AU2774202A - Method and reagent for inhibiting the expression of disease related genes - Google Patents

Description

S&F Ref: 349267D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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

2950 Wilderness Place Boulder Colorado 80301 United States of America Dan T. Stinchcomb, Bharat Chowrira, Anthony Direnzo, Kenneth G. Draper, Lech W. Dudycz, Susan Grimm, Alexander Karpeisky, Kevin Kisich, Jasenka Matulic- Adamic, James A. McSwiggen, Anil Modak, Pamela Pavco, Leonid Biegelman, Sean M. Sullivan, David Sweedler, James D. Thompson, Danuta Tracz, Nassim Usman, Francine E. Wincott, Tod Woolf Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Method and Reagent for Inhibiting the Expression of Disease Related Genes Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c METHOD AND REAGENT FOR INHIBITING THE EXPRESSION OF DISEASE RELATED GENES Background of the Invention This invention relates to reagents useful as inhibitors of gene expression relating to diseases such as inflammatory or autoimmune disorders, chronic myelogenous leukemia, or respiratory tract illness.

Summary of the Invention The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting the expression of disease related genes, ICAM-1, IL-5, relA, TNF-x, p210 bcr-abl, and respiratory .0 syncytial virus genes. Such ribozymes can be used in a method for 10 treatment of diseases caused by the expression of these genes in man and other animals, including other primates.

Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.

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 1 summarizes some of the characteristics of these ribozymes.

Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.

After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. The 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, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ration of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to 15 those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site. With their catalytic activity and increased site specificity, ribozymes represent more potent and safe therapeutic molecules than antisense oligonucleotides.

20 Thus, in a first aspect, this invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave RNA species encoding ICAM-1, relA, TNF-a, p210bcr-abl, or RSV proteins. In particular, applicant describes the selection and function of ribozymes capable of cleaving these RNAs and their use to reduce levels of ICAM-1, IL-5, relA, TNF-a, p210 bor-abl or RSV proteins in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic uses.

Applicant indicates that these ribozymes are able to inhibit expression of ICAM-1, IL-5, rel A, TNF-c, p210bcr-abl, or RSV genes and that the catalytic activity of the ribozymes is required for their inhibitory effect.

Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave target ICAM-1, IL-5, rel A, TNFa, p210bcr-abl, or RSV encoding mRNAs may be readily designed and are within the invention.

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 3 cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.

By "gene" is meant to refer to either the protein coding regions of the cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.

By "enzymatic RNA molecule" it is meant an RNA molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule.

This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur.

One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By "equivalent" RNA to a virus is meant to include those naturally occurring viral encoded RNA molecules associated with viral caused diseases in various animals, including humans, cats, simians, and other primates. These viral or viralencoded RNAs have similar structures and equivalent genes to each other.

By "complementarity" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson- S. Crick or other non-traditional types (for examplke, Hoogsteen type) of basepaired interactions.

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 associateion 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 Retroviruse 8,183, of hairpin motifs by Hampel and Tritz, 1989 Biochemistry 28, 4929, EP 0360257 and Hampel et al., 1990, Nucleic Acids Res. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell, 35 849, 4 Neurospora VS RNA ribozyme motif is described by Collins (Seville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad.

Sci.. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795- 2799 Guo and Collins, 1995 EMBO. 14, 368) and of the Group 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 which is complementary to one or more of the target gene RNA regions, and that it has nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

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 15 to a highly conserved sequence region of a target I CAM-1, IL-5, reLA, TNF-ac, p210 bcr-abl or RSV proteins) encoding mRNA 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, 20 the ribozymes can be expressed from vectors that are delivered to specific cells. By "vectors" is meant any nucleic acid and/or viral-based technique used to deliver a desired nucleic acid.

O Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such 25 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 structrure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters Scanion, K.J. et al., 1991, Proc.

Natl. Acad, Sci,. USA, 88, 10591-5; Kashani-Sabet, et al.,1992, Antisense Res, Dev,, 2, 3-15; Dropoulic, et al., 1992, J.Virol 66, 1432- 41; Weerasinghe, et al., 191, 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 would realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from tlhe primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa, et al., 1992, Nucleic Acids Symp.

Se. 27, 15-6; Taira, K. et al., Nucleic Acids Res., 19, 5125-30; Ventura, M., et al., 1993, Nucleic Acids Res., 21, 3249-55, Chowrira et al., 1994 J. Biol.

Chem.. 269, 25856).

By "inhibit" is meant that the activity or level of ICAM-1,Rel A, TNF-a, p210bcr-abl or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, 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.

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 ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV protein or activity in a cell or tissue. By "related" is meant that the inhibition of ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV mRNA translation, and thus reduction in the level of, ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV proteins 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 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. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 2,3,6-9, 11, 13, 15-23, 27, 28, 31, 33, 34, 36 and 37.

Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. 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 mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

r 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 the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two basepaired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequence listed in the above identified Tables may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

In another aspect of the invention, ribozymes that cleave target 15 molecules and inhibit ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV gene expression are expressed from transcription units inserted into DNA, RNA, or viral vectors. Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the 20 ribozyme sequences are driven from a promoter for eukaryotic 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 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, 25 silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl.

Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). 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. USA, 90, 6340-4; L'Huiller et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl.

Acad. Sci. 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).

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

Description Of The Preferred Embodiments The drawings will first briefly be described.

Drawings: Figure 1 is a diagrammatic representation of the hammerhead .i 10 ribozyme domain known in the art. Stem II can be 2 base-pair long.

Figure 2(a) is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2(b) is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 15 2(c) is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2(d) is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, NucI. Acids. Res., 17, 1371-1371) into two portions.

Figure 3 is a diagrammatic representation of the general' structure of a 20 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 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases r is 1 base).

Helix 1, 4 or 5 may also be 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 the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size o and p is each independently from 0 to any number, e.g. 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be 8 modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. is 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases.

refers to a covalent bond.

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

Figure 5 is a representation of the general structure of the selfcleaving VS RNA ribozyme domain.

Figure 6 is a diagrammatic representation of the genetic map of RSV strain A2.

15 Figure 7 is a diagrammatic representation of the solid-phase synthesis of RNA.

Figure 8 is a diagrammatic representation of exocyclic amino protecting groups for nucleic acid synthesis.

Figure 9 is a diagrammatic representation of the deprotection of RNA.

20 Figure 10 is a graphical representation of the cleavage of an RNA substrate by ribozymes synthesized, deprotected and purified using the improved methods described herein.

Figure 11 is a schematic representation of a two pot deprotection protocol. Base deprotection is carried out with aqueous methyl amine at °C for 10 min. The sample is dried in a speed-vac for 2-24 hours depending on the scale of RNA synthesis. Silyl protecting group at the 2'hydroxyl position is removed by treating the sample with 1.4 M anhydrous HF at 650C for 1.5 hours.

Figure 12 is a schematic representation of a one pot deprotection of RNA synthesized using RNA phosphoramidite chemistry. Anhydrous methyl amine is used to deprotect bases at 65°C for 15 min. The sample is allowed to cool for 10 min before adding TEA-3HF reagent, to the same pot, to remove protecting groups at the 2'-hydroxyl position. The deprotection is carried out for 1.5 hours.

Figs. 13a b is a HPLC profile of a 36 nt long ribozyme, targeted to site B. The RNA is deprotected using either the two pot or the one pot deprotection protocol. The peaks corresponding to full-length RNA is indicated. The sequence for site B is CCUGGGCCAGGGAUUA

AUGGAGAUGCCCACU.

Figure 14 is a graph comparing RNA cleavage activity of ribozymes deprotected by two pot vs one pot deprotection protocols.

S 10 Figure 15 is a schematic representation of an improved method of synthesizing RNA containing phosphorothioate linkages.

Figure 16 shows RNA cleavage reaction catalyzed by ribozymes containing phosphorothioate linkages. Hammerhead ribozyme targeted to site C is synthesized such that 4 nts at the 5' end contain phosphorothioate 15 linkages. P=O refers to ribozyme without phosphorothioate linkages. P=S refers to ribozyme with phosphorothioate linkages. The sequence for site C is UCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG.

Figure 17 is a schematic representation of synthesis of 2'-Nphtalimido-nucleoside phosphoramidite.

Figure 18 is a diagrammatic representation of a prior art method for the solid-phase synthesis of RNA using silyl ethers, and the method of this invention using SEM as a 2'-protecting group.

Figure 19 is a diagrammatic representation of the synthesis of 2'- SEM-protected nucleosides and phosphoramidites useful for the synthesis of RNA. B is any nucleotide base as exemplified in the Figure, P is purine and I is inosine. Standard abbreviations are used throughout this application, well known to those in the art.

Figure 20 is a diagrammatic representation of a prior art method for deprotection of RNA using TBDMS protection of the 2'-hydroxyl group.

Figure 21 is a diagrammatic representation of the deprotection of RNA having SEM protection of the 2'-hydroxyl group.

Figure 22 is a representation of an HPLC chromatogram of a fully deprotected 10-mer of uridylic acid.

Figs. 23 25 are diagrammatic representations of hammerhead, hairpin or hepatitis delta virus ribozyme containing self-processing

RNA

transcript. Solid arrows indicate self-processing sites. Boxes indicate the sites of nucleotide substitution. Solid lines are drawn to show the binding sites of primers used in a primer-extension assay. Lower case letters indicate vector sequence present in the RNA when transcribed from a Hindlll-linearized plasmid. (23) HH Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hammerhead ribozyme. The structure of the hammerhead ribozyme is based on phylogenetic and mutational analysis (reviewed by Symons, 1992 supra).

The trans ribozyme domain extends from nucleotide 1 through 49. After 3'end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (UC at positions 50 and 51) at its 3' end. The 3' processing ribozyme is comprised of nucleotides 44 through 96. Roman numerals I, II and III, indicate the three helices that contribute to the structure of the 3' cis-acting hammerhead ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20, 3252).

Substitution of G70 and A71 to U and G respectively, inactivates the hammerhead ribozyme (Ruffner et al., 1990 Biochemistry 29, 10695) and generates the HH(mutant) construct. (24) HP Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hairpin ribozyme. The structure of the hairpin ribozyme is based on phylogenetic and mutational analysis (Berzal-Herranz et al., 1993 EMBO. J 25 12, 2567). The trans-ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 5 non-ribozyme nucleotides (UGGCA at positions 50 to 54) at its 3' end. The 3' cis-acting ribozyme is comprised of nucleotides 50 through 115. The transcript named HP(GU) was constructed with a potential wobble base pair between G52 and U77; HP(GC) has a Watson-Crick base pair between G52 and C77. A shortened helix 1 (5 base pairs) and a stable tetraloop (GAAA) at the end of helix 1 was used to connect the substrate with the catalytic domain of the hairpin ribozyme (Feldstein Bruening, 1993 Nucleic Acids Res. 21, 1991; Altschuler et al., 1992 supra). (25) HDV Cassette, transcript containing the trans-acting hammerhead ribozyme linked to a 3' cis-acting hepatitis delta virus (HDV) ribozyme. The secondary structure of the HDV ribozyme is as proposed by Been and 11 coworkers (Been et al., 1992 Biochemistry 31, 11843). The trans-ribozyme domain extends from nucleotides 1 through 48. After 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (AA at positions 49 to 50) at its 3' end. The 3' cis-acting HDV ribozyme is comprised of nucleotides 50 through 114. Roman numerals I, 11, III IV, indicate the location of four helices within the 3' cis-acting HDV ribozyme (Perrota Been, 1991 Nature 350, 434). The AHDV transcript contains a 31 nucleotide deletion in the HDV portion of the transcript (nucleotides 84 through 115 deleted).

Fig. 26 is a schematic representation of a plasmid containing the insert encoding self-processing cassette. The figure is not drawn to scale.

Fig. 27 demonstrates the effect of 3' flanking sequences on RNA selfprocessing in vitro. H, Plasmid templates linearized with Hindlll restriction enzyme. Transcripts from H templates contain four non-ribozyme 15 nucleotides at the 3' end. N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from N templates contain 220 nonribozyme nucleotides at the 3' end. R, Plasmid templates linearized with Rcal restriction enzyme. Transcripts from R templates contain 450 nonribozyme nucleotides at the 3' end.

Fig. 28 shows the effect of 3' flanking sequences on the trans- S* cleavage reaction catalyzed by a hammerhead ribozyme. A 622 nt internally-labeled RNA (<10 nM) was incubated with ribozyme (1000 nM) under single turn-over conditions (Herschlag and Cech, 1990 Biochemistry 29, 10159). HH+2, HH+37, and HH+52 are trans-acting ribozymes produced by transcription from the HH, AHDV, and HH(mutant) constructs, respectively, and that contain 2, 37 and 52 extra nucleotides on the 3' end.

The plot of the fraction of uncleaved substrate versus time was fit to a double exponential curve using the KaleidaGraph graphing program (Synergy Software, Reading, PA). A double exponential curve fit was used because the data points did not fall on a single exponential curve, presumably due to varying conformers of ribozyme and/or substrate RNA.

Fig. 29 shows RNA self-processing in OST7-1 cells. In vitro lanes contain full-length, unprocessed transcripts that were added to cellular lysates prior to RNA extraction. These RNAs were either pre-incubated with MgCI2 or with DEPC-treated water prior to being hybridized with 5' end-labeled primers. Cellular lanes contain total cellular RNA from cells transfected with one of the four self-processing constructs. Cellular RNA are probed for ribozyme expression using a sequence specific primerextension assay. Solid arrows indicate the location of primer extension bands corresponding to Full-Length RNA and 3' Cleavage Products.

Figs. 30,31 are diagrammatic representations of self-processing cassettes that will release trans-acting ribozymes with defined, stable stemloop structures at the 5' and the 3' end following self-processing. shows various permutations of a hammerhead self-processing cassette. 31, shows various permutations of a hairpin self-processing cassette.

S.Figs. 32a-b Schematic representation of RNA polymerse III promoter structure. Arrow indicates the transcription start site and the direction of coding region. A, B and C, refer to consensus A, B and C box promoter sequences. I, refers to intermediate cis-acting promoter sequence. PSE, 15 refers to proximal sequence element. DSE, refers to distal sequence element. ATF, refers to activating transcription factor binding element. refers to cis-acting sequence element that has not been fully characterized.

EBER, Epstein-Barr-virus-encoded-RNA. TATA is a box well known in the art.

Figs. 33a-e Sequence of the primary tRNAimet and A3-5 transcripts.

l The A and B box are intemal promoter regions necessary for pol Ill transcription. Arrows indicate the sites of endogenous tRNA processing.

.The A3-5 transcript is a truncated version of tRNA wherein the sequence 3' of B box has been deleted (Adeniyi-Jones et al., 1984 supra). This modification renders the A 3-5 RNA resistant to endogenous tRNA processing.

Figure 34. Schematic representation of RNA structural motifs inserted into the A3-5 RNA. A3-5/HHI- a hammerhead (HHI) ribozyme was cloned at the 3' region of A3-5 RNA; S3- a stable stem-loop structure was incorporated at the 3' end of the A3-5/HHI chimera; S5- stable stem-loop structures were incorporated at the 5' and the 3' ends of A3-5/HHI ribozyme chimera; S35- sequence at the 3' end of the A3-5/HHI ribozyme chimera was altered to enable duplex formation between the 5' end and a complementary 3' region of the same RNA; S35Plus- in addition to structural alterations of S35, sequences were altered to facilitate additional 13 duplex formation within the non-ribozyme sequence of the chimera.

Figures 35 and 36. Northern analysis to quantitate ribozyme expression in T cell lines transduced with A3-5 vectors. 35) A3-5/HHI and its variants were cloned individually into the DC retroviral vector (Sullenger et al., 1990 supra). Northern analysis of ribozyme chimeras expressed in MT-2 cells was performed. Total RNA was isolated from cells (Chomczynski Sacchi, 1987 Analytical Biochemistry 162, 156-159), and transduced with various constructs described in Fig. 34. Northern analysis was carried out using standard protocols (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY). Nomenclature is same as in Figure 34. This assay measures the level of expression from the type 2 pol III S* promoter. 36) Expression of S35 constructs in MT2 cells. S35 (+ribozyme),

S

35 construct containing HHI ribozyme. S35 (-ribozyme), S35 construct o*'o 15 containing no ribozyme.

Figure 37. Ribozyme activity in total RNA extracted from transduced MT-2 cells. Total RNA was isolated from cells transduced with constructs described in Figs. 35 and 36 In a standard ribozyme cleavage reaction, 5 gg total RNA and trace amounts of 5' terminus-labeled ribozyme target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCI 2 RNAs were renatured by cooling the reaction mixture to 37 0 C for 10-15 min. Cleavage reaction was initiated by mixing the labeled substrate RNA and total cellular RNA at 37°C. The reaction was allowed to proceed for 18h, following which the samples were resolved on a 20 urea-polyacrylamide gel. Bands were visualized by autoradiography.

Figures 38 and 39. Ribozyme expression and activity levels in transduced clonal CEM cell lines. 38) Northern analysis of transduced clonal CEM cell lines. Standard curve was generated by spiking known concentrations of in vitro transcribed S5 RNA into total cellular RNA isolated from non-transduced CEM cells. Pool, contains RNA from pooled cells transduced with S35 construct. Pool (-G418 for 3 Mo), contains RNA from pooledcells that were initially selected for resistance to G418 and then grown in the absence of G418 for 3 months. Lanes A through N contain RNA from individual clones that were generated from the pooled cells transduced with S35 construct. tRNAlmet, refers to the endogenous tRNA. S35, refers to the position of the ribozyme band. M, marker lane. 39) Activity levels in S35-transduced clonal CEM cell lines.

RNA isolation and cleavage reactions were as described in Fig.37.

Nomenclature is same as in Figs. 35 and 36 except, S, 5' terminus-labeled substrate RNA. P, 8 nt 5' terminus-labeled ribozyme-mediated RNA cleavage product.

Figures 40 and 41 are proposed secondary structures of S35 and containing a desired RNA (HHI), respectively. The position of HHI ribozyme is indicated in figure 41. Intramolecular stem refers to the stem structure formed due to an intramolecular base-paired interaction between the 3' sequence and the complementary 5' terminus. The length of the stem ranges from 15-16 base-pairs. Location of the A and the B boxes are shown.

Figures 42 and 43 are proposed secondary structures of S35 plus 15 and S35 plus containing HHI ribozyme.

Figures 44, 45, 46 and 47 are the nucleotide base sequences of HHIS35, S35 Plus, and HHIS35 Plus respectively.

Figs. 48a-b is a general formula for pol III RNA of this invention.

Figure 49 is a digrammatic representation of 5T construct. In this construct the desired RNA is located 3' of the intramolecular stem.

Figures 50 and 51 contain proposed secondary structures of construct alone and 5T contruct containing a desired RNA (HHI ribozyme) respectively.

Figure 52 is a diagrammatic representation of TRZ-tRNA chimeras.

The site of desired RNA insertion is indicated.

Figure 53 shows the general structure of HHITRZ-A ribozyme chimera.

A hammerhead ribozyme targeted to site I is inserted into the stem II region of TRZ-tRNA chimera.

Figure 54 shows the general structure of HPITRZ-A ribozyme chimera.

A hairpin ribozyme targeted to site I is cloned into the indicated region of TRZ-tRNA chimera.

Figure 55 shows.a comparison of RNA cleavage activity of HHITRZ-A, HHITRZ-B and a chemically synthesized HHI hammerhead ribozymes.

Figure 56 shows expression of ribozymes in T cell lines that are stably transduced with viral vectors. M, markers; lane 1, non-transduced CEM cells; lanes 2 and 3, MT2 and CEM cells transduced with retroviral vectors; lanes 4 and 5, MT2 and CEM cells transduced with AAV vectors.

