AU6879501A - Methods and compositions for treatment of restenosis and cancer using ribozymes - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Ribozyme Phannaceuticals, Inc.

2950 Wilderness Place Boulder Colorado 80301 United States of America Dan T. Stinchcomb, Kenneth Draper, James McSwiggen, Thale Jarvis Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Methods and Compositions for Treatment of Restenosis and Cancer Using Ribozymes Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:p [P Australia Documents received on: S SP 7 SEP 21 z 5845c

DESCRIPTION

Methods and Compositions for Treatment of Restenosis and Cancer Using Ribozymes 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. 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 hyperproliferative response of these neointimal cells (Garratt, K. et al., 1991, J. Am. Coll. Cardio., 17, 442-428; Austin, G. 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, 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, et al., 1993, New Enql. J. Med., 328, 608- 613). In any case, slowing smocth muscle cell proliferation after angioplasty could prevent intimal thickening and restenosis.

The presently preferred therapeutic treatment for restenosis is the use of streptokinase, uro:kinase or other thrombolytic compounds, such as fish oil, anticoagulants, 15 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. et al., report that the current tnerapies have not significantly impacted the rates of restenosis occurrence. (Circulation, 84, 1426-1436, 1991).

Recently, :he 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, gpIIb/gpIIIa. After six months, patients with the treatment showed a 23% reduction in the occurrence of restenosis than patients receiving placebo (27 vs. p=0.001).

A number of growth factors have been shown to induce smooth muscle cell proliferation. In vitro, plateletderived growth factor (PDGF) is a potent smooth muscle cell mitogen (Ross, et al., 1974, Proc. Natl. Acad.

Sci. USA, 71, 1207-1210) and a smooth muscle cell chemoattractant (Grotendorst, 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. 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. et al., 1991, Science, 253, 1129-1132). Analysis of 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. 1989, Arteriosclerosis, 9, 269-278). In a rat model, anti-bFGF antibodies inhibit the prolifera- 15 tion 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, et al., 1991, Science, 251, 936-939.), insulin-like growth factor I (IGF-I) (Banskota, N. et al., 1989, Molec. Endocrinol., 3, 1183-1190) and endothelin (Komuro, 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-a) may indirectly affect smooth muscle cell proliferation by inducing the expression of PDGF (Hajjar, K. et al., 1987, J. Exp.

Med, 166, 235-245; Raines, E. 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 cmyb, are induced (Kindy, M. S. and Sonenshein, G. E., 1986, J. Biol. Chem., 261, 12865-12868; Brown, K. 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. et al., 1992, J. Biol. Chem., 267, 4625- 4630.). Thus, c-myb is required for the G, 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, 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, et al., 1993, Circulation, 88, 1190-5) and migration (Biro, et al., 1993, Proc. Natl. Acad. Sci. U S A, 654-8).

Ohno et al., 1994 Science 265, 781, have shown that a combination of viral thymidine kinase enzyme expression Sg.. ene erapy t: atment with anti-viral drug ganci- 15 clovir inhibits smoot:. muscle cell proliferation in pigs, following baloon angioplasty.

Epstein et al., "Inhibition of non-transformed cell proliferation using antisense oligonucleotides,"

NTIS

publication 19 discusses use of antisense oligonucleo- 20 ride: to c-my- PCNA or cyclin B. :-ng et al., PCT W091/15580, 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., W093/08845, Calabretta et al., W092/20348 and Gewirtz W093/09789 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. Patent 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, angiogenesis or growth factors for the purpose of revascularization..." Sullivan and Draper, International PCT publication WO 94/02595 describe the use of ribozymes against c-myb RNA to treat stenosis.

Summary Of The Invention This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species that are 20 required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by the oncogene, cmyb. Such ribozymes may be used to inhibit the hyperproliferation of smooth muscle cells in restenosis and of 25 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-KB, oct-i and SRF. NF-KB 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-KB. Upon phosphorylation of the I-KB molecule, the complex dissociates and NF-KB is released for transport to the nucleus, where it binds DNA and induces transcriptional activity in (NF-KB)-responsive genes. One of the (NF-KB)-responsive genes is the NF-KB gene itself. Thus, release of the NF-KB protein from the inhibitory complex results in a cascade of gene expression which is auto-induced. Early inhibition of NF-KB 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-i 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-i 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.

et al., 1994, J. Cell. Biochem., S18A, 282; Belknap, J. et al., 1994, J. Cell. Biochem., S18A, 277). Upon injury during angioplasty, the expression of oct-i 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 25 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 S. 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 complemen- 25 tarity 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 s: e of the characteristics of these ribozymes. In genera_, enzymatic nucleic acids act by first binding to a target RNA. Such oinding 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 basepairing, 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 S" 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.

20 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 treat- 25 ment 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. 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 Recroviruses 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 20 I intron by Cech et al., U.S. Patent 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 25 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 :han 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 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 Scanlon et 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kas:.ani-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 25 (Draper et al., PCT W093/23569, and Sullivan et al., PCT W094/02595, 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, 25856).

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. Ribozvmes 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 Sof the c-myb oncogene, particularly leukemias, neuro- Sblastomas, and lung, colon, and breast carcinomas. Using the methods described herein, other enzymatic RNA mole- 20 cules that cleave c-myb, c-myc, oct-1, SRF, NF-KB, PDGF receptor, bFGF receptor, angiotensin II, and endotheliumderived relaxing factor and thereby inhibit smooth muscle cell proliferation and/or tumor cell proliferation may be derived and used as described above. Specific examples 25 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 cmyb 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 explanted 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 viralbased technique used tc deliver a desired nucleic acid.

In preferred embodiments, the ribozymes have binding 25 arms which are complementary to the sequences in the tables II, XII-XXIV. Examples of such ribozymes are shown as Seq. I.D. Nos. 101-129 (table III) and in tables XII- XXIV. By complementary is thus meant that the binding arms are able to cause cleavage of a human or mouse or rat or porcine mRNA target. Examples of such ribozymes consist essentially of sequences defined in tables III, XII- XXIV. 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 c-myb 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 viral 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.

20 Drawings: Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be 2 2 base-pair long.

Figure 2a is a diagrammatic representation of the 25 hammerhead ribozyme domain known in the art; Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into Sa substrate and enzyme portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature 334, 585-591) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 20 bases, m is from 1 or more). Helix 2 and helix 5 may be covalently linked by one or rr.-e bases r is a 1 base). Helix 1, 4 or may also extended by 2 or more base pairs 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 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 o and p is each independently from 0 to any number, as ong as some base-pairing is maintained. Essential bass.. are shown as specific bases in the structure, but those -n the art will recognize that S: one or more may be modif: d chemically (abasic, base, sugar and/or phosphate moaifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, without a con- 20 necting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. is a 2 bases. The connecting "0 loop can also be replaced witr a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to S 25 pyrimidine bases. refers to a covalent bond.

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

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

Figure 6 is a schematic representation of an RNAseH accessibility assay. Specifically, the left side of Figure 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 Figure 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.

Figure 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.

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

Figure 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 ribozymeosubstrate complex. 2'-O-methyl ribo- .:00 20 zyme 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' 25 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 4 contains 2'-C-allyl substitution. Additionally, 3' end of this ribozyme contains a 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.

Figure 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.

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

Figure 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.

Figure 13 shows the effect of phosphorthioate substitutions on the catalytic activity of 2'-C-allyl 575 HH ribozyme. A) diagrammatic representation of 575 hammerhead ribozymeosubstrate 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 20 nucleotides at the 3' termini contain phosphorothioate substitutions. 5 P=S Loop ribozyme is similar to 2'-Callyl 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 phospnorothioate substitutions. Additionally, this ribozyme contains a linked inverted T at its 3' end. B) shows the ability of ribozymes described in Fig. 13A to inhibit smooth muscle cell proliferation.

Figure 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.

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

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

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

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

Figure 19 shows ribozyme-mediated inhibition of c-myb expression and cell proliferation.

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

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

0 Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al supra, Sullivan et al., supra, 20 as well as by Draper et al. "Method and reagent for treatment of arthritic conditions PCT No. PCT/US94/13129, U.S.S.N. 08/152,487, filed 11/12/93, and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, 25 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 XII-XXI (All sequences are 5' to 3' in the tables) The nucleocide b-se 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 ther. fter -signed, the human targeted sequences are of most utility. However, murine and pig targeted ribozymes may be useful to test efficacy c- action of m:e ribozyme prior to testing in humans. Tne nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

o* 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 20 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 25 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 and XII-XXIV. 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, XIII, XVI, XIX, XX, XXIII, XXIV (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 ribozyme sequences listed in Table III and XII- XXIV 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 Figure 2c), or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see 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 20 al., International Publication No. WO 93/15187; and Rossi 99' et al., International Publication No. WO 91/03162, as well as Usman, N. et al. US Patent Application 07/829,729, and Sproat, US Patent No. 5, 334, 711 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.) 30 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 inl:- orated by -eference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozymeencoding sequences into a DNA or RNA expression vector.

Transcription of the ribozyme sequences are driven from a promoter for eu.aryotic RNA polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol III) Transcripts from pol II or pol III promoters will be 20 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, provicing that the prokary- 25 otic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci.

SU 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).

30 Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells 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. 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 recombinart adenovirus or AAV particles are locally administered to the site of 20 treatment, 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 smooth eo: omuscle cells) in an appropriate liposomal vesicle.

Examples Ability Of Exoqenously-Delivered Ribozymes Directed Against c-myb To Inhibit Vascular Smooth Muscle Cell Proliferation 30 The following examples demonstrate the selection of 00. 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, i-nhibition requires the catalytic activity of the Sozymes. The cell division assay used represents a n .el 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 XII-XXIV Sites are numbered using the sequence numbers from (Westin, E. et al., 1990, Oncogene, 5, 1117-1124) (GenBank Accession No. X52125); the 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 computerbased RNA folding algorithm corresponded to accessible 25 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, respectively) and prioritizing the sites on the basis of overall nucleo- 30 tide sequence homology. Hammerhead ribozymes were designed that could bind each target (see Figure 2C) and were individually analyzed by computer folding (Jaeger, J.

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 23 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 Draper et al., International PCT publication WO 93/23569, 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 20 370 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 S' are shown in Figures 7 and 8. From these data, 25 hammerhead ribozyme sites were chosen as the most accessible (see Table III) Example 4: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of c-myb RNA 30 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 Inactive ribozymes were synthesized by substituting a U for Gs and a U for A 1 4 (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 Uhlenbeck, 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'- 0-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 liqu-i chr:-:atography (HPLC; See Usman et al., Synthesis, deprotec:_n, analysis and purification of RNA 20 and ribozymes, filed May, 18, 1994, U.S.S.N. 08/245,736 *the totality of which is hereby incorporated herein by rererence and were resuspended in water. The sequences of the chemic._ly sythesized 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 *30 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 II. All hammerhead ribozymes were synthesized with binding arm (Stems I and III; see Figure 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 II.

Ribozymes (1 pM) were incubated with 3 2 P-labeled substrate RNA (prepared as described in Example 3, approximately nM) for 60 minutes at 37 0 C using buffers described previously. Intact RNA and cleavage products were separated by electrophoresis through polyacrylamide gels. The oercentage of cleavage was determined by Phosph c Imagerquantitation of bands representing the intac: subs:rate 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 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 II. Six 20 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 cmyb RNA demonstrate the sensitivity of ribozyme action to RNA structure and suggest that even when homologous target 25 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.

30 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 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/streptomycir. -nd 2 rrm~ L-Gluta.-ne. The oth muscle cells were allowed to migrate anc grow to c- luence over a 3-4 week period. These primary cells were frozen and subsequent passages were grown at 37 c C

CO

2 in Dulbecco's modified Eagle's medium (DMEM), 10% fe:al bovine serum (FBS), and the following additives: 2 m! L- 1C Glutamine, 1% penicillin/streptomycin, 1 mM sodium pyruvate, non-essential a-mno acids (0.1 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 1x10 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.

S* 20 The cells were serum-starved by removi:. the medium, Swashing once with PBS, and incubating 48 hours in DMEM containing 0.5% FBS plus additives.

In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides 25 to cells in culture (Bennet, C. 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 trifluoroacetate) and dioleoyl phosphatidylethanolamine (DOPE)), was purchased from Life Technologies, Inc. DMRIE ditetradecyloxy propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide was obtained from VICAL. DMRIE was resuspended in CHC1 3 and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). The CHC1 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 25 0 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 0 C. The medium was then removed and DMEM containing 10% FBS, additives and 10 pM bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation. The plates were incubated at 37 0 C for 24 hours, fixed with 0.3% H 2 0 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 SBrdU 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 pM, 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 IV, demonstrate a considerable ranc in the efficacy of ribozymes directed against differen: 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 exoQenously delivered hairpin ribozvme against c-myb to inhibit vascular smooth muscle cell proliferation In addition to the hammerhead ribozymes tested above, 25 .a bipartite hairpin ribozyme (Chowrira, B. supra, 1992, Nucleic Acids Res., 20, 2835-2840) was identified Sthat 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 1% proliferation. The results of a dose-response exper.zment are shown in Table V. 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 G:C 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 VI 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% 20 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 25 binding arm sequences (stems I and III in Figure 2c) Thus, ribozyme inhibition of smooth muscle cell proliferation requires sequence-specific binding by the hammerhead arms to c-myb mRNA.

30 Example 9: Delivery of a c-myb Ribozvme With Different Cationic Lipids The experiment in Table VII 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 Figure 2C) The length of the binding arms may have an effect on both the binding and the catalytic cleavage step (Herschlag, 1991, Proc.

Natl. Acad. Sci. U S A, 88, 6921-5). For example, Table VIII 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 AM) 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 20 should be determined empirically for each ribozyme.

Towards this end, hammerhead ribozymes target with 7 nucleotide binding arms and ribczymes with 12 nucleotide binding arms (12/12) targeted to three different cleavage sites were compared.

25 Ribozymes were delivered at 0.2 AM 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 IX. As can be seen, all three ribozymes demonstrated enhanced inhibition of smooth 30 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 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; Curiel, 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 20 (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 25 effects of chloroquine on ribozyme inhibition of smooth muscle cell proliferation were assessed. A ribozyme with twelve nucleotide binding arms that cleaves c-mby RNA was S....delivered to rat smooth muscle cells as described in Example 6 (0.2 AM ribozyme complexed with DMRIE/DOPE at a 30 1:1 charge ratio). In some cases, 10 iM chloroquine was added upon stimulation of the cells. The addition of chloroquine 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 X 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®). 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 20 uninduced background was 6 1% proliferation. The results in Table XI show that inhibition is observed over *0 a similar concentration range as was seen with rat smooth muscle cells.

25 Example 13: Inhibition by direct addition of a modified, stabilized ribozyme.

A hammerhead ribozyme that cleaves site 575 was chemically synthesized with 12 nLule -de binding arms (sequence ID NO. 127, in Table III). C. -ically modified 30 nucleotides were incorporated into this Lozyme that have been shown to enhance ribozyme stability in serum without greatly impacting catalytic activity. (See Eckstein et al., Int ationa] Publication No. WO 92/07065, Perrault et al., 90, NF -re 344, 565-568 Pieken,W. et al.

1991, nce 314-317, Usman,N.; Cederc: 1992, Trends in Biochem. Sci., 17, 334-339, Usman,N. et al. US Patent Application 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 4 and U, contained 2'-amino substitutions; Ag, G12, and A. were 2'-OH ribonucleotides (numbering as in Figure An inactive ribozyme was chemically synthesized in which G 5 and A14 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 XII 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 e* smooth muscle cells from proliferating in response to 25 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 aoo.

30 delivery of the ribozyme to the cells is expected to increase the extent of inhibition.

Tables VIII and IX demonstrate one means of optimizing ribozyme activity. By altering the length of the ribozyme binding arms (stems I and III, see Figure 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 parts (see above and see Mamone, U.S.

Serial No. 07/882,689, filed May 11, 1992, hereby incorporated by reference herein) or by in vitro transcription (see Cech et al., U.S. Patent 4.987,071). Ribozymes are chemically esizec jith modifications that prevent their degradat_.. by seaim ribonucieases (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 cata tic rate, the arm length of zh ribozyme will be optimized for maximal inhibitory er ct in cells. Similarly. ne precise sequence of modified nucleotides in the abilized ribozym will affect the activity in cells. e nature of the abilizing modifications will be optimized for maximal _nhibitory effect in cells. In each case, activity of the ribozyme that 20 cleaves c-myb RNA will be compared to the activity of its catalytically inac. ve control (substitution of 2 -0methyl U for G 5 and methyl U for A 14 and to a ribozyme targeted to an irrelevant RNA (same catalytic core, with appropriate modifications, but different binding arm ooe 25 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 AM ribozyme delivered with Lipofectamine produced the same inhibitory effect as 0.3 AM 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- dioleoyloxy)-propyl]-n,n,n-trimethylammoniumchloride (DOTMA), N- [1-(2,3-dioleoyloxy)-propyl]-N,N-dimethyl-Nhydroxyethylammonium bromide (DORIE), and N- (2,3dioleoyloxy)propyl]-N,N-dimethyl-N-hydroxypropylammonium bromide (DORIE-HP). Experiments similar to those performed in Example 9 are used to determine which lioids 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 2to 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 deliv-

*S*

ery of ribozymes alone (as in Example 13), or delivery in 0* 20 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 25 include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), chloroquine, monensin, colchicine, amphipathic 0** peptides, viral proteins, and viral particles. Such compounds may be used in conjunction with ribozymes as 30 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 oy 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.

Alterna:z.ely, ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but nc- restricted to, encapsulation in lipos-mes, by iontoph.. esis or oy incorporation into other -ehicles, such -as hycrogels, cyclodextrins, biodegra-able 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, ir-ramuscular injection, aerosol inhalation, oral (tablet :pill form), topical, systemic, ocular, intraperitonea_ 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 S 25 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 hammer- 30 head (HH) ribozyme that cleaves c-myb RNA at site 575 can **oo 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. Non-limiting examples of chemically-modified ribozymes used are diagrammed in Figure 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 Svmp.

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 phosphorothioate linkages between the terminal nucleotides in each target binding arm (referred to as "2'-C-allyl

P=S")

20 Ribozymes were delivered to smooth muscle cells as cationic lipid complexes (Sullivan et al., supra). In 7' this example, the cationic lipid, Lipofectamine (GIBCO- BRL), was used at a charged lipid concentration of 3.6 pM (see Examples 6 and Active versus inactive forms of 25 each ribozyme were compared to determined whether inhibition is mediated specifically by ribozyme cleavage. As shown in Figure 9B, the ribozyme synthesized with the 2'o. 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 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 prolifer- -on Example 15: Dose res-onse 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 c= ulated by comparing the effects of the active ribozyme to the effects of the inactive ribozyme. As shown in Figure 10, the ribozyme concentration at which cell proliferation is inhibited by 50% (IC 50 is approximately 70 nM. From day to day, the IC 50 varies between and 100 nM.

Example 16: Direct comparison of the effects of ribozvmes and antisense DNA.

Ribozymes are thought to be more specific reagents for the inhibition of gene expression than antisense 25 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 nucleotide long region of the c-myb mRNA. Thus, a 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 Figure 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 20 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- 25 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-mvb 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 Figure 12). None of the inactive ribozymes inhibited cell proliferation. Active ribozymes targeting other mRNA sequences not present in cmyb or ribozymes with scrambled arm sequences also fail to inhibit smooth muscle cell proliferation (see Figure 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 20 determine the position and minimum number of phosphorothioate residues required for efficacy.

Example 18: Effect of position of phosphorothioate linkages on ribozyme inhibition.

25 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 co the ribozyme described in Examples 14 through 17 above (referred to as 10 P=S 5' and 3' in Figure 13A). One ribozyme contained only 5 phosphorothioate linkages in Stem III (5 P=S 3' in Figure 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 Figure 13A). The fourth ribozyme contained 5 phosphorothioate linkages in stem I P=S 5' in Figure 13A). The latter two ribozymes also were synthesized with the 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 Figure 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 Figure 13B, the ribozyme containing 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 20 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, 25 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 firs: and second nucleotides and proceeding 3 Each ribozyme contained the 2'-O-methyl modifications, the U 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 phosphorothioate linkages, 5 each in Stems I and III (referred to as 10 P=S in Figure 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 Figure 14.

Ribozymes with 10, 5, and 4 phosphorothioate linkages 20 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, methyl and 2'-C-allyl modifications and an inverted 30 thymidine nucleotide at the 3' end as described above.

Figure 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 Figure 2) all showed comparable efficacy. As shown in Figure 15, ribozymes with longer arm lengths tended to demonstrate more nonspecific 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 inverted thymidine at the 3' end, were synthesized with various modified nucleotides at the U 4 and

U

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 20 U4 2'-C-allyl contains a 2'-C-allyl uridine at the U 4 *o position and a 2'-O-methyl nucleotide at the U 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 S* 25 2'-O-methyl at U7. The ribozyme termed U4 6-methyl contains a 6-methyl uridine nucleotide at U4 and 2 -0methyl 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 Figure 16, all of the nucleasestable, 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 usine standard techniques and are hence within the scooe 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 20 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 The YSM cells were starved for 72 hours in 25 DMEM with 0.1% FBS. Active and inactive ribozymes (four phosphorothioate linkages at the 5 end, 2'-C-allylmodified core and inverted thymidine at the 3' end) were applied as RNA/Lipofectamine complexes as described in the above examples. Proliferation was stimulated with serum and assessed by BrdU incorporation. Figure 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 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 Figure 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-mvb ribozymes to vessels in vivo.

The ribozyme that cleaves c-myb RNA at site 575 was synthesized in two parts (Mamone, supra), the internal end was labeled with 33 P using polynucleotide kinase and the two fragments were ligated with RNA ligase. The resulting RNA was an intact ribozyme with an internal 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 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 33 label; the amount of radioactivity in each band was quantitated using a Phosphor-imaging system.

The amount of extracted and intact ribozyme was calculated 20 by direct comparison to labeled ribozyme controls run on Sthe 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 S* 25 (PBS), 3% of the 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 33 P label was t"o 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 PBSbuffered 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 33 P label could be detected in the underlying adventitia 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 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 20 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 3 P label could be detected in the media, overlying or within smooth muscle cells. Some label was 25 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 These data demonstrate that ribozymes can be locally delivered to smooth 0* 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 cmyb 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 caialytically active or inactive ribozyme.

Rat smooth muscle cells (RASMC) were treated with ribozymes as described above. Following the ribozyme treatment for 4h, cells were stimulated with 10% serum (in the presence or absence of BrdU). After 24h, 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.

i. 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 20 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 wildtype 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.

30 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 49 sample, thus allowing for a confirmation of cellular RNA's integrity and for the efficiency of RNA isolation. All quantitations for the level ofc-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 cmyb RNA by a ribozyme in RASMC.

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

20 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 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 Adenosine with a terminal 30 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 chimera was complexed with LipofectAMINE and assayed on rat aortic smooth muscle cells (RASMC) as described above.

As shown in Figure 21, when no terminal phosphate is present, the active ribozyme [575 inactive Rz+ inactive 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 tha: contains both an active ribozyme core and 5' phosphatecontining 2-5A [575 active Rz+active P shows even greater inhibition than that obtained by either mechanism individually, inhibiting the smooth muscle cell proliferation to baseline levels FBS). Thus the ribozyme and 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, 20 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, et al., 1993, Human Gene Therapy, 4, 749-758), or biopolymer stents (Slepian and Schindler, U.S. Patent 5,213,580). In the above 30 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, et al., 1987, Cancer Res., 47, 5266-5269; Slamon, D. et al., 1986, Science, 233, 203-206; Slamon, D. et al., 1984, Science, 224, 256-262; Thiele, C. et al., 1988, Mol. Cell. Biol., 8, 1677- 1683; Griffin, C. A. and Baylin, S. 1985, Cancer Res., 45, 272-275; Alitalo, 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, et al., 1991, Cancer Res., 51, 2897-2901; Raschella, et al., 1992, Cancer Res., 52, 4221-4226; Anfossi, et al., 1989, Proc. Natl. Acad.

Sci. USA, 86, 3379-3383). Furthermore, myeloid cells from 20 patients with chronic myelogenous leukemia and acute myelogenous leukemia are differentially sensitive to c-myb antisense oligonucleotides (Calabretta, 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 30 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 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 iM 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 20 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 30 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 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.

20 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 S 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 30 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 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.

i Page(s) 106- 26 are claims pages They appear after the tablelistings CV- 8 ~ou C Table I: Characteristics of Ribozymes Group I Introns Size: -200 to >1000 nucleotides Requires a U in the target sequence immediately 5' of the cleavage site Binds 4-6 nucleotides at 5' side of cleavage site.

Over 75 known members of this class. Found in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-gree algae, and others.

RNAseP RNA (Ml RNA) Size: -290 to 400 nucleotides RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA.

Roughly 10 known members of this group are all bacterial in origin.

Hammerhead Ribozyme Size: -13 to 40 nucleotides.

