C-myb targeted ribozymes

Abstract

Enzymatic nucleic acid molecules which cleave c-myb RNA or other RNAs associated with restenosis or cancer.

Description

BACKGROUND OF THE INVENTION

The present invention concerns therapeutic compositions and methods for the treatment of restenosis and cancer.

The following is a brief description of the physiology, cellular pathology and treatment of restenosis. The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.

Coronary angioplasty is one of the major surgical treatments for heart disease. Its use has been accelerating rapidly; over 450,000 procedures are performed in the U.S. annually. The short term success rate of angioplasty is 80 to 90%. However, in spite of a number of technical improvements in the procedure, post-operative occlusions of the arteries, or restenosis, still occur. Thirty-five to forty-five percent of patients who have undergone a single vessel angioplasty develop clinically significant restenosis within 6 months of the procedure. The rate of restenosis is even higher (50 to 60%) in patients who have undergone multivessel angioplasty (Califf, R. M., et al., 1990, in Textbook of Interventional Cardiology., E. J. Topol, ed., W. B. Saunders, Philadelphia, pp 363-394.).

Histopathological studies have shown that restenosis after angioplasty is characterized by migration of medial smooth muscle cells to the intima and a striking hyper-proliferative response of these neointimal cells (Garratt, K. N., et al., 1991, J. Am. Coll. Cardio., 17, 442-428; Austin, G. E., et al., 1985, J. Am. Coll. Cardiol., 6, 369-375). Smooth muscle cell proliferation could be an overly robust response to injury. Alternatively, the intimal smooth muscle cells within atherosclerotic lesions are already in an activated or "synthetic" state (Sjolund, M., et al., 1988, J. Cell. Biol., 106, 403-413 and thus may be poised to proliferate. One recent study demonstrated a positive correlation between the presence of activated smooth muscle cells in coronary lesions and the extent of subsequent luminal narrowing after atherectomy (Simons, M., et al., 1993, New Engl. J. Med., 328, 608-613). In any case, slowing smooth muscle cell proliferation after angioplasty could prevent intimal thickening and restenosis.

The presently preferred therapeutic treatment for restenosis is the use of streptokinase, urokinase or other thrombolytic compounds, such as fish oil, anticoagulants, ACE (angiotensin converting enzyme) inhibitors, aspirin and cholesterol lowering compounds. Alternative treatment includes the surgical incorporation of endoluminal stents. The occurrence of pharmacologic side-effects (particularly bleeding disorders associated with anti-coagulants and platelet inhibitors) is an issue with current therapies. Popoma, J. J., et al., report that the current therapies have not significantly impacted the rates of restenosis occurrence. (Circulation, 84, 1426-1436, 1991).

Recently, the results of a clinical trial of the efficacy of an anti-platelet therapy have been reported. Patients undergoing coronary angioplasty were given a single bolus injection followed by a 12 hour infusion of an antibody directed against the platelet adhesion molecule, gpllb/gpllla. After six months, patients with the treatment showed a 23% reduction in the occurrence of restenosis than patients receiving placebo (27 vs. 35%; p=0.001).

A number of growth factors have been shown to induce smooth muscle cell proliferation. In vitro, platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen (Ross, R., et al., 1974, Proc. Natl. Acad. Sci. USA, 71, 1207-1210) and a smooth muscle cell chemoattractant (Grotendorst, G., et al., 1982, Proc. Natl. Acad. Sci. USA, 71, 3669-3672.). In vivo, when PDGF is expressed ectopically in porcine arteries, it induces intimal hyperplasia (Nabel, E. B., et al., 1993, J. Clin. Invest., 91, 1822-1829). Furthermore, antibodies to PDGF have been shown to reduce intimal thickening after arterial injury (Ferns, G. A. A., et al., 1991, Science, 253, 1129-1132). Analysis of .sup.3 H-thymidine incorporation in the lesions indicates that the anti-PDGF antibodies primarily inhibit smooth muscle cell migration.

Basic fibroblast growth factor (bFGF) is another smooth muscle cell mitogen in vitro (Klagsbrun, M. and Edelman, E. R., 1989, Arteriosclerosis, 9, 269-278). In a rat model, anti-bFGF antibodies inhibit the proliferation of medial smooth muscle cells 24 to 48 hours after balloon catheter injury (Lidner, V. and Reidy, M. A., 1991, Proc. Natl. Acad. Sci. USA, 88, 3739-3743). In addition to bFGF, heparin binding epidermal growth factor (HB-EGF) (Higashiyama, S., et al., 1991, Science, 251, 936-939.), insulin-like growth factor I (IGF-I) (Banskota, N. K., et al., 1989, Molec. Endocrinol., 3, 1183-1190) and endothelin (Komuro, I., et al., 1988, FEBS Letters, 238, 249-252) have been shown to induce smooth muscle cell proliferation. A number of other factors (such as interleukin-1 and tumor necrosis factor-.alpha.) may indirectly affect smooth muscle cell proliferation by inducing the expression of PDGF (Hajjar, K. A., et al., 1987, J. Exp. Med., 166, 235-245; Raines, E. W., et al., 1989, Science, 243, 393-396).

When whole serum is added to serum-starved smooth muscle cells in vitro, the oncogenes, c-myc, c-fos, and c-myb, are induced (Kindy, M. S. and Sonenshein, G. E., 1986, J. Biol. Chem., 261, 12865-12868; Brown, K. E., et al., 1992, J. Biol. Chem., 267, 4625-4630) and cell proliferation ensues. Blocking c-myb with an antisense oligonucleotide prevents cells from entering S phase (Brown, K. E., et al., 1992, J. Biol. Chem., 267, 4625-4630.). Thus, c-myb is required for the G.sub.1 to S transition after stimulation by the multitude of growth factors present in serum. In vivo, a c-myb antisense oligonucleotide inhibits restenosis when applied to rat arteries after balloon angioplasty (Simons, M., et al., 1992, Nature, 359, 67-70). Similarly, an antisense oligonucleotide directed against mRNA of the oncogene c-myc was shown to inhibit human smooth muscle cell proliferation (Shi, Y., et al., 1993, Circulation, 88, 1190-5) and migration (Biro, S., et al., 1993, Proc. Natl. Acad. Sci. U S A, 90, 654-8).

Ohno et al., 1994 Science 265, 781, have shown that a combination of viral thymidine kinase enzyme expression (gene therapy) and treatment with anti-viral drug ganciclovir inhibits smooth muscle cell proliferation in pigs, following baloon angioplasty.

Epstein et al., "Inhibition of non-transformed cell proliferation using antisense oligonucleotides," NTIS publication 1992 discusses use of antisense oligonucleotides to c-myc, PCNA or cyclin B. Fung et al., PCT WO91/15580, describes gene therapy for cell proliferative disease and mentions administration of a ribozyme construct against a PGR element. Mention is made of inactivation of c-myb. Rosenberg et al., WO93/08845, Calabretta et al., WO92/20348 and Gewirtz WO93/09789 concern c-myb antisense oligonucleotides for treatment of melanoma or colorectal cancer, and administration locally. Sytkowski, PCT WO 93/02654, describe the uses of antisense oligonucleotides to inhibit c-myb gene expression in red blood cells to stimulate hemoglobin synthesis.

Nabel and Nabel, U.S. Pat. No. 5,328,470, describe a method for the treatment of diseases by delivering therapeutic reagents directly to the sites of disease. They state that--

" . . . Method is based on the delivery of proteins by catheterization to discrete blood vessel segments using genetically modified or normal cells or other vector systems . . . In addition,, catalytic RNAs, called ribozymes, can specifically degrade RNA sequences. . . . The requirements for a successful RNA cleavage include a hammerhead structure with conserved RNA sequence at the region flanking this structure . . . any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme . . . gene transfer using vectors expressing such proteins as tPA for the treatment of thrombosis and restenosis, anglogenesis or growth factors for the purpose of revascularization . . . "

SUMMARY OF THE INVENTION

This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by the oncogene, c-myb. Such ribozymes may be used to inhibit the hyper-proliferation of smooth muscle cells in restenosis and of tumor cells in numerous cancers. To block restenosis, a target molecule required for the induction of smooth muscle cell proliferation by a number of different growth factors is preferred. To this end c-myc, c-fos, and c-myb are useful targets in this invention.

Other transcription factors involved in the response to growth and proliferation signals include NF-.kappa.B, oct-1 and SRF. NF-.kappa.B protein activates cellular transcription and induces increases in cellular synthetic pathways. In a resting cell, this protein is found in the cytoplasm, complexed with its inhibitor, I-.kappa.B. Upon phosphorylation of the I-.kappa.B molecule, the complex dissociates and NF-.kappa.B is released for transport to the nucleus, where it binds DNA and induces transcriptional activity in (NF-.kappa.B)-responsive genes. One of the (NF-.kappa.B)-responsive genes is the NF-.kappa.B gene itself. Thus, release of the NF-.kappa.B protein from the inhibitory complex results in a cascade of gene expression which is auto-induced. Early inhibition of NF-.kappa.B can reduce expression of a number of genes required for growth and proliferation, such as c-myb.

Two other transcription factors, oct-1 and serum response factor (SRF) have been shown to be expressed selectively in dividing cells. Both oct-1 and SRF are expressed ubiquitously in cultured cells, including smooth muscle cells. However, R. Majack and his colleagues have recently shown that these transcription factors are not expressed by the smooth muscle cells in intact vessels. Both oct-1 and SRF are rapidly expressed upon dispersal of tissue into single cell suspensions. Thus, these transcription factors are thought to be regulated by their interactions with the extracellular matrix (Weiser, M. C. M., et al., 1994, J. Cell. Biochem., S18A, 282; Belknap, J. K., et al., 1994, J. Cell. Biochem., S18A, 277). Upon injury during angioplasty, the expression of oct-1 and SRF may be enhanced, leading to increased smooth muscle cell proliferation. Treatment with ribozymes that block the expression of these transcription factors can alleviate the smooth muscle cell proliferation associated with restenosis.

While some of the above mentioned studies demonstrated that antisense oligonucleotides can efficiently reduce the expression of factors required for smooth muscle cell proliferation, enzymatic RNAs, or ribozymes have yet to be demonstrated to inhibit smooth muscle cell proliferation. Such ribozymes, with their catalytic activity and increased site specificity (as described below), represent more potent and safe therapeutic molecules than antisense oligonucleotides. In the present invention, ribozymes that cleave c-myb mRNA are described. Moreover, applicant shows that these ribozymes are able to inhibit smooth muscle cell proliferation and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described, that other ribozymes that cleave target mRNAs required for smooth muscle cell proliferation may be readily designed and are within the invention.

By "inhibit" is meant that the activity of c-myb or level of mRNAs encoded by c-myb is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

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 RNA in that target. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By "equivalent" RNA to c-myb is meant to include those naturally occurring RNA molecules associated with restenosis and cancer in various animals, including human, rat and pig. Such a molecule will generally contain some ribonucleotides, but the other nucleotides may be substituted at the 2'-hydroxyl position and in other locations with other moeities as discussed below.

By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.

Six basic 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.

The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. 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 completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf, T. M., et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.

In preferred embodiments of this invention, 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 or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, 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 of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. 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.

In a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNAs encoding c-myb proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.

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 enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g., 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). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT WO93/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; 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, 25656).

Thus, in a first aspect, the invention features ribozymes that inhibit cell proliferation. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited.

In a preferred embodiment, the enzymatic RNA molecules cleave c-myb mRNA and inhibit smooth muscle cell proliferation. Such ribozymes are useful for the prevention of restenosis after coronary angioplasty. Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. The ribozymes, similarly delivered, also are useful for inhibiting proliferation of certain cancers associated with elevated levels of the c-myb oncogene, particularly leukemias, neuroblastomas, and lung, colon, and breast carcinomas. Using the methods described herein, other enzymatic RNA molecules that cleave c-myb, c-myc, oct-1, SRF, NF-.kappa.B, PDGF receptor, bFGF receptor, angiotensin II, and endothelium-derived relaxing factor and thereby inhibit smooth muscle cell proliferation and/or tumor cell proliferation may be derived and used as described above. Specific examples are provided below in the Tables.

Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of c-myb activity in a cell or tissue. By "related" is meant that the inhibition of c-myb mRNAs and thus reduction in the level of protein activity will relieve to some extent the symptoms of the disease or condition.

Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. 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 another aspect of the invention, ribozymes that cleave target molecules and inhibit c-myb activity are expressed from transcription units 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 ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could 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.

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

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 1-100. Examples of such ribozymes are shown as Seq. I.D. Nos. 101-129. Those in the art will recognize that while such examples are designed to mouse RNA, similar ribozymes can be made complementary to human RNA. By complementary is thus meant that the binding arms are able to cause cleavage of a human or mouse mRNA target. Examples of such ribozymes consist essentially of sequences defined as Seq. I.D. Nos. 101-129 below. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind human mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or vital vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. Once expressed, the ribozymes cleave their target mRNAs and prevent proliferation of their host cells. The recombinant vectors are preferably DNA plasmids or adenovirus vectors. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.

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

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be .gtoreq.2 base-pair long.

FIG. 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; FIG. 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and FIG. 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. 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 .gtoreq.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 .gtoreq.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.

FIG. 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.

FIG. 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.

FIG. 6 is a schematic representation of an RNAseH accessibility assay. Specifically, the left side of FIG. 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of FIG. 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane represent uncleaved target RNA; the bands unique to each lane represent the cleaved products.

FIG. 7 is a graph of the results of an RNAseH accessibility assay of murine c-myb RNA. On the abscissa is the sequence number of the DNA oligonucleotide that is homologous to the ribozyme target site. The ordinate represents the percentage of the intact transcript that was cleaved by RNAse H.

FIG. 8 is a graph of the outcome of an RNAseH accessibility assay of human c-myb mRNA. The graphs are labeled as in FIG. 7.

FIG. 9 shows the effect of chemical modifications on the catalytic activity of hammerhead ribozyme targeted to c-myb site 575. A) diagrammatic representation of 575 hammerhead ribozyme.cndot.substrate complex. 2'-O-methyl ribozyme represents a hammerhead (HH) ribozyme containing 2'-O-methyl substitutions at five nucleotides in the 5' and 3' termini. 2'-O-methyl P=S ribozyme represents a hammerhead (HH) ribozyme containing 2'-O-methyl and phosphorothioate substitutions at five nucleotides in the 5' and 3' termini. 2'-C-allyl iT ribozyme represents a hammerhead containing ribose residues at five positions. The remaining 31 nucleotide positions contain 2'-hydroxyl group substitutions, wherein 30 nucleotides contain 2'-O-methyl substitutions and one nucleotide (U.sub.4) contains 2'-C-allyl substitution. Additionally, 3' end of this ribozyme contains a 3'-3' linked inverted T. 2'-C-allyl P=S ribozyme is similar to 2'-C-allyl iT ribozyme with the following changes: five nucleotides at the 5' and 3' termini contain phosphorothioate substitutions and the ribozyme lacks the 3'-end inverted T modification. B) shows the ability of ribozymes described in FIG. 9A to inhibit smooth muscle cell proliferation.

FIG. 10 shows the effect of 2'-C-allyl P=S 575 HH ribozyme concentration on smooth muscle cell proliferation. A plot of percent inhibition of smooth muscle cell proliferation (normalized to the effect of a catalytically inactive ribozyme) as a function of ribozyme concentration is shown.

FIG. 11 shows a comparison of the effects of 2'-C-allyl P=S 575 HH ribozyme and phosphorothioate antisense DNA on the proliferation of smooth muscle cells.

FIG. 12 shows the inhibition of smooth muscle cell proliferation catalyzed by 2'-C-allyl P=S HH ribozymes targeted to sites 549, 575, and 1533 within c-myb mRNA.

FIG. 13 shows the effect of phosphorthioate substitutions on the catalytic activity of 2'-C-allyl 575 HH ribozyme. A) diagrammatic representation of 575 hammerhead ribozyme.cndot.substrate complex. 10 P=S 5' and 3' ribozyme is identical to the 2'-C-allyl P=S ribozyme described in FIG. 9. 5 P=S 3' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 3' termini contain phosphorothioate substitutions. 5 P=S Loop ribozyme is similar to 2'-C-allyl iT described in FIG. 9, with the exception that five nucleotides within loop II of this ribozyme contain phosphorothioate substitutions. 5 P=S 5' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 5' termini contain phosphorothioate substitutions. Additionally, this ribozyme contains a 3'-3' linked inverted T at its 3' end. B) shows the ability of ribozymes described in FIG. 13A to inhibit smooth muscle cell proliferation.

FIG. 14 shows the minimum number of phosphorothioate substitutions required at the 5' termini of 575 HH ribozyme to achieve efficient inhibition of smooth muscle cell proliferation.

FIG. 15 shows the effect of varying the length of substrate binding arm of 575 HH ribozyme on the inhibition of smooth muscle cell proliferation.

FIG. 16 shows the effect of various chemical modifications, at U.sub.4 and/or U.sub.7 positions within 575 HH ribozyme core, on the ability of the ribozyme to inhibit smooth muscle cell proliferation.

FIG. 17 shows the inhibition of pig smooth muscle cell proliferation by active c-myb 575 HH ribozyme.

FIG. 18 shows the inhibition of human smooth muscle cell proliferation by active c-myb 575 HH ribozyme.

FIG. 19 shows ribozyme-mediated inhibition of c-myb expression and smooth muscle cell proliferation.

FIG. 20 is digrammatic representation of an optimal c-myb HH ribozyme that can be used to treat diseases like restenosis.

FIG. 21 shows the inhibition of Rat smooth muscle cells by 2-5A containing nucleic acids.

Target sites

Targets for useful ribozymes can be determined as disclosed in Draper et al supra, Sullivan et al., supra, as well as by Draper et al., "Method and reagent for treatment of arthritic conditions U.S. Ser. No. 08/152,487, filed Nov. 12, 1993, and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. While specific examples to mouse RNA are provided, those in the art will recognize that equivalent human RNA targets can be used as described below. Thus, the same target may be used, but binding arms suitable for targetting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below.

The sequence of human, pig and murine c-myb mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables II and IV (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. While murine, pig and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, as discussed in Stinchcomb et al., "Method and Composition for Treatment of Restenosis and Cancer Using Ribozymes," filed May 18, 1994, U.S. Ser. No. 08/245,466, murine and pig targeted ribozymes may be useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

Hammerhead or hairpin ribozymes were designed that could bind and were individually analyzed by computer folding (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. 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.

The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Table III. 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. For example, stem-loop II sequence of hammerhead ribozymes listed in Table III (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Table III (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Table III may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described in this application. These include altering the length of the ribozyme binding arms (stems I and III, see FIG. 2c), or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Usman, N. et al. U.S. patent application Ser. No. 07/829,729, and Sproat, European Patent Application 92110298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. (All these publications are hereby incorporated by reference herein.),

Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes 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, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/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 ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al., supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme 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). Several investigators have demonstrated that 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). 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).

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves mRNAs encoded by c-myb is inserted into a plasmid DNA vector or an adenovirus or adeno-associated virus DNA viral vector or a retroviral RNA vector. Viral vectors have been used to transfer genes and lead to either transient or long term gene expression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi. Biotech. 3, 533). The adenovirus vector is delivered as recombinant adenoviral particles. The DNA may be delivered alone or complexed with vehicles (as described for RNA above). The recombinant adenovirus or AAV particles are locally administered to the site of treatment, e.g., through incubation or inhalation in vivo or by direct application to cells or tissues ex vivo.

In another preferred embodiment, the ribozyme is administered to the site of c-myb expression (e.g., smooth muscle cells) in an appropriate liposomal vesicle.

EXAMPLES

Ability of Exogenously-Delivered Ribozymes Directed Against c-myb to Inhibit Vascular Smooth Muscle Cell Proliferation

The following examples demonstrate the selection of ribozymes that cleave c-myb mRNA. The methods described herein represent a scheme by which ribozymes may be derived that cleave other mRNA targets required for cell division. Also provided is a description of how such ribozymes may be delivered to smooth muscle cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture. Moreover, no inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes. The cell division assay used represents a model system for smooth muscle cell hyperproliferation in restenotic lesions.

Example 1

Identification of Potential Ribozyme Cleavage Sites in Human c-myb mRNA

The sequence of human c-myb mRNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead ribozyme cleavage sites were identified. These sites are shown in Table II and are identical to Table I of Draper, "Method and Reagent for Treatment of a Stenotic Condition", U.S. Ser. No. 07/987,132. (All sequences are 5' to 3' in the tables.) In the original, the sites were identified using nucleotide numbers from (Majello, B., et al., 1986, Proc. Natl. Acad. Sci. USA, 83, 9636-9640) (GenBank Accession No. M15024). Here, we report sites using the sequence numbers from (Westin, E. H., et al., 1990, Oncogene, 5, 1117-1124) (GenBank Accession No. X52125); the latter sequence is derived from a longer c-myb cDNA isolate and thus is more representative of the full-length RNA.

Example 2

Selection of Ribozyme Cleavage Sites in Murine and Human c-myb mRNA

To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in c-myb RNA, 41 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by comparing cDNA sequences of mouse and human c-myb (GenBank Accession No. X02774 and GenBank Accession No. X52125, repsectively) and prioritizing the sites on the basis of overall nucleotide sequence homology. Hammerhead ribozymes were designed that could bind each target (see FIG. 2C) and were individually analyzed by computer folding (Jaeger, J. A., et al., 1989, Proc. Natl. Acad. Sci USA, 86, 7706-7710) 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 were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

Example 3

Screening Ribozyme Cleavage Sites by RNaseH Protection

Murine and human mRNA was screened for accessible cleavage sites by the method described generally in McSwiggen, U.S. patent application No. 07/883,849 filed May 1, 1992, entitled "Assay for ribozyme target site," hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing 41 potential hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from human or murine c-myb cDNA clones. Labeled RNA transcripts were synthesized in vitro from the two templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for the designated times at 37.degree. C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system. The results are shown in FIGS. 7 and 8. From these data, 20 hammerhead ribozyme sites were chosen as the most accessible (see Table III). Eighteen of the twenty sites chosen overlap sequences shown in Table II; thus, the RNA folding is predictive of accessible regions in the RNA.

Example 4

Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of c-myb RNA

Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433 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 >98%. Inactive ribozymes were synthesized by substituting a U for G.sub.5 and a U for A.sub.14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeok, 1989, Methods Enzymol. 180, 51). All ribozymes were 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). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Usman et al., Synthesis, deprotection, analysis and purification of RNA and ribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table III.

Example 5

Ribozyme Cleavage of Long Substrate RNA Corresponding to c-myb mRNA Target

Hammerhead-type ribozymes which were targeted to the murine c-myb mRNA were designed and synthesized to test the cleavage activity at the 20 most accessible sites in in vitro transcripts of both mouse and human c-myb RNAs. The target sequences and the nucleotide location within the c-myb mRNA are given in Table IV. All hammerhead ribozymes were synthesized with binding arm (Stems I and III; see FIG. 2C) lengths of seven nucleotides. Two hairpin ribozymes were synthesized to sites 1632 and 2231. The relative abilities of these ribozymes to cleave both murine and human RNAs is summarized in Table IV. Ribozymes (1 .mu.M) were incubated with .sup.32 P-labeled substrate RNA (prepared as described in Example 3, approximately 20 nM) for 60 minutes at 37.degree. C. using buffers described previously. Intact RNA and cleavage products were separated by electrophoresis through polyacrylamide gels. The percentage of cleavage was determined by Phosphor Imager.RTM. quantitation of bands representing the intact substrate and the cleavage products.

Five hammerhead ribozymes (directed against sites 549, 575, 1553, 1597, and 1635) and one hairpin ribozyme (directed against site 1632) were very active; they cleaved >70% of both murine and human c-myb RNA in 60 minutes. Nine of the hammerhead ribozymes (directed against sites 551, 634, 936, 1082, 1597, 1721, 1724, 1895, and 1943) were intermediate in activity, cleaving >50% of both murine and human c-myb RNA in 60 minutes. All of the sites cleaved by these active ribozymes were predicted to be accessible to ribozyme cleavage in Table 2. Six hammerhead ribozymes and one hairpin ribozyme showed low activity on at least one of the substrates. The observed differences in accessibility between the two species of c-myb RNA demonstrate the sensitivity of ribozyme action to RNA structure and suggest that even when homologous target sequences exist, ribozymes may be excluded from cleaving that RNA by structural constraints. This level of specificity minimizes non-specific toxicity of ribozymes within cells.

Example 6

Ability of Hammerhead Ribozymes to Inhibit Smooth Muscle Cell Proliferation

The ribozymes that cleaved c-myb RNA described above were assayed for their effect on smooth muscle cell proliferation. Rat vascular smooth muscle cells were isolated and cultured as follows. Aortas from adult Sprague-Dawley rats were dissected, connective tissue was removed under a dissecting microscope, and 1 mm.sup.2 pieces of the vessel were placed, intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) with the following additives: 10% FBS, 2% tryptose phosphate broth, 1% penicillin/streptomycin and 2 mM L-Glutamine. The smooth muscle cells were allowed to migrate and grow to confluence over a 3-4 week period. These primary cells were frozen and subsequent passages were grown at 37.degree. C. in 5% CO.sub.2 in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and the following additives: 2 mM L-Glutamine, 1% penicillin/streptomycin, 1 mM sodium pyruvate, non-essential amino acids (0.1 mM of each amino acid), and 20 mM Hepes pH 7.4. Cells passed four to six times were used in proliferation assays. For the cell proliferation assays, 24-well tissue culture plates were prepared by coating the wells with 0.2% gelatin and washing once with phosphate-buffered saline (PBS). RASMC were inoculated at 1.times.10.sup.4 cells per well in 1 ml of DMEM plus 10% FBS and additives and incubated for 24 hours. The cells were subconfluent when plated at this density. The cells were serum-starved by removing the medium, washing once with PBS, and incubating 48-72 hours in DMEM containing 0.5% FBS plus additives.

In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, C. F., et al., 1992, Mol. Pharmacology, 41, 1023-1033). In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) formulation of DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifiuoroacetate) and dioleoyl phosphatidylethanolamine (DOPE)), was purchased from Life Technologies, Inc. DMRIE (N-[1-(2,3-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide) was obtained from VICAL. DMRIE was resuspended in CHCl.sub.3 and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). The CHCl.sub.3 was evaporated, the lipid was resuspended in water, vortexed for 1 minute and bath sonicated for 5 minutes. Ribozyme and cationic lipid mixtures were prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives was warmed to room temperature (about 20.degree.-25.degree. C.), cationic lipid was added to the final desired concentration and the solution was vortexed briefly. RNA oligonucleotides were added to the final desired concentration and the solution was again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex was serially diluted into DMEM following the 10 minute incubation.

Serum-starved smooth muscle cells were washed twice with PBS, and the RNA/lipid complex was added. The plates were incubated for 4 hours at 37.degree. C. The medium was then removed and DMEM containing 10% FBS, additives and 10 .mu.M bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation. The plates were incubated at 37.degree. C. for 20-24 hours, fixed with 0.3% H.sub.2 O.sub.2 in 100% methanol, and stained for BrdU incorporation by standard methods. In this procedure, cells that have proliferated and incorporated BrdU stain brown; non-proliferating cells are counter-stained a light purple. Both BrdU positive and BrdU negative cells were counted under the microscope. 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incorporated BrdU (% cell proliferation) is presented. Errors represent the range of duplicate wells. Percent inhibition then is calculated from the % cell proliferation values as follows: % inhibition=100-100((Ribozyme-0% serum)/(Control-0% serum)).

Six hammerhead ribozymes, including the best five ribozymes from the in vitro RNA cleavage test (directed against sites 549, 575, 1553, 1598, and 1635) and one with intermediate cleavage levels (directed against site 1597) and their catalytically inactive controls were synthesized and purified as described above. The ribozymes were delivered at a concentration of 0.3 .mu.M, complexed with DMRIE/DOPE such that the cationic lipid charges and the anionic RNA charges were at 1:1 molar ratio. The results, shown in Table V, demonstrate a considerable range in the efficacy of ribozymes directed against different sites. Five of the six hammerhead ribozymes (directed against sites 549, 575, 1553, 1597, and 1598) significantly inhibit smooth muscle cell proliferation. The control, inactive ribozymes that cannot cleave c-myb RNA due to alterations in their catalytic core sequence fail to inhibit rat smooth muscle cell proliferation. Thus, inhibition of cell proliferation by these five hammerhead sequences is due to their ability to cleave c-myb RNA, and not because of any antisense activity. The sixth ribozyme (directed against site 1635) fails to function in smooth muscle cells. This ribozyme cleaved c-myb RNA very efficiently in vitro. In this experiment, 10% FBS (no ribozyme added) induced 64.+-.1% proliferation; 0% FBS produced a background of 9.+-.1% proliferation.

Example 7

Ability of Exogenously Delivered Hairpin Ribozyme Against c-myb to Inhibit Vascular Smooth Muscle Cell Proliferation

In addition to the hammerhead ribozymes tested above, a bipartite hairpin ribozyme (Chowrira, B. M., supra, 1992, Nucleic Acids Res., 20, 2835-2840) was identified that also cleaves c-myb RNA. The effect of this ribozyme on smooth muscle cell proliferation was tested. Ribozymes were delivered at the indicated doses with Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 87.+-.1% proliferation; 0% FBS produced 5.+-.1% proliferation. The results of a dose-response experiment are shown in Table VI. In this example, the control was an irrelevant hammerhead ribozyme. The irrelevant ribozyme control contains the same catalytic core sequences, but has binding arms that are directed to a cellular RNA that is not required for smooth muscle cell proliferation. This control failed to significantly inhibit cell proliferation, demonstrating the sequence specificity of these ribozymes. Another control that could be run is an irrelevant catalytically active ribozyme having the same GC content as the test ribozyme.

Example 8

Ribozymes Inhibit Proliferation of Rat Smooth Muscle Cells in a Dose-dependent Fashion

If the inhibition of proliferation observed in Example 6 is caused by the ribozymes, the level of inhibition should be proportional to the dose of RNA added. Rat aortic smooth muscle cells were assayed for proliferation in the presence of differing doses of two hammerhead ribozymes. The results shown in Table VII indicate that two hammerhead ribozymes that cleave c-myb RNA at sites 575 and 549 inhibit SMC proliferation in a dose-dependent fashion. Ribozymes were delivered with the cationic lipid, Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) gave 92.+-.1% proliferation; 0% FBS gave 6.+-.1% proliferation. The control is an active ribozyme directed against an irrelevant mRNA target and shows no inhibition over the dose range tested. The control ribozyme contains the same catalytic core sequences as the active ribozymes but differs in its binding arm sequences (stems I and III in FIG. 2c). Thus, ribozyme inhibition of smooth muscle cell proliferation requires sequence-specific binding by the hammerhead arms to c-myb mRNA.

Example 9

Delivery of a c-myb Ribozyme With Different Cationic Lipids

The experiment in Table VIII shows the response of rat smooth muscle cells to a hammerhead ribozyme that cleaves c-myb RNA at site 575 delivered with two different cationic lipids, DMRIE and Lipofectamine. Similar efficacy is observed with either lipid. 10% FBS (no ribozyme) induced 78.+-.2% proliferation; 0% FBS produced a background of 6.+-.1% proliferation.

