUCAA
2902

1434
UAUUGUGA G CAGCCAGA
922
UCUGGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACAAUA
2903

1437
UGUGAGCA G CCAGAAGG
923
CCUUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUCACA
2904

1445
GCCAGAAG G UUUGGCUU
924
AAGCCAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCUGGC
2905

1450
AAGGUUUG G CUUCCUGC
925
CCAGGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAACCUU
2906

1457
GGCUUCCU G CCACAUGA
926
UCAUGUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAAGCC
2907

1477
GACCAUCG G CUCUGGGG
927
CCCCAGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAUGGUC
2908

1493
GAAUCCUG G UGAAUAUA
928
UAUAUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGAUUC
2909

1502
UGAAUAUA G UGCUGCUA
929
UAGCAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUAUUCA
2910

1504
AAUAUAGU G CUGCUAUG
930
CAUAGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUAUAUU
2911

1507
AUAGUGCU G CUAUGUUG
931
CAACAUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCACUAU
2912

1512
GCUGCUAU G UUGACAUU
932
AAUGUCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAGCAGC
2913

1545
AUUAUCCU G UCCUGCAA
933
UUGCAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAUAAU
2914

1550
CCUGUCCU G CAAACUGC
934
GCAGUUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCCGG AGGACAGG
2915

1557
UGCAAACU G CAAAUAGU
935
ACUAUUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCCGG AGUUUGCA
2916

1564
UGCAAAUA G UAGUUCCU
936
AGGAACUA GGAGGAAACUCC CU UCAAGGACAUCGUCCCGG UAUUUGCA
2917

1567
AAAUAGUA G UUCCUGAA
937
UUCAGGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACUAUUU
2918

1576
UUCCUGAA G UGUUCACU
938
AGUGAACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAGGAA
2919

1578
CCUGAAGU G UUCACUUC
939
GAAGUGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUCAGG
2920

1590
ACUUCCCU G UUUAUCCA
940
UGGAUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGAAGU
2921

1619
UUUAUUUU G UUUGUUCG
941
CGAACAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAAUAAA
2922

1623
UUUUGUUU G UUCGGCAU
942
AUGCCGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACAAAA
2923

1628
UUUGUUCG G CAUACAAA
943
UUUGUAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAACAAA
2924

1656
UCUUAAUU G UAAGCAAA
944
UUUGCUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUUAAGA
2925

1660
AAUUGUAA G CAAAACUU
945
AAGUUUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUACAAUU
2926

1710
UUCUUCAU G UGUGUUUA
946
UAAACACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAAGAA
2927

1712
CUUCAUGU G UGUUUAGU
947
ACUAAACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAUGAAG
2928

1714
UCAUGUGU G UUUAGUAU
948
AUACUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACAUGA
2929

1719
UGUGUUUA G UAUCUGAA
949
UUCAGAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAACACA
2930

1743
CUCAUCUG G UGGAAACC
950
GGUUUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGAUGAG
2931

1754
GAAACCAA G UUUCAGGG
951
CCCUGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGUUUC
2932

1770
GGACAUGA G UUUUCCAG
952
CUGGAAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAUGUCC
2933

1778
GUUUUCCA G CUUUUAUA
953
UAUAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAAAAC
2934

1792
AUACACAC G UAUCUCAU
954
AUGAGAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGUGUAU
2935

17
AGUCCGCC G AGGUGCUC
1186
GAGCACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCGACUG
2936

19
UCCGCCGA G GUGCUCGG
1187
CCGAGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGCGGA
2937

26
AGGUGCUC G GUGGAGUC
1188
GACUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGCACCU
2938

29
UGCUCGGU G GAGUCAUG
1189
CAUGACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGAGCA
2939

30
GCUCGGUG G AGUCAUGG
1190
CCAUGACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCGAGC
2940

37
GGAGUCAU G GCAGUGCC
1191
GGCACUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGACUCC
2941

52
CCCUUUGU G GAAGACUG
1192
CAGUCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAAGGG
2942

53
CCUUUGUG G AAGACUGG
1193
CCAGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAAAGG
2943

56
UUGUGGAA G ACUGGGAC
1194
GUCCCAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCACAA
2944

60
GGAAGACU G GGACUUGG
1195
CCAAGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUCUUCC
2945

61
GAAGACUG G GACUUGGU
1196
ACCAAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUCUUC
2946

62
AAGACUGG G ACUUGGUG
1197
CACCAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGUCUU
2947

67
UGGGACUU G GUGCAAAC
1198
GUUUGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUCCCA
2948

79
CAAACCCU G GGAGAAGG
1199
CCUUCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGUUUG
2949

80
AAACCCUG G GAGAAGGU
1200
ACCUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGUUU
2950

81
AACCCUGG G AGAAGGUG
1201
CACCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGGGUU
2951

83
CCCUGGGA G AAGGUGCC
1202
GGCACCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCAGGG
2952

86
UGGGAGAA G GUGCCUAU
1203
AUAGGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCCCA
2953

95
GUGCCUAU G GAGAAGUU
1204
AACUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAGGCAC
2954

96
UGCCUAUG G AGAAGUUC
1205
GAACUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUAGGCA
2955

98
CCUAUGGA G AAGUUCAA
1206
UUGAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAUAGG
2956

115
CUUGCUGU G AAUAGAGU
1207
ACUCUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGCAAG
2957

120
UGUGAAUA G AGUAACUG
1208
CAGUUACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUUCACA
2958

128
GAGUAACU G AAGAAGCA
1209
UGCUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUUACUC
2959

131
UAACUGAA G AAGCAGUC
1210
GACUGCUU GGAUUAAACUCC CU UCAAGGACAUCGUCCGGG UUCAGUUA
2960

145
GUCGCAGU G AAGAUUGU
1211
ACAAUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGCGAC
2961

148
GCAGUGAA G AUUGUAGA
1212
UCUACAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCACUGC
2962

155
AGAUUGUA G AUAUGAAG
1213
CUUCAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACAAUCU
2963

160
GUAGAUAU G AAGCGUGC
1214
GCACGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGCG AUAUCUAC
2964

173
GUGCCGUA G ACUGUCCA
1215
UGGACAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACGGCAC
2965

182
ACUGUCCA G AAAAUAUU
1216
AAUAUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGACAGU
2966

193
AAUAUUAA G AAAGAGAU
1217
AUCUCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAUAUU
2967

197
UUAAGAAA G AGAUCUGU
1218
ACAGAUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUUAA
2968

199
AAGAAAGA G AUCUGUAU
1219
AUACAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUUCUU
2969

227
UAAAUCAU G AAAAUGUA
1220
UACAUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAUUUA
2970

248
AAUUCUAU G GUCACAGG
1221
CCUGUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAGAAUU
2971

255
UGGUCACA G GAGAGAAG
1222
CUUCUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGACCA
2972

256
GGUCACAG G AGAGAAGG
1223
CCUUCUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUGACC
2973

258
UCACAGGA G AGAAGGCA
1224
UGCCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUGUGA
2974

260
ACAGGAGA G AAGGCAAU
1225
AUUGCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUCCUGU
2975

263
GGAGAGAA G GCAAUAUC
1226
GAUAUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCUCC
2976

286
UUAUUUCU G GAGUACUG
1227
CAGUACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAAUAA
2977

287
UAUUUCUG G AGUACUGU
1228
ACAGUACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGAAAUA
2978

299
ACUGUAGU G GAGGAGAG
1229
CUCUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUACAGU
2979

300
CUGUAGUG G AGGAGAGC
1230
GCUCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUACAG
2980

302
GUAGUGGA G GAGAGCUU
1231
AAGCUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCACUAC
2981

303
UAGUGGAG G AGAGCUUU
1232
AAAGCUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCACUA
2982

305
GUGGAGGA G AGCUUUUU
1233
AAAAAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCCAC
2983

314
AGCUUUUU G ACAGAAUA
1234
UAUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAAAGCU
2984

318
UUUUGACA G AAUAGAGC
1235
GCUCUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCAAAA
2985

323
ACAGAAUA G AGCCAGAC
1236
GUCUGGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUUCUGU
2986

329
UAGAGCCA G ACAUAGGC
1237
GCCUAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUCUA
2987

335
CAGACAUA G GCAUGCCU
1238
AGGCAUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUGUCUG
2988

344
GCAUGCCU G AACCAGAU
1239
AUCUGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCAUGC
2989

350
CUGAACCA G AUGCUCAG
1240
CUGAGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUUCAG
2990

358
GAUGCUCA G AGAUUCUU
1241
AAGAAUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGCAUC
2991

360
UGCUCAGA G AUUCUUCC
1242
GGAAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGAGCA
2992

379
CAACUCAU G GCAGGGGU
1243
ACCCCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAGUUG
2993

383
UCAUGGCA G GGGUGGUU
1244
AACCACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCAUGA
2994

384
CAUGGCAG G GGUGGUUU
1245
AAACCACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGCCAUG
2995

385
AUGGCAGG G GUGGUUUA
1246
UAAACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGCCAU
2996

388
GCAGGGGU G GUUUAUCU
1247
AGAUAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCCUGC
2997

401
AUCUGCAU G GUAUUGGA
1248
UCCAAUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCAGAU
2998

407
AUGGUAUU G GAAUAACU
1249
AGUUAUUC GGAGGANXCUCC CU UCAAGGACAUCGUCCGGG AAUACCAU
2999

408
UGGUAUUG G AAUAACUC
1250
GAGUUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUACCA
3000

420
AACUCACA G GGAUAUUA
1251
UAAUAUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGAGUU
3001

421
ACUCACAG G GAUAUUAA
1252
UUAAUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUGAGU
3002

422
CUCACAGG G AUAUUAAA
1253
UUUAAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGUGAG
3003

434
UUAAACCA G AAAAUCUU
1254
AAGAUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUUUAA
3004

448
CUUCUGUU G GAUGAAAG
1255
CUUUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAGAAG
3005

449
UUCUGUUG G AUGAAAGG
1256
CCUUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACAGAA
3006

452
UGUUGGAU G AAAGGGAU
1257
AUCCCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCAACA
3007

456
GGAUGAAA G GGAUAACC
1258
GGUUAUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCAUCC
3008

457
GAUGAAAG G GAUAACCU
1259
AGGUUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUCAUC
3009

458
AUGAAAGG G AUAACCUC
1260
GAGGUUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUUCAU
3010

476
AAAUCUCA G ACUUUGGC
1261
GCCAAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGAUUU
3011

482
CAGACUUU G GCUUGGCA
1262
UGCCAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAGUCUG
3012

487
UUUGGCUU G GCAACAGU
1263
ACUGUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCCAAA
3013

501
AGUAUUUC G GUAUAAUA
1264
UAUUAUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAAAUACU
3014

515
AUAAUCGU G AGCGUUUG
1265
CAAACGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGAUUAU
3015

526
CGUUUGUU G AACAAGAU
1266
AUCUUGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAAACG
3016

532
UUGAACAA G AUGUGUGG
1267
CCACACAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUUCAA
3017

539
AGAUGUGU G GUACUUUA
1268
UAAAGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACAUCU
3018

563
UUGCUCCA G AACUUCUG
1269
CAGAAGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGCAA
3019

571
GAACUUCU G AAGAGAAG
1270
CUUCUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAGUUC
3020

574
CUUCUGAA G AGAAGAGA
1271
UCUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAGAAG
3021

576
UCUGAAGA G AAGAGAAU
1272
AUUCUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUCAGA
3022

579
GAAGAGAA G AGAAUUUC
1273
GAAAUUCU GGAGGAAACUCC CU UCAACGACAUCGUCCGGG UUCUCUUC
3023

581
AGAGAAGA G AAUUUCAU
1274
AUGAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUCUCU
3024

593
UUCAUGCA G AACCAGUU
1275
AACUGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAUGAA
3025

602
AACCAGUU G AUGUUUGG
1276
CCAAACAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACUGGUU
3026

609
UGAUGUUU G GUCCUGUG
1277
CACAGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACAUCA
3027

617
GGUCCUGU G GAAUAGUA
1278
UACUAUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGGACC
3028

618
GUCCUGUG G AAUAGUAC
1279
GUACUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGGAC
3029

644
UGCUCGCU G GAGAAUUG
1280
CAAUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGAGCA
3030

645
GCUCGCUG G AGAAUUGC
1281
GCAAUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCGAGC
3031

647
UCGCUGGA G AAUUGCCA
1282
UGGCAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGCGA
3032

657
AUUGCCAU G GGACCAAC
1283
GUUGGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGCAAU
3033

658
UUGCCAUG G GACCAACC
1284
GGUUGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGGCAA
3034

659
UGCCAUGG G ACCAACCC
1285
GGGUUGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUGGCA
3035

671
AACCCAGU G ACAGCUGU
1286
ACAGCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGGGUU
3036

682
AGCUGUCA G GAGUAUUC
1287
GAAUACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGACAGCU
3037

683
GCUGUCAG G AGUAUUCU
1288
AGAAUACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGACAGC
3038

692
AGUAUUCU G ACUGGAAA
1289
UUUCCAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAUACU
3039

696
UUCUGACU G GAAAGAAA
1290
UUUCUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUCAGAA
3040

697
UCUGACUG G AAAGAAAA
1291
UUUUCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUCAGA
3041

701
ACUGGAAA G AAAAAAAA
1292
UUUUUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCCAGU
3042

726
CAACCCUU G GAAAAAAA
1293
UUUUUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGGGUUG
3043

727
AACCCUUG G AAAAAAAU
1294
AUUUUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGGGUU
3044

737
AAAAAAUC G AUUCUGCU
1295
AGCAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUUUUUU
3045

776
UCUUAGUU G AGAAUCCA
1296
UGGAUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACUAAGA
3046

778
UUAGUUGA G AAUCCAUC
1297
GAUGGAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAACUAA
3047

792
AUCAGCAA G AAUUACCA
1298
UGGUAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCUGAU
3048

806
CCAUUCCA G ACAUCAAA
1299
UUUGAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAAUGG
3049

818
UCAAAAAA G AUAGAUGG
1300
CCAUCUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUUUGA
3050

822
AAAAGAUA G AUGGUACA
1301
UGUACCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUCUUUU
3051

825
AGAUAGAU G GUACAACA
1302
UGUUGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUAUCU
3052

844
CCCCUCAA G AAAGGGGC
1303
GCCCCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGAGGGG
3053

848
UCAAGAAA G GGGCAAAA
1304
UUUUGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUUGA
3054

849
CAAGAAAG G GGCAAAAA
1305
UUUUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUCUUG
3055

850
AAGAAAGG G GCAAAAAG
1306
CUUUUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUUCUU
3056

858
GGCAAAAA G GCCCCGAG
1307
CUCGGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUUGCC
3057

864
AAGGCCCC G ACUCACUU
1308
AAGUGACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGGCCUU
3058

875
UCACUUCA G GUGGUGUG
1309
CACACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAGUGA
3059

878
CUUCAGGU G GUGUGUCA
1310
UGACACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUGAAG
3060

887
GUGUGUCA G AGUCUCCC
1311
GGGAGACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGACACAC
3061

899
CUCCCAGU G GAUUUUCU
1312
AGAAAAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGGGAG
3062

900
UCCCAGUG G AUUUUCUA
1313
UAGAAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUGGGA
3063

928
UCCAAUUU G GACUUCUC
1314
GAGAAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUUGGA
3064

929
CCAAUUUG G ACUUCUCU
1315
AGAGAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAAUUGG
3065

959
CUUCUAGU G AAGAAAAU
1316
AUUUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUAGAAG
3066

962
CUAGUGAA G AAAAUGUG
1317
CACAUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCACUAG
3067

970
GAAAAUGU G AAGUACUC
1318
GAGUACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAUUUUC
3068

992
CUCAGCCA G AACCCCGC
1319
GCGGGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUGAG
3069

1004
CCCGCACA G GUCUUUCC
1320
GGAAAGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGCGGG
3070

1017
UUCCUUAU G GGAUACCA
1321
UGGUAUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAAGGAA
3071

1018
UCCUUAUG G GAUACCAG
1322
CUGGUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUAAGGA
3072

1019
CCUUAUGG G AUACCAGC
1323
GCUGGUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUAAGG
3073

1040
CAUACAUU G AUAAAUUG
1324
CAAUUUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUGUAUG
3074

1048
GAUAAAUU G GUACAAGG
1325
CCUUGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUUUAUC
3075

1055
UGGUACAA G GGAUCAGC
1326
GCUGAUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUACCA
3076

1056
GGUACAAG G GAUCAGCU
1327
AGCUGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGUACC
3077

1057
GUACAAGG G AUCAGCUU
1328
AAGCUGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUGUAC
3078

1085
CAUGUCCU G AUCAUAUG
1329
CAUAUGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGACAUG
3079

1099
AUGCUUUU G AAUAGUCA
1330
UGACUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAAGCAU
3080

1115
AGUUACUU G GCACCCCA
1331
UGGGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUAACU
3081

1124
GCACCCCA G GAUCCUCA
1332
UGAGGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGGUGC
3082

1125
CACCCCAG G AUCCUCAC
1333
GUGAGGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGGGUG
3083

1135
UCCUCACA G AACCCCUG
1334
CAGGGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGAGGA
3084

1143
GAACCCCU G GCAGCGGU
1335
ACCGCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGGUUC
3085

1149