Figs. 57a-b Schematic diagram of adeno-associated virus and adenovirues vectors for ribozyme delivery. Both vectors utilize one or more ribozyme encoding transcription units (RZ) based on RNA polymerase II or RNA polymerase III promoters. A. Diagram of an AAV-based vector containing minimal AAV sequences comprising the inverted terminal repeats (ITR) at each end of the vector genome, an optional selectable marker (Neo) driven by an exogenous promoter (Pro), a ribozyme transcription unit, and sufficient additional sequences (stuffer) to maintain a 15 vector length suitable for efficient packaging. B. Diagram of ribozyme expressing adenovirus vectors containing deletions of one or more wild type adenoviorus coding regions (cross-hatched boxes marked as El, plX, E3, and E4), and insertion of the ribozyme transcription unit at any or several of those regions of deletions.

Fig. 58 is a graph showing the effect of arm length variation on the activity of ligated hammerhead (HH) ribozymes. Nomenclature 5/5, 6/6, 7/7, 8/8 and so on refers to the number of base-pairs being formed between the ribozyme and the target. For example, 5/8 means that the HH ribozyme forms 5 bp on the 5' side and 8 bp on the 3' side of the cleavage site for a 25- total of 13 bp. -AG refers to the free energy of binding calculated for basepaired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Ann. Rev. Biophys. Chem. 17, 167). RPI A is a HH ribozyme with 6/6 binding arms.

Figs. 59 and 60 and 61 show cleavage of long substrate (622 nt) by ligated HH ribozymes.

Fig. 62 is a diagrammatic representation of a hammerhead ribozyme (HH-H) targeted against a site termed H. Variants of HH-H are also shown that contain either a 2 base-paired stem II (HH-H1 and HH-H2) or a 3 basepaired stem II (HH-H3 and HH-H4).

Figs. 63 and 64 show RNA cleavage activity of HH-1 and its variants (see Fig.62). 63) cleavage of matched substrate RNA (15 nt). 64) cleavage of long substrate RNA (613 nt).

Figs. 65a-b is a schematic representation of a method of this invention to synthesize a full length hairpin ribozyme. No splint strand is required for ligation but rather the two fragments hybridize together at helix 4 prior to ligation. The only prerequisite is that the 3' fragment is phosphorylated at its 5' end and that the 3' end of the 5' fragment have a hydroxyl group. The hairpin ribozyme is targeted against site J. H1 and H2 are intermolecular helices formed between the ribozyme and the substrate. H3 and H4 are intramolecular helices formed within the hairpin ribozyme motif. Arrow S indicates the cleavage site.

Fig. 66 shows RNA cleavage activity of ligated hairpin ribozymes targeted against site J.

15 Figs. 67a-b is a diagrammatic representation of a Site K Hairpin Ribozyme (HP-K) showing the proposed secondary structure of the hairpin ribozyme -substrate complex as described in the art (Berzal-Herranz et al., 1993 EMBO. J.12, 2567). The ribozyme has been assembled from two fragments (bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent intramolecular helix formation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme). Left panel (HP-K1) indicates 4 base- S" paired helix 2 and the right panel (HP-K2) indicates 6 base-paired helix 2.

Arrow indicates the site of RNA cleavage. All the ribozymes discussed herein were chemically synthesized by solid phase synthesis using RNA phosphoramadite chemistry, unless otherwise indicated. Those skilled in the art will recognize that these ribozymes could also be made transcriptionally in vitro and in vivo.

Figure 68 is a graph showing RNA cleavage by hairpin ribozymes targeted to site K. A plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-K2 (6 bp helix 2) cleaves a 422 target RNA to a greater extent than the HP-K1 (4 bp helix 2).

To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 422 nt region (containing hairpin site A) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of [a- 3 2 P]CTP (Chowrira Burke, 1991 supra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 p.l DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1pM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCI2) by heating to 900C for 2 min. and slow cooling to 370C for 10 min. The 15 reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 370C. Aliquots of 5 pil were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale,

CA).

Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary structure of the hairpin ribozymeosubstrate complex. The S: ribozyme was assembled from two fragments as described above. The nomenclature is the same as above.

Figure 70 shows RNA cleavage by hairpin ribozymes targeted to site L. A. plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-L2 (6 bp helix 2) cleaves a 2 KB target RNA to a greater extent than the HP-L1 (4 bp helix To make internallylabeled substrate RNA for trans-ribozyme cleavage reactions, a 2 kB region (containing hairpin site L) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. The cleavage reactions were carried out as described above.

Figs. 71a-b shows a Site M Hairpin Ribozyme (HP-M) with the proposed secondary structure of the hairpin ribozymeosubstrate complex.

The ribozyme was assembled from two fragments as described above.

Figure 72 is a graph showing RNA cleavage by hairpin ribozymes targeted to site M. The ribozymes were tested at both 20°C and at 260C.

To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 1.9 KB region (containing hairpin site M) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Cleavage reactions were carried out as described above except that 20°C and at 260C temperatures were used.

Figs. 73a-d shows various structural modifications of the present invention. A) Hairpin ribozyme lacking helix 5. Nomenclature is same as described under figure 3. B) Hairpin ribozyme lacking helix 4 and helix Helix 4 is replaced by a nucleotide loop wherein q is 2 2 bases.

15 Nomenclature is same as described under figure 3. C) Hairpin ribozyme lacking helix 5. Helix 4 loop is replaced by a linker 103"L", wherein L is a non-nucleotide linker molecule (Benseler et al., 1993 J. Am. Chem. Soc.

115, 8483; Jennings et al., WO 94/13688). Nomenclature is same as described under figure 3. D) Hairpin ribozyme lacking helix 4 and helix Helix 4 is replaced by non-nucleotide linker molecule (Benseler et al., 1993 supra; Jennings et supra). Nomenclature is same as described under figure 3.

Figs. 74a-b shows Hairpin ribozymes containing nucleotide spacer region at the indicated location, wherein s is 2 1 base. Hairpin 25 ribozymes containing spacer region, can be synthesized as one fragment or can be assembled from multiple fragments. Nomenclature is same as described under figure 3.

Figs. 75a-e shows the structures of the nucleotides. R 1 is as defined above. R is OH, H, O-protecting group, NH, or any group described by the publications discussed above, and those described below. B is as defined in the Figure or any other equivalent nucleotide base. CE is cyanoethyl, DMT is a standard blocking group.

Other abbreviations are standard in the art.

Figure 76 is a diagrammatic representation of the synthesis of alkyl-D-allose nucleosides and their phosphoramidites.

Figure 77 is a diagrammatic representation of the synthesis of alkyl-L-talose nucleosides and their phosphoramidites.

Figure 78 is a diagrammatic representation of hammerhead ribozymes targeted to site O containing 5'-C-methyl-L-talo modifications at various positions.

Figure 79 shows RNA cleavage activity of HH-O ribozymes. Fraction of target RNA uncleaved as a function of time is shown.

10 Figure 80 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al. Nucleic Acids Res. 1992, 3252) showing specific substitutions.

Figs. 81a-j shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention. R groups are alkyl groups, Z is a protecting group.

Figure 82 is a diagrammatic representation of the synthesis of 2'-Callyl uridine and cytidine.

Figure 83 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene uridine.

20 Figure 84 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene cytidine.

Figure 85 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene adenosine.

Figure 86 is a diagrammatic representation of the synthesis of 2'-Ccarboxymethylidine uridine, 2 '-C-methoxycarboxymethylidine uridine and derivatized amidites thereof. X is CH 3 or alkyl as discussed above, or another substituent.

Figure 87 is a diagrammatic representation of a synthesis of nucleoside Figure 88 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylphosphonate 3'-phosphoramidites, dimers and solid supported dimers.

Figure 89 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylene triphosphates.

Figures 90 and 91 are diagrammatic representations of the synthesis of 3'-deoxy-3'-difluoromethylphosphonates and dimers.

Figure 92 is a schematic representation of synthesizing RNA phosphoramidite of a nucleotide containing a 2'-hydroxyl group modification of the present invention.

Figs. 93a-b describes a method for deprotection of oligonucleotides containing a 2'-hydroxyl group modification of the present invention.

Figure 94 is a diagrammatic representation of a hammerhead ribozyme targeted to site N. Positions of 2'-hydroxyl group substitution is indicated.

Figure 95 shows RNA cleavage activity of ribozymes containing a 2'hydroxyl group modification of the present invention. All RNA, represents hammerhead ribozyme (HHN) with no 2'-hydroxyl group modifications. U7ala, represents HHN ribozyme containing 2'-NH-alanine modification at the U7 position. U4/U7-ala, represents HHA containing 2'-NH-alanine Smodifications at U4 and U7 positions. U4 lys, represents HHA containing 2'-NH-lysine modification at U4 position. U7 lys, represents HHA containing 2'-NH-lysine modification at U7 position. U4/U7-lys, represents HHN containing 2'-NH-lysine modification at U4 and U7 positions.

Figures 96 and 97 are schematic representations of synthesizing (solid-phase synthesis) 3' ends of RNA with modification of the present invention. B, refers to either a base, modified base or an H.

Figure 98 and 99 are schematic representations of synthesizing (solid-phase synthesis) 5' ends of RNA with modification of the present invention. B, refers to either a base, modified base or an H.

Figures 100 and 101 are general schematic representations of the invention.

21 Fig. 102a-d is a schematic representation of a method of the invention.

Fig. 103 is a graph of the results of the experiment diagrammed in figure 104.

Figure 104 is a diagrammatic representation of a fusion mRNA used in the experiment diagrammed in Fig. 102.

Figure 105 is a diagrammatic representation of a method for selection of useful ribozymes of this invention.

Figure 106 generally shows R-loop formation, and an R-loop complex. In addition, it indicates the location at which ligands can be 10 provided to target the R-loop complex to cells using at least three different procedures, such as ligand receptor interaction, lipid or calcium phosphate mediated delivery, or electroporation.

0* Figure 107 shows a method for use of self-processing ribozymes to generate therapeutic ribozymes of unit length. This method is essentially described by Draper et al., PCT WO 93/23509.

Figure 108 shows a method of linking ligands like folate, carbohydrate or peptides to R-loop forming RNA.

Ribozymes of this invention block to some extent ICAM-1, IL-5, rel A, TNF-a, p210 bc r a b l or RSV genes expression and can be used to treat 20 diseases or diagnose such diseases. Ribozymes will be delivered to cells in culture and to tissues in animal models. Ribozyme cleavage of ICAM-1, 11-5, rel A, TNF-o ,p210bc r a b l, or RSV mRNA in these systems may prevent or alleviate disease symptoms or conditions.

I. Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al PCT W093/23509, Sullivan et al., PCT W094/02595 as well as by Draper et al., PCT/US94/13129 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. While specific examples to animal and human RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below. Thus, the same target may be used, but binding arms suitable for targeting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below.

It must be established that the sites predicted by the computer-based RNA folding algorithm correspond to potential cleavage sites.

Hammerhead or hairpin ribozymes are designed that could bind and are individually analyzed by computer folding (Jaeger 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 are eliminated from consideration. Varying binding arm 15 lengths can be chosen to optimize activity. Generally, at least 5 bases on :i each arm are able to bind to, or otherwise interact with, the target RNA.

mRNA is screened for accessible cleavage sites by the method described generally in Draper et al., PCT W093/23569 hereby incorporated by reference herein. Briefly, DNA oligonucleotides 20 representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from cDNA clones. Labeled RNA transcripts are synthesized in vitro from DNA templates. The oligonucleotides and the labeled trascripts are annealed, RNaseH is 25 added and the mixtures are incubated for the designated times at 370C.

Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a phosphor imaging system. From these data, hammerhead or hairpin ribozynme sites are chosen as the most accessible.

Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences desribed above. The ribozymes are chemically synthesized. The method of synthesis used follows 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 phosphoramidites at the 3'-end. The average stepwise coupling yeilds are Inactive ribozymes are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan. and Uhlenbach, 1989, Methods Enzymol, 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'H (for a review see Usman and Cedergren, 1992 TIBS 17,34). Ribozymes are purified by gel electrophoresis using heneral methods or are purified by high pressure liquid chromatography and are resuspended in water.

Example 1: ICAM-1 o Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. ICAM-1 function can be blocked therapeutically using monoclonal antibodies. Ribozymes have the advantage of being generally immunologically inert, whereas significant neutralizing anti-lgG responses can be observed with some monoclonal antibody treatments.

The following is a brief description of the physiological role of ICAM-1.

The discussion is not meant to be complete and is provided only for 25 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.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose expression is induced by inflammatory mediators. ICAM-1 is required for adhesion of leukocytes to endothelial cells and for several immunological functions including antigen presentation, immunoglobulin production and cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell recognition and activity during transplant rejection and in animal models of rheumatoid arthritis, asthma and reperfusion injury.

Cell-cell adhesion plays a pivotal role in inflammatory and immune responses (Springer et al., 1987 Ann. Rev. Immunol. 5, 223-252). Cell adhesion is required for leukocytes to bind to and migrate through vascular endothelial cells. In addition, cell-cell adhesion is required for antigen presentation to T cells, for B cell induction by T cells, as well as for the cytotoxicity activity of T cells, NK cells, monocytes or granulocytes.

Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell interactions (Simmons et al., 1988 Nature (London) 331, 624-627).

ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J. Immunol. 137, 245-254). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, rinterferon, tumor necrosis factor-a, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface (Sringer et. al. supra; Dustin et al., supra; and Rothlein et al., 1988 J.

Immunol. 141, 1665-1669). Induction occurs via increased transcription of ICAM-1 mRNA (Simmons et al., supra). Elevated expression is detectable after 4 hours and peaks after 16 24 hours of induction.

20 ICAM-1 induction is critical for a number of inflammatory and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd,1988 Proc. Natl. Acad. Sci. USA 85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331).

Thus, ICAM-1 expression may be required for the extravasation of immune 25 cells to sites of inflammation. Antibodies to ICAM-1 also block T cell killing, mixed lymphocyte reactions, and T cell-mediated B cell differentiation, suggesting that ICAM-1 is required for these cognate cell interactions (Boyd et al., supra). The importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J.

Immunol 144, 4082-4091). Conversely, murine L cells require transfection with human ICAM-1 in addition to HLA-DR in order to present antigen to human T cells (Altmann et al., 1989 Nature (London) 338, 512-514). In summary, evidence in vitro indicates that ICAM-1 is required for cell-cell interactions critical to inflammatory responses, cellular immune responses, and humoral antibody responses.

By engineering ribozyme motifs we have designed several ribozymes directed against ICAM-1 mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance. These ribozymes cleave ICAM-1 target sequences in vitro.

The sequence of human, rat and mouse ICAM-1 mRNA can be screened for accessible sites using a compter folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables 2, 3, and 6-9. (All sequences are 5' to 3' in the tables) While rat, mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4 8 and 10. 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 and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

20 The ribozymes will be tested for function in vivo by exogenous delivery to human umbilical vein endothelial cells (HUVEC). Ribozymes will be delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors described above. Cytokine-induced ICAM-1 expression will be .25 monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. ICAM-1 mRNA levels will be assessed by Northern, by RNAse protection, by primer extension or by quantitative RT-PCR analysis.

Ribozymes that block the induction of ICAM-1 protein and mRNA by more than 90% will be identified.

As disclosed by Sullivan et al., PCT W094/02595, incorporated by reference herein, ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of ICAM- 1 mRNA and protein. The effect of the anti-ICAM-1 ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-ICAM-1 ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

Uses ICAM-1 plays a central role in immune cell recognition and function.

Ribozyme inhibition of ICAM-1 expression can reduce transplant rejection and alleviate symptoms in patients with rheumatoid arthritis, asthma or other acute and chronic inflammatory disorders. We have engineered several ribozymes that cleave ICAM-1 mRNA. Ribozymes that efficiently inhibit ICAM-1 expression in cells can be readily found and their activity measured with regard to their ability to block transplant rejection and 15 arthritis symptoms in animal models. These anti-ICAM-1 ribozymes represent a novel therapeutic for the treatment of immunological or inflammatory disorders.

The therapeutic utility of reduction of activity of ICAM-1 function is evident in the following disease targets. The noted references indicate the 20 role of ICAM-1 and the therapeutic potential of ribozymes described herein.

Thus, these targets can be therapeutically treated with agents that reduce ICAM-1 expression or function. These diseases and the studies that support a critical role for ICAM-1 in their pathology are listed below. This list is not meant to be complete and those in the art will recognize further 25 conditions and diseases that can be effectively treated using ribozymes of the present invention.

Transplant rejection ICAM-1 is expressed on venules and capillaries of human cardiac biopsies with histological evidence of graft rejection (Briscoe et al., 1991 Transplantation 51, 537-539).

Antibody to ICAM-1 blocks renal (Cosimi et al., 1990J. Immunol. 144, 4604- 4612) and cardiac (Flavin et al., 1991Transplant. Proc. 23, 533-534) graft rejection in primates.

A Phase I clinical trial of a monoclonal anti-ICAM-1 antibody showed significant reduction in rejection and a significant increase in graft function in human kidney transplant patients (Haug, et al., 1993Transplantation 55, 766-72).

*Rheumatoid arthritis ICAM-1 overexpression is seen on synovial fibroblasts, endothelial cells, macrophages, and some lymphocytes (Chin et al., 1990 Arthritis Rheum 33, 1776-86; Koch et al., 1991 Lab Invest 64, 313-20).

Soluble ICAM-1 levels correlate with disease severity (Mason et al., 1993 Arthritis Rheum 36, 519-27).

Anti-ICAM antibody inhibits collagen-induced arthritis in mice (Kakimoto et al., 1992 Cell Immunol 142, 326-37).

Anti-ICAM antibody inhibits adjuvant-induced arthritis in rats (ligo et al., 1991 J Immunol 147, 4167-71).

Myocardial ischemia, stroke, and reperfusion injury 15 Anti-ICAM-1 antibody blocks adherence of neutrophils to anoxic endothelial cells (Yoshida et al., 1992 Am J Physiol 262, H1891-8).

Anti-ICAM-1 antibody reduces neurological damage in a rabbit model of cerebral stroke (Bowes et al., 1993 Exp Neurol 119, 215-9).

Anti-ICAM-1 antibody protects against reperfusion injury in a cat model of myocardial ischemia (Ma et al., 1992Circulation 86, 937-46).

Asthma Antibody to ICAM-1 partially blocks eosinophil adhesion to endothelial cells and is overexpressed on inflamed airway endothelium and epithelium in vivo (Wegner et al., 1990 Science 247, 456-9).

In a primate model of asthma, anti-ICAM-1 antibody blocks airway eosinophilia (Wegneret al., supra) and prevents the resurgence of airway inflammation and hyper-responsiveness after dexamethosone treatment (Gundel et al., 1992 Clin Exp Allergy 22, 569-75).

Psoriasis 28 Surface ICAM-1 and a clipped, soluble version of ICAM-1 is expressed in psoriatic lesions and expression correlates with inflammation (Kellner et al., 1991 Br J Dermatol 125, 211-6; Griffiths 1989 J Am Acad Dermatol 20, 617-29; Schopf et al., 1993 Br J Dermatol 128, 34-7).

Anti-ICAM antibody blocks keratinocyte antigen presentation to T cells (Nickoloff et al., 1993J Immunol 150, 2148-59).

SKawasaki disease Surface ICAM-1 expression correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989Lancet2, 1298-302).

Soluble ICAM levels are elevated in Kawasaki disease patients; particularl' high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992Arthritis Rheum 35, 672-7; Tsuji, 1992 Arerugi41, 1507-14).

Circulating LFA-1+ T cells are depleted (presumably due to ICAM-1 mediated extravasation) in Kawasaki disease patients (Furukawa et al., 1993Scand J 15 Immunol 37, 377-80).

Example 2: Ribozymes that cleave IL-5 mRNA represent a novel therapeutic approach to inflammatory disorders like asthma. The invention features use of ribozymes to treat chronic asthma, by inhibiting the synthesis of IL-5 in lymphocytes and preventing the recruitment and activation of eosinophils.