Example 10

Effect of Varying Arm-lengths on Ribozyme Activity

The exact configuration of each ribozyme can be optimized by altering the length of the binding arms (stems I and III, see FIG. 2C). The length of the binding arms may have an effect on both the binding and the catalytic cleavage step (Herschlag, D., 1991, Proc. Natl. Acad. Sci. U S A, 88, 6921-5). For example, Table IX shows the ability of arm length variants of c-myb hammerhead 575 to inhibit SMC proliferation. Note that the dose used in this experiment (0.1 .mu.M) is 3-fold lower than in previous experiments. At this concentration, the 7/7 arm variant gives relatively little inhibition. In this case, the degree of inhibition increases with concomitant increases in arm length.

The optimum arm length may be site-specific and should be determined empirically for each ribozyme. Towards this end, hammerhead ribozymes target with 7 nucleotide binding arms (7/7) and ribozymes with 12 nucleotide binding arms (12/12) targeted to three different cleavage sites were compared.

Ribozymes were delivered at 0.2 .mu.M with the cationic lipid DMRIE at a 1:1 charge ratio of oligonucleotide to cationic lipid as described in Example 6. The data are shown below in Table X. As can be seen, all three ribozymes demonstrated enhanced inhibition of smooth muscle cell proliferation with twelve nucleotide binding arms. Each ribozyme showed greater inhibition than its catalytically inactive control, again demonstrating that the ribozymes function via their ability to cleave c-myb RNA. In this experiment, 10% stimulation resulted in 54.+-.2% cell proliferation; unstimulated cells showed 8.+-.0.5% cell proliferation.

Example 11

Effect of Chloroquine on Ribozyme Activity

A number of substances that effect the trafficking of macromolecules through the endosome have been shown to enhance the efficacy of DNA delivery to cells. These include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), chloroquine, monensin, colchicine, and viral particles (Cotten, M. et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 4033-4037; Cotten, M. et al., 1993, J. Virol., 67, 3777-3785; Cotten, M. et al., 1992, Proc. Natl. Acad. Sci USA, 89, 6094-6098; Cristiano, R. J. et al., 1993, Proc. Natl. Acad. Sci. U S A, 90, 2122-6; Cudel, D. T. et al., 1991, Proc. Nat. Acad. Sci., USA, 88, 8850-8854; Ege, T. et al., 1984, Exp. Cell Res., 155, 9-16; Harris, C. E. et al., 1993, Am. J. Respir. Cell Mol. Biol., 9, 441-7; Seth, P. et al., 1994, J. Virol., 68, 933-40; Zenke, M. et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 3655-3659). It is thought that DNA is taken up by cells by endocytosis, resulting in DNA accumulation in endosomes (Akhtar, S. and Juliano, R. L., 1992, Trends Cell Biol., 2, 139-144). Thus, the above agents may enhance DNA expression by promoting DNA release from endosomes. To determine whether such agents may augment the functional delivery of RNA and ribozymes to smooth muscle cells, the effects of chloroquine on ribozyme inhibition of smooth muscle cell proliferation were assessed. A ribozyme with twelve nucleotide binding arms that cleaves c-ruby RNA was delivered to rat smooth muscle cells as described in Example 6 (0.2 .mu.M ribozyme complexed with DMRIE/DOPE at a 1:1 charge ratio). In some cases, 10 .mu.M chloroquine was added upon stimulation of the cells. The addition of choloroquine had no effect on untreated cells (stimulation with 10% serum in the presence or absence of chloroquine resulted in 80.5.+-.1.5% and 83.+-.2% cell proliferation, respectively; unstimulated cells with and without chloroquine showed 7.+-.0.5% and 7.+-.1% cell proliferation, respectively). As shown in Table XI below, addition of chloroquine augments ribozyme inhibition of smooth muscle cell proliferation two- to three-fold.

Example 12

Effect of a Hammerhead Ribozyme on Human Smooth Muscle Cell Proliferation

The hammerhead ribozyme that cleaves human c-myb RNA at site 549 was tested for its ability to inhibit human aortic smooth muscle cell proliferation. The binding site for this ribozyme is completely conserved between the mouse and human cDNA sequences. Human aortic smooth muscle cells (AOSMC) were obtained from Clonetics and were grown in SmGM (Clonetics.RTM.). Cells from passage five or six were used for assays. Conditions for the proliferation assay were the same as for the rat cells (see Example 6), except that the cells were plated in SmGM and starved in SmBM plus 0.5% FBS. The ribozyme that cleaves site 549 was delivered at varying doses complexed with the cationic lipid DMRIE at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 57.+-.7% proliferation; the uninduced background was 6.+-.1% proliferation. The results in Table XII show that inhibition is observed over a similar concentration range as was seen with rat smooth muscle cells.

Example 13

Inhibition by Direct Addition of a Modified, Stabilized Ribozyme

A hammerhead ribozyme that cleaves site 575 was chemically synthesized with 12 nucleotide binding arms (sequence ID NO. 127, in Table III). Chemically modified nucleotides were incorporated into this ribozyme that have been shown to enhance ribozyme stability in serum without greatly impacting catalytic activity. (See Eckstein et al., International Publication No. WO 92/07065, Perrault et al., 1990, Nature, 344, 565-568, Pieken, W. et al. 1991, Science, 253, 314-317, Usman, N.; Cedergren, R. J., 1992, Trends in Biochem. Sci., 17, 334-339, Usman, N. et al. U.S. patent application Ser. No. 07/829,729, and Sproat, B. European Patent Application 92110298.4 describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.) The modifications used were as follows. All the nucleotides of the ribozyme contained 2'-O-methyl groups with the following exceptions: U.sub.4 and U.sub.7 contained 2'-amino substitutions; G.sub.5, A.sub.6, G.sub.8, G.sub.12, and A.sub.15.1 were 2'-OH ribonucleotides (numbering as in FIG. 1). An inactive ribozyme was chemically synthesized in which G.sub.5 and A.sub.14 were substituted with 2'-O-methyl U. Ribozymes were added to rat smooth muscle cells at the indicated concentrations as per Example 6 except that cationic lipids were omitted. Proliferation was assessed by BrdU incorporation and staining. Table XIII shows that the modified ribozyme is capable of inhibiting rat smooth muscle cell proliferation without addition of cationic lipids. In this experiment, 10% serum induced 45.+-.2% proliferation while uninduced cells showed a background of 2.3.+-.0.1% proliferation.

Optimizing Ribozyme Activity

As demonstrated in the above examples, ribozymes that cleave c-myb RNA are capable of inhibiting 50% of the smooth muscle cells from proliferating in response to serum. This level of inhibition does not represent the maximal effect obtainable with the ribozymes; in each dose response experiment, the highest dose produced the greatest extent of inhibition. Thus, optimizing activity of the ribozyme within the cells and/or optimizing the delivery of the ribozyme to the cells is expected to increase the extent of inhibition.

Tables IX and X demonstrate one means of optimizing ribozyme activity. By altering the length of the ribozyme binding arms (stems I and Ill, see FIG. 2c), the ability of the ribozyme to inhibit smooth muscle cell proliferation is greatly enhanced. Ribozymes with increasing arm lengths will be synthesized either chemically in one or two pads (see above and see Mamone, U.S. Ser. No. 07/882,689, filed May 11, 1992, hereby incorporated by reference herein) or by in vitro transcription (see Cech et al., U.S. Pat. No. 4,987,071). Ribozymes are chemically synthesized with modifications that prevent their degradation by serum ribonucleases (as described in Example 13, above). When synthesized in two parts, the fragments are ligated or otherwise juxtaposed as described (see original application and Mamone, supra). The effects of the ribozymes on smooth muscle cell proliferation are assessed as in Examples 6 and 12, above. As the length of stems I and III can affect both hybridization to the target and the catalytic rate, the arm length of each ribozyme will be optimized for maximal inhibitory effect in cells. Similarly, the precise sequence of modified nucleotides in the stabilized ribozyme will affect the activity in cells. The nature of the stabilizing modifications will be optimized for maximal inhibitory effect in cells. In each case, activity of the ribozyme that cleaves c-myb RNA will be compared to the activity of its catalytically inactive control (substitution of 2'-O- methyl U for G.sub.5 and a 2'-O-methyl U for A.sub.14) and to a ribozyme targeted to an irrelevant RNA (same catalytic core, with appropriate modifications, but different binding arm sequences).

Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. The data presented in Example 9 indicate that different cationic lipids can deliver active ribozymes to rat smooth muscle cells. In this example, 0.6 .mu.M ribozyme delivered with Lipofectamine produced the same inhibitory effect as 0.3 .mu.M ribozyme delivered with DMRIE. Thus, DMRIE is twice as efficacious as Lipofectamine at delivering active ribozymes to smooth muscle cells. There are a number of other cationic lipids known to those skilled in the art that can be used to deliver nucleic acid to cells, including but not limited to dioctadecylamidoglycylspermine (DOGS), dioleoxltrimetylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxy)-propyl]-n,n,n-trimethylammoniumchloride (DOTMA), N-[1-(2,3-dioleoyloxy)-propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), and N-[1-(2,3-dioleoyloxy)propyl]-N,N-dimethyl-N-hydroxypropylammonium bromide (DORIE-HP). Experiments similar to those performed in Example 9 are used to determine which lipids give optimal delivery of ribozymes to smooth muscle cells. Other such delivery methods are known in the art and can be utilized in this invention.

The data described in Example 11 show that ribozyme delivery and efficacy may be augmented by agents that disrupt or alter cellular endosome metabolism. Chloroquine was shown to increase the ability of a ribozyme to inhibit smooth muscle cell proliferation by 2- to 3-fold. Experiments similar to those described in Example 11 can be performed to determine the optimal concentration of chloroquine to be used to augment delivery of ribozymes alone (as in Example 13), or delivery in the presence different cationic lipids (as in Example 9 and described above) or with other delivery agents (as described below). Other agents that disrupt or alter endosomes known to those familiar with the art can be used to similarly augment ribozyme effects. These agents may include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), chloroquine, monensin, colchicine, amphipathic peptides, viral proteins, and viral particles. Such compounds may be used in conjunction with ribozymes as described above, may be chemically conjugated directly to ribozymes may be chemically conjugated to liposomes, or may be incorporated with ribozymes in liposome particles (see Sullivan, et al., supra, incorporated by reference herein).

The data presented in Example 13 indicate that the proliferation of smooth muscle cells can be inhibited by the direct addition of chemically stabilized ribozymes. Presumably, uptake is mediated by passive diffusion of the anionic nucleic acid across the cell membrane. In this case, efficacy could be greatly enhanced by directly coupling a ligand to the ribozyme. The ribozymes are then delivered to the cells by receptor-mediated uptake. Using such conjugated adducts, cellular uptake can be increased by several orders of magnitude without having to alter the phosphodiester linkages necessary for ribozyme cleavage activity.

Alternatively, ribozymes 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. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Alternative routes of delivery include, but are not limited to, intramuscular injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al., supra which have been incorporated by reference herein.

Example 14

Phosphorothioate Linkages Enhance the Ability of Ribozymes to Inhibit Smooth Muscle Cell Proliferation

As the applicant had shown in Example 13, the hammerhead (HH) ribozyme that cleaves c-myb RNA at site 575 can be modified to confer resistance to nucleases while maintaining catalytic activity (see also Usman et al., supra). To identify ribozymes with optimal activity in cells, several different chemically-modified ribozymes were directly compared for inhibition of rat smooth muscle cell proliferation. Chemically-modified ribozymes used are diagrammed in FIG. 9A. One ribozyme (designated "2'-O-methyl") contains ribonucleotide residues at all positions except the 5 terminal nucleotides of each target binding arm (Stems I and III). The ribozyme designated "2'-O-methyl P=S" in addition contains five phosphorothioate linkages between the terminal nucleotides in each target binding arm. The ribozyme termed "2'-C-allyl iT" contains thirty 2'-O-methyl nucleotides as specified in Example 13. The ribozyme also contains 2'-C-allyl U (Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163) at the U4 position and 2'-O-methyl U at the U7 position and a 3'-3'-linked inverted thymidine (Ortigao et al., 1992 Antisense Res. & Development 2, 129; Seliger et al., Canadian Patent Application No. 2,106,819) at the 3' end of the molecule (referred to as 2'-C-allyl iT). The fourth ribozyme contains the same 2'-O-methyl and 2'-C-allyl residues described above with the addition of 5 phosphorothioate linkages between the terminal nucleotides in each target binding arm (referred to as "2'-C-allyl P=S").

Ribozymes were delivered to smooth muscle cells as cationic lipid complexes (Sullivan et al., supra). In this example, the cationic lipid, Lipofectamine (GIBCO-BRL), was used at a charged lipid concentration of 3.6 .mu.M (see Examples 6 and 9). Active versus inactive forms of each ribozyme were compared to determined whether inhibition is mediated specifically by ribozyme cleavage. As shown in FIG. 9B, the ribozyme synthesized with the 2'-C-allyl modification and the phosphorothioate linkages demonstrated enhanced inhibition of smooth muscle cell proliferation. The catalytically inactive form of the ribozyme had little effect on cell proliferation; thus, the inhibition observed requires the catalytic activity of the ribozyme. In contrast, ribozymes without the stable 2'-O-methyl- and 2'-C-allyl-modified catalytic core (2'-O-methyl and 2'-O-methyl P=S) at best showed only modest inhibition of smooth muscle cell proliferation. The stable core chemistry alone was not sufficient to greatly enhance ribozyme-mediated inhibition; without terminal P=S linkages, the 2'-C-allyl-modified ribozyme showed very little specific inhibition when compared to its inactive ribozyme control. These results demonstrate that certain chemical modifications greatly enhance the ability of exogenously-delivered ribozymes to cleave c-myb RNA and impact cell proliferation.

Example 15

Dose Response of the Chemically Modified Ribozyme

Varying doses of the 2'-C-allyl P=S-modified ribozyme were delivered to rat aortic smooth muscle cells as described above. As in previous examples, percent inhibition was calculated by comparing the effects of the active ribozyme to the effects of the inactive ribozyme. As shown in FIG. 10, the ribozyme concentration at which cell proliferation is inhibited by 50% (IC.sub.50) is approximately 70 nM. From day to day, the IC.sub.50 varies between 25 and 100 nM.

Example 16

Direct Comparison of the Effects of Ribozymes and Antisense DNA

Ribozymes are thought to be more specific reagents for the inhibition of gene expression than antisense oligonucleotides due to their catalytic activity and strict sequence requirements around the site of cleavage (Castanotto et al., 1994 Adv. in Pharmacol. 25, 289). To test this hypothesis, ribozyme activity was directly compared to the activity of phosphorothioate DNA oligonucleotides that target the same site in the c-myb mRNA. The ribozyme used was the 2'-C-allyl P=S-modified ribozyme described in Example 14, above. This ribozyme binds to a 15 nucleotide long region of the c-myb mRNA. Thus, a 15 nucleotide antisense phosphorothioate DNA molecule was prepared. A phosphorothioate DNA oligonucleotide with a randomly scrambled sequence of the same 15 nucleotides and a 2'-C-allyl P=S-modified ribozyme with randomly scrambled target binding arm sequences were synthesized as controls (by comparison to the murine c-myb cDNA sequence, the scrambled controls would not be expected to bind any region of the c-myb mRNA). Since longer phosphorothioate DNA oligonucleotides are often utilized as antisense inhibitors (for a review see Wagner, 1994 Science 372, 333), a symmetrically placed, 25 nucleotide phosphorothioate DNA antisense oligonucleotide and its scrambled sequence control were also synthesized. The ribozymes and the antisense oligonucleotides were delivered to rat smooth muscle cells as complexes with the cationic lipid, Lipofectamine, and serum-stimulated smooth muscle cell proliferation was measured subsequently.

As shown in FIG. 11, the 2'-C-allyl P=S-modified ribozyme demonstrated greater inhibition of smooth muscle cell proliferation than either of the antisense oligonucleotides. Furthermore, the scrambled arm ribozyme and inactive ribozyme controls demonstrated less non-specific inhibition than either of the scrambled sequence antisense control oligonucleotides. In fact, the non-specific inhibition demonstrated by the 25 nucleotide phosphorothioate molecule completely masked any specific effect of the antisense molecule. Similar results have been obtained with phosphorothioate DNA targeting other sites in the c-myb mRNA. Thus, a ribozyme that cleaves c-myb RNA is a more potent and more specific inhibitor of smooth muscle cell proliferation than phosphorothioate antisense DNA molecules.

Example 17

Chemically-modified Ribozymes Targeting Different Sites in the c-myb mRNA Specifically Inhibit Smooth Muscle Cell Proliferation

If the observed inhibition of smooth muscle cell proliferation is mediated by ribozyme cleavage of c-myb mRNA, then other ribozymes that target the same mRNA should have the same effect. Two other ribozymes targeting two disparate sites in the c-myb mRNA (sites 549 and 1553, ribozyme Seq. ID Nos. 102 and 112) were synthesized with the 2'-C-allyl P=S modifications as described in Example 14. Inactive ribozyme controls also were synthesized corresponding to each new target sequence. Chemically-modified ribozymes targeting sites 549, 575, and 1553 were delivered to rat smooth muscle cells and their ability to inhibit serum-stimulated cell proliferation was assessed. Equivalent levels of inhibition are obtained with active ribozymes targeting sites 549, 575 and 1553 (see FIG. 12). None of the inactive ribozymes inhibited cell proliferation. Active ribozymes targeting other mRNA sequences not present in c-myb or ribozymes with scrambled arm sequences also fail to inhibit smooth muscle cell proliferation (see FIG. 12). Thus, inhibition of cell proliferation requires a catalytically active ribozyme that can bind to accessible c-myb mRNA sequences and is likely due to the reduction of c-myb mRNA levels by ribozyme cleavage.

Examples 18 and 19 describe experiments designed to determine the position and minimum number of phosphorothioate residues required for efficacy.

Example 18

Effect of Position of Phosphorothioate Linkages on Ribozyme Inhibition

Ribozymes targeting c-myb site 575 were synthesized with the 2'-C-allyl modification and with phosphorothioate linkages between various nucleotides in the ribozyme. One ribozyme contained a total of 10 phosphorothioate linkages, 5 in Stem I and 5 in Stem III, identical to the ribozyme described in Examples 14 through 17 above (referred to as 10 P=S 5' and 3' in FIG. 13A). One ribozyme contained only 5 phosphorothioate linkages in Stem III (5 P=S 3' in FIG. 13A). Another ribozyme contained 5 phosphorothioate linkages between the 6 nucleotides comprising the last base pair of stem II and the GAAA loop (5 P=S loop in FIG. 13A). The fourth ribozyme contained 5 phosphorothioate linkages in stem I (5 P=S 5' in FIG. 13A). The latter two ribozymes also were synthesized with the 3'-3' thymidine at the 3' end to help protect the ribozyme from 3' exonucleases (Ortigao et al., 1992 Antisense Res. & Development 2, 129; Seliger et al., Canadian Patent Application No. 2,106,819). The structure of these four different ribozymes is diagrammed in FIG. 13A. Inactive ribozyme controls were synthesized for each individual ribozyme. The active and inactive ribozymes were applied to rat smooth muscle cells as RNA/Lipofectamine complexes and their effects on cell proliferation were measured.

Referring to FIG. 13B, the ribozyme containing 5 phosphorothioate linkages in Stem I and the 3' inverted thymidine inhibited smooth muscle cell proliferation as well as the parent ribozyme with 10 total phosphorothioate linkages. None of the other ribozymes demonstrated significant differences between active and inactive controls. Therefore, the 3' inverted T can effectively substitute for the 5 phosphorothioate linkages in Stem III. Phosphorothioate linkages in the loop position lead to non-specific inhibition of smooth muscle cell proliferation, while phosphorothioate linkages in Stem I are necessary for enhanced efficacy in cells. Additionally, these results suggest that 3'-end modifications, such as iT, is desirable to minimize the amount of phosphorothioate contained in the ribozymes in order to minimize toxicity and facilitate chemical synthesis, while maintaining protection from endogenous 3'-exonuclease digestion.

Example 19

Minimizing Phosphorothioate Linkages in Stem I

Fewer phosphorothioate linkages in the ribozyme will reduce the complexity and cost of chemical synthesis. Furthermore, phosphorothioate DNA molecules are known to have some undesirable and non-specific effects on cellular functions (for a review see Wagner, supra); reducing the phosphorothioate linkages in these RNA molecules is expected to enhance their specificity. A series of ribozymes targeting c-myb were synthesized to determine how many phosphorothioate linkages in Stem I are required for optimal ribozyme activity. The ribozymes contained 5, 4, 3, 2, or 1 phosphorothioate linkage(s) in Stem I, beginning with the phosphodiester bond between the first and second nucleotides and proceeding 3'. Each ribozyme contained the 2'-O-methyl modifications, the U.sub.4 2'-C-allyl nucleotide, and the inverted T nucleotide at the 3' end as described above. Activity of each of these ribozymes was compared to the activity of the ribozyme with 10 phosphorothioate linkages, 5 each in Stems I and III (referred to as 10 P=S in FIG. 14). Active and inactive ribozymes were applied to rat smooth muscle cells as complexes with Lipofectamine and their effects on smooth muscle cell proliferation were measured in two separate experiments. The results are diagrammed in FIG. 14. Ribozymes with 10, 5, and 4 phosphorothioate linkages showed equivalent efficacy. Ribozymes with fewer than four phosphorothioate linkages also showed efficacy, but the level of inhibition of smooth muscle cell proliferation was modestly reduced.

Example 20

Varying the Length of Stems I and III

Ribozymes that cleave c-myb RNA at position 575 were synthesized with varying arm lengths. Each ribozyme contained 4 phosphorothioate linkages at the 5' end, 2'-O-methyl and 2'-C-allyl modifications and an inverted thymidine nucleotide at the 3' end as described above. FIG. 15 shows the effects of these ribozymes upon rat smooth muscle cell proliferation. Ribozymes were delivered at 100 nM with cationic lipid. Ribozymes with 6/6, 7/7 and 5/10 arms (where x/y denotes the nucleotides in Stem I/nucleotides in Stem III; see FIG. 2) all showed comparable efficacy. As shown in FIG. 15, ribozymes with longer arm lengths tended to demonstrate more non-specific inhibition (the inactive ribozyme controls with longer binding arms inhibited smooth muscle cell proliferation) when compared to ribozymes with shorter binding arms. From these data, it appears that ribozymes with 6/6, 7/7, 5/10, 10/5, 8/8 and 10/10 nucleotide arms all specifically inhibit smooth muscle cell proliferation, optimal inhibition, however, is observed with 6/6, 7/7 and 5/10 nucleotide arms.

Example 21

Ribozymes with Different Modified Nucleotides Inhibit Smooth Muscle Cell Proliferation

Ribozymes containing seven nucleotides in both Stems I and III, four phosphorothioate residues at the 5' end and a 3'-3' inverted thymidine at the 3' end, were synthesized with various modified nucleotides at the U.sub.4 and U.sub.7 positions within the core of a HH ribozyme. All of the modified catalytic core chemistries retained ribozyme activity and demonstrated enhanced stability to serum nucleases (Usman et al., 1994 supra). The ribozyme termed U4 2'-C-allyl contains a 2'-C-allyl uridine at the U.sub.4 position and a 2'-O-methyl nucleotide at the U.sub.7 position. The ribozyme termed U4,U7 2'-amino contains a 2'-amino nucleotide at both U4 and U7. The ribozyme termed U4 2'-fluoro contains a 2'-fluoro-modified nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 6-methyl contains a 6-methyl uridine nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 deoxyabasic contains a deoxyribose moeity and lacks a base at U4 (Beigelman et al., 1994 Bioorganic & Med. Chem. Letters 4, 1715) and 2'-O-methyl at U7. Active and inactive versions of each of the chemically-modified ribozymes were applied to rat smooth muscle cells using Lipofectamine as described above. As diagrammed in FIG. 16, all of the nuclease-stable, chemically-modified ribozymes demonstrated significant inhibition of rat smooth muscle cell proliferation. Thus, the requirements for ribozyme activity in smooth muscle cells appear to be a catalytically core that is modified to minimize endonucleolytic degradation and modifications at the 5' and 3' ends which may prevent exonucleolytic degradation.

Chemical modifications described in this invention are meant to be non-limiting examples, and those skilled in the art will recognize that other modifications (base, sugar and phosphate modifications) to enhance nuclease stability of a ribozyme can be readily generated using standard techniques and are hence within the scope of this invention.

Example 22

Ribozyme Inhibition of Pig Smooth Muscle Cell Proliferation

Of the commonly used animal models of intimal hyperplasia after balloon angioplasty, the pig model is believed to be most predictive of human disease (Steele et al., 1985 Circ. Res. 57, 105; Ohno et al., 1994 Science 265, 781; Baringa, 1994 Science 265, 738). Therefore, we wished to assess the ability of c-myb ribozymes to inhibit pig smooth muscle cell proliferation. Yucatan pig smooth muscle cells (YSM) were obtained from Dr. Elizabeth Nabel (University of Michigan Medical Center) and were grown in Dulbecco's modified Eagle's medium as described (see Example 6). The YSM cells were starved for 72 hours in DMEM with 0.1% FBS. Active and inactive ribozymes (four phosphorothioate linkages at the 5' end, 2'-C-allyl-modified core and 3'-3' inverted thymidine at the 3' end) were applied as RNA/Lipofectamine.RTM. complexes as described in the above examples. Proliferation was stimulated with serum and assessed by BrdU incorporation. FIG. 17 shows that a ribozyme dose of as low as 75 nM can inhibit pig smooth muscle cell proliferation by as much as 60%. The same chemical modifications of the ribozymes (2'-modified, stable core, 5' phosphorothioate linkages and 3' inverted thymidine) are required to obtain significant and reproducible inhibition of pig smooth muscle cell proliferation as were shown to be required for inhibition of rat cells in the above Examples.

Example 23

Ribozyme Inhibition of Human Smooth Muscle Cell Proliferation

In Example 12, we demonstrated that a minimally modified ribozyme directed against c-myb site 549 could significantly inhibit human smooth muscle cell proliferation. The 2'-C-allyl and phosphorothioate-modified ribozyme targeting c-myb site 575 characterized above was applied to human smooth muscle cells as RNA/Lipofectamine.RTM. complexes. Inactive ribozyme and inactive, scrambled arm ribozymes were applied as controls. At 200 nM, the active ribozyme inhibits human smooth muscle proliferation by greater than 75% while the inactive ribozyme inhibits proliferation by only 38%. The ribozyme with scrambled binding arm sequences fails to inhibit. At 100 nM, the active ribozyme still demonstrates significant inhibition while neither the inactive or scramble controls inhibit cell proliferation (see FIG. 18). Thus, the active ribozyme identified in these studies mediates significant inhibition of human smooth muscle cell proliferation and represents a novel therapeutic for restenosis and/or vascular disease.

Example 24

Delivery of c-myb Ribozymes to Vessels in vivo

The ribozyme that cleaves c-myb RNA at site 575 was synthesized in two parts (Mamone, supra), the internal 5' end was labeled with .sup.33 P using polynucleotide kinase and the two fragments were ligated with RNA ligase. The resulting RNA was an intact ribozyme with an internal .sup.33 P label. This internally-labeled ribozyme was delivered to balloon injured rat carotid arteries as described (Simons et al., 1992 Nature 359, 67). Rats were anesthetized and the carotid artery was surgically exposed. The external carotid was dissected and a 2F Fogarty balloon catheter was inserted and directed into the carotid artery. Injury was caused by repeated (3 times) inflation and retraction of the balloon. The injured region was isolated by ligatures and a cannula was inserted in the external carotid. Ribozymes alone (two rat vessels) or ribozyme/Lipofectamine.RTM. complexes (two rat vessels) were applied to the injured vessel through the cannula and were left in the vessel for twenty minutes. After application, blood flow was restored by removal of the ligatures for five minutes and the vessels were harvested and processed as described below.

Half of the vessel was frozen in liquid nitrogen, crushed into a fine powder, and RNA was extracted using standard protocols. The extracted RNA was applied to a denaturing polyacrylamide gels and subjected to electrophoresis. Autoradiography of the gel permitted detection of the .sup.33 P label; the amount of radioactivity in each band was quantitated using a Phosphor-imaging system. The amount of extracted and intact ribozyme was calculated by direct comparison to labeled ribozyme controls run on the same gel. The percentage of the ribozyme delivered intact could be estimated by quantifying the percentage of label that co-migrates with the intact ribozyme controls. After delivery of ribozymes in phosphate-buffered saline (PBS), 3% of the .sup.33 P label was recovered from the rat vessels and >90% of the label was present in the form of intact ribozyme. After delivery of ribozyme in RNA/Lipofectamine complexes, 10 to 11% of the .sup.33 P label was recovered from the rat vessels and 20 to 90% of the label was present in the form of intact ribozyme. The significant uptake of the intact ribozyme demonstrates that local delivery of modified ribozymes to arterial walls is feasible.

The other half of each vessel was fixed in PBS-buffered 2% glutaraldehyde, sectioned onto slides and coated with emulsion. After autoradiography for four days, the emulsion was developed and the sections were stained with hematoxylin and eosin by standard techniques (Simons et al., 1992 supra). Inspection of the sections showed a majority of the grains present over the medial smooth muscle cells after application of the ribozyme. Some 33p label could be detected in the underlying adventilia as well. Similar density and distribution of grains was observed when the ribozyme was delivered with or without Lipofectamine. These data demonstrate that ribozyme can penetrate the injured vessel wall and is in close apposition or within the underlying medial smooth muscle cells. Thus, therapeutic ribozymes can be locally delivered to vessels for the treatment of vascular disease.

Similar experiments were performed in pig iliofemoral vessels. After balloon injury, a ribozyme, internally labeled with .sup.33 P as described above, was delivered with a double balloon catheter device (Nabel and Nabel, supra; Ohno et al., 1994 supra). After 20 minutes, blood flow was restored by deflating the balloons. The vessels were harvested after an additional hour or the surgical injuries were sutured and the vessels harvested one day later. Harvested vessels were sectioned, subjected to autoradiography and stained. One hour after delivery, the majority of the .sup.33 P label could be detected in the media, overlying or within smooth muscle cells. Some label was also detected at the luminal surface of the vessel and in the adventitial tissue. One day after delivery, grains could be still be detected associated with remaining medial smooth muscle cells. No major differences in density or distribution was observed between ribozymes delivered with or without Lipofectamine.RTM.. These data demonstrate that ribozymes can be locally delivered to smooth muscle cells of injured vessels in a large animal model that is clinically relevant to human vascular disease.

Example 25

Ribozyme-mediated Decrease in the Level of c-myb RNA in Rat Smooth Muscle Cells

To determine whether a ribozyme catalyzes the cleavage of c-myb RNA in a mammalian cell, applicant has used a sensitive quantitative competitive polymerase chain reaction (QCPCR) to assay the level of c-myb RNA in rat smooth muscle cells treated with either catalytically active or inactive ribozyme.

Rat smooth muscle cells (RASMC) were treated with ribozymes as described above. Following the ribozyme treatment for 4 h, cells were stimulated with 10% serum (in the presence or absence of BrdU). After 24 h, cells were harvested for further analysis. Cells, that were treated with BrdU, were assayed for proliferation as described above. Cells, that were not treated with BrdU, were used for the QCPCR assay.

The following is a brief description of the QCPCR technique used to quantitate levels of c-myb mRNA from RASMC, normalizing to the housekeeping gene, GAPDH. This method was adapted from Thompson et al, Blood 79: 1692, 1992. Briefly, total RNA was isolated from RASMC using the Guanidinium isothiocyanate technique of Chomczynski and Sacchi (Analytical Biochemistry, 162: 156, 1987). In order to construct a deletion competitor and control wild-type RNA, a cDNA clone of the rat c-myb message, referred to as pc8myb, was used. The competitor RNA comprises a deletion of 50 bases, making it smaller than the wild-type cellular RNA, and spansfrom nucleotide 428 to nucleotide 753.