CUGGCAGC G GUUGGUCA
1336
UGACCAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGCG GCUGCCAG
3086

1153
CAGCGGUU G GUCAAAAG
1337
CUUUUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACCGCUG
3087

1161
GGUCAAAA G AAUGACAC
1338
GUGUCAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUGACC
3088

1165
AAAAGAAU G ACACGAUU
1339
AAUCGUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGCG AUUCUUUU
3089

1170
AAUGACAC G AUUCUUUA
1340
UAAAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGUCAUU
3090

1186
ACCAAAUU G GAUGCAGA
1341
UCUGCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUUUGGU
3091

1187
CCAAAUUG G AUGCAGAC
1342
GUCUGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUUUGG
3092

1193
UGGAUGCA G ACAAAUCU
1343
AGAUUUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAUCCA
3093

1213
CAAUGCCU G AAAGAGAC
1344
GUCUCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCAUUG
3094

1217
GCCUGAAA G AGACUUGU
1345
ACAAGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCAGGC
3095

1219
CUGAAAGA G ACUUGUGA
1346
UCACAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUUCAG
3096

1226
AGACUUGU G AGAAGUUG
1347
CAACUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAGUCU
3097

1228
ACUUGUGA G AAGUUGGG
1348
CCCAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACAAGU
3098

1234
GAGAAGUU G GGCUAUCA
1349
UGAUAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACUUCUC
3099

1235
AGAAGUUG G GCUAUCAA
1350
UUGAUAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACUUCU
3100

1245
CUAUCAAU G GAAGAAAA
1351
UUUUCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUGAUAG
3101

1246
UAUCAAUG G AAGAAAAG
1352
CUUUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUGAUA
3102

1249
CAAUGGAA G AAAAGUUG
1353
CAACUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCAUUG
3103

1261
AGUUGUAU G AAUCAGGU
1354
ACCUGAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUACAACU
3104

1267
AUGAAUCA G GUUACUAU
1355
AUAGUAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAUUCAU
3105

1286
CAACAACU G AUAGGAGA
1356
UCUCCUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUUGUUG
3106

1290
AACUGAUA G GAGAAACA
1357
UGUUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUCAGUU
3107

1291
ACUGAUAG G AGAAACAA
1358
UUGUUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUAUCAGU
3108

1293
UGAUACGA G AAACAAUA
1359
UAUUGUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUAUCA
3109

1318
UUCAAAGU G AAUUUGUU
1360
AACAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUUGAA
3110

1328
AUUUGUUA G AAAUGGAU
1361
AUCCAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAACAAAU
3111

1333
UUAGAAAU G GAUGAUAA
1362
UUAUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUCUAA
3112

1334
UAGAAAUG G AUGAUAAA
1363
UUUAUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUUCUA
3113

1337
AAAUGGAU G AUAAAAUA
1364
UAUUUUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCAUUU
3114

1348
AAAAUAUU G GUUGACUU
1365
AAGUCAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUAUUUU
3115

1352
UAUUGGUU G ACUUCCGG
1366
CCGGAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACCAAUA
3116

1359
UGACUUCC G GCUUUCUA
1367
UAGAAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAAGUCA
3117

1369
CUUUCUAA G GGUGAUGG
1368
CCAUCACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAGAAAG
3118

1370
UUUCUAAG G GUGAUGGA
1369
UCCAUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUAGAAA
3119

1373
CUAAGGGU G AUGGAUUG
1370
CAAUCCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCUUAG
3120

1376
AGGGUGAU G GAUUGGAG
1371
CUCCAAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCACCCU
3121

1377
GGGUGAUG G AUUGGAGU
1372
ACUCCAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCACCC
3122

1381
GAUGGAUU G GAGUUCAA
1373
UUGAACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUCCAUC
3123

1382
AUGGAUUG G AGUUCAAG
1374
CUUGAACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUCCAU
3124

1390
GAGUUCAA G AGACACUU
1375
AAGUGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGAACUC
3125

1392
GUUCAAGA G ACACUUCC
1376
GGAAGUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUGAAC
3126

1402
CACUUCCU G AAGAUUAA
1377
UUAAUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAAGUG
3127

1405
UUCCUGAA G AUUAAAGG
1378
CCUUUAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAGGAA
3128

1412
AGAUUAAA G GGAAGCUG
1379
CAGCUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUAAUCU
3129

1413
GAUUAAAG G GAAGCUGA
1380
UCAGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUAAUC
3130

1414