A number of cytokines besides IL-5 may also be involved in the activation of inflammation in asthmatic patients, including platelet activating factor, IL-1, IL-3, IL-4, GM-CSF, TNF-a, gamma interferon, VCAM, ILAM-1, ELAM-1 and NF-KB. In addition to these molecules, it is appreciated that any cellular receptors which mediate the activities of the cytokines are also good targets for intervention in inflammatory diseases. These targets include, but are not limited to, the IL-1R and TNF-R on keratinocytes, epithelial and endothelial cells in airways. Recent data suggest that certain neuropeptides may play a role in asthmatic symptoms. These peptides include substance P, neurokinin A and calcitonin-gene-related peptides.

These target genes may have more general roles in inflammatory diseases, but are currently assumed to have a role only in asthma.

Ribozymes of this invention block to some extent IL-5 expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of asthma (Clutterbuck et al., 1989 supra: Garssen et al., 1991 Am. Rev.

Respir. Dis. 144, 931-938; Larsen et al., 1992 J. Clin. Invest. 89, 747-752; Mauser et al., 1993 supra). Ribozyme cleavage of IL-5 mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse IL-5 mRNA were screened for accessible sites using a computer folding algorithm. Potential ~hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 11, 13, and 14, 15. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most 15 utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. (In Table 12, lower case letters indicate positions that are not conserved between the Human and the Mouse IL-5 sequences.) 20 The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 12, 14 16. 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 S 25 hammerhead ribozymes listed in Tables 12 and 14 can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form.

Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables and 16 (5'-CACGUUGUG-3') can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two basepaired stem structure can form. The sequences listed in Tables 12, 14 16 may be formed of ribonucleotides or other nucleotides or non-nucleotides.

Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes directed against IL-5 mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave IL-5 target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors. IL-5 expression will be monitored by biological assays, ELISA, by indirect immunofluoresence, and/or by FACS analysis. IL-5 mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.

Ribozymes that block the induction of IL-5 activity and/or IL-5 mRNA by more than 90% will be identified.

Uses Interleukin 5 a cytokine produced by CD4+ T helper cells and mast cells, was originally termed B cell growth factor II (reviewed by 15 Takatsu et al., 1988 Immunol. Rev. 102, 107). It stimulates proliferation of activated B cells and induces production of IgM and IgA. IL-5 plays a major role in eosinophil function by promoting differentiation (Clutterbuck et al., 1989 Blood 73, 1504-12), vascular adhesion (Walsh et al., 1990 Immunology 71, 258-65) and in vitro survival of eosinophils (Lopez et al., S" 20 1988 J. Exp. Med. 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. Exp. Med. 172, 1525-8). The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma: Several studies have shown a direct correlation between the number 25 of activated T cells and the number of eosinophils from asthmatic patients vs. normal patients (Oehling et al., 1992 J. Investig. Allergol. Clin. Immunol.

2, 295-9). Patients with either allergic asthma or intrinsic asthma were treated with corticosteroids. The bronchoalveolar lavage was monitored for eosinophils, activated T helper cells and recovery of pulmonary function over a 28 to 30 day period. The number of eosinophils and activated T helper cells decreased progressively with subsequent improvement in pulmonary function compared to intrinsic asthma patients with no corticosteroid treatment.

Bronchoalveolar lavage cells were screened for production of cytokines using in situ hybridization for mRNA. In situ hybridization signals were detected for IL-2, IL-3, IL-4, IL-5 and GM-CSF. Upregulation of mRNA was observed for IL-4, IL-5 and GM-CSF (Robinson et al., 1993 J. Allergy Clin. Immunol. 92, 313-24). Another study showed that upregulation of transcripts from allergen challenged vs. saline challenged asthmatic patients (Krishnaswamy et al., 1993 Am. J. Respir. Cell. Mol. Biol. 9, 279- 86).

An 18 patient study was performed to determine a mechanism of action for corticosteroid improvement of asthma symptoms. Improvement was monitored by methacholine responsiveness. A correlation was i* .10 observed between the methacholine responsiveness, a reduction in the :t"number of eosinophils, a reduction in the number of cells expressing IL-4 and IL-5 mRNA and an increase in number of cells expressing interferongamma.

Bronchial biopsies from 15 patients were analyzed 24 hours after 15 allergen challenge (Bentley et al., 1993 Am. J. Respir. Cell. Mol. Biol. 8, 35-42). Increased numbers of eosinophils and IL-2 receptor positive cells were found in the biopsies. No differences in the numbers of total leukocytes, T lymphocytes, elastase-positive neutrophils, macrophages or mast cell subtypes were observed. The number of cells expressing 20 and GM-CSF mRNA significantly increased.

In another patient study, the eosinophil phenotype was the same for asthmatic patients and normal individuals. However, eosinophils from asthmatic patients had greater leukotriene C4 producing capacity and migration capacity. There were elevated levels of IL-3, IL-5 and GM-CSF in the circulation of asthmatics but not in normal individuals (Bruijnzeel et al., 1992 Schweiz. Med. Wochenschr. 122, 298-301).

Efficacy of antibody to IL-5 was assessed in a guinea pig asthma model. The animals were challenged with ovalbumin and assayed for eosinophilia and the responsiveness to the bronchioconstriction substance P. A 30 mg/kg dose of antibody administered i.p. blocked ovalbumininduced increased sensitivity to substance P and blocked increases in bronchoalveolar and lung tissue accumulation of eosinophils (Mauser et al., 1993 Am. Rev. Respir. Dis. 148, 1623-7). In a separate study guinea pigs challenged for eight days with ovalbumin were treated with monoclonal antibody to IL-5. Treatment produced a reduction in the number of eosinophils in bronchoalveolar lavage. No reduction was observed for unchallenged guinea pigs and guinea pigs treated with a control antibody. Antibody treatment completely inhibited the development of hyperreactivity to histamine and arecoline after ovalbumin challenge (van Oosterhout et al., 1993 Am. Rev. Respir. Dis. 147, 548-52) Results obtained from human clinical analysis and animal studies indicate the role of activated T helper cells, cytokines and eosinophils in asthma. The role of IL-5 in eosinophil development and function makes ILa good candidate for target selection. The antibody studies neutralized 10 IL-5 in the circulation thus preventing eosinophilia. Inhibition of the production of IL-5 will achieve the same goal.

Asthma a prominent feature of asthma is the infiltration of eosinophils and deposition of toxic eosinophil proteins major basic protein, eosinophil-derived neurotoxin) in the lung. A number of T-cell- 15 derived factors like IL-5 are responsible for the activation and maintainance of eosinophils (Kay, 1991 J. Allergy Clin. Immun. 87, 893). Inhibition of expression in the lungs can decrease the activation of eosinophils and will help alleviate the symptoms of asthma.

Atopy is characterized by the developement of type I hypersensitive 20 reactions associated with exposure to certain environmental antigens. One of the common clinical manifestations of atopy is eosinophilia (accumulation of abnormally high levels of eosinophils in. the blood).

Antibodies against IL-5 have been shown to lower the levels of eosinophils in mice (Cook et al., 1993 in Immunopharmacol. Eosinophils ed. Smith and Cook, pp. 193-216, Academic, London, UK) Parasitic infection-related eosinophilia- infections with parasites like helminths, can lead to severe eosinophilia (Cook et al., 1993 suDra). Animal models for eosinophilia suggest that infection of mice, for example, can lead to blood, peritoneal and/or tissue eosinophilia, all of which seem to be lowered to varying degrees by antibodies directed against Pulmonary infiltration eosinophilia- is characterised by accumulation of high levels of eosinophils in pulmonary parenchyma (Gleich, 1990 J. Allergy Clin. Immunol. 85, 422).

33 L-Tryptophan-associated eosinophilia-myalgia syndrome (EMS)- The EMS disease is closely linked to the consumption of Ltryptophan, an essential aminoacid used to treat conditions like insomnia (for review see Varga et al., 1993 J Invest. Dermatol. 100, 97s). Pathologic and histologic studies have demonstrated high levels of eosinophils and mononuclear inflammatory cells in patients with EMS. It appears that and transforming growth factor play a significant role in the development of EMS (Varga et al., 1993 supra) by activating eosinophils and other inflammatory cells.

10 Thus, ribozymes of the present invention that cleave IL-5 mRNA and .:thereby IL-5 activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits function is described above; available cellular and activity assays are 15 numerous, reproducible, and accurate. Animal models for IL-5 function and for each of the suggested disease targets exist (Cook et al., 1993 sup o and can be used to optimize activity.

Example 3: NF-KB Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-KB. One subunit of NF-KB, the relA gene product (termed RelA or p65) is implicated specifically in the induction of inflammatory responses. Ribozyme therapy, due to its exquisite specificity, is particularly well-suited to target intracellular factors that contribute to disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A or TNF-a may represent novel therapeutics for the treatment of inflammatory and autoimmune disorders.

The nuclear DNA-binding activity, NF-icB, was first identified as a factor that binds and activates the immunoglobulin K light chain enhancer in B cells. NF-KB now is known to activate transcription of a variety of other cellular genes cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-KB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-KB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-KB (encoded by the nf-KB2 or nf-KB1 genes, respectively) are generated from the precursors NF-KB1 (p105) or NF-KB2 (p100). The p65 subunit of NF-KB (now termed Rel A is encoded by the rel A locus.

The roles of each specific transcription-activating complex now are being elucidated in cells Perkins, et al., 1992 Proc. Natl Acad. Sci U"SA 89, 1529-1533). For instance, the heterodimer of NF-KB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NF-KB2/RelA heterodimers (p49/p65) actually inhibit transcription Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)).

Conversely, heterodimers of NF-KB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-KB1/RelA (p50/p65) heterodimers have little effect Liu, N.D. Perkins, R.M. Schmid, G.J.

Nabel, J. Virol. 1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-KB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 Mol. Cell. Biol. 13, 3802-3810). Thus, the promiscuous role initially assigned to NF-KB in transcriptional activation Lenardo, D. Baltimore, 1989 Cell 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic "knock-out" mice of individual members of the rel family. Such "knockouts" show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.

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

*NF-KB is required for phorbol ester-mediated induction of IL-6 (I.

Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993 Mol. Cell. Biol. 13, 6137-46).

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

10 *NF-KB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).

.o The above studies suggest that NF-iB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators.

Two recent papers point to another connection between NF-KB and 15 inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-KB. The glucocorticoid receptor and p65 both act at NF-KB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol.

Chem. 269, 6185-6192). Glucocorticoid receptor inhibits NF-KB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl Acad. Sci USA 91, 20 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor Ribozymes of this invention block to some extent NF-KB expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of restenosis, transplant rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse re/A mRNA can be screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 17, 18 and 21-22. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targetted sequences are of most utility.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 19 22. 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 and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

10 By engineering ribozyme motifs we have designed several ribozymes directed against re/A mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave re/A target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing cytokine- 15 induced VCAM-1, ICAM-1, IL-6 and IL-8 expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA and RNA vectors. Cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Rel A mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative

RT-PCR.

Activity of NF-KB will be monitored by gel-retardation assays. Ribozymes that block the induction of NF-KB activity and/or rel A mRNA by more than will be identified.

RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1,

ICAM-

1, IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-re/A ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

37 Uses A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.

10 -Rheumatoid arthritis (RA).

Due to the chronic nature of RA, a gene therapy approach is logical.

Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the •15 synovium: high efficiency of gene transfer and expression for several months would be expected Roessler, E.D. Allen, J.M. Wilson, J.W.

Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple 20 administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint.

However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

*Restenosis.

Expression of NF-KB in the vessel wall of pigs causes a narrowing of the luminal space due to excessive deposition of extracellular matrix components. This phenotype is similar to matrix deposition that occurs subsequent to coronary angioplasty. In addition, NF-KB is required for the expression of the oncogene c-myb La Rosa, J.W. Pierce, G.E.

Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-KB induces smooth muscle proliferation and the expression of excess matrix components: both processes are thought to contribute to reocclusion of vessels after coronary angioplasty.

*Transplantation.

38 NF-KB is required for the induction of adhesion molecules (Eck et al., supra, K. O'Brien, et al., J. Clin. Invest. 92, 945-951 (1993)) that function in immune recognition and inflammatory responses. At least two potential modes of treatment are possible. In the first, transplanted organs are treated ex vivo with ribozymes or ribozyme expression vectors. Transient inhibition of NF-KiB in the transplanted endothelium may be sufficient to prevent transplant-associated vasculitis and may significantly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme expression vectors. Recipients would receive the treatment prior to transplant. Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1, VCAM-1, and/or B7 an B7-2) can induce antigen-specific anergy. Tolerance to the donor's histocompatibility antigens could result; potentially, any donor could be used for any transplantation procedure.

15 *Asthma.

Granulocyte macrophage colony stimulating factor (GM-CSF) is t hought to play a major role in recruitment of eosinophils and other inflammatory cells during the late phase reaction to asthmatic trauma.

Again, blocking the local induction of GM-CSF and other inflammatory mediators is likely to reduce the persistent inflammation observed in chronic asthmatics. Aerosol delivery of ribozymes or adenovirus ribozyme expression vectors is a feasible treatment.

*Gene Therapy.

Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

Thus, ribozymes of the present invention that cleave relA mRNA and thereby NF-B activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits

NF-KB

39 function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for NF-KB function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity.

Example 4: TNF-a Ribozymes that cleave the specific cites in TNF-o mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.

Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated leukocytes, that is a potent mediator of inflammatory reactions. Injection of TNF-a into experimental animals can simulate the symptoms of systemic and local inflammatory diseases such as septic shock or rheumatoid e arthritis.

TNF-a was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, 15 1985 Science 230, 4225-4231). TNF-a subsequently was found to be identical to cachectin, an agent responsible for the weight loss and wasting syndrome associated with tumors and chronic infections (Beutler, et al., 1985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-a have been cloned and found to be related to TNF-B (Shakhov et al., 1990 20 J. Exp. Med. 171, 35-47). Both TNF-a and TNF-1 bind to the same receptors and have nearly identical biological activities. The two TNF receptors have been found on most cell types examined (Smith, et al., 1990 Science 248, 1019-1023). TNF-a secretion has been detected from monocytes/macrophages, CD4+ and CD8+ T-cells, B-cells, lymphokine activated killer cells, neutrophils, astrocytes, endothelial cells, smooth muscle cells, as well as various non-hematopoietic tumor cell lines for a review see Turestskaya et al., 1991 in Tumor Necrosis Factor: Structure.

Function, and Mechanism of Action B. B. Aggarwal, J. Vilcek, Eds. Marcel Dekker, Inc., pp. 35-60). TNF-a is regulated transcriptionally and translationally, and requires proteolytic processing at the plasma membrane in order to be secreted (Kriegler et al., 1988 Cell 53, 45-53).

Once secreted, the serum half life of TNF-a is approximately 30 minutes.

The tight regulation of TNF-a is important due to the extreme toxicity of this cytokine. Increasing evidence indicates that overproduction of TNF-a during infections can lead to severe systemic toxicity and death (Tracey Cerami, 1992 Am. J. Trop. Med. Hyga 47, 2-7).

Antisense RNA and Hammerhead ribozymes have been used in an attempt to lower the expression level of TNF-a by targeting specified cleavage sites [Sioud et al., 1992 J. Mol. Biol. 223; 831; Sioud WO 94/10301; Kisich and co-workers, 1990 abstract (FASEB J. 4, A1860; 1991 slide presentation Leukocyte Biol sup. 2, 70); December, 1992 poster presentation at Anti-HIV Therapeutics Conference in SanDiego, CA; and "Development of anti-TNF-a ribozymes for the control of TNF-a gene expression"- Kisich, Doctoral Dissertation, 1993 University of California, Davis] listing various TNFa targeted ribozymes.

Ribozymes of this invention block to some extent TNF-a expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of septic shock and rheumatoid arthritis. Ribozyme cleavage of TNF-a mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse TNF-a mRNA can be screened for accessible sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 23, 25, and 27 28. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

(In Table 24, lower case letters indicate positions that are not conserved between the human and the mouse TNF-a sequences.) The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 24, 26 28. 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 Tables 24 and 26 can be altered (substitution, deletion, and/or insertion) to contain any 41 sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 27 and 28 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 24, 26 28 may be formed of ribonucleotides or other nucleotides or nonnucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables or AAV In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-a RNA is inserted into a plasmid DNA vector or an adenovirus DNA viral vector or AAV or alpha virus or retroviris vectors. Viral vectors have been used to transfer genes to the intact vasculature or to joints of live animals (Willard et al., 1992 Circulation, 86, 1-473.; Nabel et al., 1990 Science, 249, 1285-1288) and 15 both vectors lead to transient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, .DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, through the use of an 20 injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

Se In another preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-c RNA is inserted into a retrovirus vector for sustained expression of ribozyme(s).

By engineering ribozyme motifs we have designed several ribozymes directed against TNF-a mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave TNF-a target sequences in vitro is evaluated.

The ribozymes will be tested for function in cells by analyzing bacterial lipopolysaccharide (LPS)-induced TNF-a expression levels.

Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. TNF-a expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. TNF-a mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of TNF-a activity and/or TNF-a mRNA by more than 90% will be identified.

RNA ribozymes and/or genes encoding them will be locally delivered to macrophages by intraperitoneal injection. After a period of ribozyme uptake, the peritoneal macrophages are harvested and induced ex vivo with LPS. The ribozymes that significantly reduce TNF-a secretion are selected. The TNF-a can also be induced after ribozyme treatment with fixed Streptococcus in the peritoneal cavity instead of ex vivo. In this fashion the ability of TNF-a ribozymes to block TNF-a secretion in a 10 localized inflammatory response are evaluated. In addition, we will :i determine if the ribozymes can block an ongoing inflammatory response by delivering the TNF-a ribozymes after induction by the injection of fixed Streptococcus.

To examine the effect of anti-TNF-a ribozymes on systemic :15 inflammation, the ribozymes are delivered by intravenous injection. The ability of the ribozymes to inhibit TNF-a secretion and lethal shock caused by systemic LPS administration are assessed. Similarly, TNF-a ribozymes can be introduced into the joints of mice with collagen-induced arthritis.

Either free delivery, liposome delivery, cationic lipid delivery, adeno- 20 associated virus vector delivery, adenovirus vector delivery, retrovirus vector delivery or plasmid vector delivery in these animal model experiments can be used to supply ribozymes. One dose (or a few infrequent doses) of a stable anti-TNF-a ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate tissue damage in these inflammatory diseases.

Macrophage isolation.

To produce responsive macrophages 1 ml of sterile fluid thioglycollate broth (Difco, Detroit, MI.) was injected i.p. into 6 week old female C57bl/6NCR mice 3 days before peritoneal lavage. Mice were maintained as specific pathogen free in autoclaved cages in a laminar flow hood and given sterilized water to minimize "spontaneous" activation of macrophages. The resulting peritoneal exudate cells (PEC) were obtained by lavage using Hanks balanced salt solution (HBSS) and were plated at 2.5X10 5 /well in 96 well plates (Costar, Cambridge, MA.) with Eagles minimal essential medium (EMEM) containing 10% heat inactivated fetal bovine serum. After adhering for 2 hours the wells were washed to remove non-adherent cells. The resulting cultures were 97% macrophages as determined by morphology and staining for non-specific esterase.

Transfection of ribozymes into macrophages: The ribozymes were diluted to 2X final concentration, mixed with an equal volume of 11nM lipofectamine (Life Technologies, Gaithersburg, and vortexed. 100 ml of lipid:ribozyme complex was then added directly to the cells, followed immediately by 10 ml fetal bovine serum.

Three hours after ribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride (LPS) was added to each well to stimulate TNF .i production.

Quantitation of TNF-a in mouse macrophages: Supernatants were sampled at 0, 2, 4, 8, and 24 hours post LPS stimulation and stored at -70oC. Quantitation of TNF-a was done by a specific ELISA. ELISA plates were coated with rabbit anti-mouse TNF-a serum at 1:1000 dilution (Genzyme) followed by blocking with milk proteins and incubation with TNF-a containing supernatants. TNF-a was then detected using a murine TNF-a specific hamster monoclonal antibody (Genzyme). The ELISA was developed with goat anti-hamster IgG coupled to alkaline phosphatase.