A house-keeping gene, GAPDH, that is constitutively expressed by the RASMC, was used as an internal control for QCPCR assay. A deletion competitor and wild-type controls for GAPDH were made the same way as for c-myb. GAPDH-containing plasmid (pTri-GAPDH) was purchased from Ambion. The GAPDH competitor is also a deletion mutant, lacking 50 bases. The GAPDH competitor was used to quantitate the amount of this housekeeping gene in each sample, thus allowing for a confirmation of cellular RNA's integrity and for the efficiency of RNA isolation. All quantitations for the level of c-myb expression were normalized to the level of GAPDH expression in the same sample of cells.

Referring to FIG. 19, RASMC that were treated with a stabilized catalytically active 575 HH ribozyme did not proliferate well. There was greater than 70% inhibition of RASMC proliferation when compared with approximately 25% inhibition of cell proliferation by a catalytically inactive version of the 575 HH ribozyme. The level of inhibition of RASMC proliferation correlates very well with the greater than 70% decrease in the level of c-myb RNA. This shows that the inhibition of smooth muscle cell proliferation is directly mediated by the cleavage of c-myb RNA by a ribozyme in RASMC.

FIG. 20 shows what Applicant presently believes is an optimal ribozyme configuration.

Example 26

Inhibition of Smooth Muscle Cell Proliferation by 2-5A Antisense Chimera

By "2-5A antisense chimera" is meant, an antisense oligonucleotide containing a 5' phosphorylated 2'-5'-linked adenylate residues. 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).

RNAs containing 2'-5' Adenosine with a terminal 5' phosphate has been shown to activate RNAse L (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300). The terminal phosphate is required for efficient activation of RNAse L. Ribozymes targeting c-myb site 575 were synthesized with 2-5A moieties on the 5' end, with and without the terminal 5' phosphate. The ribozyme-2-5A chimera was complexed with LipofectAMINE and assayed on rat aortic smooth muscle cells (RASMC) as described above.

As shown in FIG. 21, when no terminal phosphate is present, the active ribozyme [575 inactive Rz+ inactive (A)4] functions similarly to a normal active ribozyme lacking a 2-5A modification (575 active Rz). An inactive ribozyme core with 5' phosphate-2-5A [575 inactive Rz+active P(A)4] shows significant inhibition relative to the controls, but has significantly lower activity when compared with an active ribozyme. A molecule that contains both an active ribozyme core and 5' phosphate-contining 2-5A [575 active Rz+active P (A)4] shows even greater inhibition than that obtained by either mechanism individually, inhibiting the smooth muscle cell proliferation to baseline levels (0% FBS). Thus the ribozyme and 2-5A anitisense chimera together show an additive effect in inhibiting RASMC proliferation.

Use of Ribozymes That Cleave c-myb RNA to Treat Restenosis.

The above discussion demonstrates, by way of example, how ribozymes that inhibit smooth muscle cell proliferation are delivered directly, or through the use of expression vectors, to vessels. Preferably, ribozymes cleaving c-myb RNA are delivered to vessels at the time of coronary angioplasty. Local delivery during intervention can be achieved through the use of double balloon catheters, porous balloon catheters, balloon catheters coated with polymers (Riessen, R., et al., 1993, Human Gene Therapy, 4, 749-758), or biopolymer stents (Slepian and Schindler, U.S. Pat. No. 5,213,580). In the above examples, ribozymes were identified that could inhibit roughly half of the smooth muscle cells in culture from proliferating in response to the growth factors present in serum. A corresponding 50% (or even lower) reduction in intimal thickening will significantly improve the outcome of patients undergoing coronary angioplasty.

Use of Ribozymes Targeting c-myb to Treat Cancer

Overexpression of the c-myb oncogene has been reported in a number of cancers, including leukemias, neuroblastomas, and lung, colon, and breast carcinomas (Torelli, G., et al., 1987, Cancer Res., 47, 5266-5269; Slamon, D. J., et al., 1986, Science, 233, 203-206; Slamon, D. J., et al., 1984, Science, 224, 256-262; Thiele, C. J., et al., 1988, Mol. Cell. Biol., 8, 1677-1683; Griffin, C. A. and Baylin, S. B., 1985, Cancer Res., 45, 272-275; Alitalo, K., et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 4534-4538). Thus, inhibition of c-myb expression can reduce cell proliferation of a number of cancers. Indeed, in tissue culture, treatment of colon adenocarcinoma, neurectodermal, and myeloid leukemia cell lines with antisense c-myb oligonucleotides inhibits their proliferation (Melani, C., et al., 1991, Cancer Res., 51, 2897-2901; Raschella, F., et al., 1992, Cancer Res., 52, 4221-4226; Anfossi, G., et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 3379-3383). Furthermore, myeloid cells from patients with chronic myelogenous leukemia and acute myelogenous leukemia are differentially sensitive to c-myb antisense oligonucleotides (Calabretta, B., et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 2351-2355). Ratajczak, et al. (1992, Proc. Natl. Acad. Sci. USA, 89, 11823-11827) treated mice bearing human leukemia cells with c-myb antisense oligonucleotides and significantly prolonged their survival and reduced their tumor burden. Thus, reduction of c-myb expression in leukemic cells in tissue culture and in vivo can reduce their proliferative potential.

While the above studies demonstrated that antisense oligonucleotides can efficiently reduce the expression of c-myb in cancer cells and reduce their ability to proliferate and spread, this invention describes the first enzymatic RNAs, or ribozymes, shown to cleave c-myb RNA. Such ribozymes, with their catalytic activity and increased site specificity (see above), are likely to represent more potent and safe therapeutic molecules than antisense oligonucleotides for the treatment of cancer as well as restenosis. In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation. From those practiced in the art, it is clear from the examples described, that the same ribozymes may be delivered in a similar fashion to cancer cells to block their proliferation.

In a preferred embodiment, autologous bone marrow from patients suffering with acute myelogenous leukemia or chronic myelogenous leukemia are treated with ribozymes that cleave c-myb RNA. Ribozymes will be delivered to the autologous bone marrow cells ex vivo at 0.1 to 50 .mu.M with or without forming complexes of the ribozymes with cationic lipids, encapsulating in liposomes or alternative delivery agents. After several days, the proliferative capacity of the leukemic cells in the patients bone marrow will be reduced. The patient's endogenous bone marrow cells will be depleted by chemical or radiation treatments and their bone marrow reconstituted with the ex vivo treated cells. In such autologous bone marrow reconstitution treatments of leukemic patients, recurrence of the disease can be caused by proliferation of leukemic cells present in the transplanted bone marrow. Significantly reducing the proliferative potential of the leukemic cells by treating with ribozymes that cleave c-myb RNA will reduce the risk of recurrent leukemia.

Diagnostic uses

Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of c-myb 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 may 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 may 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 may 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 c-myb related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

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 will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be 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 will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be 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., c-myb) 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 will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

Other embodiments are within the following claims.