20 Requires the target sequence UH immediately 5' of the cleavage site.

Binds a variable number nucleotides on both sides of the cleavage site.

14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent (Figure 1)

C

Hairpin Ribozyme Size: -50 nucleotides.

30 Requires the target sequence GUC immediately 3' of the cleavage site.

Binds 4-6 nucleotides at 5' side of the cleavage site and 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 mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent (Figure 3).

Hepatitis Delta Virus (HDV) Ribozvme Size: 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.

(Figure 4).

Found in human HDV Neurospora VS RNA Ribozyme Size: -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 (Figure r r Table II: Ribozvme catalyzed cleavaae of c-mvb RNA Hammerhead Sites Cleavage Sequence Target Sequence Site 310 549 25 551 575 634 738 839 936 1017 1082 1363 1553 1597 1598

CGUCACU

GUCUGUU

CUGUUAU

GGAGAAU

AAAACCU

UAAUGCU

CAAGCUU

UUCCUAU

UGUCCCU

AGCGAAU

UUAGAAU

CAGCUAU

ACACCAU

CACCAUU

U GGGGAAA A UUGCCAA U GCCAAGC U GGAAAAC C CUGGACA A UCAAGAA C CAGAAGA U ACCACAU C AGCCAGC A AAGGAAU U UGCAGAA C AAAAGGU U CAAACAU C AAACAUG ID No.

Cleavage Mouse Human c-myb c-myb RNA RNA 28.5 0.1 87.4 91.6 56.8 82.4 93.9 91.3 68.4 87.1 78.1 0.01 27.2 0.01 61.8 60.6 40.3 0.1 55.2 89.2 11.6 0.1 87.1 92.5 71.2 62.7 79.6 85.5 Hammerhead Sites Cleavage Sequence Site ID No.

Target Sequence 1635 1721 1724 1895 1909 1943

AUACGGU

CUGGAAU

GAAUUGU

AUAUUCU

UCCGUUU

ACAAUGU

CCCUGAA

GUUGCUG

GCUGAGU

ACAAGCU

AAUGGCA

CUCAA.AG

Cleavage Mouse Human c-mb -my RNA RNA 84.4 82.3 62.1 79.3 65.6 86 79.1 66.2 31.1 0.1 66.1 Hairpin 1632 2231 Ribozymes 99 100 ACO GUCC CCUGAAG ACA GUTG AGAGCAG 92.8 0.1 84 .6 0.1 a The nucleotide numbers given correspond to the nucleotide just 5- of the ribozyme cleavage site in the human c-myb sequence taken from Westin, et al., supra (GenBank 15 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 III: Seauences of ribozymes used in these studies.

Target Sequence Ribozyme Sequence Site ID No.

Hammrhead ribozvmes with 7 nudeotide binding arms 310 549 551 575 634 738 839 936 1017 1082 1363 101 102 103 104 105 106 107 108 109 110 111 UUUC CCCCUGAUGAGGCCGAAAGGCCGAAAGUGACG

UUGGCAACUGAUGAGGCCGAAAGGCCGAAAACAGAC

GCUUGGCCUGAUGAGGCCGAAAGGCCGAAAUAACAG

GCUUUCCCUGAUGAGGCCGAAAGGCCGAAAUUCUCC

UGUCCAGCUGAUGAGGCCGAAAGGCCGAAAGGUUUU

UUCUUGACUGAUGAGGCCGAAAGGCCGAAAGCAUUA

UCUUCUGCUGAUGAGGCCGAAAGGCCGAAAAGCUCG

AUGUGGUCUGAUGAGGCCGAAAGGCCGAAAUAGGAA

GCCGGCUCUGAUGAGCGCGAAAGCGCGAAAGGGACG

GCUCCUUCUGAUGAGGCCGAAAGGCCGAAAUUCGCU

UUCUGCACUGAUGAGGCCGAAAGGCCGAAAUUCUAA

1553 112 ACCUJIJUCUGAUGAGGCCGAAAGGCCGAAAUAGCUG 1597 113 AUGUUUGCUGAUGAGGCCGAAAGGCCGAAAUGGUGU 1598 114 CAUGUUUCUGAUGAGGCCGAAAGGCCGAAAAUGGUG 1635 115 U1CAGGGCUGAUGAGGCCGAAAGGCCGAAACCGUAU 1721 116 CAGCAACCUGAUGAGGCCGAAAGGCCGAAAJUCCAG 1724 117 ACUCAGCCUGAUGAGGCCGAAAGGCCGAAACAAUUC 1895 118 AGCUTGUCUGAUGAGGCCGAAAGGCCGAAAGAAUAU 1909 119 UGUCAUUCUGAUGAGGCCGPAAGGCCGAAAAACAGA 1943 120 CUUUGAGCUGAUGAGGCCGAAAGGCCGAAACAUUGU Bimolecular Hairpin Ribozymes 1 6 3 2 a 121 5' Fragment: UCAGGGAGAAGUAUACCAGAGAAACACACG CG 3' Fragment: CGCGUGGUACAUUACCUGGUA 2 2 3 1 a' 122 5' Fragment: GCUCUC-AGAAGtJUGACCAGAGAAACACACGCG 3' Fragment: CGCGUGGUACATJUACCUGGUA Hammerhead riboyzmes with 6, 8, 9, 10, and 12 *nucleotide binding arms 575 123 CUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUC 6 6 b 575 124 UGCUUUCCCUGAUGAGGCCGAAAGGCCGAA 8/8 AUUCUCCC :575 125 CUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA *.20 9/9 AUIJCUCCCU 575 126 ACUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA 10/10 AUUCUCCC.U 575 127 ACACUGCTJUUCCCUGAUGAGGCCGAAAGGCCGAA 12/12 AUUCUCCCtUUU 549 128 AGUGCUUGGCAACUGAUGAGGCCGAAAGGCCGAA *12/12 AACAGACCAACG 1553 129 GAUUGACCTJUUUCUGAUGAGGCCGAAAGGCCGAA 12/12 AUAGCUGGAGUU aThe hairpin ribozymes were synthesized in two pieces as indicated. The two ol igonucleot ides' were annealed and tested for activity against the c-myb RNA as described above. See Mamone, Ribozyme synthesis, filed May 11, 1992, U.S.S.N. 07/882,689, hereby incorporated by reference herein.

bDesignation of the ribozymes with different arm lengths is a/b where represents the nucleotides in s:em I and represents the nucleotides in stem III (see Figure 1).

Table IV Comoarison of the effects six hammerhead a ribozymes, that cleave c-myb RNA, on smooth muscle cell proliferation Inactive Active Inhibition Ribozyme Ribozyme Ribozyme Cell Cell (Active vs.

Site Proliferation Proliferation Inactive) 549 68 1 59.5 1.5 14 4 575 66.5 0.5 54.5 1.5 21 3 15 1553 68.5 0.5 52 1 28 1 1597 66 1 57 3 16 7 1598 67 1 58.5 0.5 15 1 1635 62.5 2.5 64 1 0 Table V: Dose Response of c-mvb Hairpin Ribozyme 1632 Control Ribozyme 1632 Ribozyme Ribozyme Inhibition Dose (pM) Proliferation Proliferation (vs. control) 0.05 86.5 1.5 88 5 0 0.15 89.5 1.5 78.5 2.5 10 0.45 87.5 1 66.5 1.5 25 4 a a Table VI: Dose Response of c-myb Hammerhead Ribozymes 575 and 549 Control Ribozyme 575 Ribozyme 549 Ribozyme Ribo- cells cells cells zyme in S in S Inhibi- in S Inhibi- Dose phase phase tion phase tion (0M) (vs. (vs.

con- control) trol) 0.05 89+5 77.5+1.5 14+8 92+1 0 0.15 90+1 68.5+1.5 26+2 84+2 9+4 0.45 91.5+0.5 59+5 38+7 76.5+2.5 18+5 Table VII: Delivery of c-mvb Ribozyme 575 by Two Different Cationic Lipids 15 Delivery with DMRIE/DOPE Inactive Active Ribozyme 575 Ribozyme 575 Ribozyme cells in S cells in S Inhibition Dose (tM) phase phase (vs. inactive) 0.075 79 6 74.5 1.5 6 6 0.15 79.5 0.5 67 1 17 4 0.30 77 1 57 2 28 Delivery with Lipofectamine Inactive Active Ribozyme 575 Ribozyme 575 Ribozyme cells in S cells in S Inhibition Dose (tM) phase phase (vs. inactive) 0.075 81 1 83 1 0 0.15 79 3 71 1 11 4 0.30 82 1 68.5 1.5 18 4 0.60 75 1 59.5 3.5 22 7 :0 0 Thh1~VTTT Arm Length Variations of c-mvb Hammerhead Table

VIII:

Ribozyme 575 Arm Length (base %cells in S %Inhibition (vs.

pairs) phase Inactive 7/7) 6/6 62 1 4 4 7/7 60 1 7 3 8/8 60.5 0.5 6 2 9/9 53.5 0.5 18 2 10/10 55 1 16 4 12/12 48 1 28 3 Table IX: Hammerhead ribozymes with 7 vs. 12-nucleotide binding arms targeting three different sites Ribozyme Length of Inactive Active Target Binding Ribozyme Ribozyme Inhibition Site Arms Cell Cell (Active Prolifera- Prolifera- vs.

tion) tion) Inactive) 575 7/7 51.5 0.5 43 0.5 24 575 12/12 50.5 3.5 37 0.5 37 4 549 7/7 49.5 0.5 44.5 1.5 21 7 20 549 12/12 48.5 1.5 35 2 41 7 1553 7/7 .49.5 +4 .0.5 43.5 2.5 23 9 11553 112/12 149 1 33.5 1.5 45 6 Table X: Effect of chloroctuine on ribozyme inhibition of 25 smooth muscle cell Proliferation Ribozyme Chioro- Inactive Active %Inhibiquine Ribozyme Ribozyme tion (AiM) Cell Cell (Active Prolifera- Prolifera- VS.

tion) tion) inactive) 575, 12/12 0 81.8 ±0.5 74 ±1 10 2 575, 12/12 10 83 ±4 62.5 ±0.5 28 6 T~hlP XT inhibition of Human Aortic Smooth Muscle Cells Table by c-myb Ribozvme 549 Inactive Active Inhibition Ribozyme Ribozyme Ribozyme Prolifera- Prolifera- (active vs.

Dose (pM) tion tion inactive) 0.075 55 2 40.5 4.5 30 13 0.15 53 10 42 1 23 23 0.30 53 7 32.5 4.5 44 22 Table XII: Inhibition of Rat Smooth Muscle Cell a a a Proliferation by Direct Addition of a Chemically-Modified c-nyb Ribozxme 575 Inactive Active Inhibition Ribozyme Ribozyme Ribozyme Prolifera- Prolifera- (active vs.

Dose (AM) tion tion inactive) 0.22 42 3 36 0.5 15 8 0.67 48 3 35 2 28 9 52 5 25 1 54 7 20 Table XIII: Human c-myb Hairpin Ribozyme and Taraet Sequences Posi- Ribozyme Sequence Target tion 104 CCCUCCCC AGAA GCGC GCGCA GCC ACCAGAGAAACACACGUUGUGGUACAUIACCUGGUA

GGGGAGGG

148 ACCGACCG AGAA GCCG CGGCA GCC ACCAGAGAAACACACGJUGUGGUACAUUACCUGGUA

CGGUCGGU

185 GCGCGGCG AGAA GCGG CCGCC GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGCCGCGC

528 ACGUTUCG AGAA GUAU AUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGAAACGU

a a a *aa.

C.

a a a a a a a Posi- Ribozyme Sequence Target tion 715 UUCGUCCA AGAA GUAG CUACU GCC ACCAGAGAAACACACGUUGUGGUACAUTJAC CUGGUA UGGACGAA 1025 AUGGCUGC AGAA GCUG CAGCU GCC ACCAGAGAA.ACACACGUUGUGGUACAUJACCUGGUA C CAC CCAU 1187 CUGGUCUG AGAA GCAA UTJGCC CAC ACCACAGAAACACACGUUGUCGUACAUUACCUGCUA CACACCAC 1532 GUTJCUAAA AGAA GUAU AUACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGCUA UTJTUAGAAC 1632 CUUCAGGG AGAA GUAU AUACG GUC ACCAGAGAAACACACGLTUGUGCUACAUUACCUGCUA CCCUCAAC 1836 GGUAUIJCA AGAA GUCC OGACA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGCUA UGAAUACC 1852 UCUGCGUG AGAA GTJUG CAACU CULT ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CACGCAGA 1861 CAGGCGAG AGAA GCGU ACOCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGCUA CUCGCCUG 1993 UGCUACAA AGAA GCAA UUGCA GCC ACCAGAGAAACACACGUUGUGGUACATJUACCUGGUA. UUGUAGCA 2231 CUGCUCUC AGAA GtJUG CAACA GULT ACCAGAGAAACACACGUUGUGGUACAUJACCUGGUA GAGAGCAG 2316 UUAGGUAA AGAA GUUA UAACA GUC ACCAGAGAAACACACGUUCUGGUACAUUACCUGGUA UUACCUAA 3068 AAU-UAUAA AGAA GUCA UGACU GUU ACCAGAGAAACACACGUUCUGGUACAUUACCUGGUA UUAUAAUU 3138 AUCCAUGC AGAA GUUC GAACU GUT ACCAGAGAAACACACGUUGUGGUACAUUACCUCCUA GCAUGGAU 3199 GUIJCUUAA AGAA GUGA UCACU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUAAGAAC 3264 UGCUACAA AGAA GUAA UUACU GCC ACCAGAGAAACACACGUUGIJGGIJACAUUACCJGGUA lUUGUAGCA 64 Table XIV: Human c-mvb Hammerhead Ribozxrme and Target Sequence 6 6* 6 @6 6 6@ 66 6 6 666* 6 6*6* 6 6666 6*6* @6 6e 6 6

S.

6666

S

*566 6 *606 nt.

Position 14 16 19 22 28 29 31 34 37 49 52 20 55 58 60 61 62 25 64 75 76 156 160 170 172 224 226 228 253 254 274 287 288 Target Sequence

CAACCUGU

AACCUGUJ

AC CUGUUU

UGUUCCU

UTJCCUCCU

CUCCUCCU

CT-CCUCCU

UCCUCcU CUCCUUcU cmUjcuccu

CUCCUCCU

CUCCUCcU

CGUGACCU

GACCUCCU

CUCCUCCU

CTJCCUCCU

CCUccUcU CUccUcUU UccUcUtU cUcUUUcU

GAGAAACU

AGAAACLU

AGCCCGGU

CGGUCGGU

CCGCGGCU

GCGGCUCU

CACAGCAU

CAGCAUAU

GCAUAUAU

UGAGGACU

GAGGACUU

CCAUGACU

GGGCUGCU

GGCUGCUU

UCCUCCUC

CCUCCUcc cUcCUCcU CUcCUCCU CUCcUUCU

CTJUCUCCU

CUCCUCCU

UCCUCCUC

CUCCUCcU

CUCCUCCG

CUCCGUGA

CGUGACCU

CUCCUCCU

CUCCUCtU

CUCUUUCU

uuucu :Ccu

UCUCCUGA

CUCCUGAG

UCCUGAGA

CUGAGAAA

CGCCCCAG

GCCCCAGC

GGUCCCCG

CCCGCGGC

UCGCGGAG

GCGGAGCC

UAUAGCAG

UAGCAGUG

GCAGUGAC

UGAGAUGU

GAGAUGUG

UGAUGGGC

CCCAAGUC

CCAAGUCU

GAGGAGGA

GGAGGAGG

AGGAGGAG

AGGAGGAG

AGAAG GAG

AGGAGAAG

AGGAGGAG

GAGGAGGA

AGGAGGAG

CGGAGGAG

UCACGGAG

AGGUCACG

AGGAGGAG

AAGAGGAG

AGAAAGAG

AGGAGAAA

UCAGGAGA

CUCAGGAG

UCUCAGGA

UUUCUCAG

CUGGGGCG

GCUGGGGC

CGGGGACC

GCCGCGGG

CUCCG CGA

GGCUCCGC

CUGCUAUA

CACUGCUA

GUCACUGC

ACAUCUCA

CACAUCUC

GCCCAUCA

GACUUGGG

AGACUUGG

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

ACAGGUJG

AACAGG-U

AAACAGGU

AGGAAACA

AG GAG GAA

AGGAGGAG

AGGAGGAG

AAG GAG GA AGAAG GAG

AGGAGAAG

AGGAGGAG

AGGAGGAG

AGGUCACG

AGGAGGUC

AGGAGGAG

AGGAGGAG

AGAGGAGG

AAGAGGAG

AAAGAGGA

AGAAAGAG

AGtJUUCUC

AAGUUUCU

ACCGGGCU

ACCGACCG

AGCCGCGG

AGAGCCGC

AUGCUGUG

AUAUGCUG

AUAUAUGC

AGUCCUCA

AAGUC CU C

AGUCAUGG

AGCAGCCC

AAGCAGCC

Ribozyme Sequence 0 0 0 0 0 0 .0 0000 0 a 0 0600 0 nt.

Position 295 306 310 392 393 395 402 403 405 414 452 455 467 470 477 480 498 509 515 20 518 526 531 540 544 25 548 549 551 562 563 575 588 603 615 623 3S 624 634 Target Sequence

UCCCAAGU

GAA-AGCGU

GCGUCACU

UGGAAAGU

GGAAAGUU

AAAGUUAU

LJTJGCCAAU

UGCCAAUU

CCAALTUAU

UCCCGAAU

CAGAAAGU

AAAGUACU

CCUGAGCU

GAGCUCAU

UCAAGGGU

AGGGUCCU

AAGAAGAU

AGAGUGAU

AUAGAGCU

GAGCUUGU

ACAGAAAU

AAUACGGU

CGAAACGU

ACGUUGGU

UGGUCUGU

GGUCUGUU

UCUGUUAU

CAAGCACU

AAGCACUU

GGGAGAAU

AACAAUGU

GGUGGCAU

ACUUGAAU

CCAGAAGU

CAGAAGLTU

GAAAACCTJ

C UGGAAAGC C ACUUGGGG U GGGGAAAA U AUUGCCAA A UUGCCAAU U GCCAAUUA U AUCUCCCG A UCUCCCGA C UCCCGAAU C GA-ACAGAU A CUAA.ACCC A AACCCUGA C AUCAAGGG C AAGGGUCC C CUUGGACC U GGACCAAA C AGAGAGUG A GAGCUUGU U GUACAGAA A CAGAAAUA A CGGUCCGA C CGAAACGU U GGUCUGUU C UGUUAUUG U AUUGCCAA A UUGCCAAG U GCCAAGCA U AAAGGGGA A AAGGGGAG U GGAAAACA A GGGAGAGG A ACCACUUG C CAGAAGUU U AAGAAAAC A AGAAAACC C CUGGACAG GCUUUCCA CUGAUGA X GAA ACUUGGGA

CCCCAAGU

UUUUCCCC

UUGGCAAU

AUUGGCAA

UAAUUGGC

CGGGAGAU

UCGGGAGA

AUUCGGGA

AUCUGUUC

GGGUUUAG

UCAGGGT-TU

CCCUUGAU

GGACCCUU

GGUCCAAG

UUUGGUCC

CACUCUCU

ACAAGCUC

UUCUGUAC

UAUUUCUG

UCGGACCG

ACGUUUCG

AACAGACC

CAAUAACA

LTUGGCAAU

CUUGGCAA

UGCUUGGC

UCCCCUUU

CUCCCCUU

UGULTUUCC

CCUCUCCC

CAAGUGGU

AACLTUCUG

GUULTUCUU

GGLTUUUCU

CUGUCCAG

CUGAUGA X GAA ACGCUUUC CUGAUGA X GA.A AGUGACGC CUGAUGA X GAA ACUUUCCA CUGAUGA X GAA AACUUUCC CUGAUGA X GAA AUAACUUU CUGAUGA X GAA AUUGGCAA CUGAUGA X GAA A.AUUGGCA CUGAUGA X GAA AUA-AUUGG CUGAUGA X GAA AUUCGGGA CUGAUGA X GAA ACUUUCUG CUGAUGA X GAA AGUACUUU CUGAUGA X GAA AGCUCAGG CUGAUGA X GA-A AUGAGCUC CUGAUGA X GAA ACCCUUGA CUGAUGA X GAA AGGACCCU CUGAUGA X GAA AUCUUCUU CUGAUGA X GA-A AUCACUCU CUGAUGA X GAA AGCUCUAU CUGAUGA X GAA ACA.AGCUC CUGAUGA X GAA AUUUCUGU CUGAUGA X GAA ACCGUAUU CUGAUGA X GAA ACGUUUCG CUGAUGA X GAA ACCAACGU CUGAUGA X GAA ACAGACCA CUGAUGA X GAA AACAGACC CUGAUGA X GAA AUAACAGA CUGAUGA X GAA AGUGCUUG CUGAUGA X GAA AAGUGCUU CUGAUGA X GAA AUUCUCCC CUGAUGA X GAA ACAUUGUU CUGAUGA X GAA AUGCCACC CUGAUGA X GAA AUUCAAGU CUGAUGA X GA-A ACUUCUGG CUGAUGA X GAA AACUUCUG CUGAUGA X GAA AGGUUUUC Ribozyme Sequence nt Posi tion 659 660 662 663 664 704 713 732 738 740 756 757 759 768 15 776 789 790 792 802 804 805 838 839 852 25 855 856 864 865 866 870 876 882 888 893 917 928 Target Sequence

GACAGAAU

ACAGAAUU

AGAAUUAU

GAAUUAUU

AAUUAJUUU

GCAGAAAU

GCAAAGCU

GAACUGAU

AUAAUGCU

AAUGCUAU

ACUGGAAU

CUGGAAU

GGAAUUCU

CAAUGCGU

CGGAAGGU

AGGAAGGU

GGAAGGUU

AAGGtJUAU G CAGGAGU

AGGAGUCU

GGAGUCUU

CACAAGCU

ACAAGCTU

AGAACAGU

ACAGUCAU

CAGUCAUUt

UGAUGGGU

GAUGGGUU

AUGGGUUU

GUULUGCU

CUCAGGCU

CUCCGCCU

CUACAGCU

GCUCAACU

CCCACUGU

AUTJUACCA

TJUUACCAG

UACCAGGC

ACCAGGCA

CCAGGCAC

GCAAAGCU

CUGCCUGG

AUGCUAUC

UCAAGAAC

AAGAACCA

CUACAAUG

UACAAUGC

CAAUGCGU

GGAAGGUC

GAACAGGA

AUCUGCAG

UCUGCAGG

UG CAGGAG

UJUCAAAAG

CAAAAGCC

AAAAG CCA

CCAGAAGA

CAGAAGAA

AUUUGAUG

UGAUGGGU

GAUGGGUU

UtJGCUCAG

UGCUCAGG

GCUCAGGC

AGGCUCCG

CGCCUACA

CAGCUCAA

AACUCCCU

CCUGCCAC

AACAACGA

UGGUAAAU

CUGGUAAA

GCCUGGUA

UGCCUGGU

GUGCCUGG

AGCTJUUG C

CCAGGCAG

GAUAGCAU

GUUCUUGA

UGGtJUCLU

CAUUGUAG

GCAUTJUGUA

ACGCAUUG

GACCUUCC

UCCUGUUC

CUGCAGAU

CCUGCAGA

CUCCUGCA

CUUUUGAA

GGCUUUUG

UGGCTUUJU

UCUUCUGG

UUCUUCUG

CAUCAAAU

ACCCAUCA

AACCCAUC

CUGAGCAA

CCUGAGCA

GCCUGAGC

CGGAGCCU

UGUAGGCG

UUGAGCtJG

AGGGAGTU

GUGGCAGG

UCGUUGUU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAhA

GAA

GAA

GAA

GAA

GAA

GAlA GAlA

GAA

GAA

GAA

GAlA

GAA

GAA

GAlA

GAA

GAA

GAlA GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA AUU CUGU C

AAIJUCUGU

AUAAUUCU

AAUAAIJUC

AAAUAAUTJ

AUUUICUG C

AGCUUUGC

AUCAGUTJC

AG CAIUAU AUAG CATJ

AUUCCAGU

AAUUCCAG

AGAAUUCC

ACGCAUUG

ACCTUCCG

AC CUU CCU AlAC CTUC C

AUAACCUU

ACUCCUGC

AGACUCCU

AAGACUCC

AGCtJUGUG

AAGCUUGU

A CUGUUC U

AUGACUGU

AAUGACUG

ACCCAUCA

AACCCAUC

AAACCCAU

AGCAAAAC

AGCCUGAG

AGGCGGAG

AGCUGUAG

AGUUGAGC

ACAGUGGG

Targe SeqenceRibozymne Sequence CAACGACU A UUCCUAUU CAAGAC A UCCAUUAAUAGGAA CUGAUGA X GAA AGUCGUUG nt Posi r-i on 930 931 934 936 937 944 945 946 962 964 969 974 975 979 15 986 991 992 1002 1004 1007 1013 1017 1037 1048 1050 1082 1090 1091 1096 1100 1103 1108 1124 1184 1203 Target SQquence

ACGACUAU

CGACUAUU

CUAUUCCU

AUUCCUAU

UUCCUAU

UACCACAU

ACCACAUJ

CCACAUUU

CAAAAUGU

AAAUGUCU

UCUCCAGU

AGUCAUGU

GUCAUGUI

UGUUCCAU

UACCCUGU

UGUAGCGU

GUAGCGLU

AUGUAAAU

GUAAAUAU

AAUAUAGU

GUCAAUGU

AUGUCCCU

GCAGCCAU

GAGACACU

GACACUAU

AAGCGAAU

AAAGGAAU

AAGGAAU

AUUAGAAU

GA.AUUGCU

UUGCUCCU

CCUAAUGU

AAUGAGCU

ACCACCAU

CCAGACCU

CCUATJUAC

C UAUUA CC

UUACCACA

AC CACATU

CCACAUUU

UCUGAAGC

CUGAAGCA

UGA.AGCAC

UCCAGUCA

CAGUCAUG

AUGUUCCA

CCAUACCC

CAUACCCU

CCCUGUAG

GCGUUACA

ACAUGUAA

CAUGUAAA

UAGUCAAU

GUCAAUGU

AAUGUCCC

CCUCAGCC

AGCCAGCU

CAGAGACA

UAAUGAUG

AUGAUGAA

AAGGAAUU

AGAAUUGC

GAAUUGCU

GCUCCUAA

CUAAUGUC

AUGUCAAC

AACCGAGA

AAAGGACA

GCCGACCA

AUGGAGAC

GUAAUAGC

GGUAAUAC

UGUGGUA)!