Assessment of reagent toxicity: Following ribozyme/lipid treatment of macrophages and harvesting of supernatants viability of the cells was assessed by incubation of the cells with 5 mg/ml of 3 4 5 -dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). This compound is reduced by the mitochondrial dihydrogenases, the activity of which correlates well with cell viability. After 12 hours the absorbance of reduced MTT is measured at 585 nm.

Uses The association between TNF-a and bacterial sepsis, rheumatoid arthritis, and autoimmune disease make TNF-a an attractive target for therapeutic intervention [Tracy Cerami 1992 supra; Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788; Jacob, 1992 J. Autoimmun. (Supp. 133-143].

Septic Shock Septic shock is a complication of major surgery, bacterial infection, and polytrauma characterized by high fever, increased cardiac output, reduced blood pressure and a neutrophilic infiltrate into the lungs and other major organs. Current treatment options are limited to antibiotics to reduce the bacterial load and non-steroidal anti-inflammatories to reduce fever. Despite these treatments in the best intensive care settings, mortality from septic shock averages 50%, due primarily to multiple organ failure and disseminated vascular coagulation. Septic shock, with an incidence of 10 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-inflammatory cytokines which are not normally detected in the serum, such as TNF-a, interleukin-1 B (IL- 13), rinterferon (IFN-y), interleukin-6

(IL-

and interleukin-8 Other non-cytokine mediators such as leukotriene b4, prostaglandin E2, C3a and C3d also reach high levels (de Boer et al., 1992 Immunopharmacology 24, 135-148).

TNF-a is detected early in the course of septic shock in a large fraction of patients (de Boer et al., 1992 suDra). In animal models, injection of TNF- 20 a has been shown to induce shock-like symptoms similar to those induced by LPS injection (Beutler et al., 1985 Science 229, 869-871); in contrast, injection of IL-113, IL-6, or IL-8 does not induce shock. Injection of TNF-a also causes an elevation of IL-11, IL-6, IL-8, PgE 2 acute phase proteins, and TxA 2 in the serum of experimental animals (de Boer et al., 1992 supra). In animal models the lethal effects of LPS can be blocked by preadministration of anti-TNF-a antibodies. The cumulative evidence indicates that TNF-a is a key player in the pathogenesis of septic shock, and therefore a good candidate for therapeutic intervention.

Rheumatoid Arthritis Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints leading to bone destruction and loss of joint function. At the cellular level, autoreactive T- lymphocytes and monocytes are typically present, and the synoviocytes often have altered morphology and immunostaining patterns. RA joints have been shown to contain elevated levels of TNF-a, IL-la and IL-1B, IL-6, GM-CSF, and TGF- B (Abney et al., 1991 Imm. Rev. 119, 105-123), some or all of which may contribute to the pathological course of the disease.

Cells cultured from RA joints spontaneously secrete all of the proinflammatory cytokines detected in vivo. Addition of antisera against TNF-a to these cultures has been shown to reduce IL-la/1 production by these cells to undetectable levels (Abney et al., 1991 Supra). Thus, TNF-a may directly induce the production of other cytokines in the RA joint. Addition of the anti-inflammatory cytokine, TGF-, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical 10 specimens clearly demonstrate the production of TNF-a, IL-la/B, and IL-6 i from macrophages near the cartilage/pannus junction when the pannus in invading and overgrowing the cartilage (Chu et al., 1992 Br J.

Rheumatology 31, 653-661). GM-CSF was shown to be produced mainly by vascular endothelium in these samples. Both TNF-a and TGF-B have 15 been shown to be fibroblast growth factors, and may contribute to the accumulation of scar tissue in the RA joint. TNF-a has also been shown to increase osteoclast activity and bone resorbtion, and may have a role in the bone erosion commonly found in the RA joint (Cooper et al., 1992 Clin.

Exp. Immunol. 89, 244-250).

20 Elimination of TNF-a from the rheumatic joint would be predicted to reduce overall inflammation by reducing induction of MHC class II, IL-la/, 11-6, and GM-CSF, and reducing T-cell activation. Osteoclast activity might also fall, reducing the rate of bone erosion at the joint. Finally, elimination of TNF-a would be expected to reduce accumulation of scar tissue within the joint by removal of a fibroblast growth factor.

Treatment with an anti-TNF-a antibody reduces joint swelling and the histological severity of collagen-induced arthritis in mice (Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788). In addition, a study of RA patients who have received i.v. infusions of anti-TNF-a monoclonal antibody reports a reduction in the number and severity of inflamed joints after treatment. The benefit of monoclonal antibody treatment in the long term may be limited by the expense and immunogenicity of the antibody.

Psoriasis Psoriasis is an inflammatory disorder of the skin characterized by keratinocyte hyperproliferation and immune cell infiltrate (Kupper, 1990 J Clin, Invest, 86, 1783-1789). It is a fairly common condition, affecting of the population. The disorder ranges in severity from mild, with small flaky patches of skin, to severe, involving inflammation of the entire epidermis. The cellular infiltrate of psoriasis includes T-lymphocytes, neutrophils, macrophages, and dermal dendrocytes. The majority of Tlymphocytes are activated CD4+ cells of the TH-1 phenotype, although some CD8+ and CD4-/CD8- are also present. B lymphocytes are typically not found in abundance in psoriatic plaques.

Numerous hypotheses have been offered as to the proximal cause of :i .10 psoriasis including auto-antibodies and auto-reactive T-cells, overproduction of growth factors, and genetic predisposition. Although there is evidence to support the involvement of each of these factors in psoriasis, they are neither mutually exclusive nor are any of them necessary and sufficient for the pathogenesis of psoriasis (Reeves, 1991 15 Semin. Dermatol. 10, 217).

The role of cytokines in the pathogenesis of psoriasis has been investigated. Among those cytokines found to be abnormally expressed were TGFa IL-la1, IL-1B, IL-1ra, IL-6, IL-8, IFN-y, and TNF-a In addition to abnormal cytokine production, elevated expression of ICAM-1, ELAM-1, 20 and VCAM has been observed (Reeves, 1991 supra). This cytokine profile is similar to that of normal wound healing, with the notable exception that cytokine levels subside upon healing. Keratinocytes themselves have recently been shown to be capable of secreting EGF, TGF-a, IL-6, and TNF-a, which could increase proliferation in an autocrine fashion (Oxholm et al., 1991 APMIS 99, 58-64).

Nickoloff et al., 1993 (J Dermatol Sci. 6, 127-33) have proposed the following model for the initiation and maintenance of the psoriatic plaque: Tissue damage induces the wound healing response in the skin.

Keratinocytes secrete IL-la, IL-18, IL-6, IL-8, TNF-a. These factors activate the endothelium of dermal capillaries, recruiting PMNs, macrophages, and T-cells into the wound site.

Dermal dendrocytes near the dermal/epidermal junction remain activated when they should return to a quiescent state, and subsequently secrete cytokines including TNF-a, IL-6, and IL-8. Cytokine expression, in turn, maintains the activated state of the endothelium, allowing extravasation of additional immunocytes, and the activated state of the keratinocytes which secrete TGF-a and IL-8. Keratinocyte IL-8 recruits immunocytes from the dermis into the epidermis. During passage through the dermis, T-cells encounter the activated dermal dendrocytes which efficiently activate the TH-1 phenotype. The activated T-cells continue to migrate into the epidermis, where they are stimulated by keratinocyteexpressed ICAM-1 and MHC class II. IFN-y secreted by the T-cells synergizes with the TNF-a from dermal dendrocytes to increase 10 keratinocyte proliferation and the levels of TGF-a, IL-8, and IL-6 production.

IFN-y also feeds back to the dermal dendrocyte, maintaining the activated phenotype and the inflammatory cycle.

Elevated serum titres of IL-6 increases synthesis of acute phase proteins including complement factors by the liver, and antibody production 15 by plasma cells. Increased complement and antibody levels increases the probability of autoimmune reactions.

Maintenance of the psoriatic plaque requires continued expression of all of these processes, but attractive points of therapeutic intervention are TNF-a expression by the dermal dendrocyte to maintain activated endothelium and keratinocytes, and IFN-y expression by T-cells to maintain S" activated dermal dendrocytes.

There are 3 million patients in the United States afflicted with psoriasis. The available treatments for psoriasis are corticosteroids. The most widely prescribed are TEMOVATE (clobetasol propionate),

LIDEX

(fluocinonide),' DIPROLENE (betamethasone propionate),

PSORCON

(diflorasone diacetate) and TRIAMCINOLONE formulated for topical application. The mechanism of action of corticosteroids is multifactorial.

This is a palliative therapy because the underlying cause of the disease remains, and upon discontinuation of the treatment the disease returns.

Discontinuation of treatment is often prompted by the appearance of adverse effects such as atrophy, telangiectasias and purpura.

Corticosteroids are not recommended for prolonged treatments or when treatment of large and/or inflamed areas is required. Altemative treatments include retinoids, such as etretinate, which has been approved for treatment of severe, refractory psoriasis. Alternative retinoid-based treatments are in advanced clinical trials. Retinoids act by converting 48 keratinocytes to a differentiated state and restoration of normal skin development. Immunosuppressive drugs such as cyclosporine are also in the advanced stages of clinical trials. Due to the nonspecific mechanism of action of corticosteroids, retinoids and immunosuppressives, these treatments exhibit severe side effects and should not be used for extended periods of time unless the condition is life-threatening or disabling. There is a need for a less toxic, effective therapeutic agent in psoriatic patients.

HIV and AIDS The human immunodeficiency virus (HIV) causes several 10 fundamental changes in the human immune system from the time of infection until the development of full-blown acquired immunodeficiency syndrome (AIDS). These changes include a shift in the ratio of CD4+ to CD8+ T-cells, sustained elevation of IL-4 levels, episodic elevation of TNFa and TNF-B levels, hypergammaglobulinemia, and lymphoma/leukemia 15 (Rosenberg Fauci, 1990 Immun. Today 11, 176; Weiss 1993 Science 260, 1273). Many patients experience a unique tumor, Kaposi's sarcoma and/or unusual opportunistic infections Pneumocystis carinii, cytomegalovirus, herpesviruses, hepatitis viruses, papilloma viruses, and tuberculosis). The immunological dysfunction of individuals with AIDS 20 suggests that some of the pathology may be due to cytokine dysregulation.

Levels of serum TNF-a and IL-6 are often found to be elevated in AIDS patients (Weiss, 1993 suDra). In tissue culture, HIV infection of monocytes isolated from healthy individuals stimulates secretion of both TNF-a and IL-6. This response has been reproduced using purified gp120, the viral coat protein responsible for binding to CD-4 (Buonaguro et al., 1992 J. Virol. 66, 7159). It has also been demonstrated that the viral gene regulator, Tat, can directly induce TNF transcription. The ability of HIV to directly stimulate secretion of TNF-a and IL-6 may be an adaptive mechanism of the virus. TNF-a has been shown to upregulate transcription of the LTR of HIV, increasing the number of HIV-specific transcripts in infected cells. IL-6 enhances HIV production, but at a post-transcriptional level, apparently increasing the efficiency with which HIV transcripts are translated into protein. Thus, stimulation of TNF-a secretion by the HIV virus may promote infection of neighboring CD4+ cells both by enhancing virus production from latently infected cells and by driving replication of the virus in newly infected cells.

The role of TNF-a in HIV replication has been well established in tissue culture models of infection (Sher et al., 1992 Immun. Rev. 127, 183), suggesting that the mutual induction of HIV replication and TNF-a replication may create positive feedback in vivo. However, evidence for the presence of such positive feedback in infected patients is not abundant.

TNF-a levels are found to be elevated in some, but not all patients tested.

Children with AIDS who were given zidovudine had reduced levels of TNFa compared to those not given zidovudine (Cremoni et al., 1993 AIDS 7, 128). This correlation lends support to the hypothesis that reduced viral 10 replication is physiologically linked to TNF-a levels. Furthermore, recently t has been shown that the polyclonal B cell activation associated with HIV infection is due to membrane-bound TNF-a. Thus, levels of secreted TNF-a may not accurately reflect the contribution of this cytokine to AIDS pathogenesis.

Chronic elevation of TNF-a has been shown to shown to result in cachexia (Tracey et al., 1992 Am. J. Trop. Med. Hyg. 47, increased autoimmune disease (Jacob, 1992 supra), lethargy, and immune suppression in animal models (Aderka et al., 1992 Isr. J. Med. Sci. 28, 126- 130). The cachexia associated with AIDS may be associated with chronically elevated TNF-a frequently observed in AIDS patients.

g Similarly, TNF-a can stimulate the proliferation of spindle cells isolated from Kaposi's sarcoma lesions of AIDS patients (Barillari et al., 1992 J Immunol 149, 3727).

A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme.that cleaves the specified sites in TNF-a mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.

*Septic shock.

Exogenous delivery of ribozymes to macrophages can be achieved by intraperitoneal or intravenous injections. Ribozymes will be delivered by incorporation into liposomes or by complexing with cationic lipids.

*Rheumatoid arthritis (RA).

Due to the chronic nature of RA, a gene therapy approach is logical.

Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several 10 months would be expected Roessler, E.D. Allen, J.M. Wilson, J.W.

Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple administrations may be necessary. Retrovirus and adeno-associated virus 15 vectors would lead to permanent gene transfer and expression in the joint.

However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

*Psoriasis The psoriatic plaque is a particularly good candidate for ribozyme or S* 20 vector delivery. The stratum corneum of the plaque is thinned, providing access to the proliferating keratinocytes. T-cells and dermal dendrocytes can be efficiently targeted by trans-epidermal diffusion Organ culture systems for biopsy specimens of psoriatic and normal skin are described in current literature (Nickoloff et al., 1993 Supra).

Primary human keratinocytes are easily obtained and will be grown into epidermal sheets in tissue culture. In addition to these tissue culture models, the flaky skin mouse develops psoriatic skin in response to UV light. This model would allow demonstration of animal efficacy for ribozyme treatments of psoriasis.

*Gene Therapy.

Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus 51 vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

Thus, ribozymes of the present invention that cleave TNF-a mRNA and thereby TNF-a activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits TNF-a function is described above; available cellular and activity assays 10 are number, reproducible, and accurate. Animal models for TNF-a function and for each of the suggested disease targets exist and can be used to optimize activity.

Example 5: 210bcr-ab Chronic myelogenous leukemia exhibits a characteristic disease 15 course, presenting initially as a chronic granulocytic hyperplasia, and invariably evolving into an acute leukemia which is caused by the clonal expansion of a cell with a less differentiated phenotype the blast crisis stage of the disease). CML is an unstable disease which ultimately progresses to a terminal stage which resembles acute leukemia. This 20 lethal disease affects approximately 16,000 patients a year.

.Chemotherapeutic agents such as hydroxyurea or busulfan can reduce the leukemic burden but do not impact the life expectancy of the patient (e.g.

approximately 4 years). Consequently, CML patients are candidates for bone marrow transplantation (BMT) therapy. However, for those patients which survive BMT, disease recurrence remains a major obstacle (Apperley et al., 1988 Br. J. Haematol. 69, 239).

The Philadelphia (Ph) chromosome which results from the translocation of the abl oncogene from chromosome 9 to the bcr gene on chromosome 22 is found in greater than 95% of CML patients and in 25% of all cases of acute lymphoblastic leukemia Fourth International Workshop on Chromosomes in Leukemia 1982, Cancer Genet. Cytogenet. 11, 316]. In virtually all Ph-positive CMLs and approximately 50% of the Ph-positive ALLs, the leukemic cells express bcrabl fusion mRNAs in which exon 2 (b2-a2 junction) or exon 3 (b3-a2 junction) from the major breakpoint cluster region of the bcr gene is spliced 52 to exon 2 of the abl gene. Heisterkamp et al., 1985 Nature 315, 758; Shtivelman et al., 1987, Blood 69, 971). In the remaining cases of Phpositive ALL, the first exon of the bcr gene is spliced to exon 2 of the abl gene (Hooberman et al., 1989 Proc. Nat. Acad. Sci. USA 86, 4259; Heisterkamp et al., 1988 Nucleic Acids Res. 16, 10069).

The b3-a2 and b2-a2 fusion mRNAs encode 210 kd bcr-abl fusion proteins which exhibit oncogenic activity (Daley et al., 1990 Science 247, 824; Heisterkamp et al., 1990 Nature 344, 251). The importance of the bcrabl fusion protein (p 2 10 bc r-abl) in the evolution and maintenance of the .i 10 leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p 2 10 b c r ab/ expression. These inhibitory molecules have been shown to inhibit the in mitro proliferation of leukemic cells in bone marrow from CML patients. Szczylik et al., 1991 Science 253, 562).

15 Reddy, U.S. Patent 5,246,921 (hereby incorporated by reference herein) describes use of ribozymes as therapeutic agents for leukemias, such as chronic myelogenous leukemia (CML) by targeting the specific junction region of bcr-abl fusion transcripts. It indicates causing cleavage S* by a ribozyme at or near the breakpoint of such a hybrid chromosome, specifically it includes cleavage at the sequence GUX, where X is A, U or G. The one example presented is to cleave the sequence 5' AGC AG i AGUU (cleavage site) CAA AAGCCCU-3'.

Scanlon WO 91/18625, WO 91/18624, and WO 91/18913 and Snyder et al., W093/03141 and W094/13793 describe a ribozyme effective to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to external stimuli.

The invention features use of ribozymes to inhibit the development or expression of a transformed phenotype in man and other animals by modulating expression of a gene that contributes to the expression of CML.

Cleavage of targeted mRNAs expressed in pre-neoplastic and transformed cells elicits inhibition of the transformed state.

The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred administration protocols can be used, either in vivo administration to reduce the tumor burden, or ex vivo treatment to 53 eradicate transformed cells from tissues such as bone marrow prior to reimplantation.

This invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of CML. The mRNA targets are present in the 425 nucleotides surrounding the fusion sites of the bcr and abl sequences in the b2-a2 and b3-a2 recombinant mRNAs. Other sequences in the 5' portion of the bcr mRNA or the 3' portion of the abl mRNA may also be targeted for ribozyme cleavage.

Cleavage at any of these sites in the fusion mRNA molecules will result in 10 inhibition of translation of the fusion protein in treated cells.

The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of CML. Such enzymatic RNA molecules can be delivered exogenously or endogenously to afflicted cells. In the preferred hammerhead motif the small size (less 15 than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced.

The smallest ribozyme delivered for any type of treatment reported to 'date (by ssi et al., 1992 sulpra) is an in iMd transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in 20 length is very difficult using automated methods, and the therapeutic cost of S.i: such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only exi vo treatments. This limits the utility of this approach. In this invention, an alternative approach uses smaller ribozyme motifs and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, as well as complementary binding of the ribozyme to the mRNA target.

The enzymatic RNA molecules of this invention can be used to treat human CML or precancerous conditions. Affected animals can be treated at the time of cancer detection or in a prophylactic manner. This timing of treatment will reduce the number of affected cells and disable cellular 54 replication. This is possible because the ribozymes are designed to disable those structures required for successful cellular proliferation.

Ribozymes of this invention block to some extent p210bcr-abl expression and can be used to treat disease or diagnose such disease.

Ribozymes will be delivered to cells in culture and to tissues in animal models of CML. Ribozyme cleavage of bcr/abl mRNA in these systems may prevent or alleviate disease symptoms or conditions.

The sequence of human bcr/abl mRNA can be screened for accessible sites using a computer folding algorithm. Regions of the mRNA 10 that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Table 29 (All sequences are 5' to 3' in the tables). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

15 The sequences of the chemically synthesized ribozymes most useful in this study are shown in Table 30. 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 20 hammerhead ribozymes listed in Table 30 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 30 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes directed against bcr-abl mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance as described above. These ribozymes cleave bcr-abl target sequences in vitro.

The ribozymes are tested for function in vivo by exogenous delivery to cells expressing bcr-abl. Ribozymes are delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. Expression of bcr-abl is monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Levels of bcr-abl mRNA are assessed by Northern analysis, RNase protection, by primer extension analysis or by quantitative RT-PCR techniques.