TABLE I______________________________________Characteristics of Ribozymes______________________________________Group I IntronsSize: .about.200 to >1000 nucleotides.Requires a U in the target sequence immediately 5' ofthe cleavage site.Binds 4-6 nucletides at 5' side of cleavage site.Over 75 known members of this class. Found in Tetrahymenathermophila rRNA, fungal mitochondria, chloroplasts,phage T4, blue-green algae, and others.RNAseP RNA (M1 RNA)Size: .about.290 to 400 nucleotides.RNA portion of a ribonucleoprotein enzyme. Cleaves tRNAprecursors to form mature tRNA.Roughly 10 known members of this group all are bacterial in origin.Hammerhead RibozymeSize: .about.13 to 40 nucleotides.Requires the target sequence UH immediately 5' ofthe cleavage site.Binds a variable number nucleotides on both sides ofthe cleavage site.14 known members of this class. Found in a number of plantpathogens (virusoids) that use RNA as the infectious agent(FIG. 1)Hairpin RibozymeSize: .about.50 nucleotides.Requires the target sequence GUC immediately 3' ofthe cleavage site.Binds 4-6 nucleotides at 5' side of the cleavage siteand a variable number to the 3' side of the cleavage site.Only 3 known member of this class. Found in three plant pathogen(satellite RNAs of the tobacco ringspot virus, arabis mosiac virusand chicory yellow mottle virus) which uses RNA as the infectiousagent (FIG. 3).Hepatitis Delta Virus (HDV) RibozymeSize: 50-60 nucleotides (at present).Cleavage of target RNAs recently demonstrated.Sequence requirements not fully determined.Binding sites and structural requirements not fully determined,although no sequences 5' of cleavage site are required.Only 1 known member of this class. Found in human HDV(FIG. 4).Neurospora VS RNA RibozymeSize: .about.144 nucleotides (at present)Cleavage of target RNAs recently demonstrated.Sequence requirements not fully determined.Binding sites and structural requirements not fully determined.Only 1 known member of this class.Found in Neurospora VS RNA (FIG. 5).______________________________________ TABLE II__________________________________________________________________________Human c-myb Target SequenceSite Target Sequence Sequence I.D. No.__________________________________________________________________________ 86 GGCGGCAGCGCCCUGCCGACGCCGGGG ID. NO. 01 162 CCGCGGCUCUCGGC ID. NO. 02 195 GCCAUGGCCCGAA ID. NO. 03 213 CGGCACAGCAUAUAUAGCAGUGACGAGGA ID. NO. 04 249 GACUUUGAGAUGUGUGACCAUGACUAUGAUGGG ID. NO. 05 295 CUGGAAAGCGUC ID. NO. 06 332 GGAAGAGGAUGAAAAACUGAAGAAG ID. NO. 07 350 GAAGAACUGGUGGAACAGAAUGGAAC ID. NO. 08 383 CUGGAAAGUUAUUGCCAA ID. NO. 09 407 CCCGAAUCGAACAGAUGUGCAG ID. NO. 10 446 GAAAGUACUAAACCCUGAG ID. NO. 11 478 CUUGGACCAAAGAAGAAGAUCAGAGAGUGAUA ID. NO. 12 518 ACAGAAAUACGGUCCGAAACGUUGGUCUG ID. NO. 13 547 UUAUUGCCAAGCACUUAAAGGGGAGAAUUGGAA ID. NO. 14 611 GAAUCCAGAAGUUAAGAA ID. NO. 15 647 GGAAGACAGAAUUAUUUACCAGGCACA ID. NO. 16 674 CAAGAGACUGGGGAACAGAU ID. NO. 17 700 AAAUCGCAAAGCUA ID. NO. 18 720 GGACGAACUGAUAAUGCUAUCAAGAACC ID. NO. 19 748 ACUGGAAUUCUACAAUGCGUCGGAAGGUCGAACA ID. NO. 20 816 CAGCCAGCAGUGGCCACAA ID. NO. 21 852 CAUUUGAUGGGUUUUGCUCAGGCUCCGCCUACA ID. NO. 22 885 GCUCAACUCCCUGCCACUGGCCAGCCC ID. NO. 23 918 AACAACGACUAUUCCUAUUACCACA ID. NO. 24 954 CAAAAUGUCUCCAGUCAUGUUCCAUACCCU ID. NO. 25 998 AAAUAUAGUCAAUGUCCCUCAGCCAGCUGCCGCA ID. NO. 261039 AGAGACACUAUAAUGAUGAAGACCCUGAGAAGGA ID. NO. 271073 AAAGCGAAUAAAGGAAUUAGAAUUG ID. NO. 281098 CUCCUAAUGUCAACCGA ID. NO. 291120 AGCUAAAAGGACAGCAGGUGCUACCAACACAGAA ID. NO. 301161 CCCGGGUGGCACAGCACCACCAUUGCCGACCACA ID. NO. 311237 AACACCACUCCACUCCAUCUCUGCCAGCGGAUCC ID. NO. 321279 UACCUGAAGAAA ID. NO. 331311 AUGAUCGUCCACCAGGGCACCAUU ID. NO. 341366 CAGAAACACUCCAAUUUA ID. NO. 351418 AAACUCAGACU ID. NO. 361434 AUGCCUUCUUUAAC ID. NO. 371480 UUACAACACCA ID. NO. 381515 ACUCAAAAGGAAAAUACUGUUUUUAGAACCC ID. NO. 391546 CAGCUAUCAAAAGGUCAAUCUUAGAAAGCU ID. NO. 401576 CUCCAAGAACUCCUACACCAUUCAA ID. NO. 411601 ACAUGCACUUGCAGCUCAAGAA ID. NO. 421630 UACGGUCCCCUGAAGAUGCUACCUCAGA ID. NO. 431657 CACCCUCUCAUCUAGUAGAAGAUCUGCAGGA ID. NO. 441693 UCAAACAGGAAUCUGAUGAAUCUGGA ID. NO. 451735 AAGAAAAUGGA ID. NO. 461751 CUUACUGAAGAAAAUCAAACAAGA ID. NO. 471780 AAUCUCCAACUGAUAAAUCAG ID. NO. 481813 GCUCACACCACUGGGA ID. NO. 491864 CCUCGCCUGUGCGAGAUGCACCGAAUAUUC ID. NO. 501913 GGCACCAGCAUCAGAAGAUGAAGAC ID. NO. 511951 CAUUUACAGUACC ID. NO. 521975 CCCUGGCGAGCCCCUUGCA ID. NO. 531994 GCCUUGUAGCAGUACCUGGGA ID. NO. 542059 GUCAAGCUCGUAAAUACGUGAA ID. NO. 552142 GAACAGUUCAA ID. NO. 562181 AUGAAACUUUUCAU ID. NO. 572304 AAAAUAAAUAACAGUC ID. NO. 582340 UGAAUUGUAGCC ID. NO. 592357 UUAAUAUCUUAAU ID. NO. 602399 AUUUAUCUGGUAUUUUAAAGGAUCCAACAGAUC ID. NO. 6,12483 CCAGUAUUUCA ID. NO. 622499 CUCGAUCACUAAACAUAUG ID. NO. 632518 CAUAUAUUUUUAAAAAUC ID. NO. 642767 UGCUAUGGUCUUAGCCU ID. NO. 652799 AGUAUCAGAGG ID. NO. 662849 UAGGUAAUUGACUAU ID. NO. 672871 UAUUUCAGACUUUUAAUUUUAUAUAUAUAUACA ID. NO. 682920 CAAUACAUUUGAAAACUUGUUUGGGAGACUCUGC ID. NO. 692964 GUGGUUUUUUUGUUAUUGUUGGUUU ID. NO. 703008 UUCUUUUUUGGGAGAU ID. NO. 713040 CUAUGUUUUGUUUUG ID. NO. 723060 AGCCUGACUGUUUUAUA ID. NO. 733089 UCGAUUUGAUC ID. NO. 743145 UGGAUCCUGUGUU ID. NO. 753184 UUGAUAGCCAGUCACUGCCUUAAGA ID. NO. 763209 ACAUUUGAUGCAAGAUGGCCAGCACU ID. NO. 773252 CGGUGUACUUACUGCC ID. NO. 78__________________________________________________________________________ TABLE III__________________________________________________________________________Sequences of ribozymes used in these studies.Target SequenceSite ID No. Ribozyme Sequence__________________________________________________________________________Hammerhead ribozymes with 7 nucleotide binding arms 310 101 UUUCCCCCUGAUGAGGCCGAAAGGCCGAAAGUGACG 549 102 UUGGCAACUGAUGAGGCCGAAAGGCCGAAAACAGAC 551 103 GCUUGGCCUGAUGAGGCCGAAAGGCCGAAAUAACAG 575 104 GCUUUCCCUGAUGAGGCCGAAAGGCCGAAAUUCUCC 634 105 UGUCCAGCUGAUGAGGCCGAAAGGCCGAAAGGUUUU 738 106 UUCUUGACUGAUGAGGCCGAAAGGCCGAAAGCAUUA 839 107 UCUUCUGCUGAUGAGGCCGAAAGGCCGAAAAGCUCG 936 108 AUGUGGUCUGAUGAGGCCGAAAGGCCGAAAUAGGAA1017 109 GCCGGCUCUGAUGAGCGCGAAAGCGCGAAAGGGACG1082 110 GCUCCUUCUGAUGAGGCCGAAAGGCCGAAAUUCGCU1363 111 UUCUGCACUGAUGAGGCCGAAAGGCCGAAAUUCUAA1553 112 ACCUUUUCUGAUGAGGCCGAAAGGCCGAAAUAGCUG1597 113 AUGUUUGCUGAUGAGGCCGAAAGGCCGAAAUGGUGU1598 114 CAUGUUUCUGAUGAGGCCGAAAGGCCGAAAAUGGUG1635 115 UUCAGGGCUGAUGAGGCCGAAAGGCCGAAACCGUAU1721 116 CAGCAACCUGAUGAGGCCGAAAGGCCGAAAUUCCAG1724 117 ACUCAGCCUGAUGAGGCCGAAAGGCCGAAACAAUUC1895 118 AGCUUGUCUGAUGAGGCCGAAAGGCCGAAAGAAUAU1909 119 UGUCAUUCUGAUGAGGCCGAAAGGCCGAAAAACAGA1943 120 CUUUGAGCUGAUGAGGCCGAAAGGCCGAAACAUUGUBimolecular Hairpin Ribozymes 1632.sup.a 121 5' Fragment: UCAGGGAGAAGUAUACCAGAGAAACACACGCG 3' Fragment: CGCGUGGUACAUUACCUGGUA 2231.sup.a 122 5' Fragment: GCUCUCAGAAGUUGACCAGAGAAACACACGCG 3' Fragment: CGCGUGGUACAUUACCUGGUAHammerhead riboyzmes with 6, 8, 9, 10, and 12 nucleotide binding arms 575 123 CUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUC 6/6.sup.b 575 124 UGUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUCCC8/8 575 125 CUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUCCCU9/9 575 126 ACUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUCCCUU10/10 575 127 ACACUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUCCCUUUU12/12 549 128 AGUGCUUGGCAACUGAUGAGGCCGAAAGGCCGAA AACAGACCAACG12/12 1553 129 GAUUGACCUUUUCUGAUGAGGCCGAAAGGCCGAA AUAGCUGGAGUU12/12__________________________________________________________________________ .sup.a The hairpin ribozymes were synthesized in two pieces as indicated. The two oligonucleotides were annealed and tested for activity against th cmyb RNA as described above. See Mamone, Ribozyme synthesis, filed May 11 1992, U.S.S.N. 07/882,689, hereby incorporated by reference herein. .sup.b Designation of the ribozymes with different arm lengths is a/b where (a) represents the nucleotides in stem I and (b) represents the nucleotides in stem III (see FIG. 1). TABLE IV______________________________________Ribozyme catalyzed cleavage of c-myb RNA % Cleavage Mouse HumanCleavage Sequence c-myb c-mybSite ID No. Target sequence RNA RNA______________________________________Hammerhead Sites 310 79 CGUCACU U GGGGAAA 28.5 0.1 549 80 GUCUGUU A UUGCCAA 87.4 91.6 551 81 CUGUUAU U GCCAAGC 56.8 82.4 575 82 GGAGAAU U GGAAAAC 93.9 91.3 634 83 AAAACCU C CUGGACA 68.4 87.1 738 84 UAAUGCU A UCAAGAA 78.1 0.01 839 85 CAAGCUU C CAGAAGA 27.2 0.01 936 86 UUCCUAU U ACCACAU 61.8 60.61017 97 UGUCCCU C AGCCAGC 40.3 0.11082 88 AGCGAAU A AAGGAAU 55.2 89.21363 89 UUAGAAU U UGCAGAA 11.6 0.11553 90 CAGCUAU C AAAAGGU 87.1 92.51597 91 ACACCAU U CAAACAU 71.2 62.71598 92 CACCAUU C AAACAUG 79.6 85.51635 93 AUACGGU C CCCUGAA 84.4 82.31721 94 CUGGAAU U GUUGCUG 62.1 79.31724 95 GAAUUGU U GCUGAGU 65.6 861895 96 AUAUUCU U ACAAGCU 79.1 66.21909 97 UCCGUUU U AAUGGCA 31.1 0.11943 98 ACAAUGU U CUCAAAG 66.1 80Hairpin Ribozymes1632 99 ACG GUCC CCUGAAG 92.8 84.62231 100 ACA GUUG AGAGCAG 0.1 0.1______________________________________ .sup.a The nucleotide numbers given correspond to the nucleotide just 5' of the ribozyme cleavage site in the human cmyb sequence taken from Westin, et al., supra (GenBank Accession No. X52125). All but two of the sequences (310; I.D. No. 79 and 2231; I.D. No. 100) overlap sequences in Table I. TABLE V______________________________________Comparison of the effects six hammerhead ribozymes, thatcleave c-myb RNA, on smooth muscle cell proliferation. Inactive Ribozyme Active RibozymeRibozyme % Cell % Cell % InhibitionSite Proliferation Proliferation (Active vs. Inactive)______________________________________ 549 68 .+-. 1 59.5 .+-. 1.5 14 .+-. 4 575 66.5 .+-. 0.5 54.5 .+-. 1.5 21 .+-. 31553 68.5 .+-. 0.5 52 .+-. 1 28 .+-. 11597 66 .+-. 1 57 .+-. 3 16 .+-. 71598 67 .+-. 1 58.5 .+-. 0.5 1.5 .+-. 11635 62.5 .+-. 2.5 64 .+-. 1 0______________________________________ TABLE VI______________________________________Dose Response of c-myb Hairpin Ribozyme 1632Ribozyme Control Ribozyme Ribozyme 1632 % InhibitionDose (.mu.M) % Proliferation % Proliferation (vs. control)______________________________________0.05 86.5 .+-. 1.5 88 .+-. 5 00.15 89.5 .+-. 1.5 78.5 .+-. 2.5 10 .+-. 50.45 87.5 .+-. 1 66.5 .+-. 1.5 25 .+-. 4______________________________________ TABLE VII______________________________________Dose Response of c-myb Hammerhead Ribozymes 575 and 549Control Ribozyme 575 Ribozyme 549 Ribozyme % Inhibi- % Inhibi-Ribozyme % cells % cells tion (vs. % cells tion (vs.Dose (.mu.M) in S phase in S phase control) in S phase control)______________________________________0.05 89 .+-. 5 77.5 .+-. 1.5 14 .+-. 8 92 .+-. 1 00.15 90 .+-. 1 68.5 .+-. 1.5 26 .+-. 2 84 .+-. 2 9 .+-. 40.45 91.5 .+-. 0.5 59 .+-. 5 38 .+-. 7 76.5 .+-. 2.5 18 .+-. 5______________________________________ TABLE VIII______________________________________ Inactive Ribozyme Active Ribozyme 575 575Ribozyme % cells in % cells in % InhibitionDose (.mu.M) S phase S phase (vs. inactive)______________________________________Delivery with DMRIE/DOPE 0.075 79 .+-. 6 74.5 .+-. 1.5 6 .+-. 60.15 79.5 .+-. 0.5 67 .+-. 1 17 .+-. 40.30 77 .+-. 1 57 .+-. 2 28 .+-. 5Delivery with Lipofectamine 0.075 81 .+-. 1 83 .+-. 1 00.15 79 .+-. 3 71 .+-. 1 11 .+-. 40.30 82 .+-. 1 68.5 .+-. 1.5 18 .+-. 40.60 75 .+-. 1 59.5 .+-. 3.5 22 .+-. 7______________________________________ TABLE IX______________________________________Arm Length Variations of c-myb Hammerhead Ribozyme 575Arm Length % Inhibition(base-pairs) % cells in S phase (vs. Inactive 7/7)______________________________________6/6 62 .+-. 1 4 .+-. 47/7 60 .+-. 1 7 .+-. 38/8 60.5 .+-. 0.5 6 .+-. 29/9 53.5 .+-. 0.5 18 .+-. 210/10 55 .+-. 1 16 .+-. 412/12 48 .+-. 1 28 .+-. 3______________________________________ TABLE X______________________________________Hammerhead ribozymes with 7 vs. 12-nucleotidebinding arms targeting three different sites. Inactive ActiveRibozyme Length of Ribozyme Ribozyme % InhibitionTarget Binding (% Cell (% Cell (Active vs.Site Arms Proliferation) Proliferation) Inactive)______________________________________575 7/7 51.5 .+-. 0.5 43 .+-. 0.5 24 .+-. 5575 12/12 50.5 .+-. 3.5 37 .+-. 0.5 37 .+-. 4549 7/7 49.5 .+-. 0.5 44.5 .+-. 1.5 21 .+-. 7659 12/12 48.5 .+-. 1.5 35 .+-. 2 41 .+-. 71553 7/7 49.5 .+-. 0.5 43.5 .+-. 2.5 23 .+-. 91553 12/12 49 .+-. 1 33.5 .+-. 1.5 45 .+-. 6______________________________________ TABLE XI______________________________________Effect of chloroquine on ribozyme inhibitionof smooth muscle cell proliferation. Inactive Active Ribozyme Ribozyme % Inhibition Chloroquine (% Cell (% Cell (Active vs.Ribozyme (.mu.M) Proliferation) Proliferation) Inactive)______________________________________575, 12/12 0 81.8 .+-. 0.5 74 .+-. 1 10 .+-. 2575, 12/12 10 83 .+-. 4 62.5 .+-. 0.5 28 .+-. 6______________________________________ TABLE XII______________________________________Inhibition of Human Aortic Smooth Muscle Cellsby c-myb Ribozyme 549Ribozyme Inactive Ribozyme Active Ribozyme % InhibitionDose (.mu.M) % Proliferation % Proliferation (active vs. inactive)______________________________________0.075 55 .+-. 2 40.5 .+-. 4.5 30 .+-. 130.15 53 .+-. 10 42 .+-. 1 23 .+-. 230.30 53 .+-. 7 32.5 .+-. 4.5 44 .+-. 22______________________________________ TABLE XIII______________________________________Inhibition of Rat Smooth Muscle Cell Proliferation by DirectAddition of a Chemically-Modified c-myb Ribozyme 575Ribozyme Inactive Ribozyme Active Ribozyme % InhibitionDose (.mu.M) % Proliferation % Proliferation (active vs. inactive)______________________________________0.22 42 .+-. 3 36 .+-. 0.5 15 .+-. 80.67 48 .+-. 3 35 .+-. 2 28 .+-. 92.0 52 .+-. 5 25 .+-. 1 54 .+-. 7______________________________________ TABLE XIV__________________________________________________________________________Human c-myb Hairpin Ribozyme and Target SequencesPosition Ribozyme Sequenc Target__________________________________________________________________________ 104 CCCUCCCC AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGCA GCC GGGGAGGG 148 ACCGACCG AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCA GCC CGGUCGGU 185 GCGCGGCG AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCGCC GCC CGCCGCGC 528 ACGUUUCG AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUACG GUC CGAAACGU 715 UUCGUCCA AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACU GCC UGGACGAA1025 AUGGCUGC AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCU GCC GCAGCCAU1187 CUGGUGUG AGAA GCAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGCC GAC CACACCAG1532 GUUCUAAA AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUACU GUU UUUAGAAC1632 CUUCAGGG AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUACG GUC CCCUGAAG1836 GGUAUUCA AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACA GUC UGAAUACC1852 UCUGCGUG AGAA GUUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAACU GUU CACGCAGA1861 CAGGCGAG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCA GAC CUCGCCUG1993 UGCUACAA AGAA GCAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGCA GCC UUGUAGCA2231 CUGCUCUC AGAA GUUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAACA GUU GAGAGCAG2316 UUAGGUAA AGAA GUUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAACA GUC UUACCUAA3068 AAUUAUAA AGAA GUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGACU GUU UUAUAAUU3138 AUCCAUGC AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAACU GUU GCAUGGAU3199 GUUCUUAA AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACU GCC UUAAGAAC3264 UGCUACAA AGAA GUAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUACU GCC UUGUAGCA__________________________________________________________________________ TABLE XV__________________________________________________________________________Human c-myb Hammerhead Ribozyme and Target Sequences(REVISED) Seq. Seq.nt. Target Seqence ID No. HH Ribozyme Sequence ID__________________________________________________________________________ No.15 AACCUGUU U CCUCCUCC 170 GGAGGAGG CUGAUGAGGCCGAAAGGCCGAA 171AGGUU16 ACCUGUUU C CUCCUCCU 172 AGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 173CAGGU19 UGUUUCCU C CUCCUCCU 174 AGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 175AAACA22 UUCCUCCU C CUCCUUCU 176 AGAAGGAG CUGAUGAGGCCGAAAGGCCGAA 177AGGAA25 CUCCUCCU C CUUCUCCU 178 AGGAGAAG CUGAUGAGGCCGAAAGGCCGAA 179AGGAG28 CUCCUCCU U CUCCUCCU 180 AGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 181AGGAG29 UCCUCCUU C UCCUCCUC 182 GAGGAGGA CUGAUGAGGCCGAAAGGCCGAA 183GAGGA31 CUCCUUCU C CUCCUCCU 184 AGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 185AGGAG34 CUUCUCCU C CUCCUCCG 186 CGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 187AGAAG37 CUCCUCCU C CUCCGUCA 188 UCACGGAG CUGAUGAGGCCGAAAGGCCGAA 189AGGAG40 CUCCUCCU C CGUCACCU 190 AGGUCACG CUGAUGAGGCCGAAAGGCCGAA 191AGGAG49 CGUGACCU C CUCCUCCU 192 AGGAGGAG CUGAUGAGGCCGAAAGGCCGAA 193UCACG52 GACCUCCU C CUCCUCUU 194 AAGAGGAG CUGAUGAGGCCGAAAGGCCGAA 195AGGUC55 CUCCUCCU C CUCUUUCU 196 AGAAAGAG CUGAUGAGGCCGAAAGGCCGAA 197AGGAG58 CUCCUCCU C UUUCUCCU 198 AGGAGAAA CUGAUGAGGCCGAAAGGCCGAA 199AGGAG60 CCUCCUCU U UCUCCUGA 200 UCAGGAGA CUGAUGAGGCCGAAAGGCCGAA 201GGAGG61 CUCCUCUU U CUCCUGAG 202 CUCAGGAG CUGAUGAGGCCGAAAGGCCGAA 203AGGAG62 UCCUCUUU C UCCUGAGA 204 UCUCAGGA CUGAUGAGGCCGAAAGGCCGAA 205GAGGA64 CUCUUUCU C CUGAGAAA 206 UUUCUCAG CUGAUGAGGCCGAAAGGCCGAA 207AAGAG75 GAGAAACU U CGCCCCAG 208 CUGGGGCG CUGAUGAGGCCGAAAGGCCGAA 209UUCUC76 AGAAACUU C GCCCCAGC 210 GCUGGGGC CUGAUGAGGCCGAAAGGCCGAA 211UUUCU170 CCGCGGCU C UCGCGGAG 212 CUCCGCGA CUGAUGAGGCCGAAAGGCCGAA 213CGCGG172 GCGGCUCU C GCGGAGCC 214 GGCUCCGC CUGAUGAGGCCGAAAGGCCGAA 215GCCGC224 CACAGCAU A UAUAGCAG 216 CUGCUAUA CUGAUGAGGCCGAAAGGCCGAA 217CUGUG226 CAGCAUAU A UAGCAGUG 218 CACUGCUA CUGAUGAGGCCGAAAGGCCGAA 219UGCUG228 GCAUAUAU A GCAGUGAC 220 GUCACUGC CUGAUGAGGCCGAAAGGCCGAA 221UAUGC253 UGAGGACU U UGAGAUGU 222 ACAUCUCA CUGAUGAGGCCGAAAGGCCGAA 223CCUCA254 GAGGACUU U GAGAUGUG 224 CACAUCUC CUGAUGAGGCCGAAAGGCCGAA 225UCCUC274 CCAUGACU A UGAUGGGC 226 GCCCAUCA CUGAUGAGGCCGAAAGGCCGAA 227CAUGG287 GGGCUGCU U CCCAAGUC 228 GACUUGGG CUGAUGAGGCCGAAAGGCCGAA 229AGCCC288 GGCUGCUU C CCAAGUCU 230 AGACUUGG CUGAUGAGGCCGAAAGGCCGAA 231CAGCC310 GCGUCAUC U GGGGAAAA 232 UUUUCCCC CUGAUGAGGCCGAAAGGCCGAA 233GACGC393 GGAAAGUU A UUGCCAAU 234 AUUGGCAA CUGAUGAGGCCGAAAGGCCGAA 235UUUCC395 AAAGUUAU U GCCAAUUA 236 UAAUUGGC CUGAUGAGGCCGAAAGGCCGAA 237ACUUU402 UUGCCAAU U AUCUCCCG 238 CGGGAGAU CUGAUGAGGCCGAAAGGCCGAA 239GGCAA403 UGCCAAUU A UCCUCCGA 240 UCGGGAGA CUGAUGAGGCCGAAAGGCCGAA 241UGGCA405 CCAAUUAU C UCCCGAAU 242 AUUCGGGA CUGAUGAGGCCGAAAGGCCGAA 243AUUGG497 AAUUAUCU C CCGAAUCG 244 CGAUUCGG CUGAUGAGGCCGAAAGGCCGAA 245UAAUU414 UCCCGAAU C GAACAGAU 246 AUCUGUUC CUGAUGAGGCCGAAAGGCCGAA 247CGGGA455 AAAGUACU A AACCCUGA 248 UCAGGGUU CUGAUGAGGCCGAAAGGCCGAA 249ACUUU467 CCUGAGCU C AUCAAGGG 250 CCCUUGAU CUGAUGAGGCCGAAAGGCCGAA 251UCAGG470 GAGCUCAU C AAGGGUCC 252 GGACCCUU CUGAUGAGGCCGAAAGGCCGAA 253AGCUC480 AGGGUCCU U GGACCAAA 254 UUUGGUCC CUGAUGAGGCCGAAAGGCCGAA 255ACCCU498 AAGAAGAU C AGAGAGUG 256 CACUCUCU CUGAUGAGGCCGAAAGGCCGAA 257UUCUU509 AGAGUGAU A GAGCUUGU 258 ACAAGCUC CUGAUGAGGCCGAAAGGCCGAA 259ACUCU515 AUAGAGCU U GUACAGAA 260 UUCUGUAC CUGAUGAGGCCGAAAGGCCGAA 261UCUAU526 ACAGAAAU A CGGUCCGA 262 UCGGACCG CUGAUGAGGCCGAAAGGCCGAA 263UCUGU549 GGUCUGUU A UUGCCAAG 264 CUUGGCAA CUGAUGAGGCCGAAAGGCCGAA 265AGACC551 UCUGUUAU U GCCAAGCA 266 UGCUUGGC CUGAUGAGGCCGAAAGGCCGAA 267ACAGA562 CAAGCACU U AAAGGGGA 268 UCCCCUUU CUGAUGAGGCCGAAAGGCCGAA 269GCUUG563 AAGCACUU A AAGGGGAG 270 CUCCCCUU CUGAUGAGGCCGAAAGGCCGAA 271UGCUU575 GGGAGAAU U GGAAAACA 272 UGUUUUCC CUGAUGAGGCCGAAAGGCCGAA 273AUCCC603 GGUGGCAU A ACCACUUG 274 CAAGUGGU CUGAUGAGGCCGAAAGGCCGAA 275CCACC610 UAACCACU U GAAUCCAG 276 CUGGAUUC CUGAUGAGGCCGAAAGGCCGAA 277GGUUA615 ACUUGAAU C CAGAAGUU 278 AACUUCUG CUGAUGAGGCCGAAAGGCCGAA 279CAAGU624 CAGAAGUU A AGAAAACC 280 GGUUUUCU CUGAUGAGGCCGAAAGGCCGAA 281UUCUG634 GAAAACCU C CUGGACAG 282 CUGUCCAG CUGAUGAGGCCGAAAGGCCGAA 283UUUUC659 GACAGAAU U AUUUACCA 284 UGGUAAAU CUGAUGAGGCCGAAAGGCCGAA 285CUGUC660 ACAGAAUU A UUUACCAG 286 CUGGUAAA CUGAUGAGGCCGAAAGGCCGAA 287UCUGU662 AGAAUUAU U UACCAGGC 288 GCCUGGUA CUGAUGAGGCCGAAAGGCCGAA 289AUUCU663 GAAUUAUU U ACCAGGCA 290 UGCCUGGU CUGAUGAGGCCGAAAGGCCGAA 291AAUUC664 AAUUAUUU A CCAGGCAC 292 GUGCCUGG CUGAUGAGGCCGAAAGGCCGAA 293UAAUU704 GCAGAAAU C GCAAAGCU 294 AGCUUUGC CUGAUGAGGCCGAAAGGCCGAA 295UCUGC713 GCAAAGCU A CUGCCUGG 296 CCAGGCAG CUGAUGAGGCCGAAAGGCCGAA 297UUUGC732 GAACUGAU A AUGCUAUC 298 GAUAGCAU CUGAUGAGGCCGAAAGGCCGAA 299AGUUC738 AUAAUGCU A UCAAGAAC 300 GUUCUUGA CUGAUGAGGCCGAAAGGCCGAA 301AUUAU740 AAUGCUAU C AAGAACCA 302 UGGUUCUU CUGAUGAGGCCGAAAGGCCGAA 303GCAUU756 ACUGGAAU U CUACAAUG 304 CAUUGUAG CUGAUGAGGCCGAAAGGCCGAA 305CCAGU757 CUGGAAUU C UACAAUGC 306 GCAUUGUA CUGAUGAGGCCGAAAGGCCGAA 307UCCAG759 GGAAUUCU A CAAUGCGU 308 ACGCAUUG CUGAUGAGGCCGAAAGGCCGAA 309AUUCC790 GGAAGGUU A UCUGCAGG 310 CCUGCAGA CUGAUGAGGCCGAAAGGCCGAA 311CUUCC792 AAGGUUAU C UGCAGGAG 312 CUCCUGCA CUGAUGAGGCCGAAAGGCCGAA 313ACCUU804 AGGAGUCU U CAAAAGCC 314 GGCUUUUG CUGAUGAGGCCGAAAGGCCGAA 315CUCCU805 GGAGUCUU C AAAAGCCA 316 UGGCUUUU CUGAUGAGGCCGAAAGGCCGAA 317ACUCC838 CACAAGCU U CCAGAAGA 318 UCUUCUGG CUGAUGAGGCCGAAAGGCCGAA 319UUGUG839 ACAAGCUU C CAGAAGAA 320 UUCUUCUG CUGAUGAGGCCGAAAGGCCGAA 321CUUGU855 ACAGUCAU U UGAUGGGU 322 ACCCAUCA CUGAUGAGGCCGAAAGGCCGAA 323ACUGU856 CAGUCAUU U GAUGGGUU 324 AACCCAUC CUGAUGAGGCCGAAAGGCCGAA 325GACUG865 GAUGGGUU U UGCUCAGG 326 CCUGAGCA CUGAUGAGGCCGAAAGGCCGAA 327CCAUC866 AUGGGUUU U GCUCAGGC 328 GCCUGAGC CUGAUGAGGCCGAAAGGCCGAA 329CCCAU870 GUUUUGCU C AGGCUCCG 330 CGGAGCCU CUGAUGAGGCCGAAAGGCCGAA 331AAAAC876 CUCAGGCU C CGCCUACA 332 UGUAGGCG CUGAUGAGGCCGAAAGGCCGAA 333CUGAG882 CUCCGCCU A CAGCUCAA 334 UUGAGCUG CUGAUGAGGCCGAAAGGCCGAA 335CGGAG888 CUACAGCU C AACUCCCU 336 AGGGAGUU CUGAUGAGGCCGAAAGGCCGAA 337UGUAG893 GCUCAACU C CCUGCCAC 338 GUGGCAGG CUGAUGAGGCCGAAAGGCCGAA 339UGAGC918 CCACUGUU A ACAACGAC 340 GUCGUUGU CUGAUGAGGCCGAAAGGCCGAA 341AGUGG928 CAACGACU A UUCCUAUU 342 AAUAGGAA CUGAUGAGGCCGAAAGGCCGAA 343CGUUG930 ACGACUAU U CCUAUUAC 344 GUAAUAGG CUGAUGAGGCCGAAAGGCCGAA 345GUCGU931 CGACUAUU C CUAUUACC 346 GGUAAUAG CUGAUGAGGCCGAAAGGCCGAA 347AGUCG934 CUAUUCCU A UUACCACA 348 UGUGGUAA CUGAUGAGGCCGAAAGGCCGAA 349AAUAG936 AUUCCUAU U ACCACAUU 350 AAUGUGGU CUGAUGAGGCCGAAAGGCCGAA 351GGAAU937 UUCCUAUU A CCACAUUU 352 AAAUGUGG CUGAUGAGGCCGAAAGGCCGAA 353AGGAA944 UACCACAU U UCUGAAGC 354 GCUUCAGA CUGAUGAGGCCGAAAGGCCGAA 355UGGUA945 ACCACAUU U CUGAAGCA 356 UGCUUCAG CUGAUGAGGCCGAAAGGCCGAA 357GUGGU946 CCACAUUU C UGAAGCAC 358 GUGCUUCA CUGAUGAGGCCGAAAGGCCGAA 359UGUGG964 AAAUGUUC C CAGUCAUG 360 CAUGACUG CUGAUGAGGCCGAAAGGCCGAA 361CAUUU975 GUCAUGUU C CAUACCCU 362 AGGGUAUG CUGAUGAGGCCGAAAGGCCGAA 363AUGAC979 UGUUCCAU A CCCUGUAG 364 CUACAGGG CUGAUGAGGCCGAAAGGCCGAA 365GAACA992 GUAGCGUU A CAUGUAAA 366 UUUACAUG CUGAUGAGGCCGAAAGGCCGAA 367GCUAC998 UUACAUGU A AAUAUAGU 368 ACUAUAUU CUGAUGAGGCCGAAAGGCCGAA 369UGUAA1002 GUAAAUAU A GUCAAUGU 370 AUUGACUA CUGAUGAGGCCGAAAGGCCGAA 371UACAU1004 GUAAAUAU A GUCAAUGU 372 ACAUUGAC CUGAUGAGGCCGAAAGGCCGAA 373UUUAC1017 AUGUCCCU C AGCCAGCU 374 AGCUGGCU CUGAUGAGGCCGAAAGGCCGAA 375GACAU1037 GCAGCCAU U CAGAGACA 376 UGUCUCUG CUGAUGAGGCCGAAAGGCCGAA 377GCUGC1038 CAGCCAUU C AGAGACAC 378 GUGUCUCU CUGAUGAGGCCGAAAGGCCGAA 379GGCUG1048 GAGACACU A UAAUGAUG 380 CAUCAUUA CUGAUGAGGCCGAAAGGCCGAA 381GUCUC1050 GACACUAU A AUGAUGAA 382 UUCAUCAU CUGAUGAGGCCGAAAGGCCGAA 383GUGUC1082 AAGCGAAU A AAGGAAUU 384 AAUUCCUU CUGAUGAGGCCGAAAGGCCGAA 385CGCUU1090 AAAGGAAU U AGAAUUGC 386 GCAAUUCU CUGAUGAGGCCGAAAGGCCGAA 387CCUUU1091 AAGGAAUU A GAAUUGCU 388 AGCAAUUC CUGAUGAGGCCGAAAGGCCGAA 389UCCUU1096 AUUAGAAU U GCUCCUAA 390 UUAGGAGC CUGAUGAGGCCGAAAGGCCGAA 391CUAAU1100 GAAUUGCU C CUAAUGUC 392 GACAUUAG CUGAUGAGGCCGAAAGGCCGAA 393AAUUC1103 UUGCUCCU A AUGUCAAC 394 GUUGACAU CUGAUGAGGCCGAAAGGCCGAA 395AGCAA1124 AAUGAGCU A AAAGGACA 396 UGUCCUUU CUGAUGAGGCCGAAAGGCCGAA 397UCAUU1159 AUGCAGCU A CCCCGGGU 398 ACCCGGGG CUGAUGAGGCCGAAAGGCCGAA 399UGCAU1184 ACCACCAU U GCCGACCA 400 UGGUCGGC CUGAUGAGGCCGAAAGGCCGAA 401GUGGU1203 CCAGACCU C AUGGAGAC 402 GUCUCCAU CUGAUGAGGCCGAAAGGCCGAA 403UCUGG1224 CACCUGUU U CCUGUUUG 404 CAAACAGG CUGAUGAGGCCGAAAGGCCGAA 405AGGUG1225 ACCUGUUU C CUGUUUGG 406 CCAAACAG CUGAUGAGGCCGAAAGGCCGAA 