AAUGUGGt

AAAUGUGC

GCIJUCAGP

UGCTJUCAG-

GUGCUUCP

UGACUGGP

CAUGACUC

UGGAACAL

GGGUAUGC

AGGGUAUC

CUACAGGG

UGUAACGC

UUACAUGU

UUUACAUC-

AUUGACUP

ACAUUGAC

GGACAUU

GGCUGAGG

AGCUGGCU

UGUCUCUG

CAUCAUUA

UUCAUCAU

AAUUCCUU

GCAAU-UCUC

AGCAAUTUC

UEJAGGAGC

GACAUUAC

GUUGACAL

UCUCGGUI

UGUCCUJI

UGGUCGGC

GUCUC CAT

AAACAGG;

CUGAUGA

CUGAUGA

CUGAUGA

J CUGAUGA

;CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

I CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

ICUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

FCUGAUGA

FCUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

I CUGAUGA

JCUGAUGA

I CUGAUGA CtJGAUGA I CUGAUGA LCtJGAUGA KGAA AUAGUCGU K GAA AAUAGUCG GAA AGGAAUAG GAA AUAGGAAU GALA AAUAGGAA GAA AUGUGGUA GAA AAUGUGGU CGAA AAAUGUGG CGAA ACAUUUG CGAA AGACAUUU GAA ACUGGAGA GAA ACAUGACU GAA AACAUGAC GAA AUGGAACA GAA ACAGGGUA LGAA ACGCUACA GAA AACGCUAC GAA AUUUACAU GAA AUAUUUAC GAA ACUAUAUIJ GAA ACAUUGAC GAA AGGGACAU GAA AUGGCUGC LGAA AGUGUCUC LGAA AUAGUGUC GAA AUUCGCUU GAA AUUCCUUU GAA AAUUCCUU GAA AUUCUAAU GAA AGCALATUC GAA AGGAGCAA GAA ACAUUAGG IGAA AGCUCAUU IGAA AUGGUGGU IGAA AGGUCUGG C GAA ACAGGUGC Targe SeqenceRibozyme Sequence 1223 GCACCUGU U UCCUGULU

S

S

S

nt Posi tion 1224 1225 1230 1231J 1246 1251 1255 1257 1269 1276 1280 1297 1316 1319 15 1334 1335 1341 1346 1347 1355 1357 1358 1363 1364 25 1376 1381 1382 1383 1385 1389 1390 1392 1393 1394 1396 1397 Target Sequence

CACCUGUU

ACCUGUUU

UJUUCCUGU

UUC CUGUUI

ACACCACU

ACUCCACU

CACUCCAU

CUCCAUCU

CAGCGGAU

UCCUGGCU

GGCUCCCU

AAGCGCCU

UGCAUGAU

AUGAUCGU

GGCACCAU

GCACCAUU

tJUCUGGAU U CCUGUU-UG C CUGUTJUGG U UGGGAGAA U GGGAGAAC C CACUCCAU C CAUCUCUG C UCUGCCAG C UGCCAGCG C CUGGCUCC C CCUACCUG A CCUGAAGA C GCCAGCAA C GUCCACCA C CACCAGGG U CUGGAUAA C UGGAUAAU A AUGUUAAG GAUAAUGU U AAGAACCU AUAAUGUU A AGAACCUC AAGAACCU C UTUAGAALU GAACCUCU U AGAAIJUUG AACCUCUU A GAAUUUGC CUUAGAAU U UGCAGAAA UtJAGAAUIJ U GCAGAAAC GAAACACU C CAAUUUAU ACUCCAAU U UAUAGAUU CUCCAAUU U AUAGAtJUC UCCAAUUU A UAGAU-UCU CAAUUUAU A GAUUCUUU UUAUAGAU U CtJUUCUIJA UAUAGAUTU C UUUCUUAA UAGAUUCU U UCUUAAAC AGAUUCUU U CUUAAACA GAtJUCUUU C UUAAACAC UUCtJUUCU U AAACACtU UCUUEJCUU A AACACUUC

CAAACAGG

CCAAACAG

UUCUCCCA

GUTJCUCC C

AUGGAGUG

CAGAGAUG

CUGG CAGA

CGCUGGCA

GGAGCCAG

CAGGUAGG

UCTJTUCAGG

TJUGCUGGC

UGGUGGAC

CCCUGGUG

UIJAUCCAG

AUUAUCCA

CtJUAACAU

AGGUTUCUU

GAGGUUCU

AAUUCUAA

CAAAUUCU

GCAAAUUC

tJUUCUGCA

GUTJUCUGC

AUAAAUUG

AAUCUAUA

GAAUCUAU

AGAAUCUA

AAAGAAUC

UAAGAAAG

UUAAGAAA

GUtJUAAGA

UGLTUUAAG

GUGUUUAA

AAGUGUUU

GAAGUGLU

Targe Seqence Ribozyme Sequence CUGAUGA X GA-A AACAGGUG CUGAUGA X GAA AAACAGGU CUGAUGA X GAA ACAGGAAA CUGAUGA X GAA AACAGGAA CUGAUGA X GAA AGUGGUGU CUGAUGA X GALA AGUGGAGU CUGAUGA X GAA AUGGAGUG CUGAUGA X GAA AGAUGGAG CUGAUGA N GAA AUCCGCUG CUGAUGA X GAA AGCCAGGA CUGAUGA N GAA AGGGAGCC CUGAUGA N GAA AGGCGCJU CUGAUGA X GAA AUCAUGCA CUGAUGA X GAA ACGAUCAU CUGAUGA X GAA AUGGUGCC CUGAUGA X GAA AAUGGUGC CUGAUGA X GAA AUCCAGAA CUGAUGA X GAA ACAtJUAUC CUGAUGA X GAA AACAUUAU CUGAUGA X GAA AGGUTUCUU CUGAUGA X GAA AGAGGUUC CUGAUGA X GAA AAGAGGJU CUGAUGA X GAA AUUCUAAG CUGAUGA X GAA AAUUCUAA CUGAUGA X GAA AGUGUUUC CUGAUGA X GAA AUUGGAGU CUGAUGA X GAA AAUUGGAG CUGAUGA X GAA AAAUUGGA CUGAUGA X GAA AUAAAUUG CUGAUGA N GAA AUCUAUAA CUGAUGA X GAA AAUCUAUA CUGAUGA X GAA AGAAUCUA CUGAUGA X GAA AAGAAUCU CUGAUGA N GAA AAAGAAUC CUGAUGA N GAA AGAAAGAA CUGAUGA X GAA AAGAAAGA nt.

Posi tion 1404 1405 1410 1423 1429 1440 1441 1443 1444 1445 1449 1450 1460 1463 1 5 1467 1474 1481 1482 1492 1493 1494 1497 1518 1530 1535 1536 1537 1538 1539 1551 1553 1561 1565 1567 1568 1578 Target Sequence UAALACACU U CCAGUAAC AAACACUU C CAGUAACC CUUCCAGU A ACCAUGAA UGAAAACU C AGACUUGG CUCAGACU U CGAAAUGC AAAUGCCU U CUUUAACU AAUGCCUU C =UUAACUIJ UGCCUtJCU U UAACUUCC GCCUUCUU U AACUEJCCA CCLTUCUUU A ACUUCCAC CUUUAACU U CCACCCCC UUUAACUU C CACCCCCC ACCCCCCU C AUUGGUCA CCCCUCAU U GGUCACAA UCAUUGGU C ACAAAUTJC UCACAAAU U GACUGUUA UUGACUGU U ACAACACC UGACUGUU A CAACACCA AACACCAU U UCAUAGAG ACACCATU U CAUAGAGA CACCAUUU C AUAGAGAC CAUUUCAU A GAGACCAG UGAAAACU C AAAAGGAA AGGAAAAU A CUGUUUUU AAUACUGU U UUUAGAAC AUACUGUU U UUAGAACC UACUGUJ U UAGAACCC ACUGUUUU U AGAACCCC CUGUUUTJU A GAACCCCA CCCCAGCU A UCAAAAGG CCAGCUAU C AAAAGGUC CAAAAGGU C AAUCUUAG AGGUCAAU C UUAGAAAG GUCAAUCU U AGAAAGCU UCAAUCUU A GAAAGCUC AAAGCUCU C CAAGAACU

GUUACUGG

GGUUACUG

EJUCAUGGU

CCAAGUCU

GCAULUCC

AGUUAAAG

AAGIJUAAA

GGAAGUtJA

UGGAAGUU

GUGGAAGU

GGGGGUGG

GGGGGGUG

UGACCAAU

UUGUGACC

CAAUUJUGU

UAACAGUC

GGUGUUGU

UGGUGUUG

CUCUAUGA

UCtJCUAUG

GUCUCUAU

CUGGUCUC

UUCC*uTUu

AAAAACAG

GtJUCUAAA

GGUUCUAA

GGGUUCUA

GGGGUUCU

UGGGGUUC

CCUTUUUGA

GACCUUEU

CTJAAGAUU

CULTUCUAA

AGCUUUCU

GAGCUUTUC

AGTUUCT-UG

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA.

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

X GAA X GAA X GAA X GAA X GAA X GAA X GAA

GAA

GAA

GAA

GAlA

GAA

GAA

X GAA X GAA GAlA X GAA

GAA

GAA

GAA

GAlA

AGUGUUUA

AAGUGUUUJ

ACUGGA-AG

AGUUUUCA

AGUCUGAG

AGGCAUUU

AAGGCAUU

AGAAGGCA

AAGAAGG C

AAAGA-AGG

AGUUAAAG

AAGUUAAA

AGGGGGGU

AUGAGGGG

ACCAAUGA

AUUUGUGA

ACAGUCAA

AACAGUCA

AUGGUGUU

AAUGGUGU

AAAUGGUG

AUGAAAUG

AGUUUCA

AUtJUUCCU

ACAGUAUIJ

AACAGUAU

AAACAGUA

AAAACAGU

AAAAACAG

AGCUGGGG

AUAGCUGG

ACCUUU-UG

AtJUGACCU

AGAUUGAC

AAGAUUGA

AGAGCUUU

Ribozyme Sequence CUGAUGA X GAA CUGAUGA X GAA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

X GAlA

GAA

GAlA

GAA

GAA

GAlA X GAlA GAlA X GAlA X GAA X GAlA X GAA X GAlA nt.

Posi tion 1587 1590 1597 1598 1610 1617 1625 1626 1635 1649 1653 1663 1665 1668 15 1670 1673 1680 1694 1705 1714 1721 1724 1732 1733 1753 1754 1766 1783 1785 1794 1798 1807 1808 1810 1811 Target Seguence CAAGAACU C GAACUCCU A UACACCAU U ACACCAUU C CAUGCACU U UUCCAGCU C CAAGAAAU U AAGAAAUU A AAUACGGU C AAGAUGCU A UGCUACCU C GACACCCU C CACCCUCU C CCUCUCAU C UCUCAUCU A CAUCUAGU A UAGAAGAU C GAUGUGAU C ACAGGAAU C UGAUGAAU C UCUGGAAU U GGAAUUGU U UGCUGAGU U GCUGAGUU U ACCACCCU U CCACCCUU A AAGAAAAU C GGUGGAAU C UGGAAUCU C CAACUGAU A UGAUAAAU C AGGAAACU U GGAAACUTU C AAACUTUCU U AACLTUCLTU C

CUACACCA

CACCAUUC

CAA.ACAUG

AAACAUG C

GCAGCUCA

AAGAAAUU

AAAUACGG

AAUACGGU

CCCUGAAG

CCUCAGAC

AGACACCC

UCAUCUAG

AUCUAGUA

UAGUAGAA

GUAGAAGA

GAAGAUCU

UGCAGGAU

AAACAGGA

UGAUGAAU

UGGAAUTUG

GUUGCUGA

GCUGAGUU

UCAAGAAA

CAAGAAAA

ACUGAAGA

CUGAAGAA

AAACAAGA

UCCAACUG

CAACUGAU

AAUCAGGA

AGGAAACU

CUUCUGCU

UUCUG CU C

CUGCUCAC

UGCUCACA

UGGUGUAG

GAAUGGUG

CAUGUUUG

GCAUGUUU

UGAGCUGC

AAULJUCUU

CCGUAUUU

ACCGUAUU

CUUCAGGG

GUCUGAGG

GGGUGUCU

CUAGAUGA

UACUAGAU

LTUCUACUA

UCUUCUAC

AGAUCUUC

AUCCUGCA

UCCUGUUU

AUUCAUCA

CAAUUCCA

UCAGCAAC

AACUCAGC

UUUCUUGA

tJUUUCUUG

UCUTJCAGU

UUCUTUCAG

UCUUGUUU

CAGUUGGA

AUCAGUUG

UCCUGAUU

AG UTUC CU

AGCAGAAG

GAGCAGAA

GUGAGCAG

UGUGAGCA

CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X GAA AGUUCUUG GAA AGGAGTJUC GALA AUGGUGUA GAA AAUGGUGU GAA AGUCCAUG GAA AGCUGCAA GAA ATJUUCUUG GAA AAUUUCUU GAA ACCGUAUU GAA AGCAUCUU GAA AGGUAGCA GAA AGGGUGUC GAA AGAGGGUG GAA AUGAGAGG GAA AGAUGAGA GAA ACUAGAUG GAA AUCUUCUA GAA AUCACAUC GAA AUUCCUGU GAA AUUCAUCA GAA AUUCCAGA GAA ACAAU-UCC GAA ACUCAGCA GAA AACUCAGC GAA AGGGUGGU GAA AAGGGUGG GAA AUJUUUCUU GAA AUUCCACC GAA AGATJUCCA GAA AUCAGUUG GAA AUTJUAUCA GAA AGUUUCCU GAA AAGUUUCC GAA AGAAGUUUT GAA AAGAAGUU Targe Seqence Ribozyme Sequence 1816 CUUCUGCU C ACACCACU 1816 CUUCGCU ACACACUAGUGGUGU CUGAUGA X GAA AGCAGAAG a

S

nt..

Posi tion 1839 1845 1855 1856 1867 1890 1892 1893 1895 1896 1903 1907 1908 1909 15 1910 1924 1943 1944 1946 1954 1955 1956 1961 1965 25 1975 1990 1998 2001 2007 2023 2053 2055 2056 2061 2067 Target Sequence

GGGACAGU

GUCUGAAU

CCAACUGU

CAACUGUU

GCAGACCU

CACCGAAU

CCGAAUAU

CGAAEJAUU

AAUALUUCU

AUAUUCLU

UACAAGCU

AGCUCCGU

GCUCCGtU

CUCCGUUU

UCCGUUUU

ACCAGCAU

GACAAUGU

ACAAUGUU

AAUGUEJCU

CAAAGCAU

AAAGCAUU

AAG CAUUU

UUUACAGU

CAGUACCU

AAACAGGU

GAGCCCCU

UGCAGCCU

AGCCUUGU

GUAGCAGU

ACCUGCAU

GAUGACAU

UGACAUCU

GACAUCUU

CLTUCCAGU

GUCAAGCU

UGAAUACC

CCCAACUG

CACGCAGA

ACGCAGAC

GCCUGUGG

UUCUUACA

CUUACAAG

UUTACAAGC

ACAAGCUC

CAAGCUCC

CGTJUtJUAA tJUAAUGGC

UAAUGGCA

AAUGGCAC

AUGGCACC

AGAAGAUG

CUCAAAG C

UCAAAGCA

AAAGCAUU

UACAGUAC

ACAGUACC

CAGUACCU

CCUAAAAA

AAAACAGG

CCUGGCGA

GCAGCCUU

GUAGCAGU

GCAGUACC

CCUGGGAA

CUGUGGAA

UUCCAGUC

CCAGUCAA

CAGUCAAG

AAGCUCGU

GUAAAUAC

GGUAUUCA

CAGIJUGGG

UCUGCGUG

GUCUGCGU

CCACAGG C

UGUAAGAA

CUUTGUAAG

GCUIJGUAA

GAGCUUGU

GGAGCUUG

LTJUAAAACG

GCCAUUTAA

UGCCAUTA

GUGCCAUU

GGUGCCAU

CAUCtJUCU

GCUTJUGAG

UGCUUUGA

AAUGCUUU

GUACUGUA

GGUACUGU

AGGUACUG

UUUUTUAGG

CCUGUUU

UCGCCAGG

AAGGCUGC

ACUGCUAC

GGUACUGC

UUCCCAGG

TJUCCACAG

GACUGGAA

UUGACUGG

CUUGACUG

ACGAGCUU

GUAUUUAC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GA-A

GAA

GAAx

GA-A

GA-A

GAA

GAA

GAA

GA-A

GAA

GAA

GAA

GA-A

GA-A

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

ACUGUCCC

AUUCAGAC

ACAGUUGG

AACAGUUG

AGGUCUGC

AtJUCGG UG

AUAUUCGG

AAUAUUCG

AGAAUAUU

AAGAAUAU

AG CUUGUA

ACGGAGCU

AACGGAGC

AAACG GAG AAAA CCGA AUG CUGGU

ACAUUGUC

AACAUUGU

AGAACAUU

AUGCUTUUG

AAUGCUU

AAAUGCLTU

ACUGUAA-A

AGGUACUG

ACCUGtUU

AGGGGCUC

AGGCUGCA

ACAAGGCU

ACUGCUAC

AUGCAGGU

AUGUCAUC

AGAUGUCA

AAGAUGUC

ACUGGAAG

AGCUUGAC

Targe Seqence Ribozyme Sequence 2070 AAGCUCGU A AAUACGUG 2070 AAGCCGU AAUCGUG CACGUAUU CUGAUGA X GAA ACGAGCUU nt Posi tion 2074 2086 2087 2089 2105 2117 2118 2119 2131 2132 2137 2138 2139 2140 2149 2150 2155 2160 2166 2168 2170 2173 2174 2177 2189 2190 2191 2192 2212 2214 2215 2216 2217 2220 2226 2234 Target SQquence

UCGUAAAU

GAAUGCAU

AAUGCAU

UGCAUUCU

ACGCUGGU

UGAGACAU

GAGACAUI

AGACAUU'U

AAAAGCAU

AAAGCAJ

AUUAUGGU

IJUAUGGUTJ

UAUGGUUU

AUGGUUUU

AGAACACU

GAACACUU

CUUCAAGU

AGUIJGACU

CUUGGGAU

UGGGAUAU

GGAUAUAU

UAUAUCAU

AUAUCALU

UCAUUCCU

AUGAAACU

UGAAACLU

GAAACtUU

AAACULUU

AAGAACCU

GAACCUAU

AACCUAUU

ACCUAULU

CCUAUUUU

AUUUUTUGU

GUUGUGGU

ACAACAGU

CGUGAAUG

CUCAG CC C

UCAGCCCG

AGCCCGGA

AUGUGAGA

UCCAGAAA

CCAGAAA.A

CAGAAAAG

AUGGUUUU

UGGUUIJUC

UUCAGAAC

UCAGAACA

CAGAA CA C

AGAACACU

CAAGUUGA

AAGUUGAC

GACUUGGG

GGGAUAUA

UAUCAUTUC

UCAUUCCU

AUUCCUCA

CCUCAACA

CUCAACAU

AACAUGAA

UUCAUGAA

UCAUGAAU

CAUGAAUG

AUGAAUGG

UUUULUGUU

UUUGUUGU

UUGUUGUG

UGUUGUGG

GUUGUGGU

GUGGUACA

CAACAGUU

GAGAGCAG

CAUUCACG

GGGCUGAG

CGGGCUGA

UCCGGGCU

UCUCACAU

UJUUCUGGA

UUUUCUGG

CUULUCUG

AAAACCAU

GAAAACCA

GUUCUGAA

UGUUCUGA

GUGUUCUG

AGUGUUCU

UCAACUUG

GUCAACUU

CCCAAGUC

UAUAUCCC

GAAUGAUA

AGGAAUGA

UGAGGAAU

UGUUGAGG

AUGUUGAG

UUCAUGUU

UUCAUGAA

AUUCAUGA

CAUUCAUG

CCAUUCAU

AACAAAAA

ACAACAAA

CACAACAA

CCACAACA

ACCACAAC

UGUACCAC

AACUGLUG

CUGCUCUC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CtJGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AUUUACGA

AUG CAUIJC

PLAUGCAUU

AGAAUGCA

ACCAGCGU

AUGUCUCA

AAUGU CU C

AAAUGUCU

AUGCUUUU

AAUGCUTJ

ACCAUALAU

AACCAUAA

AAACCAUA

AAAACCAU

AGUGUUCU

AAGUGUUC

ACUUJGAAG

AGUCAACU

AUCCCAAG

AUAUCCCA

-AUAUAUCC

AUGAUAUA

AAUGAUAU

AGGAAUGA

AGUUUCAU

AAGUUUCA

AAAGUUUC

AAAAGUUU

AGGUTUCUIJ

AUAGGUUC

AAUAGGUTU

AALAUAGGU

AAAAUAGG

ACAAAA-AU

ACCACAAC

ACUGUUGU

Targe Segence Ribozyme Sequence .q nt.

Posi tion 2255 2256 2257 2260 2270 2272 2273 2275 2280 2281 2282 2291 2299 2300 15 2301 2302 2309 2313 2319 2321 2322 2326 2330 2331 2333 2334 2338 2345 2348 2355 2358 2359 2362 2364 2366 2367 Target Seqiuence AAGUGCAU U AGUGCAUU U GUGCAUUU A CAUUUAGU U AAUGAAGU C UGAAGUCU U GAAGUCUU C AGUCUUCU U UCUUGGAU U CUUGGAUU U UtJGGAUUU C ACCCAACU A AAAAGGAU U AAAGGAUU U AAGGAUUtU U AGGAUUUTU U UTUAAAAAU A AAAUAAAU A AUAACAGU C AACAGUCU U ACAGUCUU A UCUUACCU A ACCUAAAU U CCUAAAUU A UAAAUUAU U AAAUUAUU A UAUUAGGU A UAAUGAAU U UGAAUUGU A UAGCCAGU U CCAGUUGU U CAGUUGUTU A UTUGLTUAAU A GUUAAUAU C UAAUAUCU U AAUAUCUU A

UAGUUGAA

AGUUGAAU

GUUGAAUG

GAAUGAAG

TJUCUUGGA

CIJUGGAIU

UUGGAUUU

GGAUUUCA

UCACCCAA

CACCCAAC

ACCCAACU

A.AAGGAUU

UUUAAAAA

UUAAAAAU

UAAAAAUA

AAAAAUAA

AAUAACAG

ACAGUCUJ

UUACCUAA

ACCUAAAU

CCUAAAUU

AAUUAUUA

AUUAGGUA

UTUAGGUAA

AGGUAAUG

GGUAAUGA

AUGAAUUG

GUAGCCAG

GCCAGUUG

GUUAAUAU

AAUAUCUU

AUAUCUEJA

UCUUAAUG

UUAAUGCA

AAUGCAGA

AUG CAGAU

UJUCAACUA

AUUCAACU

CAUUCAAC

CUUCAUUC

UCCAAGAA

AAUCCAAG

AAAUCCAA

UGAAAUCC

UUGGGUGA

GUUGGGUG

AGUUGGGU

AAUCCUUU

UUUUUAAA

ALUtIJJAA

UAUTUUUUA

UUAUUUU

CUGUTUALU

AAGACUGU

UU AGGUAA

AUUUAGGU

AAUTUUAGG

UAAUAA-U

UACCUAAU

UUACCUAA

CAUUACCU

UCAUUACC

CAAUUCAU

CUGGCUAC

CAACUGGC

AUAUUAAC

AAGAUAUU

UAAGAUAU

CAUUJAAGA

UGCAUUAA

UCUGCAUU

AUCUGCAU

CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X.

CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X GAA AUGCACLU GAA ALAUGCACU GAA AAAUGCAC GAAk ACUAAAUC GAA ACIJUCAUU GAA AGACUUCA GAA AAGACUTJC GAA AGAAGACU GAA AUCCAAGA GAA AAUCCAAG GAA AAAUCCAA GAA AGTJUGGGU GAA AUCCUUUI GAA AAUCCUUU GAA AAAUCCUU GAA AAAAUCCU GAA AUUUU-UAA GAA AUUJUAUTU GAA ACUGUUAU GAA AGACUGUU GAA AAGACUGU GAA AGGUAAGA GAA AUUUAGGU GAA AAUUUAGG GAA AUAAUUUA GAA AAUAAUUUJ GAA ACCUAAUA GAA AUUCAUUA GAA ACAAUUCA GAA ACUGGCUA GAA ACAACUGG GAA AACAACUG GAA AUUAACAA GAA AUAUUAAC GAA AGAUAUUA GAA AAGAUAUU Ribozvme Sequence

S

nt.

Posi tion 2376 2377 2378 2379 2380 2381 2382 2393 2401 2402 2403 2405 2409 2411 15 2412 2413 2414 2421 2430 2434 2436 2437 2438 2439 25 2440 2441 2453 2454 2455 2456 2457 2463 2464 2472 2473 Target Sequence

AUGCAGAU

UGCAGAtU

GCAGAUUU

CAGAUUUU

AGAUUUUIJ

GAUUUtUU

AUIUUUUU

AAAAACAU

AAAAUGAU

AAAUGALU

AAUGAUUUI

UGAUUUAU

UUAUCUGU

AUCUGUAU

UCUGUAUU

CUGUAUUU

UGUAUUUU

UAAAGGAU

CAACAGAU

AGAUCAGU

AUCAGUAU

UCAGUAU

CAGUAUUU

AGUAUULU

GUAtJUUUU

UAUUUUUU

UGAUGGGU

GAUGGGU

AtJGGGUUU

UGGGUUU

GGGUUEUU

UTJUGAAAU

UYUGAAALU

UGACACAU

GACACAUU

UUUUUJAAA

UUUUA

UUUAAAAA

UUJAAAAAA

UAAAAAAA

AAAAAAAC

AAAUGALU

UAUCUGUA

AUCUGUAU

UCUGUAUU

UGUAUUUU

UUUTUAAAG

tUAAAGGA

UAAAGGAU

AAAGGAUC

AAGGAUCC

CAACAGAU

AGUAUUUU

uuuuuuCC

UUUUCCUG

UUUCCUGU

UUCCUGUG

UCCUGUGA

CCUGUGAU

CUGUGAUG

UUtJUGAAA

UUUGAAAU

LTUGAAAUU

UGAAAUUU

GAAAUUTUG

UGACACAU

GA CA CATU

AAAAGGUA

AAAGGUAC

UUUAAAAA

UUUUAAkA

UUIUUUUA

UUI-TUUUA

AAUCAUUJ

UACAGAUA

AUACAGAU

AAUACAGA

AAAAUACA

CUUTUAAAA

UCCUEJUAA

AUCCUUUA

GAUCCUUU

GGAUCCUJ

AUCUGUUG

AAAAUACU

GGAAAAAA

CAGGAAAA

ACAGGAAA

CACAGGAA

UCACAGGA

AUCACAGG

CAUCACAG

UUTUCAAAA

AUUJUCAAA

AAUUUTCAA

AAAUUUCA

CAAAUUUC

AUGUGUCA

AAUGUGUC

UACCUUUT

GUACCUTU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

GAA

GAlA GAlA

GAA

GAlA

GAA

GAA

GAlA

GAA

GAA

GAA

GAA

GAA

GAA

AUCUGCAU

AAUCUGCA

AAAUCUG C

AAAAUCUG

AAAAAUCU

AAAAAAU C

AAAAAAAU

AUGUUUUU

AUCAUUUU

AAUCAUU

AAAUCAUU

AUAAAUCA

ACAGAUAA

AUACAGAU

AAUACAGA

AAAUACAG

AAAAUACA

AUCCUUUA

AUCUGUUG

ACUGAUCU

AUACUGAU

AAUACUGA

AAAUACUG

AAAAUA CU

AAAAAUAC

AAAAAAUA

ACCCAUCA

AACCCAUC

AAACCCAU

AAAA C CCA

AAAAACCC

ATUUCAAA

AAUULUCAA

AUGUGUCA

AAUGUGUC

TarcetiSe~unce Ribozyme Sequence 2480 UAAAAGGU A CUCCAGUA 2480 UAAAGGU CUCAGUA UACUGGAG CUGAUGA X GAA ACCUUUUA a.

a nt.

Posi tion 248 2490 2491 2492 2496 2497 2498 2501 2505 2509 2515 2521 2523 2525 2526 2527 2528 2529 2535 2539 2547 2548 2551 2553 2559 2564 2565 2568 25B0 2581 2582 2585 2591 2594 2601 Target Seqruence

ACUCCAGU

UCCAGUAU

CCAGUAUU

CAGUAUUU

AUUUCACU

UUUCACLU

UUCACUU

ACUUUTUCU

UUCUCGAU

CGAUCACU

CUAAACAU

AUAUGCAU

AUG CAUAU

GCAUAUAU

CAUAUAUU

AUAUAUUU

UAUAUUUU

AUAUUULTU

UUAA.AAAU

AAAUCAGU

AAAAGCAU

AAAGCAUJ

GCAUUACU

AUTUACUCU

CUAAGUGU

UGUAGACU

GUAGACLU

GACUTUAAU

UGUGACAU

GUGACAUU

UGACAUUU

CAUUJUAAU

AUCCAGAU

CAGAUUJGU

UAAAUGCU

UUTUCACLU

UCACUUUUJ

CACUUTUC

ACUUUJCU

UUCUCGAU

UCUCGAUC

CUCGAUCA

GAUCACUA

ACUAAACA

AACAUAUG

UGCAUAUA

UAUUUUUA

UUTUUAAA

UUUAAAAA

UUAAAAAU

UAAAAAUC

AAAAAUCA

AAA.AUCAG

AGUAAAAG

AAAGCAUU

ACUCUAAG

CUCUAAGU

UAAGUGUA

AGUGUAGA

GACUUAAU

AAUACCAU

AUACCAUG

CCAUGUGA

UAAUCCAG

AAUCCAGA

AUCCAGAU

CAGAUUGU

GUAAAUGC

.AAUGCUCA

AUUUAUGG

AAGUGAAA

AAAAGUGA

GAAAAGUG

AGAAAAGU

AUCGAGAA

GAUCGAGA

UGAUCGAG

UAGUGAUC

UGUUUAGU

CAUAUGLU

UAUAUGCA

UAAAAAUA

UTUUAAAAA

UUULTUAAA

AUUUEJUAA

GAUTJUUUA

UGAUUUU

CUGAUUtU

CUULTUACU

AAUGCUUUT

CUAGAGU

ACUUAGAG

UACACUUA

UCUACACU

AUTJAAGUC

AUGGUAUU

CAUGGUAU

UCACAUGG

CUGGAUUA

UCUGGAUU

AUCUGGAU

ACAAUCUG

GCAIJUUAC

UGAGCAUU

CCAUAAAU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA ACUGGAGU GAA AUACUGGA GAA AAUACUGG GAA AAAUACUG GA-A AGUGAAAU GA-7A AAGUGAAA GAA AAAGUGAA GA.A AGAAAAGU GAA AUCGAGAA GAA AGUGAUCG GAA AUGUJTJUAG GAA AUG CAUAU GAA AUAUGCAU GAA AUAUAUGC GAA AAUAUAUG GAA AAAUAUAU GAA AAAAUAUA GAA AAAAAUAU GAA AUUTUUUAA GAA ACUGAUUU GAA AUGCUUUU GAA AAUGCUUU GAA AGUAAUGC GAA AGAGUAAU GAA ACACUHAG GAA AGUCUACA GAA AAGUCUAC GAA AUUAAGUC GAA AUGUCACA GAA AAUGUCAC GAA AAAUGUCA GAA AUEJAAAUG GAA AUCUGGAU GAA ACAAUCUG GAA AGCAUUUA Ribozxrme Sequence 2604 AUGCUCAU U UAUGGUUA 2604 AUGCCAU UAUGUUA UAACCAUA CUGAUGA X GAA AUGAGCAU nt.

Posi tion 2605 2606 2611 2612 2620 2627 2631 2632 2633 2635 2638 2648 2649 2650 2651 2657 2658 2659 2660 20 2664 2665 2669 2673 2674 2675 2681 2682 2684 2685 2690 2697 2700 2701 2702 2703 2705 Tar et Sequence

UGCUCAUU

C CUCAUUU

UUTJAUGGU

UUAUGGUU

AAUGACAU

UTJGAAGGU

AGGUACAU

GGUACAUU

GUACAUUU

ACAUUtUAU

UUUALUGU

CAAACCAU

AAACCAUU

AACCAUUUT

ACCAUUUU

UUAUGAGU

UAUGAGUUJ

AUGAGUUU

UGAGUUUEIJ

UUTUUCUGU

tJUUCUGUU

UGUUAGCU

AGCUUGCU

GCUtJGCUU

CUUGCUUUJ

UUAAAAAU

UAAAAALU

AAAAUUAU

AAAUUAUU

AUUACUGU

UAAGAAAU

GAAAUAGU

AAAUAGtU AAUAGUtU

AUAGUUUEJ

AGULTUUAU

AUGGUIJAA

UGGUTUAAU

AAUGACAU

AUGACAUU

GAAGGUAC

CAUTJUAU'U

UAUUGUAC

AUUGUACC

UUGUACCA

GUACCAAA

C CAAA CCA

TJUAUGAGU

UAUGAGUU

AUGAGULU

UGAGUTUU

UUCUGUTUA

UCUGUUAG

CUGUUAGC

UGUUAGCU

AGCUUGCU

GCUUGCTU

GCUUUAAA

UAAAAALU

AAAAAUUA

AAAAtJUAU

AUUACUGU

UIJACUGUA

ACUGUAAG

CUGUAAGA

AGAAAUAG

GUTJUUAUA

UIJAUAAAA

UAUAAAAA

AUAAAAAA

UAAAAAAU

AAAAALTUA

UUAACCAU

AUUAACCA

AUGUCAUU

AAUGUCAU

GUACCUUC

AAUAAAUG

GUACAAUA

GGUACA-AU

UGGUACAA

UUUGGUAC

UGGUIJUGG

ACUCAUAA

AACUCAUA

AAACUCAU

AAAACUCA

UAACAGAA

CUAACAGA

GCUAACAG

AGCUAACA

AGCAAGCU

AAGCAAGC

UUUAAAGC

AAUUUEJA

UAAUUULU

AUAAUTUUU

ACAGUAAU

UACAGUAA

CUUACAGU

UCIJUACAG

CUAUUUCU

UAUAAAAC

UUEUUAUAA

LTUUUUAUA

UUUUUJUAU

AUUUUTUUA

UAALTUUUU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GA.A

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AAUGAG CA AAAUGAG C

ACCAUAAA

AAC CAUAA

AUGUCAUU

ACCTJCA

AUGUACCU

AAUGUACC

AAAUGUAC

AUAAAUGU

ACAAUAAA

AUGGUUUG

AAUGGUUU

AAAUGGUU

AAAAUGGU

ACUCAUAA

AACUCAUA

AAACUCAU

AAAACUCA

ACAGAAAA

AACAGAAA

AGCUAACA

AGCAAGCU

AAGCAAGC

AAAG CAAG

AUUUUUAA

AAUIJTJUUA

AUAAUTUU

AAUAAUJU

ACAGUA-AU

AUUUCUU.A

ACUAUUUC

AACUAUUU

AAACUAUU

AAAACUAU

AUAAAACU

Targe Seqence Ribozvme Sequence nt.

Posi tion 2712 2713 2715 2717 2718 2719 2720 2721 2723 2724 2728 2731 2732 2733 2736 2737 2738 2741 2761 2762 2763 2764 2765 2772 2777 2779 2780 2787 2802 2804 2816 2822 2843 2849 2850 Target Seq~uence

UAAAAAAU

AAAAAAUU

AAAAUUAU

AAUUAUAU

AUTUAUALU

UTJAUAUUU

UAUAUULU

AUAIUUU

AtUUUUAU

UUUUEJAUU

UAUUCAGU

UCAGUAAU

CAGUAALTU

AGUAAUUtJ

AAUUUAAU

AUUTUAALU

UTJUAAUUU

AAUUUUGU

AAAAACGU

AAAACGUU

AAACGULTU

AACGUIUU

ACGUUUUU

UUGCUGCU

GCUAUGGU

UAUGGUCU

AUGGUCUU

UAGCCUGU

CUGCUAGU

C CUAGUAU

GGGGCAGU

GUAGAGCU

AAGAAACU

CUUGGUGU

UUGGUGUU

AUAUULUU

UAUJLJLtA

UUUUUAUU

UEJUAUUCA

UUAUtJCAG

UATJUCAGU

AUUCAGUA

UUCAGUAA

CAGUAAUU

AGUAAUTU

AUUTJAAUU

UAAUtUUC

AAUUUTUGU

AUUtJUGUA

UUGUAAAU

UGUAAAUG

GUAAAUGC

AAUGCCAA

UUUEJGCUG

UUUGCUGC

UETJGCUGCU

UGCtJGCUA

GCUGCUAU

UGGUCUUA

UtJAGCCUG

AGCCUGUA

GCCUGUAG

GACAUGCU

UCAGAGGG

AGAGGGGC

GAGCUUGG

GGACAGAA

GGUGUUAG

AGGUAALU

GGUAALUUG

AAAAAUAU

UAAAAAUA

AAUAAAAA

UGAAUAAA

CtJGAAUAA

ACUGAAUA

UACUGAAU

UEJACUGAA

AAUTUACUG

AAAUUACU

AATJUAAAU

CAAAAUTUA

ACAAAAUU

UACAAAAU

AUT-UACAA

CAUUACA

GCAtUUAC

UTJGGCAUU

CAGCAAAA

GCAGCAAA

AGCAGCAA

UAGCAGCA

AUAGCAGC

UAAGACCA

CAGGCUAA

UACAGGCU

CUACAGGC

AGCAUGUC

CCCUCUGA

GCCCCUCU

CCAAGCUC

UUCUGUCC

CUAACACC

AAUUACCU

CAALTUACC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA AUUUUUUA GAA AAUUUIJ GAA AUAAUJU GAA AUAUAAUU GAA AAUAUAAU GAA AAAUAUAA GAA AAAAUAUA GAA AAAAAUAU GAA AUAA.AAAU GAA AAUAAAAA GAA ACUGAAUA GAA AUUACUGA GAA AAUUACUG GAA AAAUUACU GAA AUUAAAUTJ GAA AAUUAAAU GAA AAAtJUAAA GAA ACAAAAUU GAA ACGUULUU GAA AACGUUUU GAA AAACGULU GAA AAAACGUU GAA AAAAACGU GAA AGCAGCAA GAA ACCAUAGC GAA AGACCAUA GAA AAGACCAU GAA ACAGGCUA GAA ACUAGCAG GAA AUACUAGC GAA ACUGCCCC GAA AGCUCUAC GAA AGUUUCUU GAA ACACCAAG GAA AACACCAA Ribozyme Sequence

S

S.

S

2854 UGtTUAGGU A AUUGACUA 2854 U~tUAGU A UUGCUAUAGUCAAU CUGAUGA X GAA ACCUAACA nt., Posi tion 2857 2862 2869 2872 2874 2875 2876 2882 2883 2884 2885 2886 2889 2890 2891 2892 2894 2896 2898 20 2900 2902 2906 2907 2908 25 2909 2910 2 911 2912 2913 2916 2917 2924 2928 2929 2937 Target Sequence UAGCUAAU U AAUUGACU A UAUGCACU A GCACUAGU A ACUAGUAU U CUACUAUU U UAGUAUUI C UUCAGACU U UCAGACUU U CAGACUUU U AGACULTUU U GACUUUU. A UUUUUAAU U UUUUAAUU U UUUAAUU U EUAAUUU'U A AAUUUUAU A UIJUUAUAU A UUJAUAUAU A AUAUAUAU A GACUAUG C

UGCACUAG

GUAUUUCA

UUUCAGAC

UCAGACUJ

CAGACUUU

AGACUUU

UUUAALUU

UTUAAUUUU

UAAUUUJUA

AAIUUUAU

AUIJUUAUA

UTJAUAUAU

UAUAUAUA

AUAUAUAU

UAUAUAUA

UAUAUAUA

UAUAUACA

UAUACAUJ

UACAULTUU

GCAUAGUC

CUAGUGCA

UGAAAUAC

GUCUGAAA

AAGUCUGA

AAAGUCUG

AAAAGUCU

AAAUUJAAA

AAAAUUAA

UAAAAUUA

AUAAAAUU

UAUAAAAU

AUAUAUAA

UAUAUAUA

AUAUAUAU

UAUAUAUA

UAUAUAUA

UGUAUAUA

AAUGUAUA

AAAAUGUA

AAAAAAUG

GGAAAAAA

AGGAAAAA

AAGGAA7AA

GAAGGAAA

AGAAGGAA

CAGAAGGA

GCAGAAGG

UGCAGAAG

UAUTJGCAG

GUAUUGCA

LTUCAAAUG

AGUUUUCA

AAGUTUUUC

UCCCAAAC

CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA, X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AUIUA C CUA

AGUCAAUU

AGUG CAUA

ACUAGUGC

AUACUAGU

AAUACUAG

AAAUACUA

AGUCUGAA

AAGUCUGA

AAAGUCUG

AAAAGUCU

AAAAAGUC

AUUAAAAA

AAUUAAAA

AAAUTUAAA

AAAAUUAA

AUAAAAUUI

AUAUAAAA

AUAUAUAA

AUAUAUAU

AUAUAUAU

AUGUAUAU

AAUGUAUA

AAAUGUAU

AAAAUGUA

AAAAAUGU

AAAAAAUG

AAAAAAAU

AAAAAAAA

AGGAAAAA

AAGGAAA

AUUGCAGA

AUGUALJUG

AAUGUAU

AGLTUUUCA

Targe SeqenceRibozyme Sequence AUAUAUAU A CAUUUTUUU AUAUACAU U TUULUCC UAUACAUU U UUUUUCCU AUACAUUTU U LULTUUCCU UACAUUUU U tUUCCUTUC ACAUUUUU U UUCCUUCU CAUUUUTJU U UCCUUCUG AUTJUUUUU U CCUUCUGC UUUUUUULU C CUUCUGCA UUUUUCCU U CUGCAAUA UUUUCCT-JU C UGCAAUAC UCUGCAAU A CAUUUGAA CAAUACAU U UGAAAACU AAUACAUTU U GAAAACLU UGAAAACU U GUUUGGGA 2940 AAACUUGU U UGGGAGAC 2940 AAACUGU UGGAGACGUCUCCCA CUGAUGA X GAA ACAAGUUU nt Position 2941 2950 2956 2957 2958 2959 2960 2961 2969 2970 2971 2972 2973 2974 2977 2978 2980 2983 2987 2988 2989 2991 3003 3009 25 3010 3012 3013 3014 3015 3016 3030 3033 3039 3042 3046 3047 Target Sequence

AACUUGUU

GGGAGACU

CUCUGCAU

UCUGCAUU

CUGCAUU

UG CAUUUU G CALUUJUU

CAUIUUUU

AUUGUGGU

UUGUGGUJ

UGUGGUUU

GUGGUUUJ

UGGUTUUU

GGULUUU

UUUUEJUGU

UUUUUGUUJ

UUUGUUAU

GUUALTUGU

UUGUUGGU

UGUTUGGUUI

GUUJGGUUU

UGGUUUAU

GCAUGCGU

GUUGCACU

UUGCACUU

GCACEJUCU

CACUU CLU

ACUUCUUU

CUPTJUUu UUCUUTUUu

AUGUGUGU

UGUGtJUGU

GUUGAUGU

GAUGUUCU

UUCUAUGU

UCUAUGUU

GGGAGACU

UG CAUUUU

UUTUAUJG

UUUAUUGU

UUAUUGUG

UATJUGUGG

AUUGUGGU

UUGUGGUU

UUUUUGUU

UUUUGUUA

UUIJGLUAU

UUGUUAUU

UGUUAUUG

GUUAUUGU

AUTUGUUGG

UTJGUUGGU

GUUGGUULI

GGUUUAUA

UAUACAAG

AUACAAGC

UACAAGCA

CAAGCAUG

G CACUUCU CUUUUtJUG

LUUUUUGG

UUUUGGGA

UUtUGGGAG

UTJGGGAGA

UGGGAGAU

GGGAGAUG

GUUGAUGU

GAUGUUCU

CUAUGUUU

UGUUUUGU

UUGUUUUG

UGUUUUGA

AGUCUCCC

AAAAUGCA

CAAUAAAA

ACAAUAA-A

CACAAUAA

CCACAAUA

ACCACAAU

AACCACAA

AACAAAAA

UAACAAAA

AUAACAAA

A.AUAACAA

CAAUAACA

ACAAUAAC

CCAACAAU

ACCAACAA

AAACCAAC

UAUAAACC

CUUGUAUA

GCUTUGUAU

UGCUUGUA

CAUGCUUG

AGAAGUG C

CAAAAAAG

CCAAAAAA

UCCCAAAA

CUCCCAAA

UCUCCCAA

AUCUCCCA

CAUCUCCC

ACAUCAAC

AGAACAUC

AAACAUAG

ACAAAACA

CAAAACAA

UCAAAACA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AACA.AGUU

AGUCUCCC

AUGCAGAG

AAUGCAGA

AAAUGCAG

AAAAUG CA AAAAAUG C

AAAAAAUG

ACCACAAU

AACCACAA

AAACCACA

AAAACCAC

AAAA.ACCA

AAAAAAC C

ACAAAAAA

AACAAAAA

AUAACAAA

ACAAUAAC

ACCAACAA

AACCAACA

AAACCAAC

AUAAACCA

ACGCAUGC

AGUGCAAC

AAGUG CAA AGAAGUG C

AAGAAGUG

AA.AGAAGU

AAAAGAAG

AAAAAGAA

ACACACAU

ACAACACA

ACAUCAAC

AGAACAUC

ACAUAGAA

AACAUAGA

Tarqe SeqenceRibozxrme Sequence Target Sequence Targe Seqence Ribozyme Sequence 9 9 9@ 9* 9 9 9 *e9.

Posi tion 3048 3051 3052 3053 3060 3071 3072 3073 3074 3076 3079 3080 3087 3094 3095 3099 3105 3115 3116 20 3117 3119 3125 3130 3132 3141 3150 3157 3158 3185 3189 3196 3204 3205 3213 3214 3240

CUAUGUUU

UGUUUUGU

GUUUTUGU

UIJEUGUUU

IUGAGUGU

CUGACUGU

UGACUGUU

GACUGUUU

ACUGtUUU

UGLUUUAU

UUUAUAAU

UUAUAAUU

UUGG GAG U

UUCUGCAU

UCUGCAUtJ

CALUJUGAU

AUCCGCAU

CCUGUGGU

CUGUGGU

UGUGGUU

UGGUUUCU

CUAAGUGU

UGUAUGGU

UAUGGUCU

AGAACUGU

GCAUGGAU

UCCUGUGU

CCUGUGUU

ACUGUGGU

UGGUUGAU

UAGCCAGU

CACUGCCU

ACUGCCUTJ

AAGAACAU

AGAACAUU

ACUGAACU

U GUtJUUGAG U UUGAGUGU U UGAGUGUA U GAGUGUAG A GCCUGACU U UUAUAAUU U UAUAAUJU U AUAAUUUG A UAALTUUGG A AUUUGGGA U UGGGAGUU U GGGAGUUC U CUGCAJUU U UGAUCCGC U GAUCCGCA C CGCAUCCC C CCCUGUGG U UCUAAGUG U CUAAGUGU C UAAGUGUA A AGUGUAUG A UGGUCUCA C UCAGAACU C AGAACUGU U GCAUGGAU C CUGUGUUU U UGCAACUG U GCAACUGG U GAUAGCCA A GCCAGUCA C ACUGCCUU U AAGAACAU A AGAACALU U UGAUGCAA U GAUGCA1AG U UUGAGAUA

CUCAA.AAC

ACACUCAA

UACACUCA

CUACACUC

AGUCAGG C

AAUUAUAA

AAAUUAUA

CAAAUUTAU

CCAAALTUA

UCCCAAAU

AACUCCCA

GAACUCCC

AAAUGCAG

GCGGAUCA

UGCGGAUC

GGGAUGCG

CCACAGGG

CACIJUAGA

ACACUUAG

UACACUA

CAUACACU

UGAGACCA

AGUUCUGA

ACAGUUCU

AUCCAUGC

AAACACAG

CAGUUGCA

CCAGtJUGC

UGGCUAUC

UGACUGGC

AAGGCAGU

AUGUUCUU

AAUGUUCU

UUGCAUCA

CUTUGCAUC

UAUCUCAA

CUGAUGA X GA-A AAACAUAG CUGAUGA X GAA ACAAAACA CUGAUGA X GAA A.ACAAAAC CUGAUGA X GAA AAACAAAA CUGAUGA X GAA ACACUCAA CUGAUGA X GAA ACAGUCAG CUGAUGA X GAA AACAGUCA CUGAUGA X GAA AAACAGUC CUGAUGA X GAA AAAACAGU CUGAUGA X GAA AUAAAACA CUGAUGA X GA-A AUUAUAA-A CUGAUGA X GAA AAUUAUAA CUGAUGA X GAA ACUCCCPA CUGAUGA X GAA AUG CAGAA CUGAUGA X GAA AAUGCAGA CUGAUGA X GAA AUCAAAUG CUGAUGA X GAA AUGCGGAU CUGAUGA X GAA ACCACAGG CUGAUGA X GAA AACCACAG CUGAUGA X GAA AAACCACA CUGAUGA X GAA AGAAACCA CUGAUGA X GAA ACACUUAG CUGAUGA X GAA ACCAUACA CUGAUGA X GAA AGACCAUA CUGAUGA X GALA ACAGUUCU CUGAUGA X GAA AUCCAUGC CUGAUGA X GAA ACACAGGA CUGAUGA X GAA AACACAGG CUGAUGA X GAA ACCACAGU CUGAUGA X GAA AUCAACCA CUGAUGA X GAA ACUGGCUA CUGAUGA X GAA AGOCAGUG CUGAUGA X GAA AAGGCAGU CUGAUGA X GA-A AUGUUCUU CUGAUGA X GAA AAUGUUCU CUGAUGA X GAA AGUJUCAGU nt.