Ribozymes that block the induction of p210bcr-ab) protein and mRNA by more than 20% are identified.

Example 6: RSV This invention relates to the use of ribozymes as inhibitors of respiratory syncytial virus (RSV) production, and in particular, the inhibition of RSV replication.

I" RSV is a member of the virus family paramyxoviridae and is classified 10 under the genus Pneumovirus (for a review see Mclntosh and Chanock, 1990 in Virology ed. B.N. Fields, pp. 1045, Raven Press Ltd. NY). The infectious virus particle is composed of a nucleocapsid enclosed within an envelope. The nucleocapsid is composed of a linear negative singlestranded non-segmented RNA associated with repeating subunits of 15 capsid proteins to form a compact structure and thereby protect the RNA from nuclease degradation. The entire nucleocapsid is enclosed by the envelope. The size of the virus particle ranges from 150 300 nm in diameter. The complete life cycle of RSV takes place in the cytoplasm of infected cells and the nucleocapsid never reaches the nuclear j20 compartment (Hall, 1990 in Principles and Practice of Infectious Diseases ed. Mandell et al., Churchill Livingstone,

NY).

The RSV genome encodes ten viral proteins essential for viral N production. RSV protein products include two structural glycoproteins

(G

and F) found in.the envelope spikes, two matrix proteins [M and M2 (22K)] found in the inner membrane, three proteins localized in the nucleocapsid P and one protein that is present on the surface of the infected cell and two nonstructural proteins [NS1 (1C) and NS2 found only in the infected cell. The mRNAs for the 10 RSV proteins have similar and 3' ends. UV-inactivation studies suggest that a single promoter is used with multiple transcription initiation sites (Barik et al., 1992 J. Virol. 66, 6813). The order of transcription corresponding to the protein assignment on the genomic RNA is 1C, 1B, N, P, M, SH, G, F, 22K and L genes (Huang et al., 1985 Virus Res. 2, 157) and transcript abundance corresponds to the order of gene assignment (for example the 1C and 1B mRNAs are much more abundant than the L mRNA. Synthesis of viral message begins immediately after RSV infection of cells and reaches a maximum at 14 hours post-infection (Mclntosh and Chanock, supra).

There are two antigenic subgroups of RSV, A and B, which can circulate simultaneously in the community in varying proportions in different years (Mclntosh and Chanock, supra). Subgroup A usually predominates.

Within the two subgroups there are numerous strains. By the limited sequence analysis available it seems that homology at the nucleotide level is more complete within than between subgroups, although sequence divergence has been noted within subgroups as well. Antigenic 10 determinates result primarily from both surface glycoproteins, F and G. For F, at least half of the neutralization epitopes have been stably maintained over a period of 30 years. For G however, A and B subgroups may be related antigenically by as little as a few percent. On the nucleotide level, l however, the majority of the divergence in the coding region of G is found in the sequence for the extracellular domain (Johnson et al., 1987, Proc.

o o Natl. Acad. Sci. USA 84, 5625).

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract illness during infancy and childhood (Hall, supra) and as such is associated with an estimated 90,000 hospitalizations and 4500 ~20 deaths in the United States alone (Update: respiratory syncytial virus activity United States, 1993, Mmwr Morb Mortal Wkly Rep, 42, 971).

Infection with RSV generally outranks all other microbial agents leading to both pneumonia and bronchitis. While primarily affecting children under two years of age, immunity is not complete and reinfection of older children and adults, especially hospital care givers (Mclntosh and Chanock, supra), is not uncommon. Immunocompromised patients are severely affected and RSV infection is a major complication for patients undergoing bone marrow transplantation.

Uneventful RSV respiratory disease resembles a common cold and recovery is in 7 to 12 days. Initial symptoms (rhinorrhea, nasal congestion, slight fever, etc.) are followed in 1 to 3 days by lower respiratory tract signs of infection that include a cough and wheezing. In severe cases, these mild symptoms quickly progress to tachypnea, cyanosis, and listlessness and hospitalization is required. In infants with underlying cardiac or respiratory disease, the progression of symptoms is especially rapid and can lead to respiratory failureby the second or third day of illness. With modem intensive care however, overall mortality is usually less than 5% of hospitalized patients (Mclntosh and Chanock, supra).

At present, neither an efficient vaccine nor a specific antiviral agent is available. An immune response to the viral surface glycoproteins can provide resistance to RSV in a number of experimental animals, and a subunit vaccine has been shown to be effective for up to 6 months in children previously hospitalized with an RSV infection (Tristam et al., 1993, J. Infect. Dis. 167, 191). An attenuated bovine RSV vaccine has also been shown to be effective in calves for a similar length of time (Kubota et a., 1992 J. Vet. Med. Sci. 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in subsequent natural infections with RSV (Connors et al., 1992 J. Virol. 66, 7444).

Se: The current treatment for RSV infection requiring hospitalization is the use of aerosolized ribavirin, a guanosine analog [Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J. Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ltd., Ribavirin therapy is associated with a decrease in the severity of the symptoms, improved arterial oxygen and a decrease in the amount of viral shedding at the end of the treatment period. It is not certain, however, whether ribavirin therapy actually shortens the patients' hospital stay or diminishes the need for supportive therapies (Mclntosh and Chanock, supra). The benefits of ribavirin therapy are especially clear for high risk infants, those with the most serious symptoms or for patients with underlying bronchopulmonary or cardiac disease. Inhibition of the viral polymerase complex is supported as the main mechanism for inhibition of RSV by ribavirin, since viral but not cellular polypeptide synthesis is inhibited by ribavirin in RSV-infected cells (Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J.

Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ltd., NY]. Since ribavirin is at least partially effective against RSV infection when delivered by aerosolization, it can be assumed that the target cells are at or near the epithelial surface. In this regard, RSV antigen had not spread any deeper than the superficial layers of the respiratory epithelium in autopsy studies of fatal pneumonia (Mclntosh and Chanock, supra).

Jennings et al., WO 94/13688 indicates that targets for specific types of ribozymes include respiratory syncytical virus.

The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting production of respiratory syncytial virus (RSV). Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals. The invention also features cleavage of the genomic RNA and mRNA of these viruses by use of ribozymes. In particular, the ribozyme molecules described are targeted to the NS1 NS2 (1B) and N viral genes.

These genes are known in the art (for a review see Mclntosh and Chanock, 1990 supra).

10 Ribozymes that cleave the specified sites in RSV mRNAs represent a novel therapeutic approach to respiratory disorders. Applicant indicates that ribozymes are able to inhibit the activity of RSV and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in RSV mRNAs encoding 1C, 1B and N proteins may be readily designed and are within the invention.

Also, those of ordinary skill in the art, will find that it is clear from the examples described that ribozymes cleaving other mRNAs encoded by RSV M, SH, G, F, 22K and L) and the genomic RNA may be readily designed and are within the invention.

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables .31, 33, 35, 37 and 38.

Examples of such ribozymes are shown in Tables 32, 34, 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. 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 mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

Ribozymes of this invention block to some extent RSV production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of respiratory disorders. Ribozyme cleavage of RSV encoded mRNAs or the genomic RNA in these systems may alleviate disease symptoms.

While all ten RSV encoded proteins (1C, 1B, N, P, M, SH, 22K, F, G, and L) are essential for viral life cycle and are all potential targets for ribozyme cleavage, certain proteins (mRNAs) are more favorable for ribozyme targeting than the others. For example RSV encoded proteins 1C, 1B, SH and 22K are not found in other members of the family paramyxoviridae and appear to be unique to RSV. In contrast the ectodomain of the G protein and the signal sequence of the F protein show significant sequence divergence at the nucleotide level among various RSV sub-groups (Johnson et 1987 supra).. RSV proteins 1C, 1B and N are highly conserved among various subtypes at both the nucleotide and .i amino acid levels. Also, 1C, 1B and N are the most abundant of all RSV proteins.

The sequence of human RSV mRNAs encoding 1C, 1B and N proteins are screened for accessible sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 31, 33, 34, 37 and 38 (All sequences are 5' to 3' in the tables.) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

20 Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are S* complementary to the target site sequences described above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am.. Chem. Soc., 109, 7845-7854 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433-5441 and makes 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 G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Hairpin ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography and are resuspended in water.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 32, 34, 36, 37 and 38. 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 10 hammerhead ribozymes listed in Tables 32 and 34(5'-GGCCGAAAGGCCcan 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 Tables 37 and 38 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 32, 34, 36, 37 and 38 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes :ee directed against RSV encoded mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro is evaluated.

Numerous common cell lines can be infected with RSV for experimental purposes. These include HeLa, Vero and several primary epithelial cell lines. A cotton rat animal model of experimental human RSV infection is also available, and the bovine RSV is quite homologous to the human viruses. Rapid clinical diagnosis is through the use of kits designed for the immunofluorescence staining of RSV-infected cells or an ELISA assay, both of which are adaptable for experimental study. RSV encoded mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of RSV activity and/or 1C, 1B and N protein encoding mRNAs by more than 90% will be identified.

Optimizing Ribozyme Activity Ribozyme activity can be optimized as described by Draper et al., PCT W093/23569. The details will not be repeated here, but include altering the length of the ribozyme binding arms 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 al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which 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.), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Sullivan, et al., PCT W094/02595, incorporated by reference herein, 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. 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, intravenous injection, intramuscular injection, subcutaneous 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., suDra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic 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 expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.

Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529- 37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl.

Acad. Sci. US A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 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 15 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 alpha virus vectors).

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves target RNA is inserted into a plasmid DNA vector, a retrovirus DNA viral vector, an adenovirus DNA viral vector or an adeno-associated virus vector or alpha virus vector. These and other vectors have been used to transfer genes to live animals (for a review see Friedman, 1989 Science 244, 1275-1281; Roemer and Friedman, 1992 Eur. J. Biochem. 208, 211-225) and leads to transient or stable gene expression. The vectors are delivered as recombinant viral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, through the use of a catheter, stent or infusion pump.

Diagnostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. 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 :i 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 mRNA associated with ICAM-1, relA, TNF-a, p210, bcr-abl or RSV related condition. Such RNA is detected 15 by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "nontargeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wildtype 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 ICAM-1, rel A, TNF=, p2lobcr-abl or RSV) 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.

II. Chemical Synthesis Of Ribozymes There follows the chemical synthesis, deprotection, and purification of RNA, enzymatic RNA or modified RNA molecules in greater than milligram quantities with high biological activity. Applicant has determined that the synthesis of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its preparation.

10 Specifically, it is important that the RNA phosphoramidites are coupled J efficiently in terms of both yield and time, that correct exocyclic amino protecting groups be used, that the appropriate conditions for the removal of the exocyclic amino protecting groups and the alkylsilyl protecting groups on the 2'-hydroxyl are used, and that the correct work-up and purification procedure of the resulting ribozyme be used.

To obtain a correct synthesis in terms of yield and biological activity of a large RNA molecule about 30 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or substituted amide protecting groups, which must be, on the one hand, stable enough 20 to survive the conditions of synthesis, and on the other hand, removable at the end of the synthesis. These requirements are met by the amide protecting groups shown in Figure 8, in particular, benzoyl for adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine, which may be removed at the end of the synthesis by incubating the RNA in NH 3 /EtOH (ethanolic ammonia) for 20 h at 65 OC. In the case of the phenoxyacetyl type protecting groups shown in Figure 8 on guanosine and adenosine and acetyl protecting groups on cytidine, an incubation in ethanolic ammonia for 4 h at 65 °C is used to obtain complete removal of these protecting groups. Removal of the alkylsilyl 2'-hydroxyl protecting groups can be accomplished using a tetrahydrofuran solution of TBAF at room temperature for 8-24 h.

The most quantitative procedure for recovering the fully deprotected RNA molecule is by either ethanol precipitation, or an anion exchange cartridge desalting, as described in Scaringe et al. Nucleic Acids Res.

1990, 18, 5433-5341. The purification of the long RNA sequences may be accomplished by a two-step chromatographic procedure in which the molecule is first purified on a reverse phase column with either the trityl group at the 5' position on or off. This purification is accomplished using an acetonitrile gradient with triethylammonium or bicarbonate salts as the aqueous phase. In the case of the trityl on purification, the trityl group may be removed by the addition of an acid and drying of the partially purified RNA molecule. The final purification is carried out on an anion exchange column, using alkali metal perchlorate salt gradients to elute the fully purified RNA molecule as the appropriate metal salts, e.g. Na Li etc. A final de-salting step on a small reverse-phase cartridge completes the purification procedure. Applicant has found that such a procedure not only fails to adversely affect activity of a ribozyme, but may improve its activity to cleave target RNA molecules.

Applicant has also determined that significant (see Tables 39-41) improvements in the yield of desired full length product (FLP) can be obtained by: 1. Using 5-S-alkyitetrazole at a delivered or effective concentration of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA (or e analogue) amidite during the coupling step. (By delivered is meant that the actual amount of chemical in the reaction mix is known. This is possible for large scale synthesis since the reaction vessel is of size sufficient to allow such manipulations. The term effective means that available amount of chemical actually provided to the reaction mixture that is able to react with the other reagents present in the mixture. Those skilled in the art will recognize the meaning of these terms from the examples provided herein.) The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.

Alkyl, as used herein, refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl grouphas 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or N(CH 3 2 amino, or SH.

The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 halogen, N(CH 3 2 amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.

Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or

N(CH

3 2 amino or SH.

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

2. Using 5-S-alkyltetrazole at an effective, or final, concentration of 0.1-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.

3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl, propyl or butyl) or NH40H/alkylamine (AMA, with the same preferred alkyl groups as noted for MA) 65 OC for 10-15 m to remove the exocyclic 67 amino protecting groups (vs 4-20 h 55-65 °C using NH40H/EtOH or NH3/EtOH, vide supra). Other alkylamines, e.g. ethylamine, propylamine, butylamine etc. may also be used.

4. Using anhydrous triethylamine*hydrogen fluoride (aHF*TEA) 65 °C for 0.5-1.5 h to remove the 2'-hydroxyl alkylsilyl protecting group (vs 8 24 h using TBAF, vide supra or TEA.3HF for 24 h (Gasparutto et al.

Nucleic Acids Res. 1992, 20, 5159-5166). Other alkylamine*HF complexes may also be used, e.g. trimethylamine or diisopropylethylamine.

The use of anion-exchange resins to purify and/or analyze the 10 fully deprotected RNA. These resins include, but are not limited to, quartenary or tertiary amino derivatized stationary phases such as silica or polystyrene. Specific examples include Dionex-NA100®, Mono-Q®, Poros- Thus, the invention features an improved method for the coupling of 15 RNA phosphoramidites; for the removal of amide or substituted amide protecting groups; and for the removal of 2'-hydroxyl alkylsilyl protecting groups. Such methods enhance the production of RNA or analogs of the type described above with substituted 2'-groups), and allow efficient synthesis of large amounts of such RNA. Such RNA may also have enzymatic activity and be purified without loss of that activity. While specific examples are given herein, those in the art will recognize that equivalent chemical reactions can be performed with the alternative chemicals noted above, which can be optimized and selected by routine experimentation.

In another aspect, the invention features an improved method for the purification or analysis of RNA or enzymatic RNA molecules 28-70 nucleotides in length) by passing said RNA or enzymatic RNA molecule over an HPLC, reverse phase and/or an anion exchange chromatography column. The method of purification improves the catalytic activity of enzymatic RNAs over the gel purification method (see Figure Draper et al., PCT W093/23569, incorporated by reference herein, disclosed reverse phase HPLC purification. The purification of long RNA molecules may be accomplished using anion exchange chromatography, particularly in conjunction with alkali perchlorate salts. This system may be used to purify very long RNA molecules. In particular, it is advantageous to use a Dionex NucleoPak 100© or a Pharmacia Mono Q® anion exchange column for the purification of RNA by the anion exchange method. This anion exchange purification may be used following a reverse-phase purification or prior to reverse phase purification. This method results in the formation of a sodium salt of the ribozyme during the chromatography.

Replacement of the sodium alkali earth salt by other metal salts, e.g., lithium, magnesium or calcium perchlorate, yields the corresponding salt of the RNA molecule during the purification.

In the case of the 2-step purification procedure, in which the first step 10 is a reverse phase purification followed by an anion exchange step, the reverse phase purification is best accomplished using polymeric, e.g.

polystyrene based, reverse-phase media, using either a 5'-trityl-on or trityl-off method. Either molecule may be recovered using this reversephase method, and then, once detritylated, the two fractions may be pooled and then submitted to an anion exchange purification step as described above.

The method includes passing the enzymatically active RNA molecule over a reverse phase HPLC column; the enzymatically active S* RNA molecule is produced in a synthetic chemical method and not by an enzymatic process; and the enzymatic RNA molecule is partially blocked, and the partially blocked enzymatically active RNA molecule is passed over a reverse phase HPLC column to separate it from other RNA molecules.

In more preferred embodiments, the enzymatically active RNA molecule, after passage over the reverse phase HPLC column, is deprotected and passed over a second reverse phase HPLC column (which may be the same as the reverse phase HPLC column), to remove the enzymatic RNA molecule from other components. In addition, the column is a silica or organic polymer-based C4, C8 or C18 column having a porosity of at least 125 A, preferably 300 A, and a particle size of at least 2 p.m, preferably 5 pm.

Activation The synthesis of RNA molecules may be accomplished chemically or enzymatically. In the case of chemical synthesis the use of tetrazole as an activator of RNA phosphoramidites is known (Usman et al. J. Am. Chem.

Soc. 1987, 109, 7845-7854). In this, and subsequent reports, a 0.5 M solution of tetrazole is allowed to react with the RNA phosphoramidite and couple with the polymer bound 5'-hydroxyl group for 10 m. Applicant has determined that using 0.25-0.5 M solutions of 5-S-alkyltetrazoles for only min gives equivalent or better results. The following exemplifies the procedure.

Example 7: Synthesis of RNA and Ribozymes Using as Activating Agent The method of synthesis used follows the general procedure for RNA 10 synthesis as described in Usman et al., 1987supra and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The major difference used Swas the activating agent, 5-S-ethyl or -methyltetrazole 0.25 M concentration for 5 min.

All small scale syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 p.mol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 m coupling step for methylated RNA. A 6.5-fold excess (162.5 .L of 0.1 M 32.5 p.mol) of 20 phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 .L of 0.25 .i M 100 lpmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

All large scale syntheses were conducted on a modified (eight amidite port capacity) 390Z (ABI) synthesizer using a 25 pmol scale protocol with a 5-15 min coupling step for alkylsilyl protected RNA and 7.5 m coupling step for 2'-O-methylated RNA. A six-fold excess (1.5 mL of 0.1 M 150 imol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of 0.25 M 1125 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 390Z, determined by colorimetric quantitation of the trityl fractions, was 95.0-96.7%.

Oligonucleotide synthesis reagents for the 390Z: Detritylation solution was 2% DCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25-0.5 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

Deprotection The first step of the deprotection of RNA molecules may be \e accomplished by removal of the exocyclic amino protecting groups with i o either NH 4 0H/EtOH:3/1 (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845- 7854) or NH3/EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18, 5433- 5341) for -20 h 55-65 Applicant has determined that the use of methylamine or NH40H/methylamine for 10-15 min 55-65 °C gives equivalent or better results. The following exemplifies the procedure.

Example 8: RNA and Ribozyme DeDrotection of Exocyclic Amino 20 Protecting Groups Using Methylamine (MA) or NH40H/Methylamine

(AMA)

The polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or (AMA) 55-65 °C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 4 mL glass screw top vial. NH 4 0H and aqueous methylamine were pre-mixed in equal volumes. 4 mL of the resulting reagent was added to the vial, equilibrated for 5 m at RT and then heated at or 65 OC for 5-15 min. After cooling to -20 the supernatant was removed from the polymer support. The support was washed with 1.0 mL of EtOH:MeCN:H 2 0/3:1:1, vortexed and the supematant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The same procedure was followed for the aqueous methylamine reagent.