407CAGGU1231 UUCCUGUU U GGGAGAAC 408 GUUCUCCC CUGAUGAGGCCGAAAGGCCGAA 409AGGAA1246 ACACCACU C CACUCCAU 410 AUGGAGUG CUGAUGAGGCCGAAAGGCCGAA 411GGUGU1251 ACUCCACU C CAUCUCUG 412 CAGAGAUG CUGAUGAGGCCGAAAGGCCGAA 413GGAGU1255 CACUCCAU C UCUGCCAG 414 CUGGCAGA CUGAUGAGGCCGAAAGGCCGAA 415GAGUG1257 CUCCAUCU C UGCCAGUG 416 CGCUGGCA CUGAUGAGGCCGAAAGGCCGAA 417UGGAG1269 CAGCGGAU C CUGGCUCC 418 GGAGCCAG CUGAUGAGGCCGAAAGGCCGAA 419CGCUG1276 UCCUGGCU C CCUACCUG 420 CAGGUAGG CUGAUGAGGCCGAAAGGCCGAA 421CAGGA1280 GGCUCCCU A CCUGAAGA 422 UCUUCAGG CUGAUGAGGCCGAAAGGCCGAA 423GAGCC1297 AAGCGCCU C GCCAGCAA 424 UUGCUGGC CUGAUGAGGCCGAAAGGCCGAA 425CGCUU1316 UGCAUGAU C GUCCACCA 426 UGGUGGAC CUGAUGAGGCCGAAAGGCCGAA 427AUGCA1334 GGCACCAU U CUGGAUAA 428 UUAUCCAG CUGAUGAGGCCGAAAGGCCGAA 429GUGCC1335 GCACCAUU C UGGAUAAU 430 AUUAUCCA CUGAUGAGGCCGAAAGGCCGAA 431GGUGC1341 UUCUGGAU A AUGUUAAG 432 CUUAACAU CUGAUGAGGCCGAAAGGCCGAA 433CAGAA1347 AUAAUGUU A AGAACCUC 434 GAGGUUCU CUGAUGAGGCCGAAAGGCCGAA 435AUUAU1355 AAGAACCU C UUAGAAUU 436 AAUUCUAA CUGAUGAGGCCGAAAGGCCGAA 437UUCUU1357 GAACCUCU U AGAAUUUG 438 CAAAUUCU CUGAUGAGGCCGAAAGGCCGAA 439GGUUC1358 AACCUCUU A GAAUUUGC 440 GCAAAUUC CUGAUGAGGCCGAAAGGCCGAA 441AGGUU1363 CUUAGAAU U UGCAGAAA 442 UUUCUGCA CUGAUGAGGCCGAAAGGCCGAA 443CUAAG1364 UUAGAAUU U GCAGAAAC 444 GUUUCUGC CUGAUGAGGCCGAAAGGCCGAA 445UCUAA1376 GAAACACU C CAAUUUAU 446 AUAAAUUG CUGAUGAGGCCGAAAGGCCGAA 447GUUUC1381 ACUCCAAU U UAUAGAUU 448 AAUCUAUA CUGAUGAGGCCGAAAGGCCGAA 449GGAGU1382 CUCCAAUU U AUAGAUUC 450 GAAUCUAU CUGAUGAGGCCGAAAGGCCGAA 451UGGAG1383 UCCAAUUU A UAGAUUCU 452 AGAAUCUA CUGAUGAGGCCGAAAGGCCGAA 453UUGGA1385 CAAUUUAU A GAUUCUUU 454 AAAGAACU CUGAUGAGGCCGAAAGGCCGAA 455AAUUG1389 UUAUAGAU U CUUUCUUA 456 UAAGAAAG CUGAUGAGGCCGAAAGGCCGAA 457UAUAA1390 UAUAGAUU C UUUCUUAA 458 UUAAGAAA CUGAUGAGGCCGAAAGGCCGAA 459CUAUA1392 UAGAUUCU U UCUUAAAC 460 GUUUAAGA CUGAUGAGGCCGAAAGGCCGAA 461AUCUA1393 AGAUUCUU U CUUAAACA 462 UGUUUAAG CUGAUGAGGCCGAAAGGCCGAA 463AAUCU1394 GAUUCUUU C UUAAACAC 464 GUGUUUAA CUGAUGAGGCCGAAAGGCCGAA 465GAAUC1396 UUCUUUCU U AAACACUU 466 AAGUGUUU CUGAUGAGGCCGAAAGGCCGAA 467AAGAA1397 UCUUUCUU A AACACUUC 468 GAAGUGUU CUGAUGAGGCCGAAAGGCCGAA 469AAAGA1404 UAAACACU U CCAGUAAC 470 GUUACUGG CUGAUGAGGCCGAAAGGCCGAA 471GUUUA1404 AAACACUU C CAGUAACC 472 GGUUACUG CUGAUGAGGCCGAAAGGCCGAA 473UGUUU1423 UGAAAACU C AGACUUGG 474 CCAAGUCU CUGAUGAGGCCGAAAGGCCGAA 475UUUCA1429 CUCAGACU U GGAAAUGC 476 GCAUUUCC CUGAUGAGGCCGAAAGGCCGAA 477CUGAG1440 AAAUGCCU U CUUUAACU 478 AGUUAAAG CUGAUGAGGCCGAAAGGCCGAA 479CAUUU1441 AAUGCCUU C UUUAACUU 480 AAGUUAAA CUGAUGAGGCCGAAAGGCCGAA 481GCAUU1443 UGCCUUCU U UAACUUCC 482 GGAAGUUA CUGAUGAGGCCGAAAGGCCGAA 483AGGCA1444 GCCUUCUU U AACUUCCA 484 UGGAAGUU CUGAUGAGGCCGAAAGGCCGAA 485AAGGC1445 CCUUCUUU A ACUUCCAC 486 GUGGAAGU CUGAUGAGGCCGAAAGGCCGAA 487GAAGG1449 CUUUAACU U CCACCCCC 488 GGGGGUGG CUGAUGAGGCCGAAAGGCCGAA 489UAAAG1450 UUUAACUU C CACCCCCC 490 GGGGGGUG CUGAUGAGGCCGAAAGGCCGAA 491UUAAA1460 ACCCCCCU C AUUGGUCA 492 UGACCAAU CUGAUGAGGCCGAAAGGCCGAA 493GGGGU1463 CCCCUCAU U GGUCACAA 494 UUGUGACC CUGAUGAGGCCGAAAGGCCGAA 495AGGGG1474 UCACAAAU U GACUGUUA 496 UAACAGUC CUGAUGAGGCCGAAAGGCCGAA 497UGUGA1482 UGACUGUU A CAACACCA 498 UGGUGUUG CUGAUGAGGCCGAAAGGCCGAA 499AGUCA1492 AACACCAU U UCAUAGAG 500 CUCUAUGA CUGAUGAGGCCGAAAGGCCGAA 501GUGUU1493 ACACCAUU U CAUAGAGA 502 UCUCUAUG CUGAUGAGGCCGAAAGGCCGAA 503GGUGU1494 CACCAUUU C AUAGAGAC 504 GUCUCUAU CUGAUGAGGCCGAAAGGCCGAA 505UGGUG1497 CAUUUCAU A GAGACCAG 506 CUGGUCUC CUGAUGAGGCCGAAAGGCCGAA 507AAAUG1518 UGAAAACU C AAAAGGAA 508 UUCCUUUU CUGAUGAGGCCGAAAGGCCGAA 509UUUCA1530 AGGAAAAU A CUGUUUUU 510 AAAAACAG CUGAUGAGGCCGAAAGGCCGAA 511UUCCU1536 AUACUGUU U UUAGAACC 512 GGUUCUAA CUGAUGAGGCCGAAAGGCCGAA 513AGUAU1537 UACUGUUU U UAGAACCC 514 GGGUUCUA CUGAUGAGGCCGAAAGGCCGAA 515CAGUA1538 ACUGUUUU U AGAACCCC 516 GGGGUUCU CUGAUGAGGCCGAAAGGCCGAA 517ACAGU1539 CUGUUUUU A GAACCCCA 518 UGGGGUUC CUGAUGAGGCCGAAAGGCCGAA 519AACAG1551 CCCCAGCU A UCAAAAGG 520 CCUUUUGA CUGAUGAGGCCGAAAGGCCGAA 521UGGGG1553 CCAGCUAU C AAAAGGUC 522 GACCUUUU CUGAUGAGGCCGAAAGGCCGAA 523GCUGG1565 AGGUCAAU C UUAGAAAG 524 CUUUCUAA CUGAUGAGGCCGAAAGGCCGAA 525GACCU1567 GUCAAUCU U AGAAAGCA 526 AGCUUUCU CUGAUGAGGCCGAAAGGCCGAA 527UUGAC1568 UCAAUCUU A GAAAGCUC 528 GAGCUUUC CUGAUGAGGCCGAAAGGCCGAA 529AUUGA1576 AGAAAGCU C UCCAAGAA 530 UUCUUGGA CUGAUGAGGCCGAAAGGCCGAA 531UUUCU1578 AAAGCUCU C CAAGAACU 532 AGUUCUUG CUGAUGAGGCCGAAAGGCCGAA 533GCUUU1587 CAAGAACU C CUACACCA 534 UGGUGUAG CUGAUGAGGCCGAAAGGCCGAA 535UCUUG1590 GAACUCCU A CACCAUUC 536 GAAUGGUG CUGAUGAGGCCGAAAGGCCGAA 537AGUUC1597 UACACCAU U CAAACAUG 538 CAUGUUUG CUGAUGAGGCCGAAAGGCCGAA 539GUGUA1598 ACACCAUU C AAACAUGC 540 GCAUGUUU CUGAUGAGGCCGAAAGGCCGAA 541GGUGU1610 CAUGCACU U GCAGCUCU 542 UGAGCUGC CUGAUGAGGCCGAAAGGCCGAA 543GCAUG1617 UUGCAGCU C AGGAAAUU 544 AAUUUCUU CUGAUGAGGCCGAAAGGCCGAA 545UGCAA1625 CAAGAAAU U AAAUACGG 546 CCGUAUUU CUGAUGAGGCCGAAAGGCCGAA 547UCUUG1626 AAGAAAUU A AAUACGGU 548 ACCGUAUU CUGAUGAGGCCGAAAGGCCGAA 549UUCUU1630 AAUUAAAU A CGGUCCCC 550 GGGGACCG CUGAUGAGGCCGAAAGGCCGAA 551UAAUU1649 AAGAUGCU A CCUCAGAC 552 GUCUGAGG CUGAUGAGGCCGAAAGGCCGAA 553AUCUU1653 UGCUACCU C AGACACCC 554 GGGUGUCU CUGAUGAGGCCGAAAGGCCGAA 555UAGCA1663 GACACCCU C UCAUCUAG 556 CUAGAUGA CUGAUGAGGCCGAAAGGCCGAA 557GUGUC1665 CACCCUCU C AUCUAGUA 558 UACUAGAU CUGAUGAGGCCGAAAGGCCGAA 559GGGUG1668 CCUCUCAU C UAGUAGAA 560 UUCUACUA CUGAUGAGGCCGAAAGGCCGAA 561AGAGG1670 UCUCAUCU A GUAGAAGA 562 UCUUCUAC CUGAUGAGGCCGAAAGGCCGAA 563UGAGA1680 UAGAAGAU C UGCAGGAU 564 AUCCUGCA CUGAUGAGGCCGAAAGGCCGAA 565UUCUA1694 GAUGUGAU C AAACAGGA 566 UCCUGUUU CUGAUGAGGCCGAAAGGCCGAA 567ACAUC1705 ACAGGAAU C UGAUGAAU 568 AUUCAUCA CUGAUGAGGCCGAAAGGCCGAA 569CCUGU1714 UGAUGAAU C UGGAAUUG 570 CAAUUCCA CUGAUGAGGCCGAAAGGCCGAA 571CAUCA1721 UCUGGAAU U GUUGCUGA 572 UCAGCAAC CUGAUGAGGCCGAAAGGCCGAA 573CCAGA1733 GCUGAGUU U CAAGAAAA 574 UUUUCUUG CUGAUGAGGCCGAAAGGCCGAA 575UCAGC1734 CUGAGUUU C AAGAAAAU 576 AUUUUCUU CUGAUGAGGCCGAAAGGCCGAA 577CUCAG1753 ACCACCCU U ACUGAAGA 578 UCUUCAGU CUGAUGAGGCCGAAAGGCCGAA 579GUGGU1754 CCACCCUU A CUGAAGAA 580 UUCUUCAG CUGAUGAGGCCGAAAGGCCGAA 581GGUGG1766 AAGAAAAU C AAACAAGA 582 UCUUGUUU CUGAUGAGGCCGAAAGGCCGAA 583UUCUU1783 GGUGGAAU C UCCAACUG 584 CAGUUGGA CUGAUGAGGCCGAAAGGCCGAA 585CCACC1785 UGGAAUCU C CAACUGAU 586 AUCAGUUG CUGAUGAGGCCGAAAGGCCGAA 587UUCCA1794 CAACUGAU A AAUCAGGA 588 UCCUGAUU CUGAUGAGGCCGAAAGGCCGAA 589AGUUG1798 UGAUAAAU C AGGAAACU 590 AGUUUCCU CUGAUGAGGCCGAAAGGCCGAA 591UAUCA1807 AGGAAACU U CUUCUGCU 592 AGCAGAAG CUGAUGAGGCCGAAAGGCCGAA 593UUCCU1808 GGAAACUU C UUCUGCUC 594 GAGCAGAA CUGAUGAGGCCGAAAGGCCGAA 595UUUCC1810 AAACUUCU U CUGCUCAC 596 GUGAGCAG CUGAUGAGGCCGAAAGGCCGAA 597AGUUU1811 AACUUCUU C UGCUCACA 598 UGUGAGCA CUGAUGAGGCCGAAAGGCCGAA 599AAGUU1816 CUUCUGCU C ACACCACU 600 AGUGGUGU CUGAUGAGGCCGAAAGGCCGAA 601AGAAG1845 GUCUGAAU A CCCAACUG 602 CAGUUGGG CUGAUGAGGCCGAAAGGCCGAA 603CAGAC1856 CAACUGUU C ACGCAGAC 604 GUCUGCGU CUGAUGAGGCCGAAAGGCCGAA 605AGUUG1867 GCAGACCU C GCCUGUGG 606 CCACAGGC CUGAUGAGGCCGAAAGGCCGAA 607UCUGC1890 CACCGAAU A UUCUUACA 608 UGUAAGAA CUGAUGAGGCCGAAAGGCCGAA 609CGGUG1892 CCGAAUAU U CUUACAAG 610 CUUGUAAC CUGAUGAGGCCGAAAGGCCGAA 611UUCGG1893 CGAAUAUU C UUACAAGC 612 GCUUGUAA CUGAUGAGGCCGAAAGGCCGAA 613AUUCG1895 AAUAUUCU U ACAAGCUC 614 GAGCUUGU CUGAUGAGGCCGAAAGGCCGAA 615AUAUU1896 AUAUUCUU A CAAGCUCC 616 GGAGCUUG CUGAUGAGGCCGAAAGGCCGAA 617AAUAU1903 UACAAGCU C CGUUUUAA 618 UUAAAACG CUGAUGAGGCCGAAAGGCCGAA 619UUGUA1908 GCUCCGUU U UAAUGGCA 620 UGCCAUUA CUGAUGAGGCCGAAAGGCCGAA 621GGACG1909 CUCCGUUU U AAUGGCAC 622 GUGCCAUU CUGAUGAGGCCGAAAGGCCGAA 623CGGAG1910 UCCGUUUU A AUGGCACC 624 GGUGCCAU CUGAUGAGGCCGAAAGGCCGAA 625ACGGA1924 ACCAGCAU C AGAAGAUG 626 CAUCUUCU CUGAUGAGGCCGAAAGGCCGAA 627CUGGU1944 ACAAUGUU C UCAAAGCA 628 UGCUUUGA CUGAUGAGGCCGAAAGGCCGAA 629AUUGU1946 AAUGUUCU C AAAGCAUU 630 AAUGCUUU CUGAUGAGGCCGAAAGGCCGAA 631ACAUU1954 CAAAGCAU U UACAGUAC 632 GUACUGUA CUGAUGAGGCCGAAAGGCCGAA 633CUUUG1955 AAAGCAUU U ACAGUACC 634 GGUACUGU CUGAUGAGGCCGAAAGGCCGAA 635GCUUU1956 AAGCAUUU A CAGUACCU 636 AGGUACUG CUGAUGAGGCCGAAAGGCCGAA 637UGCUU1965 CAGUACCU A AAAACAGG 638 CCUGUUUU CUGAUGAGGCCGAAAGGCCGAA 639UACUG1990 GAGCCCCU U GCAGCCUU 640 AAGGCUGC CUGAUGAGGCCGAAAGGCCGAA 641GGCUC1998 UGCAGCCU U GUAGCAGU 642 ACUGCUAC CUGAUGAGGCCGAAAGGCCGAA 643CUGCA2023 ACCUGCAU C CUGUGGAA 644 UUCCACAG CUGAUGAGGCCGAAAGGCCGAA 645CAGGU2053 GAUGACAU C UUCCAGUC 646 GACUGGAA CUGAUGAGGCCGAAAGGCCGAA 647UCAUC2055 UGACAUCU U CCAGUCAA 648 UUGACUGG CUGAUGAGGCCGAAAGGCCGAA 649UGUCA2056 GACAUCUU C CAGUCAAG 650 CUUGACUG CUGAUGAGGCCGAAAGGCCGAA 651AUGUC2061 CUUCCAGU C AAGCUCGU 652 ACGAGCUU CUGAUGAGGCCGAAAGGCCGAA 653GGAAG2067 GUCAAGCU C GUAAAUAC 654 GUAUUUAC CUGAUGAGGCCGAAAGGCCGAA 655UUGAC2074 UCGUAAAU A CGUGAAUG 656 CAUUCACG CUGAUGAGGCCGAAAGGCCGAA 657UACGA2086 GAAUGCAU U CUCAGCCC 658 GGGCUGAG CUGAUGAGGCCGAAAGGCCGAA 659CAUUC2087 AAUGCAUU C UCAGCCCG 660 CGGGCUGA CUGAUGAGGCCGAAAGGCCGAA 661GCAUU2089 UGCAUUCU C AGCCCGGA 662 UCCGGGCU CUGAUGAGGCCGAAAGGCCGAA 663AUGCA2117 UGAGACAU U UCCAGAAA 664 UUUCUGGA CUGAUGAGGCCGAAAGGCCGAA 665UCUCA2188 GAGACAUU U CCAGAAAA 666 UUUUCUGG CUGAUGAGGCCGAAAGGCCGAA 667GUCUC2119 AGACAUUU C CAGAAAAG 668 CUUUUCUG CUGAUGAGGCCGAAAGGCCGAA 669UGUCU2131 AAAAGCAU U AUGGUUUU 670 AAAACCAU CUGAUGAGGCCGAAAGGCCGAA 671CUUUU2132 AAAGCAUU A UGGUUUUC 672 GAAAACCA CUGAUGAGGCCGAAAGGCCGAA 673GCUUU2138 UUAUGGUU U UCAGAACA 674 UGUUCUGA CUGAUGAGGCCGAAAGGCCGAA 675CAUAA2139 UAUGGUUU U CAGAACAC 676 GUGUUCUG CUGAUGAGGCCGAAAGGCCGAA 677CCAUA2140 AUGGUUUU C AGAACACU 678 AGUGUUCU CUGAUGAGGCCGAAAGGCCGAA 679ACCAU2149 AGAACACU U CAAGUUGA 680 UCAACUUG CUGAUGAGGCCGAAAGGCCGAA 681GUUCU2150 GAACACUU C AAGUUGAC 682 GUCAACUU CUGAUGAGGCCGAAAGGCCGAA 683UGUUC2155 CUUCAAGU U GACUUGGG 684 CCCAAGUC CUGAUGAGGCCGAAAGGCCGAA 685UGAAG2160 AGUUGACU U GGGAUAUA 686 UAUAUCCC CUGAUGAGGCCGAAAGGCCGAA 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CUGAUGAGGCCGAAAGGCCGAA 1115CACA2972 GUGGUUUU U UUGUUAUU 1116 AAUAACAA CUGAUGAGGCCGAAAGGCCGAA 1117CCAC2973 UGGUUUUU U UGUUAUUG 1118 CAAUAACA CUGAUGAGGCCGAAAGGCCGAA 1119ACCA2974 GGUUUUUU U GUUAUUGU 1120 ACAAUAAC CUGAUGAGGCCGAAAGGCCGAA 1121AACC2977 UUUUUUGU U AUUGUUGG 1122 CCAACAAU CUGAUGAGGCCGAAAGGCCGAA 1123AAAA2978 UUUUUGUU A UUGUUGGU 1124 ACCAACAA CUGAUGAGGCCGAAAGGCCGAA 1125AAAA2980 UUUGUUAU U GUUGGUUU 1126 AAACCAAC CUGAUGAGGCCGAAAGGCCGAA 1127CAAA2988 UGUUGGUU U AUACAAGC 1128 GCUUGUAU CUGAUGAGGCCGAAAGGCCGAA 1129AACA2989 GUUGGUUU A UACAAGCA 1130 UGCUUGUA CUGAUGAGGCCGAAAGGCCGAA 1131CAAC2991 UGGUUUAU A CAAGCAUG 1132 CAUGCUUG CUGAUGAGGCCGAAAGGCCGAA 1133ACCA3009 GUUGCACU U CUUUUUUG 1134 CAAAAAAG CUGAUGAGGCCGAAAGGCCGAA 1135CAAC3010 UUGCACUU C UUUUUUGG 1136 CCAAAAAA CUGAUGAGGCCGAAAGGCCGAA 1137GCAA3012 GCACUUCU U UUUUGGGA 1138 UCCCAAAA CUGAUGAGGCCGAAAGGCCGAA 1139GUGC3013 CACUUCUU U UUUGGGAG 1140 CUCCCAAA CUGAUGAGGCCGAAAGGCCGAA 1141AGUG3014 ACUUCUUU U UUGGGAGA 1142 UCUCCCAA CUGAUGAGGCCGAAAGGCCGAA 1143AAGU3015 CUUCUUUU U UGGGAGAU 1144 AUCUCCCA CUGAUGAGGCCGAAAGGCCGAA 1145GAAG3016 UUCUUUUU U GGGAGAUG 1146 CAUCUCCC CUGAUGAGGCCGAAAGGCCGAA 1147AGAA3040 UUGAUGUU C UAUGUUUU 1148 AAAACAUA CUGAUGAGGCCGAAAGGCCGAA 1149UCAA3042 GAUGUUCU A UGUUUUGU 1150 ACAAAACA CUGAUGAGGCCGAAAGGCCGAA 1151CAUC3047 UCUAUGUU U UGUUUUGA 1152 UCAAAACA CUGAUGAGGCCGAAAGGCCGAA 1153UAGA3048 CUAUGUUU U GUUUUGAG 1154 CUCAAAAC CUGAUGAGGCCGAAAGGCCGAA 1155AUAG3052 GUUUUGUU U UGAGUGUA 1156 UACACUCA CUGAUGAGGCCGAAAGGCCGAA 1157AAAC2053 UUUUGUUU U GAGUGUAG 1158 CUACACUC CUGAUGAGGCCGAAAGGCCGAA 1159AAAA3072 UGACUGUU U UAUAAUUU 1160 AAAUUAUA CUGAUGAGGCCGAAAGGCCGAA 1161GUCA3073 GACUGUUU U AUAAUUUG 1162 CAAAUUAU CUGAUGAGGCCGAAAGGCCGAA 1163AGUC3074 ACUGUUUU A UAAUUUGG 1164 CCAAAUUA CUGAUGAGGCCGAAAGGCCGAA 1165CAGU3076 UGUUUUAU A AUUUGGGA 1166 UCCCAAAU CUGAUGAGGCCGAAAGGCCGAA 1167AACA3079 UUUAUAAU U UGGGAGUU 1168 AACUCCCA CUGAUGAGGCCGAAAGGCCGAA 1169UAAA3080 UUAUAAUU U GGGAGUUC 1170 GAACUCCC CUGAUGAGGCCGAAAGGCCGAA 1171AUAA3088 UGGGAGUU C UGCAUUUG 1172 CAAAUGCA CUGAUGAGGCCGAAAGGCCGAA 1173CCCA3094 UUCUGCAU U UGAUCCGA 1174 GCGGAUCA CUGAUGAGGCCGAAAGGCCGAA 1175AGAA3095 UCUGCAUU U GAUCCGCA 1176 UGCGGAUC CUGAUGAGGCCGAAAGGCCGAA 1177CAGA3099 CAUUUGAU C CGCAUCCC 1178 GGGAUGCG CUGAUGAGGCCGAAAGGCCGAA 1179AAUG3105 AUCCGCAU C CCCUGUGG 1180 CCACAGGG CUGAUGAGGCCGAAAGGCCGAA 1181GGAU3116 CUGUGGUU U CUAAGUGU 1182 ACACUUAG CUGAUGAGGCCGAAAGGCCGAA 1183ACAG3117 UGUGGUUU C UAAGUGUA 1184 UACACUUA CUGAUGAGGCCGAAAGGCCGAA 1185CACA3119 UGGUUUCU A AGUGUAUG 1186 CAUACACU CUGAUGAGGCCGAAAGGCCGAA 1187ACCA3132 UAUGGUCU C AGAACUGU 1188 ACAGUUCU CUGAUGAGGCCGAAAGGCCGAA 1189CAUA3150 GCAUGGAU C CUGUGUUU 1190 AAACACAG CUGAUGAGGCCGAAAGGCCGAA 1191AUGC3157 UCCUGUGU U UGCAACUA 1192 CAGUUGCA CUGAUGAGGCCGAAAGGCCGAA 1193AGGA3158 CCUGUGUU U GCAACUGG 1194 CCAGUUGC CUGAUGAGGCCGAAAGGCCGAA 1195CAGG3189 UGGUUGAU A GCCAGUCA 1196 UGACUGGC CUGAUGAGGCCGAAAGGCCGAA 1197ACCA3204 CACUGCCU U AAGAACAU 1198 AUGUUCUU CUGAUGAGGCCGAAAGGCCGAA 1199AGUG3205 ACUGCCUU A AGAACAUU 1200 AAUGUUCU CUGAUGAGGCCGAAAGGCCGAA 1201CAGU3213 AAGAACAU U UGAUGCAA 1202 UUGCAUCA CUGAUGAGGCCGAAAGGCCGAA 1203UCUU3214 AGAACAUU U GAUGCAAG 1204 CUUGCAUC CUGAUGAGGCCGAAAGGCCGAA 1205UUCU3240 ACUGAACU U UUGAGAUA 1206 UAUCUCAA CUGAUGAGGCCGAAAGGCCGAA 1207CAGU3241 CUGAACUU U UGAGAUAU 1208 AUAUCUCA CUGAUGAGGCCGAAAGGCCGAA 1209UCAG3242 UGAACUUU U GAGAUAUG 1210 CAUAUCUC CUGAUGAGGCCGAAAGGCCGAA 1211UUCA3248 UUUGAGAU A UGACGGUG 1212 CACCGUCA CUGAUGAGGCCGAAAGGCCGAA 1213CAAA3261 GGUGUACU U ACUGCCUU 1214 AAGGCAGU CUGAUGAGGCCGAAAGGCCGAA 1215CACC3262 GUGUACUU A CUGCCUUG 1216 CAAGGCAG CUGAUGAGGCCGAAAGGCCGAA 1217ACAC3269 UACUGCCU U GUAGCAAA 1218 UUUGCUAC CUGAUGAGGCCGAAAGGCCGAA 1219AGUA3280 AGCAAAAU A AAGAUGUG 1220 CACAUCUU CUGAUGAGGCCGAAAGGCCGAA 1221UGCU3293 UGUGCCCU U AUUUUACC 1222 GGUAAAAU CUGAUGAGGCCGAAAGGCCGAA 1223CACA3294 GUGCCCUU A UUUUACCU 1224 AGGUAAAA CUGAUGAGGCCGAAAGGCCGAA 1225GCAC__________________________________________________________________________ TABLE XVI__________________________________________________________________________Mouse c-myb Hammerhead Ribozyme and Target Sequences(REVISED) Seq. Seq.nt. HH Ribozyme Sequence ID No. Target ID__________________________________________________________________________ No.10 UCCGCCAA CUGAUGAGGCCGAAAGGCCGAA AGCCCCGG 1226 CCGGGGCUC UUGGCGGA 122712 GCUCCGCC CUGAUGAGGCCGAAAGGCCGAA AGAGCCCC 1228 GGGGCUCUU GGCGGAGC 122933 GCCAUGGC CUGAUGAGGCCGAAAGGCCGAA AGGCGGGC 1230 GCCCGCCUC GCCAUGGC 123163 CUACUGUA CUGAUGAGGCCGAAAGGCCGAA AUGCUGUG 1232 CACAGCAUC UACAGUAG 123365 CGCUACUG CUGAUGAGGCCGAAAGGCCGAA AGAUGCUG 1234 CAGCAUCUA CAGUAGCG 123570 UUCAUCGC CUGAUGAGGCCGAAAGGCCGAA ACUGUAGA 1236 UCUACAGUA GCGAUGAA 123793 CACAUCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCUUC 1238 GAAGACAUU GAGAUGUG 1239113 GCCCAUCG CUGAUGAGGCCGAAAGGCCGAA AGUCAUGG 1240 CCAUGACUA CGAUGGGC 1241134 GCUUUCCA CUGAUGAGGCCGAAAGGCCGAA AUUUGGGC 1242 GCCCAAAUC UGGAAAGC 1243145 CCCCAAGU CUGAUGAGGCCGAAAGGCCGAA ACGCUUUC 1244 GAAAGCGUC ACUUGGGG 1245149 UUUUCCCC CUGAUGAGGCCGAAAGGCCGAA AGUGACGC 1246 GCGUCACUU GGGGAAAA 1247160 UGUCCACC CUGAUGAGGCCGAAAGGCCGAA AGUUUUCC 1248 GGAAAACUA GGUGGACA 1249231 UUGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACUUUCCA 1250 UGGAAAGUC AUUGCCAA 1251234 UAAUUGGC CUGAUGAGGCCGAAAGGCCGAA AUGACUUU 1252 AAAGUCAUU GCCAAUUA 1253241 GGGCAGAU CUGAUGAGGCCGAAAGGCCGAA AUUGGCAA 1254 UUGCCAAUU AUCUGCCC 1255242 UGGGCAGA CUGAUGAGGCCGAAAGGCCGAA AAUUGGCA 1256 UGCCAAUUA UCUGCCCA 1257244 GUUGGGCA CUGAUGAGGCCGAAAGGCCGAA AUAAUUGG 1258 CCAAUUAUC UGCCCAAC 1259264 UGGCACUG CUGAUGAGGCCGAAAGGCCGAA ACAUCUGU 1260 ACAGAUGUA CAGUGCCA 1261306 CCUUUGAU CUGAUGAGGCCGAAAGGCCGAA AGUUCAGG 1262 CCUGAACAC AUCAAAGG 1263309 GGACCUUU CUGAUGAGGCCGAAAGGCCGAA AUGAGUUC 1264 GAACUCAUC AAAGGUCC 1265316 GGUCCAGG CUGAUGAGGCCGAAAGGCCGAA ACCUUUGA 1266 UCAAAGGUC CCUGGACC 1267337 GACUCUCU CUGAUGAGGCCGAAAGGCCGAA AUCUUCUU 1268 AAGAAGAUC AGAGAGUC 1269345 AGCUUUAU CUGAUGAGGCCGAAAGGCCGAA ACUCUCUG 1270 CAGAGAGUC AUAAAGCU 1271348 ACAAGCUU CUGAUGAGGCCGAAAGGCCGAA AUGACUCU 1272 AGAGUCAUA AAGCUUGU 1273354 UUCUGGAC CUGAUGAGGCCGAAAGGCCGAA AGCUUUAU 1274 AUAAAGCUU GUCCAGAA 1275357 UAUUUCUG CUGAUGAGGCCGAAAGGCCGAA ACAAGCUU 1276 AAGCUUGUC CAGAAAUA 1277365 UCGGACCA CUGAUGAGGCCGAAAGGCCGAA AUUUCUGG 1278 CCAGAAAUA UGGUCCGA 1279370 ACGCUUCG CUGAUGAGGCCGAAAGGCCGAA ACCAUAUU 1280 AAUAUGGUC CGAAGCGU 1281379 AACAGACC CUGAUGAGGCCGAAAGGCCGAA ACGCUUCG 1282 CGAAGCGUU GGUCUGUU 1283383 CAAUAACA CUGAUGAGGCCGAAAGGCCGAA ACCAACGC 1284 GCGUUGGUC UGUUAUUG 1285387 UUGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACAGACCA 1286 UGGUCUGUU AUUGCCAA 1287388 CUUGGCAA CUGAUGAGGCCGAAAGGCCGAA AACAGACC 1288 GGUCUGUUA UUGCCAAG 1289390 UGCUUGGC CUGAUGAGGCCGAAAGGCCGAA AUAACAGA 1290 UCUGUUAUU GCCAAGCA 1291401 UCCCUUUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUUG 1292 CAAGCACUU AAAAGGGA 1293402 CUCCCUUU CUGAUGAGGCCGAAAGGCCGAA AAGUGCUU 1294 AAGCACUUA AAAGGGAG 1295414 UGCUUUCC CUGAUGAGGCCGAAAGGCCGAA AUUCUCCC 1296 GGGAGAAUU GGAAAGCA 1297427 CCUCUCCC CUGAUGAGGCCGAAAGGCCGAA ACACUGCU 1298 AGCAGUGUC GGGAGAGG 1299448 UGGAUUCA CUGAUGAGGCCGAAAGGCCGAA AUGGUUGA 1300 ACAACCAUU UGAAUCCA 1301449 CUGGAUUC CUGAUGAGGCCGAAAGGCCGAA AAUGGUUG 1302 CAACCAUUU GAAUCCAG 1303454 AACUUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCAAAU 1304 AUUUGAAUC CAGAAGUU 1305462 GUUUUCUU CUGAUGAGGCCGAAAGGCCGAA ACUUCUGG 1306 CCAGAAGUU AAGAAAAC 1307463 GGUUUUCU CUGAUGAGGCCGAAAGGCCGAA AACUUCUG 1308 CAGAAGUUA AGAAAACC 1309473 CUGUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUUUUC 1310 GAAAACCUC CUGGACAG 1311498 UGGUAAAU CUGAUGAGGCCGAAAGGCCGAA AUUCUGUC 1312 GACAGAAUC AUUUACCA 1313501 GCCUGGUA CUGAUGAGGCCGAAAGGCCGAA AUGAUUCU 1314 AGAAUCAUU UACCAGGC 1315502 UGCCUGGU CUGAUGAGGCCGAAAGGCCGAA AAUGAUUC 1316 GAAUCAUUU ACCAGGCA 1317503 GUGCCUGG CUGAUGAGGCCGAAAGGCCGAA AAAUGAUU 1318 AAUCAUUUA CCAGGCAC 1319520 GUUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACGCUUGU 1320 ACAAGCGUC UGGGGAAC 1321543 AGCUUUGC CUGAUGAGGCCGAAAGGCCGAA AUCUCUGC 1322 GCAGAGAUC GCAAAGCU 1323571 GAUAGCAU CUGAUGAGGCCGAAAGGCCGAA AUCAGUCC 1324 GGACUGAUA AUGCUAUC 1325577 GUUCUUGA CUGAUGAGGCCGAAAGGCCGAA AGCAUUAU 1326 AUAAUGCUA UCAAGAAC 1327579 UGGUUCUU CUGAUGAGGCCGAAAGGCCGAA AUAGCAUU 1328 AAUGCUAUC AAGAACCA 1329595 CAUGGUGG CUGAUGAGGCCGAAAGGCCGAA AUUCCAGU 1330 ACUGGAAUU CCACCAUG 1331596 GCAUGGUG CUGAUGAGGCCGAAAGGCCGAA AAUUCCAG 1332 CUGGAAUUC CACCAUGC 1333607 CACCUUGC CUGAUGAGGCCGAAAGGCCGAA ACGCAUGG 1334 CCAUGCGUC GCAAGGUG 1335629 UCUGCAGG CUGAUGAGGCCGAAAGGCCGAA AGCCUUCC 1336 GGAAGGCUA CCUGCAGA 1337643 GGCUUUGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUCU 1338 AGAAGCCUU CCAAAGCC 1339644 UGGCUUUG CUGAUGAGGCCGAAAGGCCGAA AAGGCUUC 1340 GAAGCCUUC CAAAGCCA 1341677 UCUUCUGG CUGAUGAGGCCGAAAGGCCGAA AGCUCGUG 1342 CACGAGCUU CCAGAAGA 1343678 UUCUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCUCGU 1344 ACGAGCUUC CAGAAGAA 1345691 CAUCAAAU CUGAUGAGGCCGAAAGGCCGAA AUUGUUCU 1346 AGAACAAUC AUUUGAUG 1347694 CCCCAUCA CUGAUGAGGCCGAAAGGCCGAA AUGAUUGU 1348 ACAAUCAUU UGAUGGGG 1349695 ACCCCAUC CUGAUGAGGCCGAAAGGCCGAA AAUGAUUG 1350 CAAUCAUUU GAUGGGGU 1351704 CAUGCCCA CUGAUGAGGCCGAAAGGCCGAA ACCCCAUC 1352 GAUGGGGUU UGGGCAUG 1353705 GCAUGCCC CUGAUGAGGCCGAAAGGCCGAA AACCCCAU 1354 AUGGGGUUU GGGCAUGC 1355716 AUGGAGGU CUGAUGAGGCCGAAAGGCCGAA AGGCAUGC 1356 GCAUGCCUC ACCUCCAU 1357721 CUGAGAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGAGG 1358 CCUCACCUC CAUCUCAG 1359725 AGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AUGGAGGU 1360 ACCUCCAUC UCAGCUCU 1361727 AGAGAGCU CUGAUGAGGCCGAAAGGCCGAA AGAUGGAG 1362 CUCCAUCUC AGCUCUCU 1363732 CUUGGAGA CUGAUGAGGCCGAAAGGCCGAA AGCUGAGA 1364 UCUCAGCUC UCUCCAAG 1365734 CACUUGGA CUGAUGAGGCCGAAAGGCCGAA AGAGCUGA 1366 UCAGCUCUC UCCAAGUG 1367736 GCCACUUG CUGAUGAGGCCGAAAGGCCGAA AGAGAGCU 1368 AGCUCUCUC CAAGUGGC 1369749 UGACGGAG CUGAUGAGGCCGAAAGGCCGAA ACUGGCCA 1370 UGGCCAGUC CUCCGUCA 1371752 UGUUGACG CUGAUGAGGCCGAAAGGCCGAA AGGACUGG 1372 CCAGUCCUC CGUCAACA 1373756 UCGCUGUU CUGAUGAGGCCGAAAGGCCGAA ACGGAGGA 1374 UCCUCCGUC AACAGCGA 1375767 AAUAGGGA CUGAUGAGGCCGAAAGGCCGAA AUUCGCUG 1376 CAGCGAAUA UCCCUAUU 1377769 GUAAUAGG CUGAUGAGGCCGAAAGGCCGAA AUAUUCGC 1378 GCGAAUAUC CCUAUUAC 1379773 UGUGGUAA CUGAUGAGGCCGAAAGGCCGAA AGGGAUAU 1380 AUAUCCCUA UUACCACA 1381775 GAUGUGGU CUGAUGAGGCCGAAAGGCCGAA AUAGGGAU 1382 AUCCCUAUU ACCACAUC 1383776 CGAUGUGG CUGAUGAGGCCGAAAGGCCGAA AAUAGGGA 1384 UCCCUAUUA CCACAUCG 1385783 GCUUCGGC CUGAUGAGGCCGAAAGGCCGAA AUGUGGUA 1386 UACCACAUC GCCGAAGC 1387801 UGACUGGA CUGAUGAGGCCGAAAGGCCGAA AUGUUUUG 1388 CAAAACAUC UCCAGUCA 1389803 CGUGACUG CUGAUGAGGCCGAAAGGCCGAA AGAUGUUU 1390 AAACAUCUC CAGUCACG 1391808 GGGAACGU CUGAUGAGGCCGAAAGGCCGAA ACUGGAGA 1392 UCUCCAGUC ACGUUCCC 1393813 GGAUAGGG CUGAUGAGGCCGAAAGGCCGAA ACGUGACU 1394 AGUCACGUU CCCUAUCC 1395814 AGGAUAGG CUGAUGAGGCCGAAAGGCCGAA AACGUGAC 1396 GUCACGUUC CCUAUCCU 1397818 CGACAGGA CUGAUGAGGCCGAAAGGCCGAA AGGGAACG 1398 CGUUCCCUA UCCUGUCG 1399820 UGCGACAG CUGAUGAGGCCGAAAGGCCGAA AUAGGGAA 1400 UUCCCUAUC CUGUCGCA 1401825 UGCAAUGC CUGAUGAGGCCGAAAGGCCGAA ACAGGAUA 1402 UAUCCUGUC GCAUUGCA 1403830 UAACAUGC CUGAUGAGGCCGAAAGGCCGAA AUGCGACA 1404 UGUCGCAUU GCAUGUUA 1405837 ACUAUAUU CUGAUGAGGCCGAAAGGCCGAA ACAUGCAA 1406 UUGCAUGUU AAUAUAGU 1407838 GACUAUAU CUGAUGAGGCCGAAAGGCCGAA AACAUGCA 1408 UGCAUGUUA AUAUAGUC 1409841 GUUGACUA CUGAUGAGGCCGAAAGGCCGAA AUUAACAU 1410 AUGUUAAUA UAGUCAAC 1411843 ACGUUGAC CUGAUGAGGCCGAAAGGCCGAA AUAUUAAC 1412 GUUAAUAUA GUCAACGU 1413846 GGGACGUU CUGAUGAGGCCGAAAGGCCGAA ACUAUAUU 1414 AAUAUAGUC AACGUCCC 1415852 GGCUGAGG CUGAUGAGGCCGAAAGGCCGAA ACGUUGAC 1416 GUCAACGUC CCUCAGCC 1417856 AGCCGGCU CUGAUGAGGCCGAAAGGCCGAA AGGGACGU 1418 ACGUCCCUC AGCCGGCU 1419876 UGUCUCUG CUGAUGAGGCCGAAAGGCCGAA AUGGCUGC 1420 GCAGCCAUC CAGAGACA 1421887 CGUCGUUA CUGAUGAGGCCGAAAGGCCGAA AGUGUCUC 1422 GAGACACUA UAACGACG 1423889 UUCGUCGU CUGAUGAGGCCGAAAGGCCGAA AUAGUGUC 1424 GACACUAUA ACGACGAA 1425921 AGCUCCUU CUGAUGAGGCCGAAAGGCCGAA AUUCGCUU 1426 AAGCGAAUA AAGGAGCU 1427935 UCAGGACG CUGAUGAGGCCGAAAGGCCGAA ACUCCAGC 1428 GCUGGAGUU GCUCCUGA 1429939 GACAUCAG CUGAUGAGGCCGAAAGGCCGAA AGCAACUC 1430 GAGUUGCUC CUGAUGUC 1431947 UCUCUGUU CUGAUGAGGCCGAAAGGCCGAA ACAUCAGG 1432 CCUGAUGUC AACAGAGA 1433980 GUGUUGGU CUGAUGAGGCCGAAAGGCCGAA AUGCCUGC 1434 GCAGGCAUU ACCAACAC 1435981 UGUGUUGG CUGAUGAGGCCGAAAGGCCGAA AAUGCCUG 1436 CAGGCAUUA CCAACACA 14371000 GUAGCUGC CUGAUGAGGCCGAAAGGCCGAA AGUGUGGU 1438 ACCACACUU GCAGCUAC 14391007 ACCCGGGG CUGAUGAGGCCGAAAGGCCGAA AGCUGCAA 1440 UUGCAGCUA CCCCGGGU 14411028 CCACAAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGCUG 1442 CAGCACCUC CAUUGUGG 14431032 UGGUCCAC CUGAUGAGGCCGAAAGGCCGAA AUGGAGGU 1444 ACCUCCAUU GUGGACCA 14451051 AUCCCCAU CUGAUGAGGCCGAAAGGCCGAA AGGUCUGG 1446 CCAGACCUC AUGGGGAU 14471060 AGGUGCAC CUGAUGAGGCCGAAAGGCCGAA AUCCCCAU 1448 AUGGGGAUA GUGCACCU 14491071 AAACAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGUGC 1450 GCACCUGUU UCCUGUUU 14511072 CAAACAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGUG 1452 CACCUGUUU CCUGUUUG 14531073 CCAAACAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGU 1454 ACCUGUUUC CUGUUUGG 14551078 UUCUCCCA CUGAUGAGGCCGAAAGGCCGAA ACAGGAAA 1456 UUUCCUGUU UGGGAGAA 14571079 GUUCUCCC CUGAUGAGGCCGAAAGGCCGAA AACAGGAA 1458 UUCCUGUUU GGGAGAAC 14591103 CAGGCAGA CUGAUGAGGCCGAAAGGCCGAA AUGGGGUG 1460 CACCCCAUC UCUGCCUG 14611105 UGCAGGCA CUGAUGAGGCCGAAAGGCCGAA AGAUGGGG 1462 CCCCAUCUC UGCCUGCA 14631117 GGAGCCGG CUGAUGAGGCCGAAAGGCCGAA AUCUGCAG 1464 CAGCAGAUC CCGGCUCC 14651124 CAGGUAGG CUGAUGAGGCCGAAAGGCCGAA AGCCGGGA 1466 UCCCGGCUC CCUACCUG 14671128 UCUUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGGAGCC 1468 GGCUCCCUA CCUGAAGA 14691145 UUGCUGGU CUGAUGAGGCCGAAAGGCCGAA AGGCACUU 1470 AAGUGCCUC ACCAGCAA 14711164 UGGUGGAC CUGAUGAGGCCGAAAGGCCGAA AUCAUGCA 1472 UGCAUGAUC GUCCACCA 14731167 CCCUGGUG CUGAUGAGGCCGAAAGGCCGAA ACGAUCAU 1474 AUGAUCGUC CACCAGGG 14751182 UUGUCCAG CUGAUGAGGCCGAAAGGCCGAA AUGGUGCC 1476 GGCACCAUU CUGGACAA 14771183 AUUGUCCA CUGAUGAGGCCGAAAGGCCGAA AAUGGUGC 1478 GCACCAUUC UGGACAAU 14791194 AGGUUCUU CUGAUGAGGCCGAAAGGCCGAA ACAUUGUC 1480 GACAAUGUU AAGAACCU 14811195 GAGGUUCU CUGAUGAGGCCGAAAGGCCGAA AACAUUGU 1482 ACAAUGUUA AGAACCUC 14831203 AAUUCUAA CUGAUGAGGCCGAAAGGCCGAA AGGUUCUU 1484 AAGAACCUC UUAGAAUU 14851205 CAAAUUCU CUGAUGAGGCCGAAAGGCCGAA AGAGGUUC 1486 GAACCUCUU AGAAUUUG 14871206 GCAAAUUC CUGAUGAGGCCGAAAGGCCGAA AAGAGGUU 1488 AACCUCUUA GAAUUUGC 14891211 UUUCUGCA CUGAUGAGGCCGAAAGGCCGAA AUUCUAAG 1490 CUUAGAAUU UGCAGAAA 14911212 