Posi tion 3241 3242 3248 3258 3261 3262 3269 3272 3280 3293 3294 Target Sequence Ribozyme Sequence CUGAACUU U UGAACUUU U UUIJGAGAU A GACGGUGU A GGUGUACU U GUGUACUU A UACUGCCU U UGCCUUGU A AGCAAAAU A UGUGCCCU U GUGCCCUU A

UGAGAUAU

GAGAUAUG

UGACGGUG

CUUACUGC

ACUC C CTU

CUGCCUUG

GUAGCAAA

GCAAAAUA

AAGAUGUG

AUUUUACC

UUTJUACCU

AUAUCUCA

CAUAUCUC

CACCGUCA

C CAG HAAG

AAGGCAGU

CAAGGCAG

UUUGCUAC

UAUUUTUGC

CACAUCUU

GGUAAAAU

AGGUAAAA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAlA

GAA

GAlA

GAA

GAA

GA-A

GAA

GAA

GAA

GAA

GAA

AAGUUCAG

AAAGIJUCA

AUCUCAAA

ACACCGUC

AGUACACC

AAGUACAC

AGGCAGUA

ACAAGGCA

AUUUUGCU

AGGGCACA

AAGGG CAC Where IIX" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252) The length of stem II may be 2 base-pairs.

Table XV: Mouse c-myb Hammerhead Ribozxrme and Target *0 0 00..0 sees* Sequence nt. T Position

C

12 G 33 G 63 C

C

U

93 G 113 C 134 G 145 G 149 G 160 G 231 U 234 A 241 U arget Sequence 'CGGGGCUC tJUGGCGGA GGGCUCUU GGCGGAGC CCCGCCUC GCCAUGGC ACAGCAUC UACAGUAG AGCAUCUA CAGUAC CUACAGUA GCGAUGAA AAGACAUU GAGAUGUG CAUGACUA CGAUGGGC CCCAAAUC UGGAAAGC AAAGCGUC ACTJUGGGG CGUCACUU GGGGAAAA GAAAACUA GGUGGACA GGAAAGUC AUUGCCAA AAGUCAUU GCCAAUUA UGCCAAUU AUCUGCCC Ribozyme Sequence UCCGCCAA CUGAUGA X GCUCCGCC CUGA(JGA X GCCAUGGC CUGAUGA X CUACUGUA CUGAUGA X CGCUACUG CUGAUGA X UUCAUCGC CUGAUGA X CACAUCUC CUGAUGA X GCCCAUCG CUGAUGA X GCUUUCCA CUGAUGA X CCCCAAGU CUGAUGA X tJUUUCCCC CUGAUGA X UGUCCACC CUGAUGA X UUGGCAAU CUGAUGA X UAAUUGGC CUGAUGA X GGGCAGAU CUGAUGA X GAA AGCCCCGG GAA AGAGCCCC GAA AGGCGGGC GAA AUG CUGUG GAA AGAUCCUG GAA ACUGUAGA GAA AUGUCUUC GAA AGUCAUGG GAA AUUUGGGC GAA ACGCUUUC GAA AGUGACGC GAA AGUUUJCC GAA ACUUUCCA GAA AUGACULU GAA AUUGGCAA

S.

0@ S

S

OOOS

SO

S.

S

OS

SS S

S.

S.

S S *5*S

S

5.55 0 *555 S S S. S @005 S

*.SS

0@

S

0005 0O@@ nt.

Position 242 244 264 306 309 316 337 345 348 354 357 365 370 379 383 387 388 390 401 20 402 414 427 448 449 25 454 462 463 473 498 30 501 502 503 520 543 571 577 Target Seguence UG CCAAUUA

CCAAUUAUC

ACAGAUGHA

CCUGAACUC

GAACUCAUC

UCAA.AGGUC

AAGAAGAUC

CAGAGAGUC

AGAGUCAUA

AUAAAG CUU

AAGCIJUGUC

CCAGAAAUA

AAUAUGGUC

CGAAGCGUU

GCGUUGGUC

UGGUCUGUU

GGUCUGUUA

UCUGUUAUU

CAAGCACUU

AAGCACUUA

GGGAGAAU

AGCAGUGUC

ACAACCAUU

CAACCAUU

AULJUGAAUC

CCAGAAGUU

CAGAAGUUJA

GAAAACCUC

GACAGAAUC

AGAAUCAUU

GAAUCAUUU

AAUCAUUUA

ACAAGCGUC

GCAGAGAUC

GGACUGAUA

AUAAUGCUA

UCUGCCCA

UGCCCAAC

CAGUGCCA

AUCAAAGG

AAAGGUCC

CCUGGACC

AGAGAGUC

AUAAAGCU

AAGCUUGU

GUCCAGAA

CAGAAAUA

UGGUCCGA

CGAAGCGU

GGUCUGUU

UGUUAUUG

AUUGCCAA

UUGCCAAG

GCCAAGCA

AAAAGGGA

AAAGGGAG

GGAA-AGCA

GGGAGAGG

UGAAUCCA

GAAUCCAG

CAGAAGUU

AAGAAAAC

AGAAAACC

CUGGACAG

AUUUACCA

UACCAGGC

ACCAGGCA

CCAGGCAC

UGGGGAAC

GCAAAGCU

AUG CUAUC

UCAAGAAC

UGGG CAGA

GUUGGGCA

UGGCACUG

CCUUUGAU

GGACCIUU

GGUCCAGG

GACUCUCU

AGCUTJUAU

ACAAGCLU

IJUCUGGAC

UAUUUCUG

UCGGACCA

ACGCUUCG

AACAGACC

CAAUAACA

UTUGGCAAU

CUUGG CAA

UGCUUGGC

UCCCUUJUu CUCCCUU~u

UGCUUUCC

cCUCUCCC

UGGAUUCA

CUGGAUUC

AACUTJCUG

GUUUUCUU

GGIUUUCU

CUGUCCAG

UGGUAAAU

GCCUGGUA

UGCCUGGU

GUGCCUGG

GUUCCCCA

AGCUUUGC

GAUAGCAU

GUUCLTUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AAUJGG CA

AUAAUUGG

ACAUCUGU

AGIJUCAGG

AUGAGUUJC

AC CUUUGA

AUCUUCU-U

ACUCUCUG

AUGACUCU

AGCUtJUAU

ACAAGCUU

AUUUCUGG

ACCAUAUU

ACGCEJUCG

ACCAACGC

ACAGACCA

AACAGACC

AUAACAGA

AGUGCUUG

AAGUGCUU

AUUCUCCC

ACACUGCU

AUGGUUGU

AAUGGtJUG

AUUTCAAAU

ACUUCUGG

AACUUTCUG

AGGUUUC

AUUCUGUC

AUGAUIJCU

AAUGAtJUC

AAAUGALU

ACGCUUGU

AUCUCUGC

AUCAGUCC

AGCAUUAU

Targe Seqence Ribozyme Sequence nt.

Position 579 595 596 607 629 643 644 677 678 691 694 695 704 705 716 721 725 727 732 20 734 736 749 752 756 25 767 769 773 775 776 783 801 803 808 813 814 Target Sequence AAUGCUAUC AAGAACCA ACUGGAAtJU CCACCAUG CUGGAAUUC CACCAUGC CCAUGCGUC GCAAGGUG GGAAGGCUA CCUGCAGA AGAAGCCUU CCAAAGCC GAAGCCUUC CAAAGCCA CACGAGCUU CCAGAAGA ACGAGCUUC CAGAAGAA AGAACAAUC AUUUGAUG ACAAUCAUU UGAUGGGG CAAUCAUUU GAUGGGGTJ GAUGGGGUU UGGGCAUG AUGGGGUtJU GGGCAUGC GCAUGCCUC ACCUCCAU CCUCACCUC CAUCUCAG ACCUCCAUC UCAGCUCU CUCCAUCUC AGCUCUCU UCUCAGCUC UCUCCAAG UCAGCUCUC UCCAAGUG AGCUCtJCUC CAAGUGGC UGGCCAGUC CUCCGUCA CCAGUCCUC CGUCAACA UCCUCCGUC AACAGCGA CAGCGAAUA UCCCUAUU GCGAAUAUC CCUAUUAC AUAUCCCUA UUACCACA AUCCCUAUU ACCACAUC UCCCUAUUA CCACAUCG UACCACAUC GCCGAAGC CAAAACAUC UCCAGUCA AAACAUCUC CAGUCACG UCUCCAGUC ACGtJUCCC AGUCACGUU CCCUAUCC GUCACGUUC CCUAUCCU Ribozyme Sequence UGGUUCIJU CUGAUGA X GAA AUAGCAUU

S.

S

S

.55.

CAUGGUGG

GCAUGGUG

CACCUUG C

UCUGCAGG

GGCUUUGG

UGGCUUUG

UCUUCUGG

UUCUUCUG

CAUCAAAU

C CC CAU CA

ACCCCAUC

CAUGCCCA

GCAUGCCC

AUGGAGGU

CUGAGAUG

AGAGCUGA

AGAGAGCU

CUUJGGAGA

CACUUGGA

GCCACUUG

UGACGGAG

UGUUGACG

UCGCUGUU

AAUAGGGA

GUAAUAGG

UGUGGUAA

GAUGUGGU

CGAUGUGG

GCUUCGGC

UGACUGGA

CGUGACUG

GGGAACGU

GGAUAGGG

AGGAUAGG

CUGAUGA X GAA ATJUCCAGU CUGAUGA X GAA AAUUCCAG CUGAUGA X GAA ACGCAUGG CUGAUGA X GAA AGCCUUCC CUGAUGA X GAA AGGCUIJCU CUGAUGA X GAA AAGGCUUC CUGAUGA X GAA AGCUCGUG CUGAUGA X GAA AAGCUCGU CUGAUGA X GAA AUUGIJUCU CUGAUGA X GAA AUGAUUGU CUGAUGA X GAA AAUGAUtJG CUGAUGA X GAA ACCCCAUC CUGAUGA X GAA AACCCCAU CUGAUGA X GAA AGGCAUGC CUGAUGA X GAA AGGUGAGG CUGAUGA X GAA AUGGAGGU CUGAUGA X GAA AGAUGGAG CUGAUGA X GAA AGCUGAGA CUGAUGA X GAA AGAGCUGA CUGAUGA X GAA AGAGAGCU CUGAUGA X GAA ACUGGCCA CUGAUGA X GAA AGGACUGG CUGAUGA X GAA ACGGAGGA CUGAUGA X GAA AULTCGCUG CUGAUGA X GAA AUAUUCGC CUGAUGA X GAA AGGGAUAU CUGAUGA X GAA AUAGGGAU CUGAUGA X GAA AAUAGGGA CUGAUGA X GAA AUGUGGUA CUGAUGA X GAA AUGUUUUG CUGAUGA X GAA AGAUGUUU CUGAUGA X GAA ACUGGAGA CUGAUGA X GAA ACGUGACU CUGAUGA X GAA AACGUGAC 818 CGUUCCCUA UCCUGUCG 818 GUUCCUAUCCUUCG CGACAGGA CUGAUGA X GAA AGGGAACG nt.

Position 820 825 830 837 838 841 843 846 852 856 876 887 889 921 935 939 947 980 981 20 1000 1007 1028 1032 1051 1060 1071 1072 1073 1078 1079 1103 1105 1117 1124 1128 1145 Target Sequence Ribozyme Sequence

UUCCCUAUC

UAUCCUGUC

UGUCGCAUU

UGCAUGUU

UGCAUGTJUA

AUG UUAAUA

GUUAAUAUA

AAUAUAGUC

GUCAACGUC

ACGUCCCUC

GCAGCCAUC

GAGACACUA

GACACUAUA

A.AGCGAAUA

GCUGGAGtU

GAGUUGCUC

CCUGAUGUC

GCAGGCAUU

CAGGCAUUA

ACCACACUU

UUGCAGCUA

CAGCACCUC

ACCUCCA-U

CCAGACCUC

AUGGGGAUA

GCACCUGUU

CACCUGUUU

ACCUGUUUC

UTJUCCUGIU

UUCCUGUUU

CACCCCAUC

CCCCAUCUC

CUGCAGAUC

UCCCGGCUC

GGCUCCCUA

AAGUGCCUC

CUGUCGCA

GCAUUGCA

C CAUGUUA

AAUAUAGU

AUAUAGUC

UAGUCAAC

GUCAACGU

AACGUCCC

CCUCAGCC

AGCCGGCU

CAGAGACA

UAACGACG

ACGACGAA

AAGGAGCU

GCUCCUGA

CUGAUGUC

AACAGAGA

ACCAACAC

CCAACACA

GCAGCUAC

CCCCGGGU

CALTUGUGG

GUGGACCA

AUGGGGAU

GUGCACCU

UCCUGUUU

CCUGUUUG

CUGUUUGG

UGGGAGAA

GGGAGAAC

UCUGCCUG

UGCCUGCA

CCGGCUCC

CCUACCUG

CCUGAAGA

ACCAGCAA

UGCGACAG

UGCAAUGC

UAACAUG C

ACUAUAUU

GACUAUAU

GUUGACUA

ACGUUGAC

GGGACGUIJ

GGCUGAGG

AGCCGGCU

UGUCUCUG

CGUCGUUA

UUCGUCGU

AGCUCCLUU

UCAGGAGC

GACAUCAG

UCUCUGUU

GUGUUGGU

UGUGUUGG

GUAGCUGC

ACCCGGGG

CCACAAUG

UGGUCCAC

AUCCCCAU

AGGUGCAC

AAACAGGA

CAAACAGG

CCAAACAG

UU)CU CC CA C UU CUCC C

CAGGCAGA

UGCAGGCA

GGAGCCGG

CAGGUAGG

UCUUCAGG

UUGCUGGU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA AUAGGGALA GAA ACAGGAUA GALA AUGCGACA GALA ACAUGCAA GALA AACAUGCA GAA AUUAACAU GAA AUAUUAAC GAA ACUAUAUU GA-A ACGUTJGAC GAA AGGGACGU GAA AUGGCUGC GAA AGUGUCUC GAA AUAGUGUC GAA AUUCGCUU GALA ACUCCAGC GAA AGCAACUC GAA ACAUCAGO GAA AUGCCUGC GAA AAUGCCUG GAA AGUGUGGU GAA AGCUGCAA GAA AGGUGCUG GAA AUGGAGGU GAA AGGUCUGG GAA AUCCCCAU GAA ACAGGUGC GAA AACAGGUG GAA AAACAGGU GAA ACAGGAA-A GA-A AACAGGAA GAA AUGGGGUG GAA AGAUGGGG GAA AUCUGCAG GAlA AGCCGGGA GAA AGGGAGCC GAA AGGCACUU nt. Target Sequence Pos i- Ribozxrme Sequence tion 1164 1167 1182 1183 1194 1195 1203 1205 1206 1211 1212 1224 1229 1230 1231 1233 1237 1238 1240 1241 1242 1244 1252 1253 25 1271 1277 1278 1288 1292 1293 1298 1303 1306 1308 1311 1315 UG CAUCAUC

AUGAUCGUC

GGCACCAUU

GCACCAUJC

GACAAUGUU

ACAAUGUUJA

AAGAAC CU C

GAACCUCUU

AACCUCUTUA

CUTUAGAAUU

TJUAGAAUUU

GAAACACUC

ACUCCAGJU

CUCCAGUUU

UCCAGIJUUA

CAGUUUAUA

UUAUAGAUUJ

UAUAGAUUC

UAGAtJUCUU

AGAUTUCUUU

GAtJUCUUUC UUcuutTCUU

UGAACACUU

GAACACUTUC

UGAAAACUC

CUCGGGCUU

UCGGGCUUA

AUGCACCUA

ACCUACCUU

CCUACCUUA

CUUACCCUC

CCUCCACUC

CCACUCCUC

ACUCCUCUC

CCUCUCAUU

UCAUUGGUC

GUCCACCA

CACCAGGG

CUGGACAA

UGGACAAU

AAGAACCU

AGAACCUC

UUAGAAUU

AGAAUUUG

GAAUUUGC

UGCAGAAA

GCAGAAAC

CAGUUUAU

UAUAGAUU

AUAGAUUC

UAGAUUCU

GAUUCUUU

CtJUUCUUG

UUUCUUGA

UCtJUGAAC

CUUGAACA

UUGAACAC

GAACACUU

CCAGCAAC

CAGCAACC

GGGCUUAG

AGAUGCAC

GAUGCACC

C CUtJACC C

ACCCUCCA

CCCUCCAC

CACUCCUC

CUCUCAUU

UCAUUGGU

AUEJGGUCA

GGUCACAA

ACAAACUG

UGGUGGAC

CCCUGGUG

TJUGUCCAG

ATJUGUCCA

AGGUUCUU

GAGGUUCU

AAUTUCUAA

CAAAUUCU

GCAAAUUC

UUUCUGCA

GUUUCUG C

AUAAACUG

AAUCUAUA

GAAUCUAU

AGAAUCUA

AAAGAAUC

CAAGAAAG

UCAAGAAA

GUTUCAAGA

UGLUCAAG

GUGUUCAA

AAGUGUUC

GUUGCUGG

GGUUGCUG

CUAAG CC C

GUGCAUCU

GGUGCAUC

GGGUAAGG

UGGAGGGU

GUGGAGGG

GAGGAGUG

A.AUGAGAG

ACCAAUGA

UGACCAAU

UUGUGACC

CAGUUEUGU

*CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA AUCAUCCA GAA ACCAUCADJ GAA AUGGUGCC GAA AAUGGUGC GAA ACAUUGUC GAA AACATUGU GAA AGGUUCU-U GAA AGAGGUUC GAA AAGAGGUIJ GAA AUUCUAAG GAA AAUUCUAA GAA AGUGUIJUC GAA ACUGGAGU GAA AACUGGAG GAA AAACUGGA GAA AUAAACUG GAA AUCUAUAA GAA AAUCUAUA GAA AGAAUCUA GAA AAGAAUCU GAA AAAGAAUC GAA AGAAAGAA GAA AGUGUUCA GAA AAGUGUUC GAA AGUUUUCA GAA AGCCCGAG GAA AAGCCCGA GAA AGGUGCAU GAA AGGUAGGU GAA AAGGUAGG GAA AGGGUAAG GAA AGUGGAGG GAA AGGAGUGG GAA AGAGGAGU GAA AUGAGAGG GAA ACCAAUGA nt.

Position 1333 1366 1367 1371 1373 1374 1375 1381 1387 1389 1397 1401 1404 1412 1414 1417 1423 1433 1434 1446 1453 1461 1462 1466 1471 1485 1489 1499 1518 1530 1531 1541 1550 1557 1560 Target Sequence Ribozyme Sequence

CACCAUGUC

AGGAAAAUU

GGAAAAUUC

AAUUCCAUC

UUC CAUCUUJ UC CAUCUUU

CCAUCUUUA

UIJAGAACUC

CUCCAGCUA

CCAGCUAUC

CAAAAGGUC

AGGUCAAUC

UCAAUCCUC

CGAAAGCUC

AAAGCUCUC

GCUCUCCUC

CUCGAACUC

CACACCAUU

ACACCAUUC

CAUGCCCUU

UUGCAGCUC

CAAGAAAUU

AAGAAAUUA

AAUUAAAUA

AAUACGGUC

AAGAUGCUA

UGCUACCUC

GACCCCCUC

GAGGACCUA

GAUGUGAU

AUGUGAUUA

GCGGGAAUC

GGAUGAAUC

UCUGGAAUU

GGAALTUGUU

GAGACCAG

CCAUCUUU

CAUCUUUA

TUUAGAAC

UAGAACUC

AG AAC UC C

GAACUCCA

CAG CUAUC

UCAAAAGG

AAAAGGUC

AAUCCUCG

CUCGAAAG

GAAAGCUC

UCCUCGAA

CUCGAACU

GAACUCCC

CCACACCA

CAAACAUG

AAACAUGC

GCAGCUCA

AAGAAAUU

AAAUACGG

AAUACGGU

CGGUCCCC

CCCUGAAG

CCUCAGAC

AGACCCCC

CCAUGCAG

CAAGAUGU

AAGCGGGA

AGCGGGAA

GGAUGAAU

UGGAALTUG

GUUGCUGA

GCUGAGUU.

CUGGUCUC

AAAGAUGG

UAAAGAUG

GUTUCUAAA

GAGUUCUA

GGAGIJUCU

UGGAGUUC

GAUAGCUG

CCUUUUGA

GACCUUUU

CGAGGAUU

CUJUUCGAG

GAG CUUUC

UUCGAGGA

AGUUCGAG

GGGAGUUC

UGGUGUGG

CAUGUtJUG

GCAUGUUU

UGAGCUGC

AAUIJUCUU

CCGUAUUU

ACCGUAUU

GGGGACCG

CUUCAGGG

GUCUGAGG

GGGGGUCU

CUGCAUGG

ACAUCUUG

UCCCGCUU

UUCCCGCU

AUUCAUCC

CAAUUCCA

UCAGCAAC

AACUCAGC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

COGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAlA

GAA

GAlA GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

GAA

GAlA GAlA GAlA GAlA

GAA

GAA

GAA

GAA

GAA

GAlA GAlA

GAA

GAA

GAlA GAlA GAlA GAlA GAlA

GAA

GAlA

ACAUGGUG

AULUUC CU AAUUUUC C AUG GALAUT

AGAUGGAA

AAGAUGGA

AAAGAUGG

AGUTUCUAA

AG CUGGAG

AUAGCUGG

ACCUJLTUG

AUUGACCU

AGGAUUGA

AG CUUUCG

AGAGCUUU

AGGAGAG C

AGUUCGAG

AUGGUGUG

AAUGGUGU

AGGGCAUG

AG CUGCAA

AUTUUCLTUG

AAUUUCTU

AUUUAAUU

ACCGUAU

AGCAUCUU

AGGUAGCA

AGGGGGUC

AGGUCCUC

AUCACAUC

AAUCACAU

AUUCCCGC

AUUCAUCC

AUUCCAGA

ACAAUUCC

1568 UGCUGAGUU UCAAGAGA 1568 UGCUAGUUUCAAAGA UCUCUUGA CUGAUGA X GAA ACUCAGCA nt.

Posi tion 1569 1570 1589 1590 1602 1619 1634 1643 1644 1646 1647 1652 1691 1692 1694 1703 1705 1726 1728 20 1729 1731 1732 1739 1743 25 1744 1745 1746 1758 1760 30 1779 1782 1790 1791 1792 1797 1801 Target Sequence Targe Seqence Ribozyne Sequence a C CUGAGUUU

CUGAGUUUC

ACCACCGU

CCACCGUUA

AAAAAAAUC

GGUGGAGUC

UGAGAAAUC

GGGAAACUTJ

GGAAACUUJC

AAACUUCUU

AACUUCUUC

CUUCUGCUC

CCAACUGUU

CAACUGUUC

ACUGtJUCUC

GCAGGCGUC

AGGCGUCUC

CCC CAAAUA

CCAAAUAUU

CAAAUAUUC

AAUALTUCLU

AUALTUCtJUA

UACAAGCUC

AGCUCUGUU

GCUCUGtUU CUCUGUUtU

UCUGUUUUA

ACACCUGUA

ACCUGUAUC

GACAAUGUC

AAUGUCCUC

CAA.AGCCUU

AAAGCCUUTJ

AAGCCUUUA

UTUUACCGUA

CCGUACCUA

CAAGAGAG

AAGAGAGU

ACUGAAAA

CUGAAAAA

AAGCAGGC

GCCAACUG

GGGAAACU

CUUCUGCU

UUCUGCUC

CUGCUCAA

UG CUCAAA

AAACCACU

CUCGCAGG

UCGCAGGC

GCAGGCGU

UCCUGUGG

CUGUGGCA

UUCLTUACA

CUTUACAAG

UTUACAAGC

ACAAGCUC

CAAGCUCU

UGUUtJUAA

UTUAAUGAC

UAAUGACA

AAUGACAC

AUGACACC

UCAGAAGA

AGAAGAUG

CUCAAAC

AAAGCCLTUJ

UACCGUAC

AC CGUAC C

CCGUACCU

CCUAAGAA

AGAACAGG

CUCUCUUG

ACUCUCUU

UUUUCAGU

LUUUUCAG

C CCUGCLU

CAGUJCC

AGUUUCCC

AGCAGAAG

GAGCAGAA

UTUGAGCAG

UUUGAGCA

AGUGGUUU

CCUGCGAG

GCCUGCGA

ACGCCUGC

CCACAGGA

UGCCACAG

UGUAAGAA

CUTUGUAAG

GCUUGUAA

GAGCUUGU

AGAGCUUG

UUAAAACA

GUCAUUAA

UGUCAUUA

GUGUCAUU

GGUGUCAU

UCUUCUGA

CAUCUUCU

GCUUUGAG

AAGGCULU

GUACGGUA

GGUACGGU

AGGUACGG

LTUCUAGG

CCUGUUCU

CUGAUGA

CUGAUGA

CUGAtJGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAEJGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GA-A

GAA

GA-A

GAA

GAA

GAA

GAA

GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

GAA

GAlA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAlA

AACUCAGC

AAACUCAG

ACGGUGGU

AlA C CCUG C

AUULUUUUU

ACUCCACC

AUUUCU CA AGUtJUCCC

AAGUTUUCC

AGAAGUUU

AAGAAGUUJ

AGCAGAAG

ACAGUTUGG

AACAGUUG

AGAACAGU

ACGCCUGC

AGACGCCU

AUtJUGGGG

AUAUUUGG

AAUAULJG

AGAAUAUIJ

AAGAAUAU

AGCUUGUA

ACAGAG CU

AACAGAGC

AAACAGAG

AAAACAGA

ACAGGUGU

AUACAGGU

ACAUUGUC

AGGACAUU

AGGCUUUG

AAGGCUUU

AAAGG CUl

ACGGUAAA

AGGUACGG

nt.