AUUAAAGG G AAGCUGAU
1381
AUCAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUUAAU
3131

1420
GGGAAGCU G AUUGAUAU
1382
AUAUCAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUCCC
3132

1424
AGCUGAUU G AUAUUGUG
1383
CACAAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUCAGCU
3133

1432
GAUAUUGU G AGCAGCCA
1384
UGGCUGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAUAUC
3134

1441
AGCAGCCA G AAGGUUUG
1385
CAAACCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUGCU
3135

1444
AGCCAGAA G GUUUGGCU
1386
AGCCAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGGCU
3136

1449
GAAGGUUU G GCUUCCUG
1387
CAGGAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACCUUC
3137

1464
UGCCACAU G AUCGGACC
1388
GGUCCGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUGGCA
3138

1468
ACAUGAUC G GACCAUCG
1389
CGAUGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUCAUGU
3139

1469
CAUGAUCG G ACCAUCGG
1390
CCGAUGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAUCAUG
3140

1476
GGACCAUC G GCUCUGGG
1391
CCCAGAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUGGUCC
3141

1482
UCGGCUCU G GGGAAUCC
1392
GGAUUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGCCGA
3142

1483
CGGCUCUG G GGAAUCCU
1393
AGGAUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGAGCCG
3143

1484
GGCUCUGG G GAAUCCUG
1394
CAGGAUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGAGCC
3144

1485
GCUCUGGG G AAUCCUGG
1395
CCAGGAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAGAGC
3145

1492
GGAAUCCU G GUGAAUAU
1396
AUAUUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAUUCC
3146

1495
AUCCUGGU G AAUAUAGU
1397
ACUAUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGGAU
3147

1515
GCUAUGUU G ACAUUAUU
1398
AAUAAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAUAGC
3148

1531
UCUUCCUA G AGAAGAUU
1399
AAUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAGGAAGA
3149

1533
UUCCUAGA G AAGAUUAU
1400
AUAAUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUAGGAA
3150

1536
CUAGAGAA G AUUAUCCU
1401
AGGAUAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCUAG
3151

1573
UAGUUCCU G AAGUGUUC
1402
GAACACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAACUA
3152

1627
GUUUGUUC G GCAUACAA
1403
UUGUAUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAACAAAC
3153

1670
AAAACUUU G GGGAAAGG
1404
CCUUUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAGUUUU
3154

1671
AAACUUUG G GGAAAGGA
1405
UCCUUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAAGUUU
3155

1672
AACUUUGG G GAAAGGAU
1406
AUCCUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAAAGUU
3156

1673
ACUUUGGG G AAAGGAUG
1407
CAUCCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAAAGU
3157

1677
UGGGGAAA G GAUGAAUA
1408
UAUUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCCCCA
3158

1678
GGGGAAAG G AUGAAUAG
1409
CUAUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUCCCC
3159

1681
GAAAGGAU G AAUAGAAU
1410
AUUCUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUUUC
3160

1686
GAUGAAUA G AAUUCAUU
1411
AAUGAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUUCAUC
3161

1696
AUUCAUUU G AUUAUUUC
1412
GAAAUAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUGAAU
3162

1725
UAGUAUCU G AAUUUGAA
1413
UUCAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUACUA
3163

1731
CUGAAUUU G AAACUCAU
1414
AUGAGUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUUCAG
3164

1742
ACUCAUCU G GUGGAAAC
1415
GUUUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUGAGU
3165

1745
CAUCUGGU G GAAACCAA
1416
UUGGUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGAUG
3166

1746
AUCUGGUG G AAACCAAG
1417
CUUGGUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCAGAU
3167

1760
AAGUUUCA G GGGACAUG
1418
CAUGUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAACUU
3168

1761
AGUUUCAG G GGACAUGA
1419
UCAUGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAACU
3169

1762
GUUUCAGG G GACAUGAG
1420
CUCAUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGAAAC
3170

1763
UUUCAGGG G ACAUGAGU
1421
ACUCAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGAAA
3171

1768
GGGGACAU G AGUUUUCC
1422
GGAAAACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUCCCC
3172

Input Sequence = AF016582. Cut Site = G/.

Stem Length = 8. Core Sequence = GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG

AF016582 (Homo sapiens checkpoint kinase Chk1 (CHK1) mRNA; 1821 bp)

Method and reagent for the inhibition of checkpoint kinase-1 (Chk1) enzyme

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Provisional Applications (1)