GUUUCUGC CUGAUGAGGCCGAAAGGCCGAA AAUUCUAA 1492 UUAGAAUUU GCAGAAAC 14931224 AUAAACUG CUGAUGAGGCCGAAAGGCCGAA AGUGUUUC 1494 GAAACACUC CAGUUUAU 14951229 AAUCUAUA CUGAUGAGGCCGAAAGGCCGAA ACUGGAGU 1496 ACUCCAGUU UAUAGAUU 14971230 GAAUCUAU CUGAUGAGGCCGAAAGGCCGAA AACUGGAG 1498 CACCAGUUU AUAGAUUC 14991231 AGAAUCUA CUGAUGAGGCCGAAAGGCCGAA AAACUGGA 1500 UCCAGUUUA UAGAUUCU 15011233 AAAGAAUC CUGAUGAGGCCGAAAGGCCGAA AUAAACUG 1502 CAGUUUAUA GAUUCUUU 15031237 CAAGAAAG CUGAUGAGGCCGAAAGGCCGAA AUCUAUAA 1504 UUAUAGAUU CUUUCUUG 15051238 UCAAGAAA CUGAUGAGGCCGAAAGGCCGAA AAUCUAUA 1506 UAUAGAUUC UUUCUUGA 15071240 GUUCAAGA CUGAUGAGGCCGAAAGGCCGAA AGAAUCUA 1508 UAGAUUCUU UCUUGAAC 15091241 UGUUCAAG CUGAUGAGGCCGAAAGGCCGAA AAGAAUCU 1510 AGAUUCUUU CUUGAACA 15111242 GUGUUCAA CUGAUGAGGCCGAAAGGCCGAA AAAGAAUC 1512 GAUUCUUUC UUGAACAC 15131244 AAGUGUUC CUGAUGAGGCCGAAAGGCCGAA AGAAAGAA 1514 UUCUUUCUU GAACACUU 15151252 GUUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGUGUUCA 1516 UGAACACUU CCAGCAAC 15171253 GGUUGCUG CUGAUGAGGCCGAAAGGCCGAA AAGUGUUC 1518 GAACACUUC CAGCAACC 15191271 CUAAGCCC CUGAUGAGGCCGAAAGGCCGAA AGUUUUCA 1520 UGAAAACUC GGGCUUAG 15211277 GUGCAUCU CUGAUGAGGCCGAAAGGCCGAA AGCCCGAG 1522 CUCGGGCUU AGAUGCAC 15231278 GGUGCAUC CUGAUGAGGCCGAAAGGCCGAA AAGCCCGA 1524 UCGGGCUUA GAUGCACC 15251288 GGGUAAGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCAU 1526 AUGCACCUA CCUUACCC 15271292 UGGAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUAGGU 1528 ACCUACCUU ACCCUCCA 15291293 GUGGAGGG CUGAUGAGGCCGAAAGGCCGAA AACCUAGG 1530 CCUACCUUA CCCUCCAC 15311298 GAGGAGUG CUGAUGAGGCCGAAAGGCCGAA AGGGUAAG 1532 CUUACCCUC CACUCCUC 15331303 AAUGAGAG CUGAUGAGGCCGAAAGGCCGAA AGUGGAGG 1534 CCUCCACUC CUCUCAUU 15351306 ACCAAUGA CUGAUGAGGCCGAAAGGCCGAA AGGAGUGG 1536 CCACUCCUC UCAUUGGU 15371308 UGACCAZU CUGAUGAGGCCGAAAGGCCGAA AGAGGAGU 1538 ACUCCUCUC AUUGGUCA 15391311 UUGUGACC CUGAUGAGGCCGAAAGGCCGAA AUGAGAGG 1540 CCUCUCAUU GGUCACAA 15411315 CAGUUUGU CUGAUGAGGCCGAAAGGCCGAA ACCAAUGA 1542 UCAUUGGUC ACAAACUG 15431333 CUGGUCUC CUGAUGAGGCCGAAAGGCCGAA ACAUGGUG 1544 CACCAUGUC GAGACCAG 15451366 AAAGAUGG CUGAUGAGGCCGAAAGGCCGAA AUUUUCCU 1546 AGGAAAAUU CCAUCUUU 15471367 UAAAGAUG CUGAUGAGGCCGAAAGGCCGAA AAUUUUCC 1548 GGAAAAUUC CAUCUUUA 15491371 GUUCUAAA CUGAUGAGGCCGAAAGGCCGAA AUGGAAUU 1550 AAUUCCAUC UUUAGAAC 15511373 GAGUUCUA CUGAUGAGGCCGAAAGGCCGAA AGAUGGAA 1552 UUCCAUCUU UAGAACUC 15531374 GGAGUUCU CUGAUGAGGCCGAAAGGCCGAA AAGAUGGA 1554 UCCAUCUUU AGAACUCC 15551375 UGGAGUUC CUGAUGAGGCCGAAAGGCCGAA AAAGAUGG 1556 CCAUCUUUA GAACUCCA 15571381 GAUAGCUG CUGAUGAGGCCGAAAGGCCGAA AGUUCUAA 1558 UUAGAACUC CAGCUAUC 15591387 CCUUUUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGGAG 1560 CUCCAGCUA UCAAAAGG 15611389 GACCUUUU CUGAUGAGGCCGAAAGGCCGAA AUAGCUGG 1562 CCAGCUAUC AAAAGGUC 15631397 CGAGGAUU CUGAUGAGGCCGAAAGGCCGAA ACCUUUUG 1564 CAAAAGGUC AAUCCUCG 15651401 CUUUCGAG CUGAUGAGGCCGAAAGGCCGAA AUUGACCU 1566 AGGUCAAUC CUCGAAAG 15671404 GAGCUUUC CUGAUGAGGCCGAAAGGCCGAA AGGAUUGA 1568 UCAAUCCUC GAAAGCUC 15691412 UUCGAGGA CUGAUGAGGCCGAAAGGCCGAA AGCUUUCG 1570 CGAAAGCUC UCCUCGAA 15711414 AGUUCGAG CUGAUGAGGCCGAAAGGCCGAA AGAGCUUU 1572 AAAGCUCUC CUCGAACU 15731417 GGGAGUUC CUGAUGAGGCCGAAAGGCCGAA AGGAGAGC 1574 GCUCUCCUC GAACUCCC 15751423 UGGUGUGG CUGAUGAGGCCGAAAGGCCGAA AGUUCGAG 1576 CUCGAACUC CCACACCA 15771433 CAUGUUUG CUGAUGAGGCCGAAAGGCCGAA AUGGUGUG 1578 CACACCAUU CAAACAUG 15791434 GCAUGUUU CUGAUGAGGCCGAAAGGCCGAA AAUGGUGU 1580 ACACCAUUC AAACAUGC 15811446 UGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AGGGCAUG 1582 CAUGCCCUU GCAGCUCA 15831453 AAUUUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUGCAA 1584 UUGCAGCUC AAGAAAUU 15851461 CCGUAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUCUUG 1586 CAAGAAAUU AAAUACGG 15871462 ACCGUAUU CUGAUGAGGCCGAAAGGCCGAA AAUUUCUU 1588 AAGAAAUUA AAUACGGU 15891466 GGGGACCG CUGAUGAGGCCGAAAGGCCGAA AUUUAAUU 1590 AAUUAAAUA CGGUCCCC 15911471 CUUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACCGUAUU 1592 AAUACGGUC CCCUGAAG 15931485 GUCUGAGG CUGAUGAGGCCGAAAGGCCGAA AGCAUCUU 1594 AAGAUGCUA CCUCAGAC 15951489 GGGGGUCU CUGAUGAGGCCGAAAGGCCGAA AGGUAGCA 1596 UGCUACCUC AGACCCCC 15971599 CUGCAUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGGUC 1598 GACCCCCUC CCAUGCAG 15991518 ACAUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCCUC 1600 GAGGACCUA CAAGAUGU 16011530 UCCCGCUU CUGAUGAGGCCGAAAGGCCGAA AUCACAUC 1602 GAUGUGAUU AAGCGGGA 16031531 UUCCCGCU CUGAUGAGGCCGAAAGGCCGAA AAUCACAU 1604 AUGUGAUUA AGCGGGAA 16051541 AUUCAUCC CUGAUGAGGCCGAAAGGCCGAA AUUCCCGC 1606 GCGGGAAUC GGAUGAAU 16071550 CAAUUCCA CUGAUGAGGCCGAAAGGCCGAA AUUCAUCC 1608 GGAUGAAUC UGGAAUUG 16091557 UCAGCAAC CUGAUGAGGCCGAAAGGCCGAA AUUCCAGA 1610 UCUGGAAUU GUUGCUGA 16111560 AACUCAGC CUGAUGAGGCCGAAAGGCCGAA ACAAUUCC 1612 GGAAUUGUU GCUGAGUU 16131568 UCUCUUGA CUGAUGAGGCCGAAAGGCCGAA ACUCAGCA 1514 UGCUGAGUU UCAAGAGA 16151569 CUCUCUUG CUGAUGAGGCCGAAAGGCCGAA AACUCAGC 1616 GCUGAGUUU CAAGAGAG 16171570 ACUCUCUU CUGAUGAGGCCGAAAGGCCGAA AAACUCGA 1618 CUGAGUUUC AAGAGAGU 16191589 UUUUCAGU CUGAUGAGGCCGAAAGGCCGAA ACGGUGGU 1620 ACCACCGUU ACUGAAAA 16211590 UUUUUCAG CUGAUGAGGCCGAAAGGCCGAA AACGGUGG 1622 CCACCGUUA CUGAAAAA 16231602 GCCUGCUU CUGAUGAGGCCGAAAGGCCGAA AUUUUUUU 1624 AAAAAAAUC AAGCAGGC 16251619 CAGUUGGC CUGAUGAGGCCGAAAGGCCGAA ACUCCACC 1626 GGUGGAGUC GCCAACUG 16271634 AGUUUCCC CUGAUGAGGCCGAAAGGCCGAA AUUUCUCA 1628 UGAGAAAUC GGGAAACU 16291643 AGCAGAAG CUGAUGAGGCCGAAAGGCCGAA AGUUUCCC 1630 GGGAAACUU CUUCUGCU 16311644 GAGCAGAA CUGAUGAGGCCGAAAGGCCGAA AAGUUUCC 1632 GGAAACUUC UUCUGCUC 16331646 UUGAGCAG CUGAUGAGGCCGAAAGGCCGAA AGAAGUUU 1634 AAACUUCUU CUGCUCAA 16351647 UUUGAGCA CUGAUGAGGCCGAAAGGCCGAA AAGAAGUU 1636 AACUUCUUC UGCUCAAA 16371652 AGUGGUUU CUGAUGAGGCCGAAAGGCCGAA AGCAGAAG 1638 CUUCUGCUC AAACCACU 16391691 CCUGCGAG CUGAUGAGGCCGAAAGGCCGAA ACAGUUGG 1640 CCAACUGUU CUCGCAGG 16411692 GCCUGCGA CUGAUGAGGCCGAAAGGCCGAA AACAGUUG 1642 CAACUGUUC UCGCAGGC 16431694 ACGCCUGC CUGAUGAGGCCGAAAGGCCGAA AGAACAGU 1644 ACUGUUCUC GCAGGCGU 16451703 CCACAGGA CUGAUGAGGCCGAAAGGCCGAA ACGCCUGC 1646 GCAGGCGUC UCCUGUGG 16471705 UGCCACAG CUGAUGAGGCCGAAAGGCCGAA AGACGCCU 1648 AGGCGUCUC CUGUGGCA 16491726 UGUAAGAA CUGAUGAGGCCGAAAGGCCGAA AUUUGGGG 1650 CCCCAAAUA UUCUUACA 16511728 CUUGUAAG CUGAUGAGGCCGAAAGGCCGAA AUAUUUGG 1652 CCAAAUAUU CUUACAAG 16531729 GCUUGUAA CUGAUGAGGCCGAAAGGCCGAA AAUAUUUG 1654 CAAAUAUUC UUACAAGC 16551731 GAGCUUGU CUGAUGAGGCCGAAAGGCCGAA AGAAUAUU 1656 AAUAUUCUU ACAAGCUC 16571732 AGAGCUUG CUGAUGAGGCCGAAAGGCCGAA AAGAAUAU 1658 AUAUUCUUA CAAGCUCU 16591739 UUAAAACA CUGAUGAGGCCGAAAGGCCGAA AGCUUGUA 1660 UACAAGCUC UGUUUUAA 16611743 GUCAUUAA CUGAUGAGGCCGAAAGGCCGAA ACAGAGCU 1662 AGCUCUGUU UUAAUGAC 16631744 UGUCAUUA CUGAUGAGGCCGAAAGGCCGAA AACAGAGC 1664 GCUCUGUUU UAAUGACA 16651745 GUGUGAUU CUGAUGAGGCCGAAAGGCCGAA AAACAGAG 1666 CUCUGUUUU AAUGACAC 16671746 GGUGUCAU CUGAUGAGGCCGAAAGGCCGAA AAAACAGA 1668 UCUGUUUUA AUGACACC 16691758 UCUUCUGA CUGAUGAGGCCGAAAGGCCGAA ACAGGUGU 1670 ACACCUGUA UCAGAAGA 16711760 CAUCUUCU CUGAUGAGGCCGAAAGGCCGAA AUACAGGU 1672 ACCUGUAUC AGAAGAUG 16731779 GCUUUGAG CUGAUGAGGCCGAAAGGCCGAA ACAUUGUC 1674 GACAAUGUC CUCAAAGC 16751782 AAGGCUUU CUGAUGAGGCCGAAAGGCCGAA AGGACAUU 1676 AAUGUCCUC AAAGCCUU 16771790 GUACGGUA CUGAUGAGGCCGAAAGGCCGAA AGGCUUUG 1678 CAAAGCCUU UACCGUAC 16791791 GGUACGGU CUGAUGAGGCCGAAAGGCCGAA AAGGCUUU 1680 AAAGCCUUU ACCGUACC 16811792 AGGUACGG CUGAUGAGGCCGAAAGGCCGAA AAAGGCUU 1682 AAGCCUUUA CCGUACCU 16831797 UUCUUAGG CUGAUGAGGCCGAAAGGCCGAA ACGGUAAA 1684 UUUACCGUA CCUAAGAA 16851801 CCUGUUCU CUGAUGAGGCCGAAAGGCCGAA AGGUACGG 1686 CCGUACCUA AGAACAGG 16871822 CUGCAAGG CUGAUGAGGCCGAAAGGCCGAA ACCCACCA 1688 UGGUGGGUC CCUUGCAG 16891826 AUGGCUGC CUGAUGAGGCCGAAAGGCCGAA AGGGACCC 1690 GGGUCCCUU GCAGCCUA 16911859 UCCCACAG CUGAUGAGGCCGAAAGGCCGAA AUGCUGGC 1692 GCCAGCAUC CUGUGGGA 16931892 CCGGACCG CUGAUGAGGCCGAAAGGCCGAA AGGCCGUC 1694 GACGGCCUC CGGUCCGG 16951897 CCGAGCCG CUGAUGAGGCCGAAAGGCCGAA ACCGGAGG 1696 CCUCCGGUC CGGCUCGG 16971903 GUAUUUCC CUGAUGAGGCCGAAAGGCCGAA AGCCGGAC 1698 GUCCGGCUC GGAAAUAC 16991910 CGUUCACG CUGAUGAGGCCGAAAGGCCGAA AUUUCCGA 1700 UCGGAAAUA CGUGAACG 17011922 GAGCUGAG CUGAUGAGGCCGAAAGGCCGAA ACGCGUUC 1702 GAACGCGUU CUCAGCUC 17031923 CGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AACGCGUU 1704 AACGCGUUC UCAGCUCG 17051925 UUCGAGCU CUGAUGAGGCCGAAAGGCCGAA AGAACGCG 1706 CGCGUUCUC AGCUCGAA 17071930 CAGAGUUC CUGAUGAGGCCGAAAGGCCGAA AGCUGAGA 1708 UCUCAGCUC GAACUCUG 17091936 CAUGACCA CUGAUGAGGCCGAAAGGCCGAA AGUUCGAG 1710 CUCGAACUC UGGUCAUG 17111941 UCUCACAU CUGAUGAGGCCGAAAGGCCGAA ACCAGAGU 1712 ACUCUGGUC AUGUGAGA 17131953 UUUCUGGA CUGAUGAGGCCGAAAGGCCGAA AUGUCUCA 1714 UGAGACAUU UCCAGAAA 17151954 UUUUCUGG CUGAUGAGGCCGAAAGGCCGAA AAUGUCUC 1716 GAGACAUUU CCAGAAAA 17171955 CUUUUCUG CUGAUGAGGCCGAAAGGCCGAA AAAUGUCU 1718 AGACAUUUC CAGAAAAG 17191967 AAAACCAU CUGAUGAGGCCGAAAGGCCGAA AUGCUUUU 1720 AAAAGCAUU AUGGUUUU 17211968 GAAAACCA CUGAUGAGGCCGAAAGGCCGAA AAUGCUUU 1722 AAAGCAUUA UGGUUUUC 17231973 GUUCUGAA CUGAUGAGGCCGAAAGGCCGAA ACCAUAAU 1724 AUUAUGGUU UUCAGAAC 17251974 UGUUCUGA CUGAUGAGGCCGAAAGGCCGAA AACCAUAA 1726 UUAUGGUUU UCAGAACA 17271975 GUGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAACCAUA 1728 UAUGGUUUU CAGAACAC 17291976 AGUGUUCU CUGAUGAGGCCGAAAGGCCGAA AAAACCAU 1730 AUGGUUUUC AGAACACU 17311985 CAACUUUU CUGAUGAGGCCGAAAGGCCGAA AGUGUUCU 1732 AGAACACUU AAAAGUUG 17331986 UCAACUUU CUGAUGAGGCCGAAAGGCCGAA AAGUGUUC 1734 GAACACUUA AAAGUUGA 17351992 CGAAAGUC CUGAUGAGGCCGAAAGGCCGAA ACUUUUAA 1736 UUAAAAGUU GACUUUCG 17371997 UGUGUCGA CUGAUGAGGCCGAAAGGCCGAA AGUCAACU 1738 AGUUGACUU UCGACACA 17391998 AUGUGUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCAAC 1740 GUUGACUUU CGACACAU 17411999 CAUGUGUC CUGAUGAGGCCGAAAGGCCGAA AAAGUCAA 1742 UUGACUUUC GACACAUG 17432911 ACGCUGAG CUGAUGAGGCCGAAAGGCCGAA AGCCAUGU 1744 ACAUGGCUC CUCAGCGU 17452014 UCCACGCU CUGAUGAGGCCGAAAGGCCGAA AGGAGCCA 1746 UGGCUCCUC AGCGUGGA 17472028 CAGCCAUG CUGAUGAGGCCGAAAGGCCGAA AGCGCUCC 1748 GGAGCGCUC CAUGGCUG 17492052 CACAACAA CUGAUGAGGCCGAAAGGCCGAA AUCAGGCU 1750 AGCCUGAUU UUGUUGUG 17512053 CCACAACA CUGAUGAGGCCGAAAGGCCGAA AAUCAAAC 1752 GCCUGAUUU UGUUGUGG 17532054 ACCACAAC CUGAUGAGGCCGAAAGGCCGAA AAAUCAGG 1754 CCUGAUUUU GUUGUGGU 17552057 UGUACCAC CUGAUGAGGCCGAAAGGCCGAA ACAAAAUC 1756 GAUUUUGUU GUGGUACA 17572063 AACUGUUG CUGAUGAGGCCGAAAGGCCGAA ACCACAAC 1758 GUUGUGGUA CAACAGUU 17592071 CUGCUCUC CUGAUGAGGCCGAAAGGCCGAA ACUGUUGU 1760 ACAACAGUU GAGAGCAG 17612092 CAACUAAA CUGAUGAGGCCGAAAGGCCGAA AUGCACUU 1762 AAGUGCAUU UUUAGUUG 17632093 GCAACUAA CUGAUGAGGCCGAAAGGCCGAA AAUGCACU 1764 AGUGCAUUU UUAGUUGC 17652094 AGCAACUA CUGAUGAGGCCGAAAGGCCGAA AAAUGCAC 1766 GUGCAUUUU UAGUUGCU 17672095 AAGCAACU CUGAUGAGGCCGAAAGGCCGAA AAAAUGCA 1768 UGCAUUUUU AGUUGCUU 17692096 CAAGCAAC CUGAUGAGGCCGAAAGGCCGAA AAAAAUGC 1770 GCAUUUUUA GUUGCUUG 17712099 UCUCAAGC CUGAUGAGGCCGAAAGGCCGAA ACUAAAAA 1772 UUUUUAGUU GCUUGAGA 17732103 GAGAUCUC CUGAUGAGGCCGAAAGGCCGAA AGCAACUA 1774 UAGUUGCUU GAGAUCUC 17752109 UCAAGUGA CUGAUGAGGCCGAAAGGCCGAA AUCUCAAG 1776 CUUGAGAUC UCACUUGA 17772111 AAUCAAGU CUGAUGAGGCCGAAAGGCCGAA AGAUCUCA 1778 UGAGAUCUC ACUUGAUU 17792115 GUGAAAUC CUGAUGAGGCCGAAAGGCCGAA AGUGAGAU 1780 AUCUCACUU GAUUUCAC 17812119 UUGUGUGA CUGAUGAGGCCGAAAGGCCGAA AUCAAGAG 1782 CACUUGAUU UCACACAA 17832120 GUUGUGUG CUGAUGAGGCCGAAAGGCCGAA AAUCAAGU 1784 ACUUGAUUU CACACAAC 17852121 AGUUGUGU CUGAUGAGGCCGAAAGGCCGAA AAAUCAAG 1786 CUUGAUUUC ACACAACU 17872130 AUCCUUUU CUGAUGAGGCCGAAAGGCCGAA AGUUGUGU 1788 ACACAACUA AAAAGGAU 17892139 AAAAAAAA CUGAUGAGGCCGAAAGGCCGAA AUCCUUUU 1790 AAAAGGAUU UUUUUUUU 17912140 UAAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAUCCUUU 1792 AAAGGAUUU UUUUUUUA 17932141 UUAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAUCCUU 1794 AAGGAUUUU UUUUUUAA 17952142 UUUAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAUCCU 1796 AGGAUUUUU UUUUUAAA 17972143 UUUUAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAUCC 1798 GGAUUUUUU UUUUAAAA 17992144 UUUUUAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAUC 1800 GAUUUUUUU UUUAAAAA 18012145 AUUUUUAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAAU 1802 AUUUUUUUU UUAAAAAU 18032146 UAUUUUUA CUGAUGAGGCCGAAAGGCCGAA AAAAAAAA 1804 UUUUUUUUU UAAAAAUA 18052147 UUAUUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAAAA 1806 UUUUUUUUU AAAAAUAA 18072148 AUUAUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAAAA 1808 UUUUUUUUA AAAAUAAU 18092154 AUUAUUAU CUGAUGAGGCCGAAAGGCCGAA AUUUUUAA 1810 UUAAAAAUA AUAAUAAU 18112157 UUCAUUAU CUGAUGAGGCCGAAAGGCCGAA AUUAUUUU 1812 AAAAUAAUA AUAAUGAA 18132160 UUAUUCAU CUGAUGAGGCCGAAAGGCCGAA AUUAUUAU 1814 AUAAUAAUA AUGAAUAA 18152167 AAGACUGU CUGAUGAGGCCGAAAGGCCGAA AUUCAUUA 1816 UAAUGAAUA ACAGUCUU 18172173 UUAGGUAA CUGAUGAGGCCGAAAGGCCGAA ACUGUUAU 1818 AUAACAGUC UUACCUAA 18192175 AUUUAGGU CUGAUGAGGCCGAAAGGCCGAA AGACUGUU 1820 AACAGUCUU ACCUAAAU 18212176 AAUUUAGG CUGAUGAGGCCGAAAGGCCGAA AAGACUGU 1822 ACAGUCUUA CCUAAAUU 18232180 UAAUAAUU CUGAUGAGGCCGAAAGGCCGAA AGGUAAGA 1824 UCUUACCUA AAUUAUUA 18252184 UACCUAAU CUGAUGAGGCCGAAAGGCCGAA AUUUAGGU 1826 ACCUAAAUU AUUAGGUA 18272185 UUACCUAA CUGAUGAGGCCGAAAGGCCGAA AAUUUAGG 1828 CCUAAAUUA UUAGGUAA 18292187 CAUUACCU CUGAUGAGGCCGAAAGGCCGAA AUAAUUUA 1830 UAAAUUAUU AGGUAAUG 18312188 UCAUUACC CUGAUGAGGCCGAAAGGCCGAA AAUAAUUU 1832 AAAUUAUUA GGUAAUGA 18332192 CAAUUCAU CUGAUGAGGCCGAAAGGCCGAA ACCUAAUA 1834 UAUUAGGUA AUGAAUUG 18352199 AUGGUCAC CUGAUGAGGCCGAAAGGCCGAA AUUCAUUA 1836 UAAUGAAUU GUGACCAU 18372208 UAUUAACA CUGAUGAGGCCGAAAGGCCGAA AUGGUCAC 1838 GUGACCAUU UGUUAAUA 18392209 AUAUUAAC CUGAUGAGGCCGAAAGGCCGAA AAUGGUCA 1840 UGACCAUUU GUUAAUAU 18412212 AUGAUAUU CUGAUGAGGCCGAAAGGCCGAA ACAAAUGG 1842 CCAUUUGUU AAUAUCAU 18432213 UAUGAUAU CUGAUGAGGCCGAAAGGCCGAA AACAAAUG 1844 CAUUUGUUA AUAUCAUA 18452216 GAUUAUGA CUGAUGAGGCCGAAAGGCCGAA AUUAACAA 1846 UUGUUAAUA UCAUAAUC 18472218 CUGAUUAU CUGAUGAGGCCGAAAGGCCGAA AUAUUAAC 1848 GUUAAUAUC AUAAUCAG 18492221 AAUCUGAU CUGAUGAGGCCGAAAGGCCGAA AUGAUAUU 1850 AAUAUCAUA AUCAGAUU 18512224 AAAAAUCU CUGAUGAGGCCGAAAGGCCGAA AUUAUGAU 1852 AUCAUAAUC AGAUUUUU 18532229 UUUUAAAA CUGAUGAGGCCGAAAGGCCGAA AUCUGAUU 1854 AAUCAGAUU UUUUAAAA 18552230 UUUUUAAA CUGAUGAGGCCGAAAGGCCGAA AAUCUGAU 1856 AUCAGAUUU UUUAAAAA 18572231 UUUUUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUCUGA 1858 UCAGAUUUU UUAAAAAA 18592232 UUUUUUUA CUGAUGAGGCCGAAAGGCCGAA AAAAUCUG 1860 CAGAUUUUU UAAAAAAA 18612233 UUUUUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAUCU 1862 AGAUUUUUU AAAAAAAA 18632234 AUUUUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAAUC 1864 GAUUUUUUA AAAAAAAU 18652243 AAUCAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUUUUU 1866 AAAAAAAUA AAAUGAUU 18672251 UACAAAUA CUGAUGAGGCCGAAAGGCCGAA AUCAUUUU 1868 AAAAUGAUU UAUUUGUA 18692252 AUACAAAU CUGAUGAGGCCGAAAGGCCGAA AAUCAUUU 1870 AAAUGAUUU AUUUGUAU 18712253 AAUACAAA CUGAUGAGGCCGAAAGGCCGAA AAAUCAUU 1872 AAUGAUUUA UUUGUAUU 18732255 AAAAUACA CUGAUGAGGCCGAAAGGCCGAA AUAAAUCA 1874 UGAUUUAUU UGAUUUUU 18752256 UAAAAUAC CUGAUGAGGCCGAAAGGCCGAA AAUAAAUC 1876 GAUUUAUUU GUAUUUUA 18772259 CUCUAAAA CUGAUGAGGCCGAAAGGCCGAA ACAAAUAA 1878 UUAUUUGUA UUUUAGAG 18792261 UUCUCUAA CUGAUGAGGCCGAAAGGCCGAA AUACAAAU 1880 AUUUGUAUU UUAGAGAA 18812262 AUUCUCUA CUGAUGAGGCCGAAAGGCCGAA AAUACAAA 1882 UUUGUAUUU UAGAGAAU 18832263 UAUUCUCU CUGAUGAGGCCGAAAGGCCGAA AAAUACAA 1884 UUGUAUUUU AGAGAAUA 18852264 GUAUUCUC CUGAUGAGGCCGAAAGGCCGAA AAAAUACA 1886 UGUAUUUUA GAGAAUAC 18872271 AUCUGUUG CUGAUGAGGCCGAAAGGCCGAA AUUCUCUA 1888 UAGAGAAUA CAACAGAU 18892280 AAAAUACU CUGAUGAGGCCGAAAGGCCGAA AUCUGUUG 1890 CAACAGAUC AGUAUUUU 18912284 GUCAAAAA CUGAUGAGGCCGAAAGGCCGAA ACUGAUCU 1892 AGAUCAGUA UUUUUGAC 18932286 CAGUCAAA CUGAUGAGGCCGAAAGGCCGAA AUACUGAU 1894 AUCAGUAUU UUUGACUG 18952287 ACAGUCAA CUGAUGAGGCCGAAAGGCCGAA AAUACUGA 1896 UCAGUAUUU UUGACUGU 18972288 CACAGUCA CUGAUGAGGCCGAAAGGCCGAA AAAUACUG 1898 CAGUAUUUU UGACUGUG 18992289 CCACAGUC CUGAUGAGGCCGAAAGGCCGAA AAAAUACU 1900 AGUAUUUUU GACUGUGG 19012303 UUUUUUUA CUGAUGAGGCCGAAAGGCCGAA AUUCACCA 1902 UGUGGAAUU UAAAAAAA 19032304 UUUUUUUU CUGAUGAGGCCGAAAGGCCGAA AAUUCACC 1904 GGUGAAUUU AAAAAAAA 19052305 UUUUUUUU CUGAUGAGGCCGAAAGGCCGAA AAAUUCAC 1906 GUGAAUUUA AAAAAAAA 19072316 UUUGUGUA CUGAUGAGGCCGAAAGGCCGAA AUUUUUUU 1908 AAAAAAAUU UACACAAA 19092317 CUUUGUGU CUGAUGAGGCCGAAAGGCCGAA AAUUUUUU 1910 AAAAAAUUU ACACAAAG 19112318 UCUUUGUG CUGAUGAGGCCGAAAGGCCGAA AAAUUUUU 1912 AAAAAUUUA CACAAAGA 19132330 UACUGGGA CUGAUGAGGCCGAAAGGCCGAA AUUUCUUU 1914 AAAGAAAUA UCCCAGUA 19152332 AAUACUGG CUGAUGAGGCCGAAAGGCCGAA AUAUUUCU 1916 AGAAAUAUC CCAGUAUU 19172338 ACAUGGAA CUGAUGAGGCCGAAAGGCCGAA ACUGGGAU 1918 AUCCCAGUA UUCCAUGU 19192340 AUACAUGG CUGAUGAGGCCGAAAGGCCGAA AUACUGGG 1920 CCCAGUAUU CCAUGUAU 19212341 GAUACAUG CUGAUGAGGCCGAAAGGCCGAA AAUACUGG 1922 CCAGUAUUC CAUGUAUC 19232347 GACUGAGA CUGAUGAGGCCGAAAGGCCGAA ACAUGGAA 1924 UUCCAUGUA UCUCAGUC 19252349 GUGACUGA CUGAUGAGGCCGAAAGGCCGAA AUACAUGG 1926 CCAUGUAUC UCAGUCAC 19272351 UAGUGACU CUGAUGAGGCCGAAAGGCCGAA AGAUACAU 1928 AUGUAUCUC AGUCACUA 19292355 UGUUUAGU CUGAUGAGGCCGAAAGGCCGAA ACUGAGAU 1930 AUCUCAGUC ACUAAACA 10312359 UGUAUGUU CUGAUGAGGCCGAAAGGCCGAA AGUGACUG 1932 CAGUCACUA AACAUACA 10332365 UCUCUGUG CUGAUGAGGCCGAAAGGCCGAA AUGUUUAG 1934 CUAAACAUA CACAGAGA 19352377 UUUUUAAA CUGAUGAGGCCGAAAGGCCGAA AUCUCUCU 1936 AGAGAGAUU UUUAAAAA 19372378 GUUUUUAA CUGAUGAGGCCGAAAGGCCGAA AAUCUCUC 1938 GAGAGAUUU UUAAAAAC 19392379 GGUUUUUA CUGAUGAGGCCGAAAGGCCGAA AAAUCUCU 1940 AGAGAUUUU UAAAAACC 19412380 UGGUUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAUCUC 1942 GAGAUUUUU AAAAACCA 19432381 CUGGUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAUCU 1944 AGAUUUUUA AAAACCAG 19452399 UUCAAAAU CUGAUGAGGCCGAAAGGCCGAA AUGCUUCU 1946 AGAAGCAUU AUUUUGAA 19472400 AUUCAAAA CUGAUGAGGCCGAAAGGCCGAA AAUGCUUC 1948 GAAGCAUUA UUUUGAAU 19482402 ACAUUCAA CUGAUGAGGCCGAAAGGCCGAA AUAAUGCU 1950 AGCAUUAUU UUGAAUGU 19512403 AACAUUCA CUGAUGAGGCCGAAAGGCCGAA AAUAAUGC 1952 GCAUUAUUU UGAAUGUU 19532404 UAACAUUC CUGAUGAGGCCGAAAGGCCGAA AAAUAAUG 1954 CAUUAUUUU GAAUGUUA 19552411 AUUUAGCU CUGAUGAGGCCGAAAGGCCGAA ACAUUCAA 1956 UUGAAUGUU AGCUAAAU 19572412 GAUUUAGC CUGAUGAGGCCGAAAGGCCGAA UACAUUCA 1958 UGAAUGUUA GCUAAAUC 19592416 UUGGGAUU CUGAUGAGGCCGAAAGGCCGAA AGCUAACA 1960 UGUUAGCUA AAUCCCAA 19612420 UUACUUGG CUGAUGAGGCCGAAAGGCCGAA AUUUAGCU 1962 AGCUAAAUC CCAAGUAA 19632427 UUAAGUAU CUGAUGAGGCCGAAAGGCCGAA ACUUGGGA 1964 UCCCAAGUA AUACUUAA 19652430 GCAUUAAG CUGAUGAGGCCGAAAGGCCGAA AUUACUUG 1966 CAAGUAAUA CUUAAUGC 19672433 GUUGCAUU CUGAUGAGGCCGAAAGGCCGAA AGUAUUAC 1968 GUAAUACUU AAUGCAAC 19692434 GGUUGCAU CUGAUGAGGCCGAAAGGCCGAA AAGUAUUA 1970 UAAUACUUA AUGCAACC 19712445 AGCUCCUA CUGAUGAGGCCGAAAGGCCGAA AGGGUUGC 1972 GCAACCCUC UAGGAGCU 19732447 UGAGCUCC CUGAUGAGGCCGAAAGGCCGAA AGAGGGUU 1974 AACCCUCUA GGAGCUCA 19752454 CCACAAAU CUGAUGAGGCCGAAAGGCCGAA AGCUCCUA 1976 UAGGAGCUC AUUUGUGG 19772457 UAGCCACA CUGAUGAGGCCGAAAGGCCGAA AUGAGCUC 1978 GAGCUCAUU UGUGGCUA 19792458 UUAGCCAC CUGAUGAGGCCGAAAGGCCGAA AAUGAGCU 1980 AGCUCAUUU GUGGCUAA 19812465 AAGAUUAU CUGAUGAGGCCGAAAGGCCGAA AGCCACAA 1982 UUGUGGCUA AUAAUCUU 19832468 UCCAAGAU CUGAUGAGGCCGAAAGGCCGAA AUUAGCCA 1984 UGGCUAAUA AUCUUGGA 19852471 AUUUCCAA CUGAUGAGGCCGAAAGGCCGAA AUUAUUAG 1986 CUAAUAAUC UUGGAAAU 19872473 AUAUUUCC CUGAUGAGGCCGAAAGGCCGAA AGAUUAUU 1988 AAUAAUCUU GGAAAUAU 19892480 AAUAAAGA CUGAUGAGGCCGAAAGGCCGAA AUUUCCAA 1990 UUGGAAAUA UCUUUAUU 19912482 AUAAUAAA CUGAUGAGGCCGAAAGGCCGAA AUAUUUCC 1992 GGAAAUAUC UUUAUUAU 19932484 AUAUAAUA CUGAUGAGGCCGAAAGGCCGAA AGAUAUUU 1994 AAAUAUCUU UAUUAUAU 19952485 UAUAUAAU CUGAUGAGGCCGAAAGGCCGAA AAGAUAUU 1996 AAUAUCUUU AUUAUAUA 19972486 CUAUAUAA CUGAUGAGGCCGAAAGGCCGAA AAAGAUAU 1998 AUAUCUUUA UUAUAUAG 19992488 UGCUAUAU CUGAUGAGGCCGAAAGGCCGAA AUAAAGAU 2000 AUCUUUAUU AUAUAGCA 20012489 AUGCUAUA CUGAUGAGGCCGAAAGGCCGAA AAUAAAGA 2002 UCUUUAUUA UAUAGCAU 20032491 AAAUGCUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAAA 2004 UUUAUUAUA UAGCAUUU 20052493 AUAAAUGC CUGAUGAGGCCGAAAGGCCGAA AUAUAAUA 2006 UAUUAUAUA GCAUUUAU 20072498 UCCUCAUA CUGAUGAGGCCGAAAGGCCGAA AUGCUAUA 2008 UAUAGCAUU UAUGAGGA 20092499 CUCCUCAU CUGAUGAGGCCGAAAGGCCGAA AAUGCUAU 2010 AUAGCAUUU AUGAGGAG 20112500 UCUCCUCA CUGAUGAGGCCGAAAGGCCGAA AAAUGCUA 2012 UAGCAUUUA UGAGGAGA 20132510 GACAACAA CUGAUGAGGCCGAAAGGCCGAA AUCUCCUC 2014 GAGGAGAUU UUGUUGUC 20152511 UGACAACA CUGAUGAGGCCGAAAGGCCGAA AAUCUCCU 2016 AGGAGAUUU UGUUGUCA 20172512 CUGACAAC CUGAUGAGGCCGAAAGGCCGAA AAAUCUCC 2018 GGAGAUUUU GUUGUCAG 20192515 AAGCUGAC CUGAUGAGGCCGAAAGGCCGAA ACAAAAUC 2020 GAUUUUGUU GUCAGCUU 20212518 AGCAAGCU CUGAUGAGGCCGAAAGGCCGAA ACAACAAA 2022 UUUGUUGUC AGCUUGCU 20232523 UUUCAAGC CUGAUGAGGCCGAAAGGCCGAA AGCUGACA 2024 UGUCAGCUU GCUUGAAA 20252527 UAACUUUC CUGAUGAGGCCGAAAGGCCGAA AGCAAGCU 2026 AGCUUGCUU GAAAGUUA 20272534 UACAUAAU CUGAUGAGGCCGAAAGGCCGAA ACUUUCAA 2028 UUGAAAGUU AUUAAGUA 20292535 AUACAUAA CUGAUGAGGCCGAAAGGCCGAA AACUUUCA 2030 UGAAAGUUA UUAAGUAU 20312537 UCAUACAU CUGAUGAGGCCGAAAGGCCGAA AUAACUUU 2032 AAAGUUAUU AAGUAUGA 20332538 UUCAUACA CUGAUGAGGCCGAAAGGCCGAA AAUAACUU 2034 AAGUUAUUA AGUAUGAA 20352542 ACUAUUCA CUGAUGAGGCCGAAAGGCCGAA ACAUAAUA 2036 UAUUAUGUA UGAAUAGU 20372548 AAUAAAAC CUGAUGAGGCCGAAAGGCCGAA AUUCAUAC 2038 GUAUGAAUA GUUUUAUU 20392551 UUCAAUAA CUGAUGAGGCCGAAAGGCCGAA ACUAUUCA 2040 UGAAUAGUU UUAUUGAA 20412552 UUUCAAUA CUGAUGAGGCCGAAAGGCCGAA AACUAUUC 2042 GAAUAGUUU UAUUGAAA 20432553 UUUUCAAU CUGAUGAGGCCGAAAGGCCGAA AAACUAUU 2044 AAUAGUUUU AUUGAAAA 20452554 UUUUUCAA CUGAUGAGGCCGAAAGGCCGAA AAAACUAU 2046 AUAGUUUUA UUGAAAAA 20472556 AUUUUUUA CUGAUGAGGCCGAAAGGCCGAA AUAAAACU 2048 AGUUUUAUU GAAAAAAU 20492565 AAAAAUAU CUGAUGAGGCCGAAAGGCCGAA AUUUUUUC 2050 GAAAAAAUU AUAUUUUU 20512566 UAAAAAUA CUGAUGAGGCCGAAAGGCCGAA AAUUUUUU 2052 AAAAAAUUA UAUUUUUA 20532568 AAUAAAAA CUGAUGAGGCCGAAAGGCCGAA AUAAUUUU 2054 AAAAUUAUA UUUUUAUU 20552570 UGAAUAAA CUGAUGAGGCCGAAAGGCCGAA AUAUAAUU 2056 AAUUAUAUU UUUAUUCA 20572571 CUGAAUAA CUGAUGAGGCCGAAAGGCCGAA AAUAUAAU 2058 AUUAUAUUU UUAUUCAG 20592572 ACUGAAUA CUGAUGAGGCCGAAAGGCCGAA AAAUAUAA 2060 UUAUAUUUU UAUUCAGU 20612573 UACUGAAU CUGAUGAGGCCGAAAGGCCGAA AAAAUAUA 2062 UAUAUUUUU AUUCAGUA 20632574 UUACUGAA CUGAUGAGGCCGAAAGGCCGAA AAAAAUAU 2064 AUAUUUUUA UUCAGUAA 20652576 AAUUACUA CUGAUGAGGCCGAAAGGCCGAA AUAAAAAU 2066 AUUUUUAUU CAGUAAUU 20672577 AAAUUACU CUGAUGAGGCCGAAAGGCCGAA AAUAAAAA 2068 UUUUUAUUC AGUAAUUU 20692581 AAUUAAAU CUGAUGAGGCCGAAAGGCCGAA ACUGAAUA 2070 UAUUCAGUA AUUUAAUU 20712584 CAAAAUUA CUGAUGAGGCCGAAAGGCCGAA AUUACUGA 2072 UCAGUAAUU UAAUUUUG 20732585 ACAAAAUU CUGAUGAGGCCGAAAGGCCGAA AAUUACUG 2074 CAGUAAUUU AAUUUUGU 20752586 UACAAAAU CUGAUGAGGCCGAAAGGCCGAA AAAUUACU 2076 AGUAAUUUA AUUUUGUA 20772589 AUUUACAA CUGAUGAGGCCGAAAGGCCGAA AUUAAAUU 2078 AAUUUAAUU UUGUAAAU 20792590 CAUUUACA CUGAUGAGGCCGAAAGGCCGAA AAUUAAAU 2080 AUUUAAUUU UGUAAAUG 20812591 GCAUUUAC CUGAUGAGGCCGAAAGGCCGAA AAAUUAAA 2082 UUUAAUUUU GUAAAUGC 20832594 UUGGCAUU CUGAUGAGGCCGAAAGGCCGAA ACAAAAUU 2084 AAUUUUGUA AAUGCCAA 20852617 UAGCAGCG CUGAUGAGGCCGAAAGGCCGAA ACACAUUU 2086 AAAUGUGUU CGCUGCUA 20872618 AUAGCAGC CUGAUGAGGCCGAAAGGCCGAA AACACAUU 2088 AAUGUGUUC GCUGCUAU 20892625 UAAAACCA CUGAUGAGGCCGAAAGGCCGAA AGCAGCGA 2090 UCGCUGCUA UGGUUUUA 20912630 UAGGCUAA CUGAUGAGGCCGAAAGGCCGAA ACCAUAGC 2092 GCUAUGGUU UUAGCCUA 20932631 AUAGGCUA CUGAUGAGGCCGAAAGGCCGAA AACCAUAG 2094 CUAUGGUUU UAGCCUAU 20952632 UAUAGGCU CUGAUGAGGCCGAAAGGCCGAA AAACCAUA 2096 UAUGGUUUU AGCCUAUA 20972633 CUAUAGGC CUGAUGAGGCCGAAAGGCCGAA AAAACCAU 2098 AUGGUUUUA GCCUAUAG 20992638 CAUGACUA CUGAUGAGGCCGAAAGGCCGAA AGGCUAAA 2100 UUUAGCCUA UAGUCAUG 21012640 AGCAUGAC CUGAUGAGGCCGAAAGGCCGAA AUAGGCUA 2102 UAGCCUAUA GUCAUGCU 21032643 AGCAGCAU CUGAUGAGGCCGAAAGGCCGAA ACUAUAGG 2104 CCUAUAGUC AUGCUGCU 21052652 ACACUAGC CUGAUGAGGCCGAAAGGCCGAA AGCAGCAU 2106 AUGCUGCUA GCUAGUGU 21072656 CCUGACAC CUGAUGAGGCCGAAAGGCCGAA AGCUAGCA 2108 UGCUAGCUA GUGUCAGG 21092661 UGCCCCCU CUGAUGAGGCCGAAAGGCCGAA ACACUAGC 2110 GCUAGUGUC AGGGGGCA 21112672 CUAAGCUC CUGAUGAGGCCGAAAGGCCGAA AUUGCCCC 2112 GGGGCAAUA GAGCUUAG 21132678 UUCCAUCU CUGAUGAGGCCGAAAGGCCGAA AGCUCUAU 2114 AUAGAGCUU AGAUGGAA 21152679 UUUCCAUC CUGAUGAGGCCGAAAGGCCGAA AAGCUCUA 2116 UACAGCUUA GAUGGAAA 21172703 CUAACACC CUGAUGAGGCCGAAAGGCCGAA AGUCUCUU 2118 AAGAGACUC GGUGUUAG 21192709 CGUUAUCU CUGAUGAGGCCGAAAGGCCGAA ACACCGAG 2120 CUCGGUGUU AGAUAACG 21212710 CCGUUAUC CUGAUGAGGCCGAAAGGCCGAA AACACCGA 2122 UCGGUGUUA GAUAACGG 21232714 UAGUCCGU CUGAUGAGGCCGAAAGGCCGAA