Position 1822 1826 1859 1892 1897 1903 1910 1922 1923 1925 1930 1936 1941 1953 1954 1955 19 67 1968 1973 1974 1975 1976 1985 1986 1992 1997 1998 1999 2011 2014 2028 2052 2053 2054 2057 2063 Target Sequence Ribozyme Sequence

UGGUGGGUC

GGGUCCCUU

GCCAGCAUC

GACGGCCUC

CCUCCGGUC

GUCCGGCUC

UCGGAAAUA

GAACGCGUU

AACGCGUCC

CGCGUUCJC

UCUCAGCUC

CUCGAACUC

ACUCUGGUC

UGAGACAT-TU

GAGACAUUU

AGACAUUUC

AAAAGCAUU

AAAGCAUUA

AUUAUGGLU

UUAUGGUUUT

UAUGGUUUU

AUGGUUUUC

AGAACACUU

GAACACLTUA

UEJAAAAGU'U

AGTJUGACUU

GUUTGACUUU

UUGACLTUUC

ACAUGGCUC

UGGCUCCUC

GGAGCGCUC

AGCCUGAUU

GCCUGAUUU

CCUGAUUUU

GAUUUUGUU

GUUGUGGUA

CCUUGCAG

GCAGCCAU

CUGUGGGA

CGGUCCGG

CGGCUCGG

GGAAAUAC

CGUGAACG

CUCAGCUC

UCAGCUCG

AGCUCGAA

GAACUCUG

UGGUCAUG

AUGUGAGA

UCCAGAAA

CCAGAAA-A

CAGAAAAG

AUGGUUUU

UGGUUUUC

UUCAGAAC

UCAGAACA

CAGAACAC

AGAACACU

AAAAGUUtG

AAAGUTUGA

GACUUtJCG

UCGACACA

CGACACAU

GACACAUG

CUCAGCGU

AGCGUGGA

CAUGGCUG

UTUGUUGUG

UGUUGUGG

GLTUGUGGU

GUGGUACA

CAACAGUUL

CUGCAAGG

AUGGCUGC

UCCCACAG

CCGGACCG

CCGAGCCG

GUAUUUCC

CGUUCACG

GAGCUGAG

CGAGCUGA

UTJCGAGCU

CAGAGUUC

CAUGACCA

UCUCACAU

UT-UCUGGA

UUUUCUGG

CUUUUCEJG

AAAACCAU

GAAAACCA

GUUCUGAA

UGUUCUGA

GUGUUCUG

AGUGUUCU

CAACULUU

UCAACUUt.J

CGAAAGUC

UGUGUCGA

AUGUGUCG

CAUGUGUC

ACGCUGAG

UCCACGCU

CAGCCAUG

CACAACAA

CCACAACA

ACCACAAC

UGUACCAC

AACUGUUG

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GA-A

GAA

GAA

GA-A

GA-A

GA-A

GAA

GAA

GAA

GAA

GAA

GA-A

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AC CCAC CA

AGGGACCC

AUGCUGGC

AGGCCGUC

ACCGGAGG

AGCCGGAC

AUJUC CGA ACG CGLJUC

AACGCGUU

AGAACG CG AG CUGAGA

AGIJUCGAG

ACCAGAGU

AUGUCUCA

AAUGU CU C

AAAUGUCU

AUGCUUUU

AAUGCUUU

ACCAUAAU

AACCAUAA

AAACCAUA

AAAACCAU

AG UGUU CU

AAGUGUUC

ACUUUUAA

AGUCAACU

AAGUCAAC

AAAGUCAA

AGCCAUGU

AGGAGCCA

AGCGCUCC

AUCAGGCU

AAUCAGGC

AAAUCAGG

ACAAAAUC

ACCACAAC

nt.

Position 2071 2092 2093 2094 2095 2096 2099 2103 2109 2111 2115 2119 2120 2121 2130 2139 2140 2141 2142 2143 2144 2145 2146 2147 2148 2154 2157 2160 2167 30 2173 2175 2176 2180 2184 2185 2187 Target Sequence Ribozyme Sequence

ACAACAGUU

AAGUGCAUU

AGUGCAUUU

GUGCAUUUU

UGCATUUUU

GCAULUUUA

UUUUAGUU

UAGUUGCUU

CUUGAGAUC

UGAGAUCUC

AUCUCACUU

CACUUGAUU

ACULTUGAUUU

CtJUGAUUUC

ACACAACUA

AAAAGGAUU

AA.AGGAUUUT

AAGGAUUTU

AGGALTUUUU

GGALULUUUU

GALUUUUU

ALTEUtUUUU

LUUUUUUU

LUUUUUULU

UULUUULUUA

LUUAAAAAUA

AAAAUAAUA

AUAAUAAUA

UAAUGAAUA

AUAACAGUC

AACAGUCUU

ACAGUCtJUA

UCUUACCUA

ACCUAAALUU

CCUAAAUTUA

UAAAUUAUU

GAGAG CAG

UUUAGUUG

UUAGUTJGC

UAGUUG CU

AGUUGCLTU

GtJUG CTUG C CUUJGAGA

GAGAUCUC

UCACUUGA

ACUUGAUU

GAUUUCAC

UCACACAA

CACACAAC

ACACAACU

AAAAGGAU

tULTUUUU

LUUUUUUA

LTUUUUUAA

UUtJUUAAA

UUUUAAAA

UUUAAAAA

UUAAAAAU

UAAAAAUA

AAAAAUAA

AAAAUAAU

AUAAUAAU

AUAAUGAA

AUGAAUAA

ACAGUCUU

LUUACCUAA

ACCUAAAU

CCUAAAUU

AAtJUATJA

AUUAGGUA

UUAGGUAA

AGGUAAUG

CUGCUCUC

CAACUAAA

GCAACUAA

AGCAACUA

AAG CAACU

CAAGCAAC

UCUCAAGC

GAGAUCUC

UCAAGUGA

AAUCAAGU

GUGAA.AUC

UUGUGUGA

GUtJGUGUG

AGTJUGUGU

AUCCUUUU

AAAAAAAA

UAAAAAAA

UUAAAAAA

LTUUAAAAA

UUUUAAAA

UUUUUAAA

ALUUUUAA

UAtUUTLUUA

UALTUUU

AUUALUJUU

AUUAUUAU

UUCAUUAU

UUAUUCAU

AAGACUGTJ

tJUAGGUAA

AUUUAGGU

AAUUUAGG

UAAUAAUU

UACCUAAU

UUACCUAA

CALTUACCU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA ACUGUUGU GAA AUGCACUU GAA A.AUGCACU GAA AAAUGCAC GAA AAAAUGCA GAA AAAALAUGC GAA ACUAAAAA GAA AGCAACUA GAA AUCUCAAG GAlA AGAUCUCA GAlA AGUGAGAU GAA AUCAAGUG GAA AAUCAAGU GAA AAAUCAAG GAA AGUUGUGU GAA AUCCUUUU GAA AAUCCU-U GAA AAAUCCUU GAA AAAAUCCU GAA AAAAAUCC GAA AAAAAAUC GAA AAAAAAAU GAA AAAAAAAA GAA AAAAAAAA GAA AAAAAAAA GAA AUUUUTUAA GAA AUUAUUUTJ GAA AUUAUUAU GAA AUUCAUUA GAA ACUGUUAU GAA AGACUGU GAA AAGACUGU GAA AGGUAAGA GAA AUUUAGGU GAA AAUUUAGG GAA AUAAUUUA nt.

Posi tion 2188 2192 2199 2208 2209 2212 2213 2216 2218 2221 2224 2229 2230 2231 2232 2233 2234 2243 2251 20 2252 2253 2255 2256 2259 2261 2262 2263 2264 2271 30 2280 2284 2286 2287 2288 2289 2303 Target Sequence

AAAUUAUUA

UAUUAGGUA

UAAUGAAUU

GUGACCAUU

UGACCAUUU

CCAUUTUGIJU

CAUUUGJUUA

UUGUUAAUA

GUUAAUAUC

AAUAUCAUA

AUCAUAAUC

AAUCAGAUU

AUCAGAtUU

UCAGAUUUU

CAGAtUUUU

AGAUULUUU

GAULTJUUJA

AAAAAAAUA

AAAAUGAUUJ

AAAUGAULU

AAUGAUtJUA

UGAUUTUAUU

GAUUTUAULU

UUAtUUGUA

AUIJUGUALU

UUUGUAUUtJ LTTGUAtUUU

UGUAUUEJUA

UAGAGAAUA

CAACAGAUC

AGAUCAGUA

AUCAGUAUU

UCAGUAUUU

CAGUAULUU

AGUALUUU

UGGUGAAUU

GGUAAUGA

AUGAAIUC

GUGACCAU

UGUUAAUA

GUUAAUAU

AAUAUCAU

AUAUCAUA

UCAUAAUC

AUAAUCAG

AUCAGAUU

AGAIJUUUU

UUUUAAAA

tJUUAAAAA

UTJAAAAAA

UAAAAAAA

AAAAAAAA

AAAAAAAU

AAAUGAUIJ

UAULTUGUA

AUUUGUAU

ULTUGUALU

UGUAUUUU

GUALTJUA

UUUUAGAG

UUAGAGAA

UAGAGAAU

AGAGAAUA

GAGAAUAC

CAACAGAU

AGUAUUUU

UUUUUGAC

UUUGACUG

UUGACUGU

UGACUGUG

GACUGUGG

UAAAAA7A UCAtJUACC

CAAUUCAU

AUGGUCAC

UAUUAACA

AUAUUAAC

AUGAUAUU

UAUGAUAU

GAUUAUGA

CUGAUUAU

AAUCUGAU

AAAAAUCU

tJUUUAAAA

TUUUUAAA

ULUUUAA

UUUUUUUA

AUUUULUU

AAUCAUUU

UACAAAUA

AUACAAAU

AAUACAAA

AAAAUACA

UAAAAUAC

CUCUAAAA

LTUCUCUAA

AUUCUCUA

UAUUCUCU

GUAUUCUC

AUCUGUUG

AAAAUACU

GUCAAAAA

CAGUCAAA

ACAGUCAA

CACAGUCA

CCACAGUC

UUULTUUUA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAZA

GAA

GAA

GAA

GAA

GAZA

GAZA

GAA

GAA

GAA

GAA

GAA

GAZA

GAA

GAA

GAA

GAA

GAA

GAA

GAZA

GAA

GAA

GAZA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AAUAALUU

ACCUAAUA

AUJUCAUUA

AUGGUCAC

AAUGGUCA

ACAAAUGG

AACAAAUG

AtJUAACAA

AUAUUTAAC

AUGAUAUU

AIJUAUGAU

AUCUGAUU

AAUCUGAU

AAAUCUGA

AAAAUCUG

AAAAAUCU

AAAAAAUC

AUUtUULU

AUCAUUUTJ

AAUCAUUU

AAAUCALU

AUAAAUCA

AAUAAAUC

ACAAAUAA

AUACAAAU

AAUACA.AA

AAAUACAA

AAAAUACA

AUUCUCUA

AUCUGUUG

ACUGAUCU

AUACUGAU

AAUACUGA

AAAUACUG

AAAAUACU

AUUCACCA

Targe Seqence Ribozyme Sequence nt. Target Sequence RizmeSune Ribozyme Sequence Position 2304 2305 2316 2317 2318 2330 2332 2338 2340 2341 2347 2349 2351 2355 2359 2365 2377 2378 2379 20 2380 2381 2399 2400 2402 2403 2404 2411 2412 2416 2420 2427 2430 2433 2434 2445

GGUGAAUU

GUGAAUUUA

AAAAAAAUU

AAAAAAUUU

AAAAAUUUA

AAAGAAAUA

AGAAAUAUC

AUCCCAGUA

CCCAGUAULI

CCAGUAUUC

UUCCAUGUA

CCAUGUAUC

AUGUAUCUC

AUCUCAGUC

CAGUCACUA

CUAAACAUA

AGAGAGAUU

GAGAGAUUU

AGAGAUUUU

GAGALUUU

AGAUUUTTJA

AGAAGCAUU

GAAGCAUUA

AGCAUUALU

GCAUUAUUU

CAUUAUUUEJ

UUGAAUGLU

UGAAUGUUTA

UGUUAGCUA

AGCUAAAUC

UCCCAAGUA

CAAGUAAUA

GUAAUACUU

UAAUACTJUA

GCAACCCUC

UACACAAA

ACACAAAG

CA CAAAG A

UCCCAGUA

CCAGUAU

UUCCAUGU

CCAUGUAU

CAUGUAUC

UCUCAGUC

UCAGUCAC

AGUCACUA

ACUAAACA

AACAUACA

CACAGAGA

UUUAAAAA

UUAAAAAC

UAAAAACC

AAAAACCA

AAAACCAG

AUUUUGAA

UUUUGAAU

UUGAAUGU

UGAAUGUU

GAAUGUUTA

AGCUAAAU

G CUAAAUC

AAUCCCAA

CCAAGUAA

AUACUUAA

CUUAAUG C

AAUGCAAC

AUGCAACC

UAGGAGCU

UUUGUGUA

CUTUGUGU

UCUITLJGUG

UACUGGGA

AAUACUGG

ACAUGGAA

AUACAUGG

GAUACAUG

GACUGAGA

GUGACUGA

UAGUGACU

UGUUUAGU

UGUAUGUU

UCUCUGUG

UUUUUAAA

GULUUUAA

GGtUUUUA

UGGUUUUU

CUGGUUUUI

UUCAAAAU

AUUCAAAA

ACAUUCAA

AACAUUJCA

UAACAUTUC

ATJUUAGCU

GAUUUAGC

TJUGGGAUU

UIJACUUGG

UUAAGUAU

G CAUUAAG

GUUGCAU

GGUUGCAU

AGCUCCUA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GA-A

GA-A

GAA

GA-A

GA-A

GAA

GAA

GAA

GAA

GAA

GA-A

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AAUUCACC

APATUCAC

AUUUUUU

AAUUUUUU

AAAUUUU

AUUUCUUU

AUAUUC U

ACUGGGAU

AUACUGGG

AAUACUGG

ACAUGGAA

AUACAUGG

AGAUACAU

ACUGAGAU

AGUGACUG

AUGUUEJAG

AUCUCUCU

AAUCUCUC

AAAUCUCU

AAAAUCUC

AAAAAUCU

AUG CtUCU

AAUGCUTUC

AUAAUGCU

AAUAAUG C

AAAUAAUG

ACAUUJCAA

AACAUTUCA

AGCUAACA

AUUUAGCU

AcUUGGGA

ATJUACUUG

AGUAtJUAC

AAGUAUUA

AGGGUUGC

2447 AACCCUCUA GGAGCUCA 2447 AACCUCUAGGAGUCA UGAGCUCC CUGAUGA X GAA AGAGGGUU

S

*5

S

S

S

nt.

Position 2454 2457 2458 2465 2468 2471 2473 2480 2482 2484 2485 2486 2488 2489 2491 2493 2498 2499 2500 20 2510 2511 2512 2515 2518 2523 2527 2534 2535 2537 2538 2542 2548 2551 2552 2553 2554 Target Sequence

UAGGAGCUC

GAG CUCAUU

AGCUCAUU

TJUGUGGCUA

UGG CUAAUA

CUAAUAAUC

AAUAAUCUU

UtJGGAAAUA

GGAAAUAUC

AAAUAUCLU

AAUAUCUUJ

AUAUCUJUA

AUCUUUALU

UCtJUUAUUA

UUUAUUAUA

UAUUAUAUA

UAUAGCAUU

AUAGCAtUU

UAGCAUUUA

GAGGAGAUU

AGGAGAUUU

GGAGAtUUU

GAUUUUGUU

UUUTGUUGUC

UGUCAGCUU

AGCtJUGCUU

UUGAAAGUU

UGAAAGUUA

AAAGUUAUU

AAGtJUATJUA

UAUUAUGUA

GUAUGAAUA

UGAAUAGUU

GAAUAGUUU

AAUAGUULT

AUAGUULTUA

AUEJUGUGG

UGUGGCUA

GUGGCUAA

AUAAUCUU

AUCUUGGA

UUGGAAAU

GGAAAUAU

UCLTUUALU

UUUAUTUAU

UAUTUAUAU

AUTUAUAUA

UUAUAUAG

AUAUAGCA

UAUAGCAU

UAGCAUUU

GCAUtJUAU

UAUGAGGA

AUGAGGAG

UGAGGAGA

tJUGLTUGUC

UGUUGUCA

GUUGUCAG

GUCAGCUU

AGCUUGCU

GCUUGAAA

GAAAGUUA

AUUAUGUA

UUAUGUAU

AUGUAUGA

UGUAUGAA

UGAAUAGU

GUUUUAUU

UUAUUGAA

UAUUJGAAA

AUTUGAAAA

UUGAAAAA

C CACAAAU UAG CCACA

UUAGCCAC

AAGAUUAU

UCCAAGAU

AULUCCAA

AUAUUUCC

AAUAAAGA

AUAAUAAA

AUAUAAUA

UAUAUAAU

CUAUAUAA

UGCUAUAU

AUG CUAUA AAAUG CUA

AUAAAUGC

UCCUCAUA

CUCCUCAU

UCUCCUCA

GACAACAA

UGACAACA

CUGACAAC

AAGCUGAC

AGCAAGCU

UUUCAAG C

UAACUUUC

UACAUAAU

AUACAUAA

UCAUACAU

UUCAUACA

ACUAUUCA

AAUAAAAC

UUCAAUAA

UtJUCAAUA

UUUUCAAU

UUULTJCAA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GA-A

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AGCUCCUA

AUGAG CUC AAUGAG CU

AGCCACAA

AUUAG CCA

AUUAUTUAG

AGAUTJAUU

AUUUCCAA

AUAUUU CC

AGAUAUUIJ

AAGAUAUU

AAAGAUAU

AUAAAGAU

AAUAAAGA

AUAAUAAA

AUAUAAUA

AUG CUAUA

AAUGCUAU

AAAUG CUA

AUCUCCUC

AAUCUCCU

AAAU CUC C

ACAAAAUC

ACAACAAA

AGCUGACA

AGCAAGCU

ACUUUCAA

AACUUUCA

AUAACUJU

AAUAACUU

ACAUAAUA

AUUCAUAC

ACUAUTUCA

AACUAUTUC

AAACUAUU

AA.AACUAU

Targe Seqence Ribozyme Sequence nt. Target Sequence Posi- Ribozymre Sequence tion 2556 2565 2566 2568 2570 2571 2572 2573 2574 2576 2577 2581 2584 2585 2586 2589 2590 2591 2594 2617 2618 2625 2630 2631 2632 2633 2638 2640 2643 2652 2656 2661 2672 2678 2679 2703

AGUUUJUAUTJ

GAAAAAAUU

AAAAA.AUUA

AAAAUUAUA

AAUUAUALU

ALTUAUAUJTU

UTUAUAUUUUJ

UAUAUUUU

AUAtUUUUA

AUUUTUUALU

UUUUUAUTUC

UAUUCAGUA

UCAGUAAUII

CAGUAALUU

AGUAAUUUA

AAUUUAAUU

AUUUAAtUU

UUUAALTUUU

AAUUUUGUA

AAAUGUGUU

AAUGUGUUC

UCGCUGCUA

GCUAUGGUU

CUAUGGUU

UAUGGtUUU

AUGGUUUUA

UUUAGCCUA

UAG CCUAUA

CCUAUAGUC

AUGCUGCUA

UGCUAGCUA

GCUAGUGUC

GGGGCAAUA

AUAGAGCUU

UAGAGCUUA

AAGAGACUC

GAAAAAAU

AUAUUUTJTU

UAUUTUUUA

UUULTUALU

UUUAUTUCA

UUAUUCAG

UAUUCAGU

AUUCAGUA

UtJCAGUAA

CAGUAAUU

AGUAAJUU

AULTUAALTU

UAAUUUTUG

AALUTUGU

AUUTUUGUA

UTUGUAAAU

UGUAAAUG

GUAAAUGC

AAUGCCAA

CGCUGCUA

GCUGCUAU

UGGLUEJUA

UUAGCCUA

UAGCCUAU

AGCCUAUA

GCCUAUAG

UAGUCAUG

GUCAUGCU

AUGCUGCU

G CUAGUGU

GUGUCAGG

AGGGGGCA

GAGCUUAG

AGAUGGAA

GAUGGAAA

GGUGUUAG

AUIJUUUTJC

AAAAAUAU

UAAAAAUA

AAUAAPAA

UGAAUAAA

CUGAAUAA

ACUGAAUA

UACUGAAU

UtJACUGAA

AAUUACUG

AAAUUACU

AAUUAAAU

CAAAAUUA

ACAAAAUU

UACAAAAU

ATJUUACAA

CAtJUUACA

GCALTUUAC

UUGGCAUU

UAGCAGCG

AUAGCAGC

UAAAACCA

UAGGCUAA

AUAGGCUA

UAUAGGCU

CUAUAGGC

CAUGACUA

AGCAUGAC

AGCAGCAU

ACACUAGC

CCUGACAC

UGCCCCCU

CUAAGCUC

UUCCAUCU

UUTUCCAUC

CUAACACC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA.

GAA AUAAAACU GAA AUUUIJUUC GA.A AAUUIUUuu GAA AUAAUUULJ GAA AUAUAAUIJ GA.A AAUAUAAU GAA AAAUAUAA GAA AAAAUAUA GAA AAAA-AUAU GAA AUAAAAAU GAA AAUAAAA-A GAA ACUGAAUA GAA AUUACUGA GAA AAUUACUG GAA AAAUUACU GAA AUUAAAUU GAA AAUUAAAU GAA AAAUUA-AA GAA ACAAAAUU GAA ACACAUUU GAA AACACAUU GAA AGCAGCGA GAA ACCAUAGC GAA AACCAUAG GAA AAACCAUA GAA AAAACCAU GAA AGGCUAA-A GAA AUAGGCUA GAA ACUAUAGG GAA AGCAGCAU GAA AGCUAGCA GAA ACACUAGC GAA AUUGCCCC GAA AGCUCUAU GAA AAGCUCUA GAA AGUCUCUU nt.