Table 40 is a summary of the results obtained using the improvements outlined in this application for base deprotection.

The second step of the deprotection of RNA molecules may be accomplished by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845- 7854). Applicant has determined that the use of anhydrous TEA*HF in Nmethylpyrrolidine (NMP) for 0.5-1.5 h 55-65 °C gives equivalent or better results. The following exemplifies this procedure.

Example 9: RNA and Ribozyme Deprotection of 2'-Hydroxyl Alkylsily Protecting Groups Using Anhydrous TEA*HF To remove the alkylsilyl protecting groups, the ammonia-deprotected 10 oligoribonucleotide was resuspended in 250 pL of 1.4 M anhydrous

HF

solution (1.5 mL N-methylpyrrolidine, 750 gL TEA and 1.0 mL TEA-3HF) and heated to 65 OC for 1.5 h. 9 mL of 50 mM TEAB was added to quench the reaction. The resulting solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) prewashed with 10 mL of 50 mM TEAB.

After washing the cartridge with 10 mL of 50 mM TEAB, the RNA was eluted with 10 mL of 2 M TEAB and dried down to a white powder.

Table 41 is a summary of the results obtained using the improvements outlined in this application for alkylsilyl deprotection.

Example 10: HPLC Purification. Anion Exchange column 20 For a small scale synthesis, the crude material was diluted to 5 mL with diethylpyrocarbonate treated water. The sample was injected onto either a Pharmacia Mono Q® 16/10 or Dionex NucleoPac® column with 100% buffer A (10 mM NaCIO 4 A gradient from 180-210 mM NaC0O 4 at a rate of 0.85 mM/void volume for a Pharmacia Mono Q® anion-exchange column or 100-150 mM NaCIO 4 at a rate of 1.7 mM/void volume for a Dionex NucleoPac® anion-exchange column was used to elute the RNA.

Fractions were analyzed by a HP-1090 HPLC with a Dionex NucleoPac® column. Fractions containing full length product at 80% by peak area were pooled.

For a trityl-off large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose® Fast Flow column. The column was thoroughly washed with 10 mM sodium perchlorate buffer. The oligonucleotide was eluted from the column with 300 mM sodium perchlorate. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material in the synthesis.

The eluent was diluted four fold in sterile H 2 0 to lower the salt concentration and applied to a Pharmacia Mono Q® 16/10 column. A gradient from 10-185 mM sodium perchlorate was run over 4 column volumes to elute shorter sequences, the full length product was then eluted in a gradient from 185-214 mM sodium perchlorate in 30 column volumes.

The fractions of interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPac® column. Fractions containing over 85% full length material were pooled. The pool was applied to a Pharmacia RPC® column for desalting.

0o For a trityl-on large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose® Fast Flow column. The column was thoroughly washed with 20 mM NH 4

CO

3

CH

3 CN buffer. The oligonucleotide was eluted from the column with 1.5 M

NH

4

CO

3 H/10% acetonitrile. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material present in the synthesis. The oligonucleotide was then applied to a Pharmacia Resource RPC column. A gradient from 20-55% B (20 mM NH 4 C0 3 H/25% CH 3

CN,

buffer A 20 mM NH 4

CO

3 H/10% CH 3 CN) was run over 35 column volumes. The fractions of interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPac® column. Fractions containing over 60% full length material were pooled. The pooled fractions were then submitted to manual detritylation with 80% acetic acid, dried down immediately, resuspended in sterile H 2 0, dried down and resuspended in H 2 0 again. This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac® column. The material was purified by anion exchange chromatography as in the trityl-off scheme (vide supra).

Example 11 Ribozyme Activity Assay Purified 5'-end labeled RNA substrates (15-25-mers) and purified end labeled ribozymes (-36-mers) were both heated to 95 OC, quenched on ice and equilibrated at 37 separately. Ribozyme stock solutions were 1 iM, 200 nM, 40 nM or 8 nM and the final substrate

RNA

concentrations were 1 nM. Total reaction volumes were 50 igL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCl 2 Reactions were initiated by mixing substrate and ribozyme solutions at t 0. Aliquots of gL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each aliquot was quenched in formamide loading buffer and loaded onto a denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

Example 12: One pot deprotection of RNA Applicant has shown that aqueous methyl amine is an efficient reagent to deprotect bases in an RNA molecule. However, in a time consuming step (2-24 hrs), the RNA sample needs to be dried completely prior to the deprotection of the sugar 2'-hydroxyl groups. Additionally, 7deprotection of RNA synthesized on a large scale 100 gmol) becomes challenging since the volume of solid support used is quite large.

In an attempt to minimize the time required for deprotection and to simplify .oi the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol (Fig. 12). According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 min and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA.3HF reagent.

The reaction is quenched with 16 mM TEAB solution.

S"Referring to Fig. 13, hammerhead ribozyme targeted to site B is synthesized using RNA phosphoramadite chemistry and deprotected using Seither a two pot or a one pot protocol. Profiles of these ribozymes on an HPLC column are compared. The figure shows that RNAs deprotected by either the one pot or the two pot protocols yield similar full-length product profiles. Applicant has shown that using a one pot deprotection protocol, time required for RNA deprotection can be reduced considerably without compromising the quality or the yield of full length RNA.

Referring to Fig. 14, hammerhead ribozymes targeted to site B (from Fig. 13 are tested for their ability to cleave RNA. As shown in the figure 14 ribozymes that are deprotected using one pot protocol have catalytic activity comparable to ribozymes that are deprotected using a two pot protocol.

Example 12a:Improved orotocol for the synthesis of phosphorothioate containing RNA and ribozymes using 5-S-Alkvltetrazoles as Activating Agent The two sulfurizing reagents that have been used to synthesize ribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu and Hirschbein, 1991 Tetrahedron Letter31, 3005), and 3H-1,2-benzodithiol-3one 1,1-dioxide (Beaucage reagent; Vu and Hirschbein, 1991 supra).

TETD requires long sulfurization times (600 seconds for DNA and 3600 seconds for RNA). It has recently been shown that for sulfurization of DNA oligonucleotides,- Beaucage reagent is more efficient than TETD (Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med. Chem. 4, 1519).

Beaucage reagent has also been used to synthesize phosphorothioate oligonucleotides containing 2'-deoxy-2'-fluoro modifications wherein the ::wait time is 10 min (Kawasaki et al., 1992 J. Med. Chem).

The method of synthesis used follows the procedure for RNA synthesis as described herein and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The sulfurization step for RNA described in the literature is a 8 second delivery and 10 min wait steps (Beaucage and lyer, 1991 Tetrahedron 49, 6123). These conditions produced about 95% sulfurization as measured by HPLC analysis (Morvan et al., 1990 Tetrahedron Letter 31, 7149). This 5% contaminating oxidation could arise from the presence of oxygen dissolved in solvents and/or slow release of traces of iodine adsorbed on the inner surface of delivery lines during previous synthesis.

A major improvement is the use of an activating agent, ethyltetrazole or 5-S-methyltetrazole at a concentration of 0.25 M for 5 min.

Additionally, for those linkages which are phosporothioate, the iodine solution is replaced with a 0.05 M solution of 3 H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) in acetonitrile. The delivery time for the sulfurization step is reduced to 5 seconds and the wait time is reduced to 300 seconds.

RNA synthesis is conducted on a 394 (ABI) synthesizer using a modified 2.5 gmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 gL of 0.1 M 32.5 gmol) of phosphoramidite and a 40-fold excess of S--ethyl tetrazole (400 UL of 0.25 M 100 gmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle.

Average coupling yields on the 394 synthesizer, determined by colorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394 synthesizer: detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2 ,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems. Sulfurizing reagent was obtained from Glen Research.

Average sulfurization efficiency (ASE) is determined using the formula: ASE (PS/Total)l/n-1 15 where, PS integrated 31P NMR values of the P=S diester Total integration value of all peaks n length of oligo Referring to tables 42 and 43, effects of varying the delivery and the wait time for sulfurization with Beaucage's reagent is described. These 20 data suggest that 5 second wait time and 300 second delivery time is.the condition under which ASE is maximum.

Using the above conditions a 36 mer hammerhead ribozyme is synthesized which is targeted to site C. The ribozyme is synthesized to contain phosphorothioate linkages at four positions towards the 5' end.

RNA cleavage activity of this ribozyme is shown in Fig. 16. Activity of the phosphorothioate ribozyme is comparable to the activity of a ribozyme lacking any phosphorothioate linkages.

Example 13: Protocol for the synthesis of 2'-N-phtalimido-nucleoside phosphoramidite The 2'-amino group of a 2 '-deoxy-2'-amino nucleoside is normally protected with N-( 9 -flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supraj Pieken et al., 1991 Science 253, 314). This protecting group is not stable in CH3CN solution or even in dry form during prolonged storage at -20 oC. These problems need to be overcome in order to achieve large scale synthesis of RNA.

Applicant describes the use of alternative protecting groups for the 2'amino group of 2'-deoxy-2'-amino nucleoside. Referring to Figure 17.

phosphoramidite 17 was synthesized starting from 2'-deoxy-2'aminonucleoside (12) using transient protection with Markevich reagent (Markiewicz J. Chem. Res. 1979, S, 24). An intermediate 13 was obtained in 50% yield, however subsequent introduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960 Nature 185, 306), desilylation dimethoxytrytilation (16) and phosphitylation led to phosphoramidite 17.

Since overall yield of this multi-step procedure was low applicant investigated some alternative approaches, concentrating on selective introduction of N-phtaloyl group without acylation of 5' and 3' hydroxyls.

o. When 2 '-deoxy-2'-amino-nucleoside was reacted with 1.05 15 equivalents of Nefkens reagent in DMF overnight with subsequent treatment with Et3N (1 hour) only 10-15% of N and 5'(3')-bis-phtaloyl derivatives were formed with the major component being N-Pht-derivative The N,O-bis by-products could be selectively and quantitively converted to N-Pht derivative 15 by treatment of crude reaction mixture 20 with cat. KCN/MeOH.

A convenient "one-pot" procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCI/Et3N and resulting in the preparation of DMT derivative 16 in 85% overall yield as follows. Standard phosphytilation of 16 produced phosphoramidite 17 in 87% yield. One gram of 2'-amino nucleoside, for example 2'-amino uridine (US Biochemicals® part 77140) was co-evaporated twice from dry dimethyl formamide (Dmf) and dried in vacuo overnight. 50 mis of Aldrich sure-seal Dmf was added to the dry 2'-amino uridine via syringe and the mixture was stirred for 10 minutes to produce a clear solution. 1.0 grams (1.05 eq.) of Ncarbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) was added and the solution was stirred overnight. Thin layer chromatography (TLC) showed 90% conversion to a faster moving products (10% ETOH in

CHCI

3 and 57 Il of TEA (0.1 eq.) was added to effect closure of the phthalimide ring. After 1 hour an additional 855 pl (1.5 eq.) of TEA was added followed by the addition of 1.53 grams (1.1 eq.) of DMT-CI WO 95/23225 77 (Lancaster Synthesis®, The reaction mixture was left to stir overnight and quenched with ETOH after TLC showed greater than desired product. Dmf was removed under vacuum and the mixture was washed with sodium bicarbonate solution aq., 500 mis) and extracted with ethyl acetate (2x 200 mls). A 25mm x 300mm flash column (75 grams Merck flash silica) was used for purification. Compound eluted at 80 to ethyl acetate in hexanes (yield: 80% purity: >95% by 1HNMR).

Phosphoramidites were then prepared using standard protocols described above.

With phosphoramidite 17 in hand applicant synthesized several ribozymes with 2'-deoxy-2'-amino modifications. Analysis of the synthesis demonstrated coupling efficiency in 97-98% range. RNA cleavage activity of ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/or U7 positions (see Figure wherein the 2'-amino positions were either protected with Fmoc or Pht, was identical. Additionally, complete deprotection of 2'-deoxy-2'-amino-Uridine was confirmed by basecomposition analysis. The coupling efficiency of phosphoramidite 17 was not effected over prolonged storage (1-2 months) at low temperatures.

Protecting 2' Position with a SEM Group There follows a method using the 2 '-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base deprotection. At the same time, this.group should also be readily removed when desired. To that end the t-butyldimethylsilyl group has been efficacious (Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.

1990, 18, 5433-5441). However, long exposure times to tetra-nbutylammonium fluoride (TBAF) are generally required to fully remove this protecting group from the 2'-hydroxyl. In addition, the bulky alkyl substituents can prove to be a hindrance to coupling thereby necessitating longer coupling times. Finally, it has been shown that the TBDMS group is base labile and is partially deprotected during treatment with ethanolic ammonia (Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res. 1990, 18, 5433-5441 and Stawinski,J.; Stromberg,R.; Thelin,M.; Westman,E.

Nucleic Acids Res. 1988, 16, 9285-9298).

The (trimethylsilyl)ethoxymethyl ether (SEM) seems a suitable substitute. This protecting group is stable to base and all but the harshest acidic conditions. Therefore it is stable under the conditions required for oligonucleotide synthesis. It can be readily introduced and the oxygen carbon bond makes it unable to migrate. Finally, the SEM group can be removed with BF 3 *OEt 2 very quickly.

There follows a method for synthesis of RNA by protecting the 2'position of a nucleotide during RNA synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group. The method can involve use of standard RNA synthesis conditions as discussed below, or any other equivalent steps. Those in the art are familiar with such steps. The "15 nucleotide used can be any normal nucleotide or may be substituted in 15 various positions by methods well known in the art, as described by Eckstein et al., International Publication No. WO 92/07065, Perrault et al., Nature 1990, 344, 565-568, Pieken et al., Science 1991, 253, 314-317, Usman,N.; Cedergren,R.J. Trends in Biochem. Sci. 1992, 17, 334-339, Usman et al., PCT W093/15187, and Sproat,B. European Patent Application 92110298.4.

This invention also features a method for covalently linking a SEM group to the 2'-position of a nucleotide. The method involves contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions. In a preferred embodiment, the conditions are dibutyltin oxide, tetrabutylammonium fluoride and SEM-CI. Those in the art, however, will recognize that other equivalent conditions can also be used.

In another aspect, the invention features a method for removal of an SEM group from a nucleoside molecule or an oligonucleotide. The method involves contacting the molecule or oligonucleotide with boron trifluoride etherate (BF 3 .OEt 2 under SEM removing conditions, in acetonitrile.

Referring to Figure 18, there is shown the method for solid phase synthesis of RNA. A 2 ',5'-protected nucleotide is contacted with a solid phase bound nucleotide under RNA synthesis conditions to form a dinucleotide. The protecting group at the 2 '-position in prior art methods can be a silyl ether, as shown in the Figure. In the method of the present invention, an SEM group is used in place of the silyl ether.

Otherwise RNA synthesis can be performed by standard methodology.

Referring to Figure 19, there is shown the synthesis of 2'-O-SEM protected nucleosides and phosphoramadites. Briefly, a nucleoside is protected at the or 3'-position by contacting with a derivative of SEM under appropriate conditions. Specifically, those conditions include contacting the nucleoside with dibutyltin oxide and SEM chloride. The 2 regioisomers are separated by chromatography and the 2'- •o 10 protected moiety is converted into a phosphoramidite by standard procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.

:i Referring to Figure 20, a prior art method for deprotection of RNA using silyl ethers is shown. This contrasts with the method shown in Figure 21 in 15 which deprotection of RNA containing an SEM group is performed. In step 1, the base protecting groups and cyanoethyl groups are removed by standard procedure. The SEM group is then removed as shown in the Figure. The details of the synthesis of phosphoramidites and SEM protected nucleosides and their use in synthesis of oligonucleotides and subsequent deprotection of Example 14: Synthesis of 2'-O-((trimethvlsilvl)ethoxvmethvl)-5'-O- Dimethoxytrityl Uridine (2) Referring to.Figure 19, 5'-O-dimethoxytrityl uridine 1 (1.0 g, 1.83 mmol) in CH 3 CN (18 mL) was added dibutyltin oxide (1.0 g, 4.03 mmol) and TBAF (1 M, 2.38 mL, 2.38 mmol). The mixture was stirred for 2 h at RT (about 20-25 0 C) at which time (trimethylsilyl)ethoxymethyl chloride (SEM- Cl) (487 gL, 2.75 mmol) was added. The reaction mixture was stirred overnight and then filtered and evaporated. Flash chromatography hexanes in ethyl acetate) yielded 347 mg of 2'-hydroxyl protected nucleoside 2 and 314 mg of 3'-hydroxyl protected nucleoside 3.

Example 15: Synthesis of 2 '--((trimethylsilyl)ethoxymethyl) Uridine (4) Nucleoside 2 was detritylated following standard methods, as shown in Figure 19.

Example 16: Synthesis of 2'-O-((trimethylsilyl)ethoxymethvl)-5'.3'-O-Acetyl Uridine Nucleoside 4 was acetylated following standard methods, as shown in Figure 19.

Example 17: Synthesis of 5'.3'-O-Acetyl Uridine (6) Referring to Figure 19. the fully protected uridine 5 (32 mg, 0.07 mmol) was dissolved in CH 3 CN (700 gL) and BF 3 *OEt 2 (17.5 iL, 0.14 mmol) was added. The reaction was stirred 15 m and MeOH was added to quench the reaction. Flash chromatography MeOH in CH 2

CI

2 gave 'i 10 20 mg of SEM deprotected nucleoside 6.

Example 18: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-3'-O- Succinyl-5'-O- Dimethoxytrityl Uridine (2) Nucleoside 3 was succinylated and coupled to the support following standard procedures, as shown in Figure 19.

15 Example 19: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-5'-O- Dimethoxvtrityl Uridine 3'-(2-Cvanoethvl NN-diisopropylphosDhoramidite) Nucleoside 3 was phosphitylated following standard methods, as shown in Figure 19.

20 Example 20: Synthesis of RNA Using 2'-0-SEM Protection Referring to Figure 18, the method of synthesis used follows the general procedure for RNA synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.

1990, 18, 5433-5441. The phosphoramidite 8 was coupled following standard RNA methods to.provide a 10-mer of uridylic acid. Syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 pmol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 uL of 0.1 M 32.5 pmol) of phosphoramidite and a 80-fold excess of tetrazole (400 gL of 0.5 M 200 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, were 98-99%.

Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N- 81 Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle.

Referring to Figure 21, the homopolymer was base deprotected with

NH

3 /EtOH at 65 The solution was decanted and the support was washed twice with a solution of 1:1:1 H20:CH3CN:MeOH. The combined solutions were dried down and then diluted with CH 3 CN (1 mL). BF 3 .OEt 2 gL, 30 imol) was added to the solution and aliquots were removed at 10 ten time points. The results indicate that after 30 min deprotection is complete, as shown in Figure 22.

Vectors Expressing Ribozymes There follows a method for expression of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In 15 general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a decreased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful for scaling up production of a ribozyme, which may be either modified or l unmodified, in situ or in vitro. Such vector systems can be used to express a desired ribozyme in a specific cell, or can be used in an in vitro system to allow productiuon of large amounts of a desired riboqyne, The vectors of this invention allow a higher yield synthesis of a ribozyme in the form of an RNA transcript which is cleaved in situ or in vitro before or after transcript isolation.

Thus, this invention is distinct from the prior art in that a single ribozyme is used to process the 3' and 5' ends of each therapeutic, transacting or desired ribozyme instead of processing only one end, or only one ribozyme. This allows smaller vectors to be derived with multiple transacting ribozymes released by only one other ribozyme from the mRNA transcript. Applicant has also provided methods by which the activity of such ribozymes is increased compared to those in the art, by designing ribozyme-encoding vectors and the corresponding transcript such that 82 folding of the mRNA does not interfere with processing by the releasing ribozyme.

The stability of the ribozyme produced in this method can be enhanced by provision of sequences at the termini of the ribozymes as described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein.