AUCUAACA 2124 UGUUAGAUA ACGGACUA 21252722 CUAGUGCA CUGAUGAGGCCGAAAGGCCGAA AGUCCGUU 2126 AACGGACUA UGCACUAG 21272729 UGGAAUAC CUGAUGAGGCCGAAAGGCCGAA AGUGCAUA 2128 UAUGCACUA GUAUUCCA 21292732 GUCUGGAA CUGAUGAGGCCGAAAGGCCGAA ACUAGUGC 2130 UCACUAGUA UUCCAGAC 21312734 AAGUCUGG CUGAUGAGGCCGAAAGGCCGAA AUACUAGU 2132 ACUAGUAUU CCAGACUU 21332735 AAAGUCUG CUGAUGAGGCCGAAAGGCCGAA AAUACUAG 2134 CUAGUAUUC CAGACUUU 21352742 AAAUAAAA CUGAUGAGGCCGAAAGGCCGAA AGUCUGGA 2136 UCCAGACUU UUUUAUUU 21372743 AAAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAGUCUGG 2138 CCAGACUUU UUUAUUUU 21392744 AAAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAGUCUG 2140 CAGACUUUU UUAUUUUU 21412745 AAAAAAUA CUGAUGAGGCCGAAAGGCCGAA AAAAGUCU 2142 AGACUUUUU UAUUUUUU 21432746 UAAAAAAU CUGAUGAGGCCGAAAGGCCGAA AAAAAGUC 2144 GACUUUUUU AUUUUUUA 21452747 AUAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAGU 2146 ACUUUUUUA UUUUUUAU 21472749 AUAUAAAA CUGAUGAGGCCGAAAGGCCGAA AUAAAAAA 2148 UUUUUUAUU UUUUAUAU 21492750 UAUAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAAAAA 2150 UUUUUAUUU UUUAUAUA 21512751 AUAUAUAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAAA 2152 UUUUAUUUU UUAUAUAU 21532752 UAUAUAUA CUGAUGAGGCCGAAAGGCCGAA AAAAUAAA 2154 UUUAUUUUU UAUAUAUA 21552753 AUAUAUAU CUGAUGAGGCCGAAAGGCCGAA AAAAAUAA 2156 UUAUUUUUU AUAUAUAU 21572754 CAUAUAUA CUGAUGAGGCCGAAAGGCCGAA AAAAAAUA 2158 UAUUUUUUA UAUAUAUG 21592756 UACAUAUA CUGAUGAGGCCGAAAGGCCGAA AUAAAAAA 2160 UUUUUUAUA UAUAUGUA 21612758 GGUACAUA CUGAUGAGGCCGAAAGGCCGAA AUAUAAAA 2162 UUUUAUAUA UAUGUACC 21632760 AAGGUACA CUGAUGAGGCCGAAAGGCCGAA AUAUAUAA 2164 UUAUAUAUA UGUACCUU 21652764 GGAAAAGG CUGAUGAGGCCGAAAGGCCGAA ACAUAUAU 2166 AUAUAUGUA CCUUUUCC 21672768 AAAAGGAA CUGAUGAGGCCGAAAGGCCGAA AGGUACAU 2168 AUGUACCUU UUCCUUUU 21692769 CAAAAGGA CUGAUGAGGCCGAAAGGCCGAA AAGGUACA 2170 UGUACCUUU UCCUUUUG 21712770 ACAAAAGG CUGAUGAGGCCGAAAGGCCGAA AAAGGUAC 2172 GUACCUUUU CCUUUUGU 21732771 GACAAAAG CUGAUGAGGCCGAAAGGCCGAA AAAAGGUA 2174 UACCUUUUC CUUUUGUC 21752774 AUUGACAA CUGAUGAGGCCGAAAGGCCGAA AGGAAAAG 2176 CUUUUCCUU UUGUCAAU 21772775 AAUUGACA CUGAUGAGGCCGAAAGGCCGAA AAGGAAAA 2178 UUUUCCUUU UGUCAAUU 21792276 CAAUUGAC CUGAUGAGGCCGAAAGGCCGAA AAAGGAAA 2180 UUUCCUUUU GUCAAUUG 2181__________________________________________________________________________ TABLE XVII - Mouse c-myb Hairpin ribozyme and target sequences (REVISED) Position Ribozyme Seq. ID No. Substrate Seq. ID No. 24 GCGAGGCG AGAA GGGGCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2182 AGCCCCG GCC CGCCUCGC 2183 28 CAUGGCGA AGAA GGCCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2184 CCGGCCC GCC UCGCCAUG 2185 122 AUUUGGGC AGAA GCCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2186 AUGGGCU GCU GCCCAAAU 2187 125 CAGAUUUG AGAA GCAGCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2188 GGCUGCU GCC CAAAUCUG 2189 216 UUCCAGUC AGAA GUUCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2190 CGGAACA GAC GACUGGAA 2191 245 UCCGGUUG AGAA GAUAAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2192 AUUAUCU GCC CAACCGGA 2193 258 CACUGUAC AGAA GUCCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2194 CCGGACA GAU GUACAGUG 2195 529 CUCUGCCC AGAA GUUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2196 GGGAACA GAU GGGCAGAG 2197 551 GUCCGGGC AGAA GCUUUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2198 CAAAGCU GCU GCCCGGAC 2199 554 UCCGUCCG AGAA GCAGCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2200 AGCUGCU GCC CGGACGGA 2201 559 AUCAGUCC AGAA GGGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2202 CUGCCCG GAC GGACUGAU 2203 563 CAUUAUCA AGAA GUCCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2204 CCGGACG GAC UGAUAAUG 2205 656 CCACUGGC AGAA GGCUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2206 CCAGCCA GAC GCCAGUGG 2207 728 UUGGAGAG AGAA GAGAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2208 CAUCUCA GCU CUCUCCAA 2209 746 UGACGGAG AGAA GGCCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2210 GUGGCCA GUC CUCCGUCA 2211 822 UGCAAUGC AGAA GGAUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2212 CUAUCCU GUC GCAUUGCA 2213 857 CCGCAGCC AGAA GAGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2214 UCCCUCA GCC GGCUGCGG 2215 861 GCUGCCGC AGAA GGCUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2216 UCAGCCG GCU GCGGCAGC 2217 941 CUGUUGAC AGAA GGAGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2218 UGCUCCU GAU GUCAACAG 2218 1040 GAGGUCUG AGAA GGUCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2220 UGGACCA GAC CAGACCUC 2221 1045 CCCAUGAG AGAA GGUCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2222 CAGACCA GAC CUCAUGGG 2223 1068 AAACAGGA AGAA GGUGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2224 UGCACCU GUU UCCUGUUU 2225 1075 UUCUCCCA AGAA GGAAAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2226 GUUUCCU GUU UGGGAGAA 2227 1106 GAUCUGCA AGAA GAGAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2228 CAUCUCU GCC UGCAGAUC 2229 1113 GAGCCGGG AGAA GCAGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2230 GCCUGCA GAU CCCGGCUC 2231 1120 AGGUAGGG AGAA GGGAUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2232 GAUCCCG GCU CCCUACCU 2233 1226 AAUCUAUA AGAA GGAGUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2234 CACUCCA GUU UAUAGAUU 2235 1340 UUUUCACA AGAA GGUCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2236 GAGACCA GAC UGUGAAAA 2237 1449 AUUUCUUG AGAA GCAAGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2238 CCUUGCA GCU CAAGAAAU 2239 1468 CUUCAGGG AGAA GUAUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2240 AAAUACG GUC CCCUGAAG 2241 1490 GGGAGGGG AGAA GAGGUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2242 UACCUCA GAC CCCCUCCC 2243 1542 CCAGAUUC AGAA GAUUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2244 GGAAUCG GAU GAAUCUGG 2245 1648 GUGGUUUG AGAA GAAGAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2246 UUCUUCU GCU CAAACCAC 2247 1672 GGUGCUCA AGAA GUUCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2248 GAGAACA GCC UGAGCACC 2249 1688 CCUGCGAG AGAA GUUGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2250 CCCAACU GUU CUCGCAGG 2251 1713 UUUGGGGC AGAA GCCACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2252 UGUGGCA GAU GCCCCAAA 2253 1740 GUCAUUAA AGAA GAGCUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2254 AAGCUCU GUU UUAAUGAC 2255 1880 AGGCCGUC AGAA GGUCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2256 AGGACCA GAU GACGGCCU 2257 1887 GGACCGGA AGAA GUCAUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2258 GAUGACG GCC UCCGGUCC 2259 1894 CCGAGCCG AGAA GGAGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2260 GCCUCCG GUC CGGCUCGG 2261 1899 UAUUUCCG AGAA GGACCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2262 CGGUCCG GCU CGGAAAUA 2263 1926 AGAGUUCG AGAA GAGAAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2264 GUUCUCA GCU CGAACUCU 2265 2048 ACAACAAA AGAA GGCUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2266 AGAGCCU GAU UUUGUUGU 2267 2068 CUGCUCUC AGAA GUUGUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2268 UACAACA GUU GAGAGCAG 2269 2170 UUAGGUAA AGAA GUUAUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2270 AAUAACA GUC UUACCUAA 2271 2225 UUUAAAAA AGAA GAUUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2272 AUAAUCA GAU UUUUUAAA 2273 2276 AAAUACUG AGAA GUUGUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2274 UACAACA GAU CAGUAUUU 2275 2519 UUCAAGCA AGAA GACAAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2276 GUUGUCA GCU UGCUUGAA 2277 2717 AGUGCAUA AGAA GUUAUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2278 GAUAACG GAC UAUGCACU 2279 2737 AUAAAAAA AGAA GGAAUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2280 UAUUCCA GAC UUUUUUAU 2281 TABLE XVIII__________________________________________________________________________Porcine c-myb (region A) Hammerhead Ribozyme and TargetSequence (266 bp; nt. 458 start; Human numbering system) (REVISED)Position HH Ribozyme Seq. ID No. Substrate Seq. ID__________________________________________________________________________ No.467 CCCUUGAU CUGAUGAGGCCGAAAGGCCGAA AGAUNAGG 2282 CCUNAUCUC AUCAAGGG 2283470 GGACCCUU CUGAUGAGGCCGAAAGGCCGAA AUGAGAUN 2284 NAUCUCAUC AAGGGUCC 2285477 GGUCCAAG CUGAUGAGGCCGAAAGGCCGAA ACCCUUGA 2286 UCAAGGGUC CUUGGACC 2287480 UUUGGUCC CUGAUGAGGCCGAAAGGCCGAA AGGACCCU 2288 AGGGUCCUU GGACCAAA 2289498 CACUCUCU CUGAUGAGGCCGAAAGGCCGAA AUCUUCUU 2290 AAGAAGAUC AGAGAGUG 2291509 ACAAGCUC CUGAUGAGGCCGAAAGGCCGAA AUCACUCU 2292 AGAGUGAUA GAGCUUGU 2293515 UUCUGUAC CUGAUGAGGCCGAAAGGCCGAA AGCUCUAU 2294 AUAGAGCUU GUACAGAA 2295518 UAUUUCUG CUGAUGAGGCCGAAAGGCCGAA ACAAGCUC 2296 GAGCUUGUA CAGAAAUA 2297526 UCGGACCG CUGAUGAGGCCGAAAGGCCGAA AUUUCUGU 2298 ACAGAAAUA CGGUCCGA 2299531 ACGUUUCG CUGAUGAGGCCGAAAGGCCGAA ACCGUAUU 2300 AAUACGGUC CGAAACGU 2301540 AACAGACC CUGAUGAGGCCGAAAGGCCGAA ACGUUUCG 2302 CGAAACGUU GGUCUGUU 2303544 CAAUAACA CUGAUGAGGCCGAAAGGCCGAA ACCAACGU 2304 ACGUUGGUC UGUUAUUG 2305548 UUGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACAGACCA 2306 UGGUCUGUU AUUGCCAA 2307549 CUUGGCAA CUGAUGAGGCCGAAAGGCCGAA AACAGACC 2308 GGUCUGUUA UUGCCAAG 2309551 UGCUUGGC CUGAUGAGGCCGAAAGGCCGAA AUAACAGA 2310 UCUGUUAUU GCCAAGCA 2311562 UCCCCUUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUUG 2312 CAAGCACUU AAAGGGGA 2313563 CUCCCCUU CUGAUGAGGCCGAAAGGCCGAA AAGUGCUU 2314 AAGCACUUA AAGGGGAG 2315575 UGUUUUCC CUGAUGAGGCCGAAAGGCCGAA AUUCUCCC 2316 GGGAGAAUU GGAAAACA 2317588 CCUCUCCC CUGAUGAGGCCGAAAGGCCGAA ACAUUGUU 2318 AACAAUGUA GGGAGAGG 2319603 CAAGUGGU CUGAUGAGGCCGAAAGGCCGAA AUGCCACC 2320 GGUGGCAUA ACCACUUG 2321610 CUGGAUUC CUGAUGAGGCCGAAAGGCCGAA AGUGGUUA 2322 UAACCACUU GAAUCCAG 2323615 AACUUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCAAGU 2324 ACUUGAAUC CAGAAGUU 2325623 GUUUUCUU CUGAUGAGGCCGAAAGGCCGAA ACUUCUGG 2326 CCAGAAGUU AAGAAAAC 2327624 GGUUUUCU CUGAUGAGGCCGAAAGGCCGAA AACUUCUG 2328 CAGAAGUUA AGAAAACC 2329634 CUGUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUUUUC 2330 GAAAACCUC CUGGACAG 2331659 UGGUAAAU CUGAUGAGGCCGAAAGGCCGAA AUUCUGUC 2332 GACAGAAUU AUUUACCA 2333660 CUGGUAAA CUGAUGAGGCCGAAAGGCCGAA AAUUCUGU 2334 ACAGAAUUA UUUACCAG 2335662 GCCUGGUA CUGAUGAGGCCGAAAGGCCGAA AUAAUUCU 2336 AGAAUUAUU UACCAGGC 2337663 UGCCUGGU CUGAUGAGGCCGAAAGGCCGAA AAUAAUUC 2338 GAAUUAUUU ACCAGGCA 2339664 GUGCCUGG CUGAUGAGGCCGAAAGGCCGAA AAAUAAUU 2340 AAUUAUUUA CCAGGCAC 2341704 AGCUUUGC CUGAUGAGGCCGAAAGGCCGAA AUUUCCGC 2342 GCGGAAAUC GCAAAGCU 2343713 CCAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUUUGC 2344 GCAAAGCUA CUGCCUGG 2345__________________________________________________________________________ TABLE XIX__________________________________________________________________________Porcine c-myb (region B) Hammerhead Ribozyme and TargetSequence (30 bp; nt. 1386 start; Human numbering system) (REVISED)Posi- Seq. Seq.tion Ribozyme ID No. Substrate ID__________________________________________________________________________ No.1394 GUGUUUAA CUGAUGAGGCCGAAAGGCCGAA AAAGAAUC 2346 GAUUCUUUC UUAAACAC 23471396 AAGUGUUU CUGAUGAGGCCGAAAGGCCGAA AGAAAGAA 2348 UUCUUUCUU AAACACUU 23491397 GAAGUGUU CUGAUGAGGCCGAAAGGCCGAA AAGAAAGA 2350 UCUUUCUUA AACACUUC 23511404 GUUAUUGG CUGAUGAGGCCGAAAGGCCGAA AGUGUUUA 2352 UAAACACUU CCAAUAAC 23531405 GGUUAUUG CUGAUGAGGCCGAAAGGCCGAA AAGUGUUU 2354 AAACACUUC CAAUAACC 23551410 UUCAUGGU CUGAUGAGGCCGAAAGGCCGAA AUUGGAAG 2356 CUUCCAAUA ACCAUGAA 23571423 CCAAGUCU CUGAUGAGGCCGAAAGGCCGAA AGUUUUCA 2358 UGAAAACUU AGACUUGG 23591424 UCCAAGUC CUGAUGAGGCCGAAAGGCCGAA AAGUUUUC 2360 GAAAACUUA GACUUGGA 23611429 GCAUUUCC CUGAUGAGGCCGAAAGGCCGAA AGUCUAAG 2362 CUUAGACUU GGAAAUGC 23631440 CGUUAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCAUUU 2364 AAAUGCCUU CUUUAACG 23651441 ACGUUAAA CUGAUGAGGCCGAAAGGCCGAA AAGGCAUU 2366 AAUGCCUUC UUUAACGU 23671443 GGACGUUA CUGAUGAGGCCGAAAGGCCGAA AGAAGGCA 2368 UGCCUUCUU UAACGUCC 23691444 UGGACGUU CUGAUGAGGCCGAAAGGCCGAA AAGAAGGC 2370 GCCUUCUUU AACGUCCA 23711445 GUGGACGU CUGAUGAGGCCGAAAGGCCGAA AAAGAAGG 2372 CCUUCUUUA ACGUCCAC 23731450 GAGGCGUG CUGAUGAGGCCGAAAGGCCGAA ACGUUAAA 2374 UUUAACGUC CACGCCUC 23751458 ACCACUGA CUGAUGAGGCCGAAAGGCCGAA AGGCGUGG 2376 CCACGCCUC UCAGUGGU 23771460 UGACCACU CUGAUGAGGCCGAAAGGCCGAA AGAGGCGU 2378 ACGCCUCUC AGUGGUCA 23791467 CAAUUUGU CUGAUGAGGCCGAAAGGCCGAA ACCACUGA 2380 UCAGUGGUC ACAAAUUG 23811474 UAACAGUC CUGAUGAGGCCGAAAGGCCGAA AUUUGUGA 2382 UCACAAAUU GACUGUUA 23831481 GGUGUUGU CUGAUGAGGCCGAAAGGCCGAA ACAGUCAA 2384 UUGACUGUU ACAACACC 23851482 UGGUGUUG CUGAUGAGGCCGAAAGGCCGAA AACAGUCA 2386 UGACUGUUA CAACACCA 23871492 CUCUAUGA CUGAUGAGGCCGAAAGGCCGAA AUGGUGUU 2388 AACACCAUU UCAUAGAG 23891493 UCUCUAUG CUGAUGAGGCCGAAAGGCCGAA AAUGGUGU 2390 ACACCAUUU CAUAGAGA 23911494 GUCUCUAU CUGAUGAGGCCGAAAGGCCGAA AAAUGGUG 2392 CACCAUUUC AUAGAGAC 23931497 CUGGUCUC CUGAUGAGGCCGAAAGGCCGAA AUGAAAUG 2394 CAUUUCAUA GAGACCAG 23951530 AAAAUAUG CUGAUGAGGCCGAAAGGCCGAA AUUUUCCU 2396 AGGAAAAUA CAUAUUUU 23971534 UUCAAAAA CUGAUGAGGCCGAAAGGCCGAA AUGUAUUU 2398 AAAUACAUA UUUUUGAA 23991536 AGUUCAAA CUGAUGAGGCCGAAAGGCCGAA AUAUGUAU 2400 AUACAUAUU UUUGAACU 24011537 GAGUUCAA CUGAUGAGGCCGAAAGGCCGAA AAUAUGUA 2402 UACAUAUUU UUGAACUC 24031538 GGAGUUCA CUGAUGAGGCCGAAAGGCCGAA AAAUAUGU 2404 ACAUAUUUU UGAACUCC 24051539 CGGAGUUC CUGAUGAGGCCGAAAGGCCGAA AAAAUAUG 2406 CAUAUUUUU GAACUCCG 24071545 GAUAGCCG CUGAUGAGGCCGAAAGGCCGAA AGUUCAAA 2408 UUUGAACUC CGGCUAUC 24091551 CCUUUUGA CUGAUGAGGCCGAAAGGCCGAA AGCCGGAG 2410 CUCCGGCUA UCAAAAGG 24111553 GACCUUUU CUGAUGAGGCCGAAAGGCCGAA AUAGCCGG 2412 CCGGCUAUC AAAAGGUC 24131561 CCAGGAUU CUGAUGAGGCCGAAAGGCCGAA ACCUUUUG 2414 CAAAAGGUC AAUCCUGG 24151565 CUUUCCAG CUGAUGAGGCCGAAAGGCCGAA AUUGACCU 2416 AGGUCAAUC CUGGAAAG 24171576 UUCUUGGA CUGAUGAGGCCGAAAGGCCGAA AGCUUUCC 2418 GGAAAGCUC UCCAAGAA 24191578 AGUUCUUG CUGAUGAGGCCGAAAGGCCGAA AGAGCUUU 2420 AAAGCUCUC CAAGAACU 24211587 CGGUGUAG CUGAUGAGGCCGAAAGGCCGAA AGUUCUUG 2422 CAAGAACUC CUACACCG 24231590 GAACGGUG CUGAUGAGGCCGAAAGGCCGAA AGGAGUUC 2424 GAACUCCUA CACCGUUC 24251597 CAUGUUUG CUGAUGAGGCCGAAAGGCCGAA ACGGUGUA 2426 UACACCGUU CAAACAUG 24271598 GCAUGUUU CUGAUGAGGCCGAAAGGCCGAA AACGGUGU 2428 ACACCGUUC AAACAUGC 24291610 UGAGCUGC CUGAUGAGGCCGAAAGGCCGAA AGUGCAUG 2430 CAUGCACUC GCAGCUCA 24311617 AAUUUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUGCGA 2432 UCGCAGCUC AAGAAAUU 24331625 CCAUAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUCUUG 2434 CAAGAAAUU AAAUAUGG 24351626 ACCAUAUU CUGAUGAGGCCGAAAGGCCGAA AAUUUCUU 2436 AAGAAAUUA AAUAUGGU 24371630 GGGGACCA CUGAUGAGGCCGAAAGGCCGAA AUUUAAUU 2438 AAUUAAAUA UGGUCCCC 24391635 CUUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACCAUAUU 2440 AAUAUGGUC CCCUGAAG 24411649 GUCUGAGG CUGAUGAGGCCGAAAGGCCGAA AGCAUCUU 2442 AAGAUGCUA CCUCAGAC 24431653 UGGUGUCU CUGAUGAGGCCGAAAGGCCGAA AGGUAGCA 2444 UGCUACCUC AGACACCA 24451663 CUAAAUGA CUGAUGAGGCCGAAAGGCCGAA AUGGUGUC 2446 GACACCAUC UCAUUUAG 24471665 UACUAAAU CUGAUGAGGCCGAAAGGCCGAA AGAUGGUG 2448 CACCAUCUC AUUUAGUA 24491668 UUCUACUA CUGAUGAGGCCGAAAGGCCGAA AUGAGAUG 2450 CAUCUCAUU UAGUAGAA 24511669 CUUCUACU CUGAUGAGGCCGAAAGGCCGAA AAUGAGAU 2452 AUCUCAUUU AGUAGAAG 24531670 UCUUCUAC CUGAUGAGGCCGAAAGGCCGAA AAAUGAGA 2454 UCUCAUUUA GUAGAAGA 24551673 AGGUCUUC CUGAUGAGGCCGAAAGGCCGAA ACUAAAUG 2456 CAUUUAGUA GAAGACCU 2457__________________________________________________________________________ TABLE XX__________________________________________________________________________Porcine c-myb (region B) Hairpin Ribozyme and Target Sequence (308 bp;nt. 1386 start; Humannumbering system) (REVISED) Seq. Seq.Posi- ID IDtion Hairpin Ribozyme No. Substrate No.__________________________________________________________________________1504 UUUUCACA AGAA GGUCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2458 GAGACCA GAC 2459AAAA1594 CAUGUUUG AGAA GUGUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2460 CUACACC GUU 2461CAUG1613 AUUUCUUG AGAA GCGAGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2462 ACUCGCA GCU 2463AAAU__________________________________________________________________________ TABLE XXI__________________________________________________________________________Porcine c-myb (region A) Hairpin Ribozyme and Target Sequence (266 bp;nt. 458 start; Humannumbering system) (REVISED) Seq. Seq.Posi- ID IDtion RZ No. Substrate No.__________________________________________________________________________528 ACGUUUCG AGAA GUAUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2464 AAAUACG GUC 2465ACGU690 UUCCGCCC AGAA GUUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2466 GGGAACA GAU 2467GGAA__________________________________________________________________________ TABLE XXII__________________________________________________________________________Rat c-myb (Region A) Hammerhead Ribozyme and TargetSequence (282 bp; nt. 428 start; Human numbering system) (REVISED)Position HH Ribozyme Seq. ID No. Substrate Seq. ID__________________________________________________________________________ No.467 CCUUUGAU CUGAUGAGGCCGAAAGGCCGAA AGCUCAGG 2468 CCUGAGCUC AUCAAAGG 2469470 GGACCUUU CUGAUGAGGCCGAAAGGCCGAA AUGAGCUC 2470 GAGCUCAUC AAAGGUCC 2471477 GGUCCAGG CUGAUGAGGCCGAAAGGCCGAA ACCUUUGA 2472 UCAAAGGUC CCUGGACC 2473498 CACUCUUU CUGAUGAGGCCGAAAGGCCGAA AUCUUCUU 2474 AAGAAGAUC AAAGAGUG 2475509 ACAAGCUC CUGAUGAGGCCGAAAGGCCGAA AUCACUCU 2476 AGAGUGAUA GAGCUUGU 2477515 UUCUGGAC CUGAUGAGGCCGAAAGGCCGAA AGCUCUAU 2478 AUAGAGCUU GUCCAGAA 2479518 UAUUUCUG CUGAUGAGGCCGAAAGGCCGAA ACAAGCUC 2480 GAGCUUGUC CAGAAAUA 2481526 UCGGACCG CUGAUGAGGCCGAAAGGCCGAA AUUUCUGG 2482 CCAGAAAUA CGGUCCGA 2483531 GCGCUUCG CUGAUGAGGCCGAAAGGCCGAA ACCGUAUU 2484 AAUACGGUC CGAAGCGC 2485544 CAAUAACA CUGAUGAGGCCGAAAGGCCGAA ACCAGCGC 2486 GCGCUGGUC UGUUAUUG 2487548 UUGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACAGACCA 2488 UGGUCUGUU AUUGCCAA 2489549 CUUGGCAA CUGAUGAGGCCGAAAGGCCGAA AACAGACC 2490 GGUCUGUUA UUGCCAAG 2491551 UGCUUGGC CUGAUGAGGCCGAAAGGCCGAA AUAACAGA 2492 UCUGUUAUU GCCAAGCA 2493562 UCCCUUUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUUG 2494 CAAGCACUU AAAAGGGA 2495563 CUCCCUUU CUGAUGAGGCCGAAAGGCCGAA AAGUGCUU 2496 AAGCACUUA AAAGGGAG 2497575 UGUUUUCC CUGAUGAGGCCGAAAGGCCGAA AUUCUCCC 2498 GGGAGAAUU GGAAAACA 2499588 CCUCUCCC CUGAUGAGGCCGAAAGGCCGAA ACAUUGUU 2500 AACAAUGUC GGGAGAGG 2501609 UGGAUUCA CUGAUGAGGCCGAAAGGCCGAA AUGGUUGU 2502 ACAACCAUU UGAAUCCA 2503610 CUGGAUUC CUGAUGAGGCCGAAAGGCCGAA AAUGGUUG 2504 CAACCAUUU GAAUCCAG 2505615 AACUUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCAAAU 2506 AUUUGAAUC CAGAAGUU 2507623 GUUUUCUU CUGAUGAGGCCGAAAGGCCGAA ACUUCUGG 2508 CCAGAAGUU AAGAAAAC 2509624 GGUUUUCU CUGAUGAGGCCGAAAGGCCGAA AACUUCUG 2510 CAGAAGUUA AGAAAACC 2511634 CUGUCCAU CUGAUGAGGCCGAAAGGCCGAA AGGUUUUC 2512 GAAAACCUC AUGGACAG 2513659 UGAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUUCUGUC 2514 GACAGAAUC AUUUAUCA 2515662 GCCUGAUA CUGAUGAGGCCGAAAGGCCGAA AUGAUUCU 2516 AGAAUCAUU UAUCAGGC 2517663 UGCCUGAU CUGAUGAGGCCGAAAGGCCGAA AAUGAUUC 2518 GAAUCAUUU AUCAGGCA 2519664 GUGCCUGA CUGAUGAGGCCGAAAGGCCGAA AAAUGAUU 2520 AAUCAUUUA UCAGGCAC 2521666 GUGUGCCU CUGAUGAGGCCGAAAGGCCGAA AUAAAUGA 2522 UCAUUUAUC AGGCACAC 2523__________________________________________________________________________ TABLE XXIII__________________________________________________________________________Rat c-myb (Region B) Hammerhead Ribozyme and TargetSequences (262 bp; nt. 1421 start; human numbering system) (REVISED)Posi- Seq. Seq.tion Ribozyme ID No. Substrate ID No.__________________________________________________________________________1429 GCGUAUCU CUGAUGAGGCCGAAAGGCCGAA AGCCCGAG 2524 CUCGGGCUU AGAUACGC 25251430 GGCGUAUC CUGAUGAGGCCGAAAGGCCGAA AAGCCCGA 2526 UCGGGCUUA GAUACGCC 25271434 AGUAGGCG CUGAUGAGGCCGAAAGGCCGAA AUCUAAGC 2528 GCUUAGAUA CGCCUACU 25291440 GGGUAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCGUAU 2530 AUACGCCUA CUUUACCC 25311443 GGAGGGUA CUGAUGAGGCCGAAAGGCCGAA AGUAGGCG 2532 CGCCUACUU UACCCUCC 25331444 UGGAGGGU CUGAUGAGGCCGAAAGGCCGAA AAGUAGGC 2534 GCCUACUUU ACCCUCCA 25351445 GUGGAGGG CUGAUGAGGCCGAAAGGCCGAA AAAGUAGG 2536 CCUACUUUA CCCUCCAC 25371450 GAGGCGUG CUGAUGAGGCCGAAAGGCCGAA AGGGUAAA 2538 UUUACCCUC CACGCCUC 25391458 ACCAAUGA CUGAUGAGGCCGAAAGGCCGAA AGGCGUGG 2540 CCACGCCUC UCAUUGGU 25411460 UGACCAAU CUGAUGAGGCCGAAAGGCCGAA AGAGGCGU 2542 ACGCCUCUC AUUGGUCA 25431463 UUGUGACC CUGAUGAGGCCGAAAGGCCGAA AUGAGAGG 2544 CCUCUCAUU GGUCACAA 25451467 CAGUUUGU CUGAUGAGGCCGAAAGGCCGAA ACCAAUGA 2546 UCAUUGGUC ACAAACUG 25471485 GUCUCGGU CUGAUGAGGCCGAAAGGCCGAA ACACGGUG 2548 CACCGUGUC ACCGAGAC 25491509 UUCCUUUU CUGAUGAGGCCGAAAGGCCGAA AGUUUUCA 2550 UGAAAACUN AAAAGGAA 25511522 UAAAGAUN CUGAUGAGGCCGAAAGGCCGAA AGUUUUCC 2552 GGAAAACUC NAUCUUUA 25531526 GUUCUAAA CUGAUGAGGCCGAAAGGCCGAA AUNGAGUU 2554 AACUCNAUC UUUAGAAC 25551528 GAGUUCUA CUGAUGAGGCCGAAAGGCCGAA AGAUNGAG 2556 CUCNAUCUU UAGAACUC 25571529 GGAGUUCU CUGAUGAGGCCGAAAGGCCGAA AAGAUNGA 2558 UCNAUCUUU AGAACUCC 25591530 UGGAGUUC CUGAUGAGGCCGAAAGGCCGAA AAAGAUNG 2560 CNAUCUUUA GAACUCCA 25611536 GAUAGCUG CUGAUGAGGCCGAAAGGCCGAA AGUUCUAA 2562 UUAGAACUC CAGCUAUC 25631542 CCUUUUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGGAG 2564 CUCCAGCUA UCAAAAGG 25651544 NACCUUUU CUGAUGAGGCCGAAAGGCCGAA AUAGCUGG 2566 CCAGCUAUC AAAAGGUN 25671552 CGAGGAUU CUGAUGAGGCCGAAAGGCCGAA ACCUUUUG 2568 CAAAAGGUN AAUCCUCG 25691556 CUUUCGAG CUGAUGAGGCCGAAAGGCCGAA AUUNACCU 2570 AGGUNAAUC CUCGAAAG 25711559 GAGCUUUC CUGAUGAGGCCGAAAGGCCGAA AGGAUUNA 2572 UNAAUCCUC GAAAGCUC 25731567 UUCUGGGA CUGAUGAGGCCGAAAGGCCGAA AGCUUUCG 2574 CGAAAGCUC UCCCAGAA 25751569 AGUUCUGG CUGAUGAGGCCGAAAGGCCGAA AGAGCUUU 2576 AAAGCUCUC CCAGAACU 25771578 UGGUGUGG CUGAUGAGGCCGAAAGGCCGAA AGUUCUGG 2578 CCAGAACUC CCACACCA 25791588 CAUGUUUG CUGAUGAGGCCGAAAGGCCGAA AUGGUGUG 2580 CACACCAUU CAAACAUG 25811589 GCAUGUUU CUGAUGAGGCCGAAAGGCCGAA AAUGGUGU 2582 ACACCAUUC AAACAUGC 25831608 AAUUUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUGCCA 2584 UGGCAGCUC AAGAAAUU 25851616 CCGUAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUCUUG 2586 CAAGAAAUU AAAUACGG 25871617 ACCGUAUU CUGAUGAGGCCGAAAGGCCGAA AAUUUCUU 2588 AAGAAAUUA AAUACGGU 25891621 GGGGACCG CUGAUGAGGCCGAAAGGCCGAA AUUUAAUU 2590 AAUUAAAUA CGGUCCCC 25911626 CUUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACCGUAUU 2592 AAUACGGUC CCCUGAAG 25931640 GUCUNAGG CUGAUGAGGCCGAAAGGCCGAA AGCAUCUU 2594 AAGAUGCUA CCUNAGAC 25951644 GGGGGUCU CUGAUGAGGCCGAAAGGCCGAA AGGUAGCA 2596 UGCUACCUN AGACCCCC 25971654 CUACAUNA CUGAUGAGGCCGAAAGGCCGAA AGGGGGUC 2598 GACCCCCUN UNAUGUAG 25991656 NACUACAU CUGAUGAGGCCGAAAGGCCGAA ANAGGGGG 2600 CCCCCUNUN AUGUAGUN 26011661 UNUNNNAC CUGAUGAGGCCGAAAGGCCGAA ACAUNANA 2602 UNUNAUGUA GUNNNANA 26031664 AGGUNUNN CUGAUGAGGCCGAAAGGCCGAA ACUACAUN 2604 NAUGUAGUN NNANACCU 26051673 ACAUCNUG CUGAUGAGGCCGAAAGGCCGAA AGGUNUNN 2606 NNANACCUN CANGAUGU 2607__________________________________________________________________________ TABLE XXIV__________________________________________________________________________Rat c-mby (Region A) Hairpin Ribozyme and Target Sequences (282 bp; nt.428 start;human numbering system)(REVISED) Seq. Seq.Posi- ID IDtion RZ No. Substrate No.__________________________________________________________________________528 GCGCUUCG AGAA GUAUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2608 AAAUACG GUC 2609GCGC690 UUCUGCCC AGAA GUUUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2610 GGAAACA GAU 2611AGAA__________________________________________________________________________ TABLE XXV__________________________________________________________________________Rat c-mby (Region B) Hairpin Ribozyme and Target Sequences (262 bp; nt.1421 start;human numbering system)(REVISED) Seq. Seq.Posi- ID IDtion RZ No. Substrate No.__________________________________________________________________________1495 UUUUCACA AGAA GGUCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2612 GAGACCA GAC 2613AAAA1604 AUUUCUUG AGAA GCCAGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2614 CCUGGCA GCU 2615AAAU1623 CUUCAGGG AGAA GUAUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 2616 AAAUACG GUC 2617GAAG__________________________________________________________________________