Position 2709 2710 2714 2722 2729 2732 2734 2735 2742 2743 2744 2745 2746 2747 2749 2750 2751 2752 2753 20 2754 2756 2758 2760 2764 25 2768 2769 2770 2771 2774 30 2775 2776 Target Sequence CUCGGUGTJU AGAUAACG UCGGUGUUA GAUAACGG UGtJUAGAUA ACGGACUA AACGGACUA UCCACUAC UAUCCACUA GUAUUCCA GCACUAGUA UIJCCACAC ACUAGUAUU CCAGACUU CUAGUAUTJC CAGACUUU UCCAGACUU UUUUAUUU CCAGACUUTJ UUUAUUUU CAGACUUUU UUAUUUUIJ AGACUUUUU UAUUULUU GACUUUUUU AUUJTUUA ACUUUtUUA UUUUJUUAU UUUUUUAULJ UUUUJAUAU UUUUUAUTJU UUUAUAUA UUUUAUtJUU UUAUAUAU UTUUALUUUtU UAUAUAUA UUAUTUUUUU AUAUAUAU UAUUUUtJUA UAUAUAUG UUtUUUAUA UAUAUGUA tUUUUAUAUA UAtJGUACC UUAUAUAUA UGUACCUU AUAUAUGUA CCUUUUCC AUGUACCUU UUCCUUUU UGUACCUUU UCCUUUUC GUACCUUUU CCUUUUGU UACCUUtJUC CUUUUGUC CtJUUUCCUU UTJGUCAAU UUtJUCCUUU UGUCAAUU UUUCCtUUU GUCAAUUG Ribozyne Sequence

CGIJUAUCU

CCC UTAUC

UAGUCCGU

CUAGUGCA

UGGAAUAC

GUCUGGAA

AAGUCUGG

AAAGUCUG

AAAUAAAA

AAAAUAAA

AAAAAUAA

AAAA.AAUA

UAAAAAAU

AUAAAAAA

AUAUAAA

UAUAUAAA

AUAUAUAA

UAUAUAUA

AUAUAUAU

CAUAUAUA

UACAUAUA

GGUACAUA

AAGGUACA

GGAAAAGG

AAAAGGAA

CAAAAGGA

ACAAAAGG

GACAAAAG

AUUGACAA

AALJUGACA

CAALUUGAC

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUCA_

CUGAUGA

CUGAUGA

CUGAUGA

GAA ACACCGAG GA-A AACACCGA GAA AUCUA-ACA GAA AGUCCGJU GAA AGUGCAUA GAA ACUAGUOC GAA AUACUAGU GAA AAUACUAG GAA AGUCUGGA GAA AAGUCUGG GAA AAAGUCUG GAA AAAAGUCU GAA AAAAAGUC GAA AAAAAAGU 0S

**OS

S.

S.

S

S.

S. 0SSO

S

554 0

*SSS

S S S. S 005w

S

*5@S *5

*SSO

0 OS 56 0 CUGAUGA X GAA AUAAAAAA CUGAUGA X GAA AAUAAAAA CUGAUGA X GAA AAAUAAAA CUGAUGA X GAA AAAAUAAA CUGAUGA X GAA AAAAAUAA CUGAUGA X GAA AAAAA.AUA CUGAUGA X GAA AUAAAAAA CUGAUGA X GAA AUAUAAAA CUGAUGA X GAA AUAUAUAA CUGAUGA X GAA ACAUAUAU CUGAUGA X GAA AGGUACAU CUGAUGA X GAA AAGGUACA CUGAUGA X GAA A.AAGGUAC CUGAUGA X GAA AAAAGGUA CUGAUGA X GAP. AGGAAAAG CUGAUGA X GAA AAGGAAAA CUGAUGA X GAA AAAGGAAA Where I"XI represents stem II region of a HH ribozyme (Hertel et al. 1992 Nucleic Acids Res. 20 3252). The length of stem II may be 2 base-pairs.

C.

C

C

Table XVI: Mouse c-mVb Hairpin ribozymre and target seguences Posi- RZ Substrate tion 24 GCGAGGCG AGAA GGGGCU AGCCCCG GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGCCUCCC 28 CAUGGCGA AGAA GGCCGG CCGGCCC GCC ACCAGAGAAACACACGUUGUGGUACAUUAC CUGGUA UCC CCAUG 122 AUEJUGGGC AGAA GCCCAU AUGGGCU GCU ACCAGAGAAACACACGtJUGUGGUACAUUACCUGGUA G CCCAAAU 125 CAGAUUUG AGAA GCAGCC GGCUGCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAAUCUG 216 UUCCAGUC AGAA GUUCCG CGGAACA GAC ACCAGAGAAACACACGUUGUGGUACAUTUACCUGGUA GACUGGAA 245 UCCGGUUG AGAA GAUAAU AUUAUCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAACCGGA 258 CACUGUAC AGAA GUCCGG CCGGACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUACAGUG 529 CUCUGCCC AGAA GUUCCC GGGAACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCAGAG 551 GUCCGGGC AGAA GCUUUG CAAAGCU GCU ACCAGAGAAACACACGUUGUGGUACAUIJACCUGGUA 'GCCCGGAC 554 UCCGUCCG AGAA GCAGCU AGCUGCU GCC ACCAGAGAAACACACGJUGUGGUACAtJUACCUGGUA CGGACGGA 559 AUCAGUCC AGAA GGGCAG CUGCCCG GAC ACCAGAGAA.ACACACGUUGUGGUACAUUACCUGGUA GGACUGAU 563 CAUUAUCA AGAA GUCCGG CCGGACG GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGAUAAUG 656 CCACUGGC AGAA GGCUGG CCAGCCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCAGUGG 728 UUGGAGAG AGAA GAGAUG CAUCUCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CUCUCCAA

746 UGACGGAG AGAA GGCCAC GUGGCCA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CUCCGUCA

822 UGCAAUGC AGAA GGAUAG CUAUCCU GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAUUGCA

C

857 CCGCAGCC AGAA GAGGGA UCCCUCA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GG CUGCGG 861 GCUGCCGC AGAA GGCUGA UCAGCCG GCU ACCAGAGAAACACACGUUGUGGUACAUTUACCUGGUA GCGGCAGC 941 CUGUUGAC*AGAA GGAGCA UGCUCCU GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUCGUA GUCAACAG 1040 GAGGUCUC AGAA GGUCCA UGGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUTACCUGGUA CAGACCUC 1045 CCCAUGAG AGAA GGUCUG CAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCAUGGG 1068 AAACAGGA AGAA GGUGCA UGCACCU GUJ ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCCUGULIJ 1075 UUCUCCCA AGAA GGAAAC GUUUCCU GU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGGAGAA 1106 GAUCUGCA AGAA GAGAUG CAUCUCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCAGAUC 1113 GAGCCGGG AGAA GCAGGC GCCUGCA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCGGCUC 1120 AGGUAGGG AGAA GGGAUC GAUCCCG GCU ACCAGAGAAACACACGJUGUGGUACAUJACCUGGUA CCCUACCU 1226 AAUCUAUA AGAA GGAGUG CACUCCA GUUl ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAUAGAUU 1340 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUGAAAA 1449 AUUUCUUG AGAA GCAAGG CCUUGCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAGAAAU 1468 CUUCAGGG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUGAAG 1490 GGGAGGGG AGAA GAGGUA .UACCUCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCCUCCC 1542 CCAGAUTUC AGAA GAUUCC GGAAUCG GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GAAUCUGG

1648 GUGGUUUG AGAA GAAGAA UUCtJUCU GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAACCAC 1672 GGUGCUCA AGAA GUIICUC GAGAACA GCC ACCAGAGAAACACACGUUGUGGUACAUJACCUGGUA UGAGCACC 1688 CCUGCGAG AGAA GUUGGG CCCAACU GUU ACCAGAGAAACACACGUUGUGGUACAUTUAC CUGGUA CUCGCAGG 1713 UUUGGGGC AGAA GCCACA UGUGGCA GAU ACCAGAGAAACACACGIJUGUGGUACAUUACCUGGUA

GCCCCAA

1740 GUCAUUkA AGAA GAGCUU AACCUCU GTJR ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UUAAUGAC

1880 AGGCCGUC AGAA GGUCCU AGGACCA GAU ACCAGAGAAACACACGTJUGUGGUACAUUACCUGGUA

GACGGCCU

1887 GGACCGGA AGAA GUCAUC GAUGACG GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UCCGGUCC

1894 CCGAGCCG AGAA GGAGGC GCCUCCG GUC ACCAGAGAAAC-ACACGUUGUGGUACAUUACCUGGUA

CGGCUCGG

1899 UAUUUCCG AGAA GGACCG CGGUCCG GCU ACCAGAGAAACACACGTJUGUGGUACAUUACCUGGUA

CGGAAAUA

1926 AGAGUUCG AGAA GAGAAC GUUCUCA GCU ACCAGAGAAACACACGUJUGUGGUACAUUACCUGGUA

CGAACUCU

2048 ACAACAA-A AGAA GGCUCU AGAGCCU GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UUUGUTUGU

2068 CUGCUCUC AGAA GUUGUA UACAACA GLU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GAGAGCAG

2170 UUAGGUAA AGAA GU-UAUTU AAUAACA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UUACCUAA

2225 UUUAAAAA AGAA GAUUAU AUAAUCA GAU ACCAGAGAAACACACGUUGUGGUACAUJACCUGGUA

UUUUUAAA

2276 AAAUACUG AGAA GUUGUA UACAACA GAU ACCAGAGAAACACACGUUGUGGUACAtJUACCUGGUA

CAGUAUUU

2519 UUCAAGCA AGAA GACAAC GUUGUCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCtJUGAA 2717 AGUGCAUA AGAA GUUAUC GAUAACG GAC ACCAGAGAAACACACGtJUGUGGUACAUUACCUGGUA

UAUGCACU

2737 AUAAAAAA~ AGAA GGAAUA UAUUCCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUL7UUUAU

C

Table XVII: Rat c-mvb (Region A) Hammerhead Ribozxrme and Tarciet Sequences (282 bp; nt 428 start; human c-mvb numbering system) S S

S.

S nt.

Position 467 470 477 498 509 515 518 526 531 544 548 549 551 562 563 575 588 609 25 610 615 623 624 634 659 662 663 664 666 Target Sequence CCUGAGCUC AUCAAAGG GAGCUCAUC AAAGGUCC UCAAAGGUC CCUGGACC AAGAAGAUC AAAGAGUG AGAGUGAUA GAGCUUGU AUAGAGCUU GUCCAGAA GAGCUTJGUC CAGAAAUA CCAGAAAUA CGGUCCGA AAUACGGUC CGAAGCGC GCGCUGGUC UGUUAUUG UGGUCUGUT) AUUGCCAA GGUCUGUUA UUGCCAAG UCUGtJUAUU GCCAAGCA CAAGCACUU AAAAGGGA AAG CACtJUA AA.AGGGAG GGGAGAAUU GGAAAACA AACAAUGUC GGGAGAGG ACAACCAUU UGAAUCCA CAACCAUUU GAAUCCAG ALUUGAAUC CAGAAGUU CCAGAAGUU AAGAAAAC CAGAAGUUA AGAAAACC GAAAACCUC AUGGACAG GACAGAAUC AUUUAUCA AGAAUCAUU UAUCAGGC GAAUCAUUU AUCAGGCA AAUCAJUUUA UCAGGCAC UCAUUUAUC AGGCACAC

CCUUUGAU

GGACCUUTJ

GGUCCAGG

CACUCUUU

ACAAGCUC

UIJCUGGAC

UAUUUCUG

UCGGACCG

GCGCUUJCG

CAAUAACA

UtJGGCAAU

CUITJGGCAA

UGCUUGGC

UCCCUUUTJ

CUCCCUTU

UGUTUUUCC

CCUCUCC

UGGAUUCA

CUGGAUUJC

AACUUCUG

GUUUELJCUUJ

GGJUUUJCU

CUGUCCAU

UGAUAAAU

GCCUGAUA

UGCCUGAU

GUG CCUGA

GUGUGCCU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

X GAA AGCUCAGG X GAA AUGAGCUC X GAA ACCUIJUGA X GAA AUCUUCUU X GAA AUCACUCU X GAA AGCUCUAU X GAA ACAAGCUC X GAA AUUTUCUGG X GAA ACCGUATU X GAA ACCAGCGC X GAA ACAGACCA GAA AACAGACC X GAA AUAACAGA GAA AGUGCUUG X GAA AAGUGCUU X GAA AUUCUCCC GAA ACAUUGUUT X GAA AUGGUUGU GAA AAUGGtJUG GAA AUUJCAAAU GAA ACUUCUGG X GAA AACUUCUG X GAA AGGUUUUC X GAA AUUCUGUC GAA AUGAUUCU GAA AAUGAUUC X GAA AAAUGATJ X GAA AUAA.AUGA a HH ribozyme 20 3252). The Ribozyme Sequence Where represents stem II region of (Hertel et, al. 1992 Nucleic Acids Res.

length of stem II may be a 2 base-pairs.

Table XVIII: Rat c-myb (Region B) Hammerhead Ribozyme and Target Seqaiences (262 bp; nt .1421 start; human c-mvb numbering system) nt. Target Sequence Ribozyme Sequence Posi tion 1429 1430 1434 1440 1443 1444 1445 1450 1458 1460 1463 1467 1485 1509 1522 1526 1528 1529 1530 1536 1542 1544 1552 1556 1559 1567 1569 1578 1588 1589 1608 1616

CUCGGGCUU

UCGGGCUUA

GC CUAGAUA

AUACGCCUA

CGCCUACUU

GCCUACUUU

CCUACtJUUA

UUUACCCUC

CCACGCCUC

ACGCCUCUC

CCUCUCAUU

UCAUUGGTJC

CACCGUGUC

UGAAAACUN

GGAAAACUC

AACUCNAUC

CUCNAUCUU

UCNAUCUUU

CNAUCUUUA

UUAGAACUC

CUCCAGCUA

CCAGCUAUC

CAAAAGGUN

AGGUNAAUC

UNAAUCCUC

CGAAAGCUC

AAAGCUCUC

CCAGAACUC

CACACCAUU

ACACCAUUC

UGGCAGCUC

CAAGAAAUU

AGAUACG C

GAUACGCC

CGCCUACU

CUUEJACCC

UACCCUCC

ACCCUCCA

CC CUC CA C

CACGCCUC

IJCAUUGGU

AUUGGUCA

GGUCACAA

ACAAACUG

ACCGAGAC

AAAAGGAA

NAUCUUUA

ULTUAGAAC

UAGAACUC

AGAACUCC

GAACUCCA

CAGCUAUC

UCAAAAGG

AAAAGGUN

AAUCCUCG

CUCGAAAG

GAAAGCUC

UCCCAGAA

CCAGAACU

CCACACCA

CAAACAUG

AAACAUGC

AAGAAAUU

AAAUACGG

GCGUAUCU

GCGUAUC

AGUAGGCG

GGGUAAAG

GGAGGGUA

UGGAGGGU

GUGGAGGG

GAGGCGUG

ACCAAUGA

UGACCAAU

UUGUGACC

CAGUTUUGU

GUCUCGGU

U.UCCLUU

UAAAGAUN

GUUCEJAAA

GAGUUCUA

GGAGUUCU

UGGAGUUC

GAUAGCUG

CCUTUUUGA

NACCUUUU

CGAGGAUU

CUtJUCGAG

GAGCUUUC

UUCUGGGA

AGUUCUGG

UGGUGUGG

CAUGUtJUG

GCAUGUUU

AAUUUCUU

CCGUAUUU

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AGCCCGAG

AAG CC CGA AUCUAAG C

AGGCGUAU

AGUAGGCG

AAGUAGG C

AAAGUAGG

AGGGUAAA

AGGCGUGG

AGAGGCGU

AUGAGAGG

ACCAAUGA

ACACGGUG

AGUUUUCA

AGUUEJUCC

AUNGAGUU

AGAUNhGAG

AAGAUNGA

AAAGAUN~G

AGUUJCUAA

AGCUGGAG

AUAGCUGG

ACCUTJG

AUUNACCU

AGGAUUNA

AGCUUUCG

AGAGCUUU

AGUUCUGG

AUGGUGUG

AAUGGUGU

AGCUGCCA

AUUUCUUG

100 nt. Target Sequence Ribozxrme Sequence Position 1429 1617 1621 1626 1640 1644 1654 1656 1661 1664 1673

CUCGGGCUU

AAGAAAUJA

AAU'UAAAUA

AAUACGGUC

AAGAUG CUA UG CUACCUN

GACCCCCUN

CCCCCUNUN

UNUNAUGUA

NAUGUAGUN

NNANACCUN

AGAUACGC

AAUACGGU

CGGUCCCC

CCCUGAAG

CCUNAGAC

AGACCCCC

TJNAUGUAG

AUGUAGUN

GUNNNANA

NNANACCU

CANGAUGU

GCGUAUCU

ACCGUALU

GGGGACCG

CUUCAGGG

GUCUNAGG

GGGGGUCU

CUACAUNA

NACUACAU

UNUNNNAC

AGGUNUNN

ACAUCNUG

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AGCCCGAG

AAUTJUCUU

AUUTUAAUUJ

ACCGUAUJ

AGCAUCUUL

AGGUAGCA

AGGGGGUC

ANAGGGGG

ACAUNANA

ACUACAJN

AGO UNUNN Where "X"I represents stem II region of (Hertel et al., 1992 Nucleic Acids Res.

length of stem II may be a 2 base-pairs.

a HH- ribozyme 20 3252) The

C

9* a..

a Table XIX: Rat c-mvb (Region A) Hairpin Ribozyme and Target Sequences (282 bp; nt. 428 start; human numbering system) Posi- RZ Substrate t ion 528 GCGCUUCG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGAAGCGC 690 tJUCUGCCC AGAA GUUUCC GGAAACA GAU ACCAGAGAAACAcACGUUGUGGUAcAUUAccUGGUA GGGCAGAA 101 Table XX: Rat c-myb (Region B) Hairpin Ribozyme and Target Sequences (262 bp; nt. 1421 start; human numbering system) Posi- RZ Substrate tion 1495 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UGUGAAA.A

1604 AUUUCUTJG AGAA GCCAGG CCUGGCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CAAGAAAU

1623 CUUCAGGG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CCCUGAAG

Table XXI: Porcine c-myb (Region A) Hammerhead Ribozyme and Target Sequence (266 bp; nt. 458 start; human c-mvb numbering svstem) 0 nt.

Position 467 470 477 480 498 509 515 518 526 531 540 544 548 549 551 562 563 575 588 603 610 Target Sequxence

CCUNAUCUC

NAUCUCAUC

UCAAGGGUC

AGGGUCCUEJ

AAGAAGAUC

AGAGUGAUA

AUAGAGCUU

GAGCUUGUA

ACAGAAAUA

AAUACGGUC

CGAA.ACGUUJ

ACGUUGGUC

UGGUCUGUU

GGUCUGLTUA

UCUGUTUAUU

CAAGCACUU

AAGCACUUA

GGGAGAALUJ

AACAAUGUA

GGUGGCAUA

UAACCACUU

AUCAAGGG

AAGGGUCC

CUtJGGACC

GGACCAAA

AGAGAGUG

GAGCUtGU

GUACAGAA

CAGAAAUA

CGGUCCGA

CGAAACGU

GGUCUGUIJ

UGLTUAUUG

AUUGCCAA

UUGCCAAG

GCCAAGCA

AAAGGGGA

AAGGGGAG

GGAAAACA

GGGAGAGG

ACCACUUG

GAAUCCAG

CCCUEJGAU

GGACCCUtJ

GGUCCAAG

UUUGGUCC

CACUCUCU

ACAAGCUC

UEJCUGUAC

UAUUUCUG

UCGGACCG

ACGUUUCG

AACAGACC

CAAUAACA

UtJGGCAAU

CUUGGCAA

UGCUUGGC

uccccuuu CUCCCCu7u UGUUUTJcc ccucuccc

CAAGUGGU

CUGGAUJUC

CUGAUGA X GAA CtJGAUGA

CEJGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

GAA

AGAUNAGG

AUGAGAUN

ACCCUUGA

AGGACCCU

AUCUUCUIJ

AUCACUCU

AGCUCUAU

ACAAGCUC

AUUUCUGU

ACCGUAtU

ACGUUUCG

ACCAACGU

ACAGACCA

AACAGACC

AUAACAGA

AGUGCUUG

AAGUGCUU

AUUCUCCC

ACAUUJGUU

AUGCCACC

AGUGGUUA

Ribozvme Sequence 102 nt.

Posi tion 615 623 624 634 659 660 662 663 664 704 713 Target Seqiuenlce ACtJUGAAUC CAGAAGUU CCAGAAGUU AAGAAAAC CAGAAGUUA AGAAAACC GAAAACCUC CUGGACAG GACAGAAUU AUUUACCA ACAGAAUUA UUUACCAG AGAAUIJAUU UACCAGGC GAAUULAUUU ACCAGGCA AAUUAUUUA CCAGGCAC GCGGAAAUC GCAAAGCU GCAAAGCUA CUGCCUGG Ribozyme Sequence AACUUCUG CUGAUGA X GAA AIJUJCAAGU GUUUIJCUU CUGAUGA X GAA ACUUCUGG GGUUUUCU CtJGAUGA X GAA A-ACUUCUG CUGUCCAG CUGAUGA X GAA AGGUUUUC UGGUAAAU CUGAUGA X GAA AUUCUGUC CUGGUAAA CUGAUGA X GAA AAUUCUGU GCCUGGUA CUGAUGA X GAA AUAAUUCU UGCCUGGU CUGAUGA X GAA AAUAAUUC GUGCCUGG CUGAUGA X GAA AAAUAAUU AGCUUUGC CUGAUGA X GAA AUJIUCCGC CCAGGCAG CUGAUGA X GAA AGCUTJUGC Where IIX' represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252).

lena: h of stem II may be a 2 base-pairs.

The

S

Table XXII: Porcine c-myb and Target Secruence (308 numbering system) 20 nt. Target Secruence Position 1394 GAUUCUUUC tJUAAACAC 1396 UUCUUUCUU AAACACUU 1397 UCUUUCUUA AACACUUC 1404 UAAACACUU CCAAUA-AC 1405 AAACACUUC CAAUAACC 1410 CUUCCAAUA ACCAUGAA 1423 UGAAAACUU AGACUUGG 1424 GAAAACUUA GACUUGGA 1429 CtJUAGACUU GGAAAUGC 1440 AAAUGCCJU CUUUAACG 1441 AAUGCCUUC UUUAACGU 1443 UGCCUUCUU UAACGUCC 1444 GCCUUCJUEJ AACGUCCA 1445 CCUUCUUUA ACGUCCAC rn- nt 1386 start; human c-myb Ribozyme Seqruence GUGUEJUAA CUGAUGA X AAGtJGUUU

GAAGUGUU

GUUAUUJGG

GGtJUAUUG

UUCAUGGU

CCAAGUCU

UCCAAGUC

G CAtJUUCC

CGUUAAAG

ACGUUAAA

GGACGUUA

UGGACGUU

GUGGACGU

CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X CUGAUGA X GAA AAAGAAUC GAA AGAAAGAA GAA AAGAAAGA GAA AGUGUUUA GA.A AAGUGUUU GAA AUUGGAAG GAA AGUULTUCA GAA AAGUUUUC GAA AGUCUAAG GAA AGGCAUUU GAA AAGGCAUU GAA AGAAGGCA GAA AAGAAGGC GAA AAAGAAGG (Region B) Hammerhead Ribozvme 103 1450 1458 1460 1467 1474 1481 1482 1492 1493 1494 1497 1530 1534 1536 1537 1538 1539 1545 1551 1553 1561 1565 1576 1578 1587 1590 1597 1598 1610 1617 1625 1626 1630 1635 1649 1653 1663 1665 1668

UUUAACGUC

CCACGCCUC

ACGCCUCUC

UCAGUGGUC

UCACAAAUU

UUGACUGUU

UGACUGUUA

AACAC CAUU

ACACCAUUJ

CACCAUJUC

CAUUUCAUA

AGGAAAAUA

AAAUACAUA

AUACAUAUU

UACAUAUJIJ

ACAUAUUUU

CAUAUUUU

TJUUGAACUC

CUCCGGCUA

CCGGCUAUC

CAAAAGGUC

AGGUCAAUC

GGAAAG CU C

AAAGCUCUC

CAAGAACUC

GAACUCCUA

UACACCGUU

ACACCGUUC

CAUGCACUC

UCGCAGCUC

CAAGAAAUU

AAGAAAUUA

AAUTUAAAUA

AAUAUGGUC

AAGAUGCUA

UGCUACCUC

GACACCAUC

CACCAUCUC

CAUCUCAUU

CACGCCUC

UCAGUGGU

AGUGGUCA

ACAAAUUG

GACUGUUA

ACAACACC

CAACACCA

UCAUAGAG

CAUAGAGA

AUAGAGAC

GAGACCAG

CAUAUU-U

UUUULUGAA

UUUTGAAC U

UUGAACUC

UGAACUCC

GAACUCCG

CGGCUAUC

UCAAAAGG

AAAAGGUC

AAUCCUGG

CUGGAAAG

UCCAAGAA

CAAGAACU

CUACACCG

CACCGtJUC

CAA.ACAUG

AAACAUG C

GCAGCUCA

AAGAAALU

AAAUAUGG

AAUAUGGU

UGGUCCCC

CCCUGAAG

CCUCAGAC

AGACACCA

UCAUUUAG

AUUUAGUA

UAGUAGAA

ACCACUGI

UGACCACT

CAAUUUGI

UA-ACAGUC

GGUGUUGt

UGGUGUUC

CUCUAUGP

UCUCUAUC

GUCUCUAU

CUGGUCUC

AAAAUAUG

UUCAAAAA

AGUUCAAA

GAG UUCAA

GGAGUUCA

CGGAGUUC

GAUAGCCG

CCUJUUGA

GACCtUUU

CCAGGAUU

CUUUCCAG

UUCUUGGA

AGUTUCUUG

CGGUGUAG

GAACGGUG

CAUGUUUG

GCAUGUUU

UGAGCUGC

AAUUUCUU

CCAUAUUU

ACCAUAUIJ

GGGGACCA

CUUCAGGG

GUCUGAGG

UGGUGUCU

CUAAAUGA

UACUAAAU

k. CUGAUGA J CUGAUGA

JCUGAUGA

CUGAUGA

JCUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA

CUGAUGA)

CUGAUGA CUGAUGA CUGAUGA CUGAUGA CUGAUGA KGAA AGGCGUGG K GAA AGAGGCGU K GAA ACCACUGA KGAP. AUIJUGUGA GAA ACAGUCALA GAP. AACAGUCA GAA AUGGUGUTJ GA.A AAUGGUGU GAA AAAUGGUG GAA AUGAAAUG GAA AUUUTLUCCU GAA AUGUAUJTJ GAA AUAUGUAU GAA AAUAUGUA GAA AAAUAUGU GAA AAAAUAUG GAA AGUUCA-AA GAA AGCCGGAG GAA AUAGCCGG GAA ACCUUUUG GAA AUUGACCU GAA AGCUUUCC GAA AGAGCUUU GAA AGUUCtJUG GAA AGGAGUUC GAA ACGGUGUA GAA AACGGUGU GAA AGUGCAUG GAA AGCUGCGA GAA AUUUCUUG GAA AAUUUCTU GAA AIJUUAAUU GAA ACCAUALU GAA AGCAUCLU GAA AGGUAGCA GAA AUGGUGUC GAA AGAUGGUG GAGGCGUG CUGAUGA X GAA ACGUTUAAA

C

UUCUACUA CUGAUGA X GAA AUGAGAUG 104 1669 1670 1673 AUCUCAUUU AGUAGAAG CUUCUACU CUCAUGA X GAA AAUGAGAU UCUCAUUUA GUAGAAGA UCUUCUAC CUGAUGA X GAA AAAUGAGA CAUTJUAGUA GAAGACCU AGGUCUUC CUGAUGA X GAA ACUAAAUG Where "X"I represents stem II region of a HH ribozyme (Hertel et al. 1992 Nucleic Acids Res. 20 3252). The length of stem II may be a 2 base-pairs.