The method of this invention is advantageous since it provides high yield synthesis of ribozymes by use of low cost transcription-based protocols, compared to existing chemical ribozyme synthesis, and can use 10 isolation techniques currently used to purify chemically synthesized oligonucleotides. Thus, the method allows synthesis of ribozymes in high yield at low cost for analytical, diagnostic, or therapeutic applications.

The method is also useful for synthesis of ribozymes in vitro for ribozyme structural studies, enzymatic studies, target RNA accessibility 15 studies, transcription inhibition studies and nuclease protection studies, much is described by Draper et al., PCT WO 93/23509 hereby incorporated by reference herein.

The method can also be used to produce ribozymes in situ either to increase the intracellular concentration of a desired therapeutic ribozyme, 20 or to produce a concatameric transcript for subsequent in vitro isolation of unit length ribozyme. The desired ribozyme can be used to inhibit gene expression in molecular genetic analyses or in infectious cell systems, and to test the efficacy of a therapeutic molecule or treat afflicted cells.

Thus, in general, the invention features a vector which includes a bacterial, viral or eucaryotic promoter within a plasmid, cosmid, phagmid, virus, viroid, virusoid or phage vector. Other vectors are equally suitable and include double-stranded, or partially double-stranded DNA, formed by an amplification method such as the polymerase chain reaction, or doublestranded, partially double-stranded or single-stranded RNA, formed by sitedirected homologous recombination into viral or viroid RNA genomes.

Such vectors need not be circular. Transcriptionally linked to the promoter region is a first ribozyme-encoding region, and nucleotide sequences encoding a ribozyme cleavage sequence which is placed on either side of a region encoding a therapeutic or otherwise desired second ribozyme.

83 Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA expression system. The desired second ribozyme may be any desired type of ribozyme, such as a hammerhead, hairpin hepatitis delta virus (HDV) or other catalytic center, and can include group I and group II introns, as discussed above. The first ribozyme is chosen to cleave the encoded cleavage sequence, and may also be any desired ribozyme, for example, a Tetrahymena derived ribozyme, which may, for example, include an imbedded restriction endonuclease site in the center of a selfrecognition sequence to aid in vector construction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector.

When the promoter of such a vector is activated an RNA transcript is produced which includes the first and second ribozyme sequences. The first ribozyme sequence is able to act, under appropriate conditions, to cause cleavage at the cleavage sites to release the second ribozyme .o sequences. These second ribozyme sequences can then act at their target RNA sites, or can be isolated for later use or analysis.

Thus, in one aspect the invention features a vector which includes a first nucleic acid sequence (encoding a first ribozyme having intramolecular cleaving activity), and a second nucleic acid sequence (encoding a second ribozyme having intermolecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA transcript encoded by the vector. The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have intermolecular cleaving activity.

As discussed above, the first ribozyme can be chosen to be any selfcleaving ribozyme, and the second ribozyme may be chosen to be any desired ribozyme. The flanking sequences are chosen to include sequences recognized by the first ribozyme. When the vector is caused to express RNA from these nucleic acid sequences, that RNA has the ability under appropriate conditions to cleave each of the flanking regions and thereby release one or more copies of the second ribozyme. If desired, several different second ribozymes can be produced by the same vector, or 84 several different vectors can be placed in the same vessel or cell to produce different ribozymes.

In preferred embodiments, the vector includes a plurality of the nucleic acid sequences encoding the second ribozyme, each flanked by nucleic acid sequences recognized by the first ribozyme. Most preferably, such a plurality includes at least six to nine or even between 60 100 nucleic acid sequences. In other preferred embodiments, the vector includes a promoter which regulates expression of the nucleic acid encoding the ribozymes from the vector; and the vector'is chosen from a plasmid, 10 cosmid, phagmid, virus, viroid or phage. In a most preferred embodiment, the plurality of nucleic acid sequences are identical and are arranged in sequential order such that each has an identical end nearest to the promoter. If desired, a poly(A) sequence adjacent to the sequence encoding the first or second ribozyme may be provided to increase stability of the RNA produced by the vector; and a restriction endonuclease site adjacent to the nucleic acid encoding the first ribozyme is provided to allow insertion of nucleic acid encoding the second ribozyme during construction of the vector.

In a second aspect, the invention features a method for formation of a ribozyme expression vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a singlestranded DNA encoding a second ribozyme, as discussed above. The single-stranded DNA is then allowed to anneal to form a partial duplex DNA which can be filled in by a treatment with an appropriate enzyme, such as a DNA polymerase in the. presence of dNTPs, to form a duplex DNA which can then be ligated to the vector. Large vectors resulting from this method can then be selected to insure that a high copy number of the single-stranded DNA encoding the second ribozyme is incorporated into the vector.

In a further aspect, the invention features a method for production of ribozymes by providing a vector as described above, expressing RNA from that vector, and allowing cleavage by the first ribozyme to release the second ribozyme.

In preferred embodiments, three different ribozyme motifs are used as cis-cleaving ribozymes. The hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs consist of small, well-defined sequences that rapidly self-cleave in vitro (Symons, 1992 Annu. Rev. Biochem. 61, 641).

While structural and functional differences exist among the three ribozyme motifs, they self-process efficiently in vivo. All three ribozyme motifs selfprocess to 87-95% completion in the absence of 3' flanking sequences. In vitro, the self-processing constructs described in this invention are significantly more active than those reported by Taira et al., 1990 suDra; and Altschuler et al., 1992 Gene 122, 85. The present invention enables the use of cis-cleaving ribozymes to efficiently truncate RNA molecules at 10 specific sites in vivo by ensuring lack of secondary structure which -prevents processing.

Isolation of Therapeutic Ribozyme The preferred method of isolating therapeutic ribozyme is by a chromatographic technique. The HPLC purification methods and reverse 15 HPLC purification methods described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein, can be used. Alternatively, the attachment of complementary oligonucleotides to cellulose or other .0 chromatography columns allows isolation of the therapeutic second ribozyme, for example, by hybridization to the region between the flanking arms and the enzymatic RNA. This hybridization will select against the .0 short flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybridization can be accomplished in the presence of a chaotropic agent to prevent nuclease degradation. The oligonucleotides on the matrix can be modified to minimize nuclease activity, for example, by provision of 2'-O-methyl RNA oligonucleotides.

Such modifications of the oligonucleotide attached to the column matrix will allow the multiple use of the column with minimal oligo degradation. Many such modifications are known in the art, but a chemically stable nonreducible modification is preferred. For example, phosphorothioate modifications can also be used.

The expressed ribozyme RNA can be isolated from bacterial or eucaryotic cells by routine procedures such as lysis followed by guanidine isothiocyanate isolation.

The current known self-cleaving site of Tetrahymena can be used in an alternative vector of this invention. If desired, the full-length Tetrahymena sequence may be used, or a shorter sequence may be used.

It is preferred that, in order to decrease the superfluous sequences in the self-cleaving site at the 5' cleavage end, the hairpin normally present in the Tetrahymena ribozyme should contain the therapeutic second ribozyme 3' sequence and its complement. That is, the first releasing ribozymeencoding DNA is provided in two portions, separated by DNA encoding the desired second ribozyme. For example, if the therapeutic second ribozyme recognition sequence is CGGACGA/CGAGGA, then CGAGGA is provided in the self-cleaving site loop such that it is in a stem structure recognized by 10 the Tetrahymena ribozyme. The loop of the stem may include a restriction endonuclease site into which the desired second ribozyme-encoding DNA .is placed.

If desired, the vector may be used in a therapeutic protocol by use of the systems described by Lechner, PCT WO 92/13070, hereby 15 incorporated by reference herein, to allow a timed expression of the therapeutic second ribozyme, as well as an appropriate shut off of cell or gene function. Thus, the vector will include a promoter which appropriately expresses enzymatically active RNA only in the presence of an RNA or another molecule which indicates the presence of an undesired organism or state. Such enzymatically active RNA will then kill or harm the cell in which it exists, as described by Lechner, id., or act to cause reduced expression of a desired protein product.

A number of suitable RNA vectors may also be used in this invention.

The vectors include plant viroids, plant viruses which contain single or double-stranded RNA genomes and animal viruses which contain RNA genomes, such as the picornaviruses, myxoviruses, paramyxoviruses, hepatitis A virus, reovirus and retroviruses. In many instances cited, use of these viral vectors also results in tissue specific delivery of the ribozymes.

Example 21: Design of self-processing cassettes In a preferred embodiment, applicant compared the in vitro and in vivo cis-cleaving activity of three different ribozyme motifs-the hammerhead, the hairpin and the hepatitis delta virus ribozyme-in order to assess their potential to process the ends of transcripts in vivo. To make a direct comparison among the three, however, it is important to design the ribozyme-containing transcripts to be as similar as possible. To this end, all the ribozyme cassettes contained the same trans-acting hammerhead ribozyme followed immediately by one of the three cis-acting ribozymes (Figure 23-25). For simplicity, applicant refers to each cassette by an abbreviation that indicates the downstream cis-cleaving ribozyme only.

Thus HH refers to the cis-cleaving cassette containing a hammerhead ribozyme, while HP and HDV refer to the cassettes containing hairpin and hepatitis delta virus cis-cleaving ribozymes, respectively. The general design of the ribozyme cassettes, as well as specific differences among the cassettes, are outlined below.

10 A sequence predicted to form a stable stem-loop structure is included at the 5' end of all the transcripts. The hairpin stem contains the T7 RNA polymerase initiation sequence (Milligan Uhlenbeck, 1989 Methods Enzymol. 180, 51) and its complement, separated be a stable tetra-loop (Antao et al., 1991 Nucleic Acids Res. 19, 5901). By incorporating the T7 15 initiation sequence into a stem-loop structure, applicant hoped to avoid nonproductive base pairing interactions with either the trans-acting ribozyme or with the cis-acting ribozyme. The presence of a hairpin at the end of a transcript may also contribute to the stability of the transcript in vivo. These are non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using a variety of promoters, initiator sequences and stem-loop structure combinations generally known in the art.

The trans-acting ribozyme used in this study is targeted to a site B 1 GACCUUC...3'). The 5' binding arm of the ribozyme, GAAGGUC-3', and the core of the ribozyme, CUGAUGAGGCCGAAAGGCCGAA-3', remain constant in all cases. In addition, all transcripts also contain a single nucleotide between the stem-loop and the first nucleotide of the ribozyme. The linker nucleotide was required to obtain the same activity in vitro that was measured with an identical ribozyme lacking the 5' hairpin. Because the three cis-cleaving ribozymes have different requirements at the site of cleavage, slight differences were unavoidable at the 3' end of the processed transcript. The junction between the trans- and cis-acting ribozyme is, however, designed so that there is minimal extraneous sequence left at the 3' end of the transcleaving ribozyme once cis-cleavage occurs. The only differences between the constructs lie in the 3' binding arm of the ribozyme, where either 6 or 7 nucleotides, complementary to the target sequence are present and where, after processing, two to five extra nucleotides remain.

The cis-cleaving hammerhead ribozyme used in the HH cassette is based on the design of Grosshans and Cech, 1991 supra. As shown in Figure 23, the 3' binding arm of the trans-acting ribozyme is included in the required base-pairing interactions of the cis-cleaving ribozyme to form stem I. Two extra nucleotides, UC, were included at the end of the 3' binding arm to form the self-processing hammerhead ribozyme site (Ruffner et al., 10 1990 suDEa) which remain on the 3' end of the trans-acting ribozyme following self-processing.

The hairpin ribozyme portion of the HP self-processing construct is based on the minimal wild-type sequence (Hampel Tritz, 1989 sura). A tetra-loop at the end of helix 1 side of the cleavage site) serves to link 15 the two portions and thus allows a minimal five nucleotides to remain at the end of the released trans-acting ribozyme following self-processing. Two variants of HP were designed: HP(GU) and HP(GC). The HP(GU) was constructed with a G-U wobble base pair in helix 2 (A52G substitution; Figure 24). This slight destabilization of helix 2 was intended to improve self-processing activity by promoting product release and preventing the reverse reaction (Berzal-Herranz et al., 1992 Genes Dev. 6, 129; Chowrira et al., 1993 Biochemistry 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing interactions in helix 2 (U77C and A52G substitution; Figure 24). Another modification to discourage the reverse ligation reaction of the hairpin ribozyme was to shorten helix 1 (Fiure 24) by one base pair relative to the wild-type sequence (Chowrira Burke, 1991 Biochemistry 30, 8518).

The HDV ribozyme self-processes efficiently when the nucleotide 5' to the cleavage site is a pyrimidine, and somewhat less so when adenosine is in that position. No other sequence requirements have been identified upstream of the cleavage site, however, we have observed some decrease in activity when a stem-loop structure was present within 2 nt of the cleavage site. The HDV self-processing construct (Fig 25) was designed to generate the trans-acting hammerhead ribozyme with only two additional nucleotides at its 3' end after self-processing. The HDV sequence used here is based on the anti-genomic sequence (Perrota Been, 1992 sura) but includes the modifications of Been et al., 1992 (Biochemistry 31, 11843) in which cis-cleavage activity of the ribozyme was improved by the substitution of a shortened helix 4 for a wild-type stem-loop (Fiure To prepare DNA inserts that encode self-processing ribozyme cassettes, partially overlapping top- and bottom-strand oligonucleotides (60-90 nucleotides) were designed to include sequences for the T7 promoter, the trans-acting ribozyme, the cis-cleaving ribozyme and appropriate restriction sites for use in cloning (see Fi. 26). The singlestrand portions of annealed oligonucleotides were converted to double- 10 strands using Sequenase® Biochemicals). Insert DNA was ligated into EcoR1/Hindlll-digested pucl8 and transformed into E. colistrain using standard protocols (Maniatis et al., 1982 in Molecular Cloning Cold :Spring Harbor Press). The identity of positive clones was confirmed by sequencing small-scale plasmid preparations.

15 Larger scale preparations of plasmid DNA for use as in vitro transcription templates and in transactions were prepared using the protocol and columns from QIAGEN Inc. (Studio City, CA) except that an additional ethanol precipitation was included as the final step.

Example 22: RNA Processing in vitro 20 Transcription reactions containing linear plasmid templates were carried out essentially as described (Milligan Uhlenbeck, 1989 Supra; Chowrira Burke, 1991 Supra). In order to prepare 5' end-labeled transcripts, standard transcription reactions were carried out in the presence of 10-20 gCi [y- 3 2 p]GTP, 200 jM each NTP and 0.5 to 1 pg of linearized plasmid template. The concentration of MgCI2 was maintained at 10 mM above the total nucleotide concentration.

To compare the ability of the different ribozyme cassettes to selfprocess in vitro, each construct was transcribed and allowed to undergo self-processing under identical conditions at 370C. For these comparisons, equal amounts of linearized DNA templates bearing the various ribozyme cassettes were transcribed in the presence of [y- 3 2 P]GTP to generate end-labeled transcripts. In this manner only the full-length, unprocessed transcripts and the released trans-ribozymes are visualized by autoradiography. In all reactions, Mg 2 was included at 10 mM above the nucleotide concentration so that cleavage by all the ribozyme cassettes would be supported. Transcription templates were linearized at several positions by digestion with different restriction enzymes so that selfprocessing in the presence of increasing lengths of downstream sequence could be compared (see Fig. 26). The resulting transcripts have either non-ribozyme nucleotides at the 3' end (Hindlll-digested template), 220 nucleotides (Ndel digested templates) or 454 nucleotides of downstream sequence (Rcal digested template).

As shown in Figure 27, all four ribozyme cassettes are capable of self- 0 processing and yield RNA products of expected sizes. Two nucleotides essential for hammerhead ribozyme activity (Ruffner et al., 1990 supra) have been changed in the HH(mutant) core sequence (see Figure 23) and so this transcript is unable to undergo self-processing (Fig. 27). This is evidenced by the lack of a released 5' RNA in the HH(mutant), although the full-length RNAs are present Comparison of the amounts of released trans-ribozyme (FiE. 27) indicate that there are differences in the ability of ~these ribozymes to self-process in vitro, especially with respect to the presence of downstream sequence. For the two HP constructs, it is clear that HP(GC) is more efficient than the HP(GU) ribozyme, both in the presence and in the absence of extra downstream sequence. In addition, the activity of HP(GU) falls off more dramatically when downstream sequence is present. The stronger G:C base pair likely contributes to the HP(GC) construct's ability to fold correctly (and/or more quickly) into the productive structure, even when as much as 216 extra nucleotides are present downstream. The HH ribozyme construct is also quite efficient at self-processing, and slightly better than the HP(GU) construct even when downstream sequence is present.

Of the three ribozyme motifs, the presence of extra downstream sequence seems to most affect the efficiency of HDV. When no extra sequence is present downstream, HDV is quite efficient and self-processes to approximately the same level as the HH and HP(GC) cassettes.

However, when extra downstream sequence is present, the self-processing activity seems to decrease almost as dramatically as is seen with the (suboptimal) HP(GU) cassette.

Example 23: Kinetics of self-processing reaction Hindlll-digested template (250 ng) was used in a standard transcription reaction mixture containing: 50 mM Tris-HCI pH 8.3; 1 mM ATP, GTP and UTP; 50 U.M CTP; 40 gCi [a- 3 2 P]CTP; 12 mM MgCI2; 10 mM DTT. The transcription/self-processing reaction was initiated by the addition of T7 RNA polymerase (15 Aliquots of 5 p.1 were taken at regular time intervals and the reaction was stopped by adding an equal volume of 2x formamide loading buffer (95% formamide, 15 mM EDTA, dyes) and freezing on dry ice. The samples were resolved on a 10 polyacrylamide sequencing gel and results were quantitated by Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Ribozyme selfcleavage rates were determined from non-linear, least-squares fits g (KaleidaGraph, Synergy Software,Reeding, PA) of the data to the equation: (Fraction Uncleaved Transcript) (1-e kt 15 where t represents time and k represents the unimolecular rate constant for cleavage (Long Uhlenbeck, 1994 Proc. Natl. Acad. Sci. USA 91, 6977).

Linear templates were prepared by digesting the plasmids with Hindlll so that transcripts will contain only four to five vector-derived nucleotides at the 3' end (see Figure 23-25). By comparison of the unimolecular rate constant determined for each construct, it is clear that HH is the most efficient at self-processing (Table 44). The HH transcript self-processes 2fold faster than HDV and 3-fold faster than HP(GC) transcripts. Although the HP(GU) RNA'undergoes self-processing, it is at least 6-fold slower than the HP(GC) construct. This is consistent with previous observations that the stability of helix 2 is essential for self-processing and trans-cleavage activity of the hairpin ribozyme (Hampel et al., 1990 supra; Chowrira Burke, 1991 sura). The rate of HH self-cleavage during transcription measured here (1.2 min-1) is similar to the rate measured by Long and Uhlenbeck 1994 suora using a HH that has a different stem I and stem III.

Self-processing rates during transcription for HP and HDV have not been previously reported. However, self-processing of the HDV ribozyme-as measured here during transcription-is significantly slower than -when tested after isolation from a denaturing gel (Been et al., 1992 supra). This decrease likely reflects the difference in protocol as well as the presence of flanking sequence in the HDV construct used here.

92 Example 24: Effect of downstream sequences on trans-cleavage in vitro Transcripts containing the trans ribozyme with or without 3' flanking sequences were assayed for their ability to cleave their target in trans. To this end, transcripts from three templates were resolved on a preparative gel and bands corresponding both to processed trans-acting ribozymes from the HH transcription reaction, and to full-length HH(mutant) and AHDV transcripts were isolated. In all three transcripts the trans-acting ribozyme portion is identical-with the exception of sequences at their 3' ends. The HH trans-acting ribozyme contains only an additional UC at its 3' end, 10 while HH(mutant) and AHDV have 52 and 37 nucleotides, respectively, at their 3' ends. A 622 nucleotide, internally-labeled target RNA was incubated, under ribozyme excess conditions, along with the three ribozyme transcripts in a standard reaction buffer.

To make internally-labeled substrate RNA for trans-ribozyme 15 cleavage reactions, a 622 nt region (containing hammerhead site P) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of [a- 3 2 P]CTP (Chowrira Burke, 1991 suDra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 pl DEPC-treated water and stored at Unlabeled ribozyme (1M) and internally labeled 622 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCl2) by heating to 90 0 C for 2 min. and slow cooling to 370C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C. Aliquots of 5 gl were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager® (Molecular Dynamics, Sunnyvale,

CA).

The HH trans-acting ribozyme cleaves the target RNA approximately faster than the AHDV transcript and greater than 20-fold faster than the HH(mutant) transcript (Figure 28). The additional nucleotides at the end of HH(mutant) form 7 base-pairs with the 3' target-binding arm of the trans-acting ribozyme (Figure 23). This interaction must be disrupted (at a cost of 6 kcal/mole) to make the trans-acting ribozyme available for binding the target sequence. In contrast, the additional nucleotides at the end of AHDV were not designed to form any strong, alternative base-pairing with the trans-ribozyme. Nevertheless, the AHDV sequences are predicted to form multiple structures involving the 3' target-binding arm of the trans ribozyme that have stabilities ranging from 1-2 kcal/mole. Thus, the 10 observed reductions in activity for the AHDV and HH(mutant) constructs are consistent with the predicted folded structures, and it reinforces the view that the flanking sequences can decrease the catalytic efficiency of a ribozyme through nonproductive interactions with either the ribozyme or the substrate or both.

Example 25: RNA self-processing in vivo Since three of the constructs (HH, HDV and HP(GC)) self-process efficiently in solution, the affect of the mammalian cellular milieu on ribozyme self-processing was next explored by applicant. A transient expression system was employed to investigate ribozyme activity in vivo. A mouse cell line (OST7-1) that constitutively expresses T7 RNA polymerase in the cytoplasm was chosen for this study (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA 87, 6743). In these cells plasmids containing a ribozyme cassette downstream of the T7 promoter will be transcribed efficiently in the cytoplasm (Elroy-Stein Moss, 1990 supra).

Monolayers of a mouse L9 fibroblast cell line (OST7-1; Elroy-Stein and Moss, 1990 sura) were grown in 6-well plates with 5x10 5 cells/well.

Cells were transfected with circular plasmids (5 gg/well) using the calcium phosphate-DNA precipitation method (Maniatis et al., 1982 supra). Cells were lysed (4 hours post-transfection) by the addition of standard lysis buffer (200 pl/well) containing 4M guanadinium isothiocyanate, 25 mM sodium citrate (pH 0.5% sarkosyl (Chomczynski and Sacchi, 1987 Anal. Biochem. 162, 156), and 50 mM EDTA pH 8.0. The lysate was extracted once with water-saturated phenol followed by one extraction with chloroform:isoamyl alcohol Total cellular RNA was precipitated with an equal volume of isopropanol. The RNA pellet was resuspended in 0.2 M ammonium acetate and reprecipitated with ethanol. The pellet was then washed with 70% ethanol and resuspended in DEPC-treated water.

Purified cellular RNA (3 gg/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90°C for 2 min. in the absence of Mg 2 and then snap-cooling on ice for at least min. This protocol allows for efficient annealing of the primer to its complementary RNA sequence. The primer was extended using Superscript II reverse transcriptase (8 U/pl; BRL) in a buffer containing mM Tris-HCI pH 8.3; 10 mM DTT; 75 mM KCi; 1 mM MgCl2; 1 mM each 10 dNTP. The extension reaction was carried out at 42°C for 10 min. The reaction was terminated by adding an equal volume of 2x formamide gel loading buffer and freezing on crushed dry ice. The samples were resolved on a 10% polyacrylamide sequencing gel. The primer sequences are as follows: HH primer, 5'-CTCCAGTTTCGAGCTTT-3'; HDV primer, AAGTAGCCCAGGTCGGACC-3'; HP primer, ACCAGGTAATATACCACAAC-3'.

As shown in Fiqure 29 specific bands corresponding to full-length precursor RNA and 3' cleavage products were detected from cells transfected with the self-processing cassettes. All three constructs, in 20 addition to being transcriptionally active, appear to self-process efficiently in the cytoplasm of OST7-1 cells. In particular, the HH and HP(GC) constructs self-process to greater than 95%. The overall extent of selfprocessing in OST7-1 cells appears to be strikingly similar to the extent of self-processing in vitro (Figure 29 "In Vitro +MgCl2" vs. "Cellular").

Consistent with the in vitro self-processing results, the HP(GU) cassette self-processed to approximately 50% in OST7-1 cells. As expected, transfection with plasmids containing the HH(mutant) cassette yielded a primer-extension product corresponding to the full-length

RNA

with no detectable cleavage products (Figure The latter result strongly suggests that the primer extension band corresponding to the 3' cleavage product is not an artifact of reverse transcription.

Applicant was concerned with the possibility that RNA self-processing might occur during cell lysis, RNA isolation and /or the primer extension assay. Two precautions were taken to exclude this possibility. First, 50 mM EDTA was included in the lysis buffer. EDTA is a strong chelator of divalent metal ions such as Mg 2 and Ca 2 that are necessary for ribozyme activity. Divalent metal ions are therefore unavailable to self-processing RNAs following cell lysis. A second precaution involved using primers in the primer-extension assay that were designed to hybridize to essential regions of the processing ribozyme. Binding of these primers should prevent the 3' cis-acting ribozymes from folding into the conformation essential for catalytic activity.

Two experiments were carried out to further eliminate the possibility that self-processing is occurring either during RNA preparations or during the primer extension analysis. The first experiment involves primer extension analysis on full-length precursor RNAs that were added to nontransfected OST7-1 lysates after cell lysis. Thus, only if self-processing is occurring at some point after lysis would cleavage products be detected.

Full-length precursor RNAs were prepared by transcribing under conditions of low Mg 2 (5 mM) and high NTP concentration (total 12 mM) in an attempt to eliminate the free Mg 2 required for the self-processing reaction (Michel et al. 1992 Genes Dev. 6, 1373). The full-length precursor RNAs were gel-purified, and a known amount was added to lysates of nontransfected OST7-1 cells. RNA was purified from these lysates and incubated for 1 hr in DEPC-treated water at 370 C prior to the standard primer extension analysis (Fiure 29in vitro "-MgCI2" control). The predominant RNA detected in all cases corresponds to the primer extension product of full-length precursor RNAs. If, instead, the purified ~RNA containing the full-length precursor is incubated in 10 mM MgCI2 prior to the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (Figure 29, in vitro "+MgCl2" control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-processing, even though the RNA in question is inherently able to undergo self-cleavage.

In a second experiment to demonstrate lack of self-processing during work up, internally-labeled precursor RNAs were prepared and added to non-transfected OST7-1 lysates as in the previous control. The internallylabeled precursor RNAs were carried through the RNA purification and primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-processing. By this analysis, the vast majority of the added full-length RNA remained intact during the entire process of RNA isolation and primer extension.

These two control experiments validate the protocols used and support applicant's conclusion that the self-processing reactions catalyzed by HH, HDV and HP(GC) cassettes are occurring in the cytoplasm of OST7-1 cells.

Sequences in figures 23 through 25 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be "readily generated using techniques generally known in the art.

10 In addition, those in the art will recognize that Applicant provides guidance through the above examples as to how to best design vectors of this invention so that secondary structure of the mRNA allows efficient cleavage by releasing ribozymes. Thus, the specific constructs are not limiting in this invention. Such constructs can be readily tested as 15 described above for such secondary structure, either by computer folding algorithms or empirically. Such constructs will then allow at least completion of release of ribozymes, which can be readily determined as described above or by methods known in the art. That is, any such secondary structure in the RNA does not reduce release of the ribozymes by more than IV. Ribozymes Expressed by RNA Polymerase

III

Applicant has determined that the level of production of a foreign RNA, using a RNA'polymerase III (pol III) based system, can be significantly enhanced by ensuring that the RNA is produced with the 5' terminus and a 3' region of the RNA molecule base-paired together to form a stable intramolecular stem structure. This stem structure is formed by hydrogen bond interactions (either Watson-Crick or non-Watson-Crick) between nucleotides in the 3' region (at least 8 bases) and complementary nucleotides in the 5' terminus of the same RNA molecule.

Although the example provided below involves a type 2 pol III gene unit, a number of other pol III promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell 29, 5S RNA (Nielsen et al., 1993, Nucleic Acids Res. 21, 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 Cell 22, 405-413), U6 snRNA (Gupta and Reddy, 1990 Nucleic Acids Res. 19, 2073-2075), vault RNA (Kickoefer et al., 1993 J.

Biol. Chem. 268, 7868-7873), telomerase RNA (Romero and Blackburn, 1991 Cell 67, 343-353), and others.

The construct described in this invention is able to accumulate RNA to a significantly higher level than other constructs, even those in which and 3' ends are involved in hairpin loops. Using such a construct the level of expression of a foreign RNA can be increased to between 20,000 and 50,000 copies per cell. This makes such constructs, and the vectors 0encoding such constructs, excellent for use in decoy, therapeutic editing and antisense protocols as well as for ribozyme formation. In addition, the .molecules can be used as agonist or antagonist RNAs (affinity RNAs).

Generally, applicant believes that the intramolecular base-paired interaction between the 5' terminus and the 3' region of the RNA should be S" in a double-stranded structure in order to achieve enhanced RNA 15 accumulation.

*o° Thus, in one preferred embodiment the invention features a pol III promoter system a type 2 system) used to synthesize a chimeric RNA molecule which includes tRNA sequences and a desired RNA a tRNA-based molecule).

The following exemplifies this invention with a type 2 pol III promoter and a tRNA gene. Specifically to illustrate the broad invention, the RNA molecule in the following example has an A box and a B box of the type 2 *pol III promoter system and has a 5' terminus or region able to base-pair with at least 8 bases of a complementary 3' end or region of the same RNA molecule. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using other pol III promoter systems and techniques generally known in the art.

By "terminus" is meant the terminal bases of an RNA molecule, ending in a 3' hydroxyl or 5' phosphate or 5' cap moiety. By "region" is meant a stretch of bases 5' or 3' from the terminus that are involved in base-paired interactions. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from Vf- -o, 98 the other end of the molecule provides a useful molecule able to be expressed at high levels.

By region" is meant a stretch of bases 3' from the terminus that are involved in intramolecular bas-paired interaction with complementary nucleotides in the 5' terminus of the same molecule. The 3' region can be designed to include the 3' terminus. The 3' region therefore is 0 nucleotides from the 3' terminus. For example, in the S35 construct described in the present invention (Fig. 40O the 3' region is one nucleotide from the 3' terminus. In another example, the 3' region is 43 nt from 3' fw o 10 terminus. These examples are not meant to be limiting. Those in the art OooqD 2 will recognize that other embodiments can be readily generated using O.o. techniques generally known in the art. Generally, it is preferred to have the 3' region within 100 bases of the 3' terminus.

By "tRNA molecule" is meant a type 2 pol III driven RNA molecule that 15 is generally derived from any recognized tRNA gene. Those in the art will eoee recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol III transcription.

By "chimeric tRNA molecule" is meant a RNA molecule that includes a pol III promoter (type 2) region. A chimeric tRNA molecule, for example, might contain an intramolecular base-paired structure between the 3' region and complementary 5' terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol III promoter boxes. Thus, such a foreign RNA may be provided at the 3' end of the B box, or may be provided in between the A and the B box, with the B box moved to an appropriate location either within the foreign RNA or another location such that it is effective to provide pol I1 transcription. In one example, the RNA molecule may include a hammerhead ribozyme with the B box of a type 2 pol III promoter provided in stem II of the ribozyme. In a second example, the B box may be provided in stem IV region of a hairpin ribozyme. A specific example of such RNA molecules is provided below. Those in the art will 9-c 99 recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist

RNA.

By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev.

Biochem. 60, 631-652). By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J.American. Med. Assoc. 260, 3030fe 3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target *5 RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the see. enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990 Cell 63, 601-608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

By "therapeutic editing RNA" is meant an antisense RNA that can bind to its cellular target (RNA or DNA) and mediate the modification of a specific base.

By "agonist RNA" is meant an RNA molecule that can bind to protein receptors with high affinity and cause the stimulation of specific cellular pathways.

By "antagonist RNA" is meant an RNA molecule that can bind to cellular proteins and prevent it from performing its normal biological function (for example, see Tsai et al., 1992 Proc. Natl. Acad. Sci. USA 89, 8864-8868).

In other aspects, the invention includes vectors encoding RNA molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this

RNA.

Thus, the invention features a transcribed non-naturally occuring RNA 10 molecule which includes a desired therapeutic RNA portion and an intramolecular stem formed by base-pairing interactions between a 3' region and complementary nucleotides at the 5' terminus in the RNA. The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs.

15 In preferred embodiments, the 5' terminus of the chimeric tRNA includes a portion of the precursor molecule of the primary tRNA molecule, of which 2. 8 nucleotides are involved in base-pairing interaction with the 3' region; the chimeric tRNA contains A and B boxes; natural sequences 3' of the B box are deleted, which prevents endogenous RNA processing; the desired RNA molecule is at the 3' end of the B box; the desired RNA Smolecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA; the molecule has an intramolecular stem resulting from a base-paired interaction between the terminus of the RNA and a complementary 3' region within the same RNA, and includes at least 8 bases; and the 5' terminus is able to base pair with at least 15 bases of the 3' region.

In most preferred embodiments, the molecule is transcribed by a RNA polymerase III based promoter system, a type 2 pol III promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases; DNA vector encoding the RNA molecule of claim 51.

In other related aspects, the invention features an RNA or DNA vector encoding the above RNA molecule, with the portions of the vector encoding the RNA functioning as a RNA pol III promoter; or a cell containing the vector or a method to provide a desired RNA molecule in a cell, by introducing the molecule into a cell with an RNA molecule as described above. The cells can be derived from animals, plants or human beings.

In order for RNA-based gene therapy approaches to be effective, sufficient amounts of the therapeutic RNA must accumulate in the appropriate intracellular compartment of the treated cells. Accumulation is 10 a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerase II (pol II) and RNA polymerase III (pol III) based expression systems have been used to produce therapeutic RNAs in cells (Sarver Rossi, 1993 AIDS Res. Human Retroviruses 9, 483-487; Yu et al., 1993 P.N.A.S.(USA) 90, 6340- 15 6344). However, pol III based expression cassettes are theoretically more attractive for use in expressing antiviral RNAs for the following reasons.

Pol II produces messenger RNAs located exclusively in the cytoplasm, oo .whereas pol III produces functional RNAs found in both the nucleus and the cytoplasm. Pol II promoters tend to be more tissue restricted, whereas pol III genes encode tRNAs and other functional RNAs necessary for basic "housekeeping" functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types. Finally, pol III transcripts from a given gene accumulate to much greater levels in cells relative to pol II genes.

Intracellular accumulation of therapeutic RNAs is also dependent on the method of gene transfer used. For example, the retroviral vectors presently used to accomplish stable gene transfer, integrate randomly into the genome of target cells. This random integration leads to varied expression of the transferred gene in individual cells comprising the bulk treated cell population. Therefore, for maximum effectiveness, the transferred gene must have the capacity to express therapeutic amounts of the antiviral RNA in the entire treated cell population, regardless of the integration site.

Pol III System The following is just one non-limiting example of the invention. A pol III based genetic element derived from a human tRNAimet gene and termed A3-5 (F;i Adeniyi-Jones et al., 1984 supra), has been adapted to express antiviral RNAs (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512- 6523). This element was inserted into the DC retroviral vector (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523) to accomplish stable gene transfer, and used to express antisense RNAs against moloney murine leukemia virus and anti-HIV decoy RNAs (Sullenger et al., 1990 Mol. Cell.

10 Biol. 10, 6512-6523; Sullenger et al., 1990 Cell 63, 601-608; Sullenger et al., 1991 J. Virol. 65, 6811-6816; Lee et al., 1992 The New Biologist 4, 66- 74). Clonal lines are expanded from individual cells present in the bulk population, and therefore express similar amounts of the therapeutic RNA in all cells. Development of a vector system that generates therapeutic levels of therapeutic RNA in all treated cells would represent a significant advancement in RNA based gene therapy modalities.

Applicant examined hammerhead (HHI) ribozyme (RNA with enzymatic activity) expression in human T cell lines using the A3-5 vector system (These constructs are termed "A3-5/HHI"; Fig. 34). On average, ribozymes were found to accumulate to less than 100 copies per cell in the bulk T cell populations. In an attempt to improve expression levels of the chimera, the applicant made a series of modified A3-5 gene units containing enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (Fig. 34). One of these modified gene units, termed gave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original A3-5/HHI vector system.

Ribozyme accumulation in individual clonal lines from the pooled T cell populations ranged from 10 to greater than 100 fold more than those achieved with the original A3-5/HHI version of this vector.

The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and antagonist RNAs. Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral V 103 vectors, including but not limited to, other stable gene transfer systems such as adeno-associated virus (AAV; Carter, 1992 Curr. Opin. Genet. Dev.

3, 74), as well as transient vector systems such as plasmid delivery and adenoviral vectors (Berkner, 1988 BioTechniques 6, 616-629).

As described below, the S35 vector encodes a truncated version of a tRNA wherein the 3' region of the RNA is base-paired to complementary nucleotides at the 5' terminus, which includes the 5' precursor portion that is normally processed off during tRNA maturation. Without being bound by any theory, Applicant believes this feature is important in the level of j 10 expression observed. Thus, those in the art can now design equivalent .0 RNA molecules with such high expression levels. Below are provided examples of the methodology by which such vectors and tRNA molecules can be made.

Vectors The use of a truncated human tRNAimet gene, termed A3-5 (Fi. 33; Adeniyi-Jones et al., 1984 supra), to drive expression of antisense RNAs, t and subsequently decoy RNAs (Sullenger et al., 1990 supra) has recently been reported. Because tRNA genes utilize internal pol Ill promoters, the antisense and decoy RNA sequences were expressed as chimeras containing tRNAi m et sequences. The truncated tRNA genes were placed into the U3 region of the 3' moloney murine leukemia virus vector LTR (Sullenger et al., 1990 supra).

Base-Paired Structures Since the A3-5 vector combination has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cloned ribozyme-encoding sequences (termed as "A3-5/HHI") into this vector to explore its utility for expressing therapeutic ribozymes. However, low ribozyme accumulation in human T cell lines stably transduced with this vector was observed To try and improve accumulation of the ribozyme, applicant incorporated various RNA structural elements (Fig. 34) into one of the ribozyme chimeras Two strategies were used to try and protect the termini of the chimeric transcripts from exonucleolytic degredation. One strategy involved the incorporation of stem-loop structures into the termini of the transcript. Two such constructs were cloned, S3 which contains a stem-loop structure at the 3' end, and S5 which contains stem-loop structures at both ends of the transcript (Figure 34). The second strategy involved modification of the 3' terminal sequences such that the 5' terminus and the 3' end sequences can form a stable base-paired stem. Two such constructs were made: in which the 3' end was altered to hybridize to the 5' leader and acceptor stem of the tRNAimet domain, and S35Plus which was identical to S35 but included more extensive structure formation within the non-ribozyme portion of the A3-5 chimeras (Fiure 34). These stem-loop structures are 10 also intended to sequester non-ribozyme sequences in structures that will prevent them from interfering with the catalytic activity of the ribozyme.

These constructs were cloned, producer cell lines were generated, and o stably-transduced human MT2 (Harada et al., 1985 supra) and CEM (Nara Fischinger, 1988 supra) cell lines were established (Curr. Protocols Mol.

Biol. 1992, ed. Ausubel et al., Wiley Sons, NY). The RNA sequences and structure of S35 and S35 Plus are provided in Figures 40-47.

Referring to Figure 48, there is provided a general structure for a chimeric RNA molecule of this invention. Each N independently represents none or a number of bases which may or may not be base paired. The A and B boxes are optional and can be any known A or B box, or a consensus sequence as exemplified in the figure. The desired nucleic acid to be expressed can be any location in the molecule, but preferably is on those places shown adjacent to or between the A and B boxes (designated by