Claims

1. An enzymatic RNA molecule which specifically cleaves c-myb RNA.

2. An enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ. ID. NOS. 1-78, wherein said enzymatic RNA molecule is in a hepatitis delta virus motif.

3. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ. ID. NOS. 2, 4-6, 9-16, 18-20, 22-45, 47-98, wherein said enzymatic RNA molecule is in a hammerhead motif.

4. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule is in a hammerhead motif.

5. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule is in a hairpin, hepatitis delta virus, VS nucleic acid, group I intron, or RNAseP nucleic acid motif.

6. The enzymatic RNA molecule of claim 5, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ. ID. NOS. 99, 100 or 130-148, wherein said enzymatic RNA molecule is in a hairpin motif.

7. The enzymatic RNA molecule of claim 5 consisting of any sequence selected from the group of SEQ. ID NOS. 121, 122, 149-167.

8. The enzymatic RNA molecule of any of claims 1, 2, 3, 4, 5, 6 or 7, wherein said enzymatic RNA molecule comprises between 12 and 100 bases complementary to said mRNA.

9. The enzymatic RNA molecule of any of claims 1, 2, 3, 4, 5, 6 or 7, wherein said enzymatic RNA molecule comprises between 14 and 24 bases complementary to said mRNA.

10. An enzymatic RNA molecule consisting of any sequence selected from the group of SEQ. ID NOS. 101-120, 123-129, 149-167, and odd numbers from 171-1225.

11. The enzymatic RNA molecule of any of claims 3 or 4 or 10, wherein said enzymatic RNA molecule comprises at least five ribose residues, and wherein said enzymatic RNA molecule comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said enzymatic RNA molecule comprises a 2'-C-allyl modification at position No. 4 of said enzymatic RNA molecule, and wherein said enzymatic RNA molecule comprises at least ten 2'-O-methyl modifications.

12. The enzymatic RNA molecule of any of claims 3 or 4 or 10, wherein said enzymatic RNA molecule comprises at least five ribose residues, and wherein said enzymatic RNA molecule comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said enzymatic RNA molecule comprises a 2'-amino modification at position No. 4 and/or at position No. 7 of said nucleic acid, wherein said enzymatic RNA molecule comprises at least ten 2'-O-methyl modifications.

13. The enzymatic RNA molecule of any of claims 3 or 4 or 10, wherein said enzymatic RNA molecule comprises at least five ribose residues, and wherein said enzymatic RNA molecule comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said enzymatic RNA molecule comprises abasic substitution at position No. 4 and/or at position No. 7 of said enzymatic RNA molecule, wherein said enzymatic RNA molecule comprises at least ten 2'-O-methyl modifications.

14. The enzymatic RNA molecule of any of claims 3 or 4 or 10, wherein said enzymatic RNA molecule comprises at least five ribose residues, and wherein said enzymatic RNA molecule comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said enzymatic RNA molecule comprises 6-methyl uridine substitutions at position No. 4 and/or at position No. 7 of the said nucleic acid molecule, wherein said enzymatic RNA molecule comprises at least ten 2'-O-methyl modifications.

15. The enzymatic RNA molecule of any of claims 3, 4 or 10, wherein said nucleic acid molecule comprises a stem II region of length greater than or equal to two base-pairs.

16. The enzymatic RNA molecule in a hairpin motif of any of claims 5, 6 or 7, wherein said enzymatic RNA molecule comprises a stem IV region of length greater than or equal to two base-pairs.

17. The enzymatic RNA molecule of any of claims 5, 6 or 7, wherein said enzymatic RNA molecule comprises a stem II region of length between three and seven base-pairs.

18. The enzymatic nucleic acid of any one of claims 1, 2, 3, 4, 5, 6 or 7, wherein said nucleic acid comprises a 3'-3' linked inverted ribose moeity at the 3' end of said nucleic acid molecule.

19. The enzymatic RNA molecule of any one of claims 1, 2, 3, 4, 5, 6 or 7, wherein said enzymatic RNA molecule comprises a 3'-3' linked inverted thymidine moeity at the 3' end of said nucleic acid molecule.

20. The enzymatic RNA molecule of any one of claims 1, 2, 3, 4, 5, 6 or 7, wherein said enzymatic RNA molecule comprises a 2'-3' linked inverted ribose or thymidine moeity at the 3' end of said enzymatic RNA molecule.

21. The enzymatic RNA molecule of any one of claims 1, 2, 3, 4, 5, 6 or 7, wherein said enzymatic RNA molecule comprises a 5'-end modification.

22. The enzymatic RNA molecule of claim 11, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted ribose moeity at the 3' end of said enzymatic RNA molecule.

23. The enzymatic RNA molecule of any one of claim 11, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted thymidine moeity at the 3' end of said molecule.

24. The enzymatic RNA molecule of claim 11, wherein said enzymatic RNA molecule further comprises a 2'-3' linked inverted ribose or thymidine moeity at the 3' end of said enzymatic RNA molecule.

25. The enzymatic RNA molecule of claim 11, wherein said enzymatic RNA molecule further comprises a 5'-end modification.

26. The enzymatic RNA molecule of claim 12, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted ribose moeity at the 3' end of said enzymatic RNA molecule.

27. The enzymatic RNA molecule of claim 12, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted thymidine moeity at the 3' end of said enzymatic RNA molecule.

28. The enzymatic RNA molecule of claim 12, wherein said enzymatic RNA molecule further comprises a 2'-3' linked inverted ribose or thymidine moeity at the 3' end of said enzymatic RNA molecule.

29. The enzymatic RNA molecule of claim 12, wherein said enzymatic RNA molecule further comprises a 5'-end modification.

30. The enzymatic RNA molecule of claim 13, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted ribose moeity at the 3' end of said enzymatic RNA molecule.

31. The enzymatic RNA molecule of any one of claim 13, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted thymidine moeity at the 3' end of said enzymatic RNA molecule.

32. The enzymatic RNA molecule of claim 13, wherein said enzymatic RNA molecule further comprises a 2'-3' linked inverted ribose or thymidine moeity at the 3' end of said enzymatic RNA molecule.

33. The enzymatic RNA molecule of claim 13, wherein said enzymatic RNA molecule further comprises a 5'-end modification.

34. The enzymatic RNA molecule of claim 14, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted ribose moeity at the 3' end of said enzymatic RNA molecule.

35. The enzymatic RNA molecule of any one of claim 14, wherein said enzymatic RNA molecule further comprises a 3'-3' linked inverted thymidine moeity at the 3' end of said enzymatic RNA molecule.

36. The enzymatic RNA molecule of claim 14, wherein said enzymatic RNA molecule further comprises a 2'-3' linked inverted ribose or thymidine moeity at the 3' end of said enzymatic RNA molecule.

37. The enzymatic RNA molecule of claim 14, wherein said enzymatic RNA molecule further comprises a 5'-end modification.

38. The enzymatic RNA molecule of claim 4, wherein the enzymatic RNA molecule molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 232.

39. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 264.

40. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 266.

41. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 272.

42. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 282.

43. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 300.

44. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 320.

45. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 350.

46. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 374.

47. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 384.

48. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 442.

49. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 522.

50. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 538.

51. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 540.

52. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 93.

53. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 572.

54. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 95.

55. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 614.

56. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 622.

57. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 98.

58. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 2 or 4.

59. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 5 or 6.

60. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 9-11.

61. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 13-16.

62. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 18-20.

63. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 22-25.

64. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 26-29.

65. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 30-33.

66. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 34-37.

67. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 38-41.

68. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 42-45.

69. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 47-50.

70. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 51-54.

71. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 55-58.

72. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 59-62.

73. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 63-66.

74. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 67-70.

75. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 71-74.

76. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 75-78.

77. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 99.

78. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NOS. 100.

79. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 130-133.

80. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 134-137.

81. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 138.

82. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 139-141.

83. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 142-145.

84. The enzymatic RNA molecule of claim 6, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 146-148.

85. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 170, 172, 174, or 176.

86. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 178, 180, 182, or 184.

87. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 186, 188, 190, or 192.

88. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 194, 196, 198, or 200.

89. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 202, 204, 206, or 208.

90. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 210, 212, 214, or 216.

91. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 218, 220, 222, or 224.

92. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 226, 228, or 230.

93. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 234, 236, 238, or 240.

94. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NO. 242, 244, 246, or 248.

95. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 250, 252, 254, or 256.

96. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 258, 260, or 262.

97. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 268 or 270.

98. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 274, 276, 278, or 280.

99. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 284, 286, or 288.

100. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 290, 292, 294, or 296.

101. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 298, 302, or 304.

102. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 306, 308, 310, or 312.

103. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 314, 316, or 318.

104. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 322, 324, 326, or 328.

105. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 330, 332, 334, or 336.

106. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 338, 340, 342, or 344.

107. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 346, 348, or 352.

108. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 354, 356, 358, or 360.

109. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 362, 364, 366, or 368.

110. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 370, 372, or 376.

111. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 378, 380, or 382.

112. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 386, 388, 390, or 392.

113. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 394, 396, 398, or 400.

114. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 402, 404, 406, or 408.

115. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 410, 412, 414, or 416.

116. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 418, 420, 422, or 424.

117. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 426, 428, 430, or 432.

118. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 434, 436, 438, or 440.

119. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 444, 446, or 448.

120. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 450, 452, 454, or 456.

121. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 458, 460, 462, or 464.

122. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 466, 468, 470, or 472.

123. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 474, 476, 478, or 480.

124. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 482, 484, 486, or 488.

125. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 490, 492, 494, or 496.

126. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 498, 500, 502, or 504.

127. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 506, 508, 510, or 512.

128. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 514, 516, 518, or 520.

129. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 524, 526, or 528.

130. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 530, 532, 534, or 536.

131. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 540, 542, or 544.

132. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 546, 548, 550, or 552.

133. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 554, 556, 558, or 560.

134. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 562, 564, 566, or 568.

135. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 570, 574, or 576.

136. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 578, 580, 582, or 584.

137. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 586, 588, 590, or 592.

138. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 594, 596, 598, or 600.

139. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 602, 604, 606, or 608.

140. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 610, 612, or 616.

141. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 618, 620, or 624.

142. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 626, 628, 630, or 632.

143. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 634, 638, 640, or 642.

144. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 644, 646, 648, or 670.

145. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 672, 674, 676, or 678.

146. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 680, 682, 684, or 686.

147. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 688, 690, 692, or 694.

148. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 696, 698, 700, or 702.

149. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 704, 706, 708, or 710.

150. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 712, 714, 716, or 718.

151. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 720, 722, 724, or 726.

152. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 728, 730, 732, or 734.

153. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 736, 738, 740, or 742.

154. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 744, 746, 748, or 750.

155. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 752, 754, 756, or 758.

156. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 760, 762, 764, or 766.

157. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 778, 780, 782, or 784.

158. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 786, 788, 790, or 792.

159. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 794, 796, 798, or 800.

160. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 802, 804, 806, or 808.

161. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 810, 812, 814, or 816.

162. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 818, 820, 822, or 824.

163. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 826, 828, 830, or 832.

164. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 834, 836, 838, or 840.

165. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 842, 844, 846, or 848.

166. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 850, 852, 854, or 856.

167. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 858, 860, 862, or 864.

168. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 866, 868, 870, or 872.

169. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 874, 876, 878, or 880.

170. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 882, 884, 886, or 888.

171. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 890, 892, 894, or 896.

172. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 898, 900, 902, or 904.

173. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 906, 908, 910, or 912.

174. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 914, 916, 918, or 920.

175. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 922, 924, 926, or 928.

176. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 930, 932, 934, or 936.

177. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 938, 940, 942, or 944.

178. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 946, 948, 950, or 952.

179. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 954, 956, 958, or 960.

180. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 962, 964, 966, or 968.

181. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 970, 972, 974, or 976.

182. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 978, 980, 982, or 984.

183. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 986, 988, 990, or 992.

184. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 994, 996, 998, or 1000.

185. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1002, 1004, 1006, 1008, or 1010.

186. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1012, 1014, 1016, or 1018.

187. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1020, 1022, 1024, or 1026.

188. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1028, 1030, 1032, or 1034.

189. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1036, 1038, 1040, or 1042.

190. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1042, 1044, 1046, or 1048.

191. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1050, 1052, 1054, or 1056.

192. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1058, 1060, 1062, or 1064.

193. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1066, 1068, 1070, or 1072.

194. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1074, 1076, 1078, or 1080.

195. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1082, 1084, 1086, or 1088.

196. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1090, 1092, 1094, or 1096.

197. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1098, 1100, 1102, or 1104.

198. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1106, 1108, 1110, or 1112.

199. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1114, 1116, 1118, or 1120.

200. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1122, 1124, 1126, or 1128.

201. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1130, 1132, 1134, or 1136.

202. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1138, 1140, 1142, or 1144.

203. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1146, 1148, 1150, or 1152.

204. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1154, 1156, 1158, or 1160.

205. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1162, 1164, 1166, or 1168.

206. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1170, 1172, 1174, or 1176.

207. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1178, 1180, 1182, or 1184.

208. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1186, 1188, 1190, or 1192.

209. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1194, 1196, 1198, or 1200.

210. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1202, 1204, 1206, or 1208.

211. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1210, 1212, 1214, or 1216.

212. The enzymatic RNA molecule of claim 4, wherein said enzymatic RNA molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1218, 1220, 1222, or 1224.

213. The enzymatic RNA molecule of claim 1, wherein the binding arms of said enzymatic RNA molecule contain sequences perfectly complementary to any of the RNA sequences defined as SEQ. ID. NOS. 1-98, 101-120, or 123-129, wherein said enzymatic RNA molecule is in a hammerhead motif.

214. The enzymatic RNA molecule of claim 1, wherein the the binding arms of said enzymatic RNA molecule contain sequences perfectly complementary to any of the RNA sequences defined as SEQ. ID. NOS. 1-8, wherein said enzymatic RNA molecule is in a hepatitis delta virus motif.

215. The enzymatic RNA molecule of claim 5, wherein the binding arms of said enzymatic RNA molecule contain sequences perfectly complementary to any of the RNA sequences defined as SEQ. ID. NOS. 99, 100, or 130-148, wherein said enzymatic RNA molecule is in a hairpin motif.

216. A mammalian cell including an enzymatic RNA molecule of any one of claims 1, 2, 3, 4, 5, 10, 6 or 7 in vitro.

217. The cell of claim 216, wherein said cell is a human cell.

218. An expression vector including a nucleic acid encoding an enzymatic RNA molecule or multiple enzymatic RNA molecules of claims 1, 2, 3, 4, 5, 10, 6 or 7 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

219. A mammalian cell including an expression vector of claim 218 in vitro.

220. The cell of claim 219, wherein said cell is a human cell.