Table XXIII: Porcine c-myb (region A) Hairpin Ribozxrme and Target Seqxuence (266bp; nt. 458 start; Human numbering system) Posi- RZ Substrate tion 528 ACGUUUCG AGAA GUAUUU

ACCAGAGAAACACACGUUGUGGUACAUJACCUGGUA

AAAUACG GUC

CGAAACGU

690 UUCCGCCC AGAA GUUCCC GGGAACA GAU 1 ____IACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GGGA

Table XXIV: Porcine c-mvb (region B) Hairpin Ribozvme and Target Secruence (308 bp; nt. 1386 start: Human numberino system) Posi- Hairpin Ribozyme Substrate t ion 1504 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGtJUGUGGUACAUUACCUGGUA UGUGAAAA 1594 CAUGUUUG AGAA GUGUAG CUACACC GU ACCAGAGAAACACACGUUGUGGUACAIJUAcCUGGUA CAAACAUG 1613 AUUUCUUG AGAA GCGAGU ACUCGCA GCU ACCAGAGAAACACACGUUGUGGUACAUtJACCUGGUA ,CAAGAAAU 105 Full disclosure of the sequence listing relied on in the present application is found in the accompanying CD in the textfile titled SEQUENCE.txt.

The sequence listing on the CD comprises 2733 sequences being sequences from SEQ ID NO:1 through to SEQ ID NO:2733.

[R:\LIBA]4249.doc: lam loG lhe claims defining the invention are as follows: 1. An enzymatic nucleic acid molecule which cleaves c-myb RNA, wherein the the binding arms of said nucleic acid contain sequences complementary to the sequences defined in Tables II, XII-XXIV.

2. An enzymatic nucleic acid molecule which cleaves RNA produced from a gene selected from one encoding c-fos, oct-1, SRF, PDGF receptor, bFGF receptor, angiotensin II, and endothelium-derived relaxing factor.

3. The enzymatic nucleic acid molecule of claims 1 or 2 wherein said nucleic acid molecule is in a hammerhead motif.

4. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said nucleic acid molecule is in a hairpin, hepatitis delta virus, VS nucleic acid, group I intron, or RNAseP nucleic acid motif.

S. 5. The enzymatic nucleic acid molecule of claim 3 or 4, wherein said nucleic acid comprises between 12 and 100 bases complementary to said mRNA.

6. The enzymatic nucleic acid molecule of claim wherein said nucle-ic acid comprises between 14 and 24 bases complementary to said mRNA.

7. Enzymatic nucleic acid molecule consisting 30 essentially of any sequence selected from the group of sequences listed in Tables III, XII-XXIV.

8. A mammalian cell including an enzymatic nucleic acid molecule of any one of claims 1 or 2.

9. The cell of claim 8, wherein said cell is a human cell.

An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of claims 1 or 2 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

0 11. A mammalian cell including an expression vector of claim 12. The cell of claim 13, wherein said cell is a human cell.

13. A method for treatment of a stenotic condition by administering to a patient an enzymatic nucleic acid molecule of claims 1 or 2, or an enzymatic nucleic acid molecule which cleaves RNA produced from the gene c-myb.

14. A method for treatment of a stenotic condition by administering to a patient an expression vector of 20 claim The method of claims 13 or 14, wherein said patient is a human.

16. A method for treatment of cancer by administering to a patient or a patient's cells an enzymatic nucleic e acid molecule of claims 1 or 2.

17. A method for treatment of cancer by administering to a patient or a patient's cells an expression vector of claim 18. The method of claims 16 or 17, wherein said patient is a human.

19. Method for administration of an enzymatic nucleic acid by mixing said nucleic acid with a chemical og8 selected from the group consisting of chloroquine, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), monensin, colchicine, amphipathic peptides, viral proteins, and viral particles.

The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the six terminal nucleotides, and wherein said nucleic acid comprises a 2'-C-allyl modification at position No. 4 of said nucleic acid, and wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a end modification.

21. The enzymatic nucleic acid of claim 20, wherein said nucleic acid comprises a linked inverted ribose moeity at said 3' end.

20 22. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises of phosphorothioate linkages at at least three of the six terminal nucleotides, and wherein said nucleic acid comprises a 2'-amino modification at position No. 4 and/or at position No. 7 of said nucleic acid, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a 3'-3' linked inverted ribose or thymidine moeity at its 3' end.

23. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the six terminal nucleotides, and wherein said nucleic acid comprises non-nucleotide substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid comprises at least ten methyl modifications, and wherein said nucleic acid comprises a linked inverted ribose or thymidine moeity at its 3' end.

24. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the six terminal nucleotides, and wherein said nucleic acid comprises 6-methyl uridine substitutions at position No.

4 and/or at position No. 7 of the said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a linked inverted ribose or thymidine moeity at its 3' end.

25. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose 20 residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of thje six terminal nucleotides, wherein said nucleic acid comprises 2'-C-allyl modification at position No. 4 of the said nucleic acid, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a linked inverted ribose or thymidine moeity at its 3' end.

26. Oligonucleotide having complementarity to c-myb 30 at at least 5 contiguous bases comprising a adenylate residue having a 27. The oligonucleotide of claim 26, having enzymatic activity on c-myb RNA.

28. The oligonucleotide of claim 26, comprising at least 20 bases able to form a hybrid with c-myb RNA.

Claims (64)

1. An enzymatic nucleic acid molecule which cleaves c-myb RNA, said nucleic acid molecule having binding arms and wherein said binding arms contain sequences complementary to the sequences defined in Tables II. XIII-XXIV.

2. The enzymatic nucleic acid molecule of claim I wherein said nucleic acid molecule is in a hammerhead motif.

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

4. The enzymatic nucleic acid molecule of claim 2 or claim 3, wherein said nucleic acid comprises between 12 and 100 bases complementary to said mRNA. An enzymatic nucleic acid molecule of claim 4. wherein said nucleic acid comprises between 14 and 24 bases complementary to said mRNA.

6. An enzymatic nucleic acid molecule of claim I consisting essentially of any sequence selected from the group of sequences listed in Tables II1. XIII-XXIV.

7. A mammalian cell including an enzymatic nucleic acid molecule of any one of claims 1-6.

8. The cell of claim 7, wherein said cell is a human cell.

9. An expression vector including nucleic acid encoding an enzymatic 20 nucleic acid molecule or multiple enzymatic molecules of any one of claims 1-6 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

10. A mammalian cell including an expression vector of claim 9. I 1. The cell of claim 10, wherein said cell is a human cell.

12. A method for treatment of a stenotic condition by administering to a 25 patient an enzymatic nucleic acid molecule of any one of claims 1-6.

13. A method for treatment of a stenotic condition by administering to a patient an expression vector of claim 9.

14. The method of claims 12 or 13, wherein said patient is a human. A method for treatment of cancer by administering to a patient or a patient's cell an enzymatic nucleic acid molecule of any one of claims 1-6. I 16. A method for treatment of cancer by administering to a patient or a patient's cells an expression vector of claim 9.

17. The method of claims 15 or 16. wherein said patient is a human.

18. Method for administration of an enzymatic nucleic acid molecule of any 3 5 one of claims 1-6 by mixing said nucleic acid molecule with a chemical selected from the group consisting of chloroquine, ammonium chloride, carbonyl cyanide p-trifluoromethoxy II:\[)AYLB\L11A102662.doc:dt1 phenyl hydrazone (FCCP), monensin, colchicine, amphipathic peptides, viral proteins, and viral particles.

19. The enzymatic nucleic acid of claim 2, wherein said nucleic acid comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said nucleic acid comprises a 2'-C-allyl modification at position No. 4 of said nucleic acid, and wherein said nucleic acid comprises at least ten 2'-O-methyl modifications. The enzymatic nucleic acid of claim 19, wherein said nucleic acid comprises a linked inverted ribose moiety at said 3' end. i, 21. The enzymatic nucleic acid of claim 2. wherein said nucleic acid comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three 5' terminal nucleotides. and wherein said nucleic acid comprises a 2'-amino modification at position No. 4 and/or at position No. 7 of said nucleic acid, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications. i 22. The enzymatic nucleic acid of claim 2. wherein said nucleic acid comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said nucleic acid comprises abasic substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications. 20 23. The enzymatic nucleic acid of claim 2. wherein said nucleic acid comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three 5' terminal nucleotides, and wherein said nucleic acid comprises 6-methyl uridine substitutions at position No. 4 and/or at position No. 7 of the said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2--methyl 2 modifications.

24. The enzymatic nucleic acid of claim 2. wherein said nucleic acid comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three 5' terminal nucleotides. and wherein said nucleic acid comprises 2'-C-allyl modification at position No. 4 of the said nucleic acid, wherein s(i said nucleic acid comprises at least ten 2'-O-methyl modifications. An enzymatic nucleic acid molecule of claim 1 having complementarity to c-myb RNA at at least 5 contiguous bases comprising a 2'-5'-linked adenylate residue having a 5'-phosphate, wherein said enzymatic nucleic acid molecule has enzymatic activity on c-myb RNA. 3 26. The enzymatic nucleic acid molecule of claim 25, comprising at least bases able to hydridize with c-myb RNA. jI:\DAYLIB\LIBA)02662.doc:tlI IZ

27. An enzymatic nucleic acid molecule which cleaves c-myb RNA at any of the RNA sequences defined in Tables II, XIII-XXIV, substantially as hereinbefore described with reference to any one of Examples 4-25.

28. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 232.

29. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 264. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 266. i,

31. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 272.

32. The enzymatic nucleic acid molecule of claim 2, wherein said enzmatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 282.

33. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic i. nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 300.

34. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 320. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 350.

36. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 374.

37. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 384.

38. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 442.

39. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 522. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 538. 'o 41. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 540.

42. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 93.

43. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic i3 nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 572. 9* 6* S S S *r 5* 5 5555 S 5*5S S *50s I :\DAYLIB\L1 BA02662.doctI( 113

44. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 614.

46. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 622.

47. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 98.

48. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1162. 1164. 1166 or 1168.

49. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1170. 1174or 1176. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1178. 1180, 1182 or 1184. *.so so.* 050 0@e *so 0 0 55 S S 0 S OSSS S 0 @000

51. nucleic acid 20 NOS. 1186.

52. nucleic acid NOS. 1194,

53. 2s nucleic acid NOS. 1202,

54. nucleic acid NOS. 1210. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic molecule specifically cleaves any of the RNA sequences defined as SEQ ID 1188, 1190 or 1192. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic molecule specifically cleaves any of the RNA sequences defined as SEQ ID 1196, 1198 or 1200 The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic molecule specifically cleaves any of the RNA sequences defined as SEQ ID 1204, 1206 or 1208. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic molecule specifically cleaves any of the RNA sequences defined as SEQ ID 1212, 1214 or 1216. M. 55. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1218, 1220, 1222 or 1224.

56. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 930, 932, 934 or 936. [I :\DAYLIL1BAj02662.doc:t1l

57. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 938, 940, 942 or 944.

58. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic Snucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 946. 948, 950 or 952.

59. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 954. 956. 958 or 960. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as.SEQ ID NOS. 962. 964. 966 or 968.

61. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID is NOS. 970. 972. 974 or 976.

62. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 978. 980, 982 or 984. .63. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 2o1 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 986. 988, 990 or 992.

64. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 994. 996, 998 or 1000. 25

65. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1002, 1004, 1006, 1008 or 1010.

66. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID .3 NOS. 1012, 1014 or 1018.

67. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1020, 1022, 1024 or 1026.

68. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1028, 1030, 1032 or 1034. I :\DAYL1e\L1BAj02662.doc:(I( i1s

69. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1036, 1038, 1040 or 1042. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1042. 1044, 1046 or 1048.

71. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1050. 1052. 1054 or 1056. i 72. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1058. 1060. 1062 or 1064.

73. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID ri NOS. 1066. 1068, 1070 or 1072.

74. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1074. 1076, 1078 or 1080. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 2 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1082, 1084, 1086 or 1088.

76. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1090. 1092, 1094 or 1096. :5 77. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1098. 1100. 1102 or 1104.

78. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1106. 1108, 1110 or 1112.

79. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1 114, 1 116. 1118 or 1120. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic .s nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1122, 1124, 1126 or 1128. I :\DAYLI B\LIBA]02662.doc:llt

81. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1130, 1132, 1134 or 1136.

82. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic Snucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 138. 1140, 1142 or 1144.

83. The enzymatic nucleic acid molecule of claim 2. wherein said cnzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 170. 172. 174 or 176.

84. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 178. 180. 182 or 184. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID is NOS. 186. 188. 190 or 192.

86. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 194, 196, 198 or 200.

87. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 2( nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID SNOS.

202. 204. 206 or 208. 88. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 210. 212, 214 or 216. 89. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 218. 220, 222 or 224. 90. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 226. 228 or 230. 91. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 234. 236, 238 or 240. 92. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic 3s nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 242, 246 or 248. II:\DAYLIB\L11A102662.doc:111 \7 93. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 250. 252, 254 or 256. 94. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 258. 260 or 262. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 268 or 270. In 96. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 274. 278 or 280. 97. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID is NOS. 284. 286 or 288. 98. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 290. 292. 294 or 296. 99. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 20 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 298. 302 or 304. 100. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 306. 308. 310 or 312. 101. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 314. 316 or 318. 102. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 322, 324, 326 or 328. 103. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 330, 332, 334 or 336. 104. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic 5 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 338, 342 or 344. I :\DA YLI B\L BA j2662.doc:lit 105. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 346, 348 or 352. 106. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 354. 356, 358 or 360. 107. The enzymatic nucleic acid molecule of claim 2. \\herein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 362. 364, 366 or 368. i, 108. The enzymatic nucleic acid molecule of claim 2. \herein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 370. 372 or 376. 109. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 380 or 382. 110. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 386, 388, 390 or 392. 111. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID S* NOS. 394, 396 or 400. 1 12. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 402, 404, 406 or 408. s 113. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 410. 412, 414 or 416. *1 14. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 418, 420, 422 or 424. 115. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 426, 428, 430 or 432. 116. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 3s nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 434, 436, 438 or 440. I :\DAYLI\LIBA]02662.docZtIt 1 17. The enzymatic nucleic acid molecule of clain 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 444, 446 or 448. 1 18. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 450, 452, 454 or 456. 119. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 458. 460. 462 or 464. 120. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 446. 468. 470 or 472. 121. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID v NOS. 474. 476. 478 or 480. 122. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 482. 484. 486 or 488. S123. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 490. 492, 494 or 496. 124. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 498. 500. 502 or 504. 125. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 506. 508, 510 or 512. 126. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequence defined as SEQ ID NOS. 514. 516, 518 or 520. 127. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 524. 526 or 528. 128. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic vs nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 530, 532, 534 or 536. II :\DAYLII\L 13A102662.doczh 120 129. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 540, 542 or 544. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic 3 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 546. 548, 550 or 552. 131. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 554. 556. 558 or 560. 1 1 1 132. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SlEQ ID NOS. 562. 564. 566 or 568. 133. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID I NOS. 570 or 574. 134. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 578. 580. 582 or 584. 135. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 20 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 586. 588. 590 or 592. 136. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 594. 596. 598 or 600. S 137. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 602. 604. 606 or 608. 138. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 610. 612 or 616. 139. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 618, 620 or 624. 140. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 626, 628, 630 or 632. II:\DA YLI\LBA 102662.doc:tI 12) 141. The enzymatic nucleic acid molecule of claini 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 634. 638, 640 or 642. 142. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 644. 646, 648 or 670. 143. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 672. 674. 676 or 678. 144. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 680. 682. 684 or 686. 145. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID i NOS. 688. 690. 692 or 694. 146. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 696. 698. 700 or 702. 147. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic 2(1 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 407. 706. 708 or 710. 148. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 712. 714, 716 or 718. -25 149. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 720. 722 724 or 726. 150. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 728. 730. 732 or 734. S151. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 736. 738. 740 or 742. 152. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 744, 746, 748 or 750. I I:A)AYI.13IIABIAI 2662.doclz 12Z 153. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 752, 754, 756 or 758. 154. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 760. 762, 764 or 766. 155. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 778. 780, 782 or 784. 'I 156. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 786. 788, 790 or 792. 157. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID SNOS. 794. 796, 798 or 800. 158. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 802, 804, 806 or 808. 59. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 810. 812, 814 or 816. 160. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 818, 820, 822 or 824. S161. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID

826. 828, 830 or 832. 162. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 834. 836, 838 or 840. 163. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 844, 846 or 848. 164. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 850, 852 or 854. 1I:\DAY LI B\LIJBA 102662 .doc:(I 123 165. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 858, 860, 862 or 864. 166. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 866, 868, 870 or 872. 167. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 874. 876. 878 or 880. I 168. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 882. 884, 886 or 888. 169. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 890. 892, 894 or 896. 170. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences identified as SEQ ID NOS. 898, 900, 902 or 904. 171. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic S. 2 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 906, 908, 910 or 912. 172. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 914, 916, 918 or 920. 173. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 922, 924, 926 or 928. 174. The enzymatic nucleic acid molecule of claim 2. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1146, 1150 or 1152. 175. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 1154, 1156, 1158 or 1160. 176. The enzymatic nucleic acid molecule of claim 3, wherein said enzymatic 3 nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID I I\DAYLII3\L11AJ02662.doc:11t 124 NOS. 99, 100 or 130-148, wherein said enzymatic nucleic acid molecule is in a hairpin motif. 177. The enzymatic nucleic acid molecule of claim 176, wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID SNO. 99. 178. The enzymatic nucleic acid molecule of claim 176. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence dclined as SEQ ID NO. 100. 179. The enzymatic nucleic acid molecule of claim 176. wherein said i" enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEO ID) NOS. 130-133. 180. The enzymatic nucleic acid molecule of claim 176. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 134-137. 181. The enzymatic nucleic acid molecule of claim 176. wherein said enzymatic nucleic acid molecule specifically cleaves the RNA sequence defined as SEQ ID NO. 138. 182. The enzymatic nucleic acid molecule of claim 176. wherein said enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as 20 SEQ ID NOS. 139-141. 183. The enzymatic nucleic acid molecule of claim 176. wherein said o. enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 142-145. 184. The enzymatic nucleic acid molecule of claim 176. wherein said s enzymatic nucleic acid molecule specifically cleaves any of the RNA sequences defined as SEQ ID NOS. 146-148. 185. The enzymatic nucleic acid molecule of claim 3 consisting of any sequence selected from the group of SEQ ID NOS. 121, 122. 149-167. 901 86. The enzymatic nucleic acid molecule of claim 1. wherein said molecule .o has binding arms and wherein the binding arms of said enzymatic nucleic acid molecule contain sequences perfectly complementary to any of the RNA sequences defined as SEQ ID. NOS. 79-98, 101-120 or 123-129, wherein said enzymatic nucleic acid molecule is in a hammerhead motif. 187. The enzymatic nucleic acid molecule of claim 3, wherein said molecule >s has binding arms and wherein the binding arms of said enzymatic nucleic acid molecule contain sequences perfectly complementary to any of the RNA sequences defined as SEQ II :\DAYLIBLIBAIO2662.doctc I ID. NOS. 99, 100 or 130-148, wherein said enzymatic nucleic acid molecule is in a hairpin motif. 188. A mammalian cell including an enzymatic nucleic acid molecule of any one of claims 19-187. 189. The cell of claim 188 wherein said cell is a human cell. 190. An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of any one of claims 19-187 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell. SI 191. A mammalian cell including an expression vector of claim 190. 192. The cell of claim 191 wherein said cell is a human cell. 193. The use of an enzymatic nucleic acid molecule of any one of claims 1-6 or 19-187. for the manufacture of a medicament for the treatment of a stenotic condition in a patient. 194. The use of an expression vector of claim 190 in the manufacture of a medicament for the treatment of a stenotic condition in a patient. 195. The use of an enzymatic nucleic acid molecule of any one of claims 1-6 or I 9- 187. for the manufacture of a medicament for the treatment of cancer in a patient. 196. The use of an expression vector of claim 190 in the manufacture of a medicament for the treatment of cancer in a patient. 197. The use of any one of claims 193-196 wherein said patient is a human. 198. An enzymatic nucleic acid molecule of any one of claims 1-6 or 19-187, when used in the treatment of a stenotic condition in a patient. 199. An expression vector of claim 190 when used in the treatment of a stenotic s condition in a patient. 200. An enzymatic nucleic acid molecule of any one of claims 1-6 or 19-187 when used in the treatment of cancer in a patient. 201. An expression vector of claim 190 when used in the treatment of cancer in a patient. 202. The nucleic acid molecule or expression vector of any one of claims 198- 201 wherein said patient is a human. 203. A method of treating a stenotic condition in a patient by administering to said patient an enzymatic nucleic acid molecule of any one of claims 19-187. 204. A method of treating a stenotic condition in a patient by administering to .s said patient an expression vector of claim 190. II \I)AY.I\III3AJ02c62,dtc:(Il 12G 205. A method of treating cancer in a patient by administering to said patient or said patient's cells an enzymatic nucleic acid molecule of any one of claims 19-187. 206. A method of treating cancer in a patient by administering to said patient or said patient's cells an expression vector of claim 190. 207. The method of any one of claims 203-206 wherein said patient is a human. 208. A method for administration of an enzymatic nucleic acid molecule of any one of claims 19-187 by mixing said nucleic acid molecule with a chemical selected from the group consisting of chloroquine, ammonium chloride, carbonyl cyanide p- trifluoromethoxy phenyl hydrazone (FCCP), monensin, colchicine, amphipathic peptides, ic viral proteins, and viral particles. 209. A formulation comprising a mixture of an enzymatic nucleic acid molecule of any one of claims 1-6 or 19-187 and a chemical selected from the group consisting of chloroquine, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), monensin, colchicine, amphipathic peptides, viral proteins, and viral 1s particles. Dated 7 September, 2001 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S II:\DAYLIB\LBA102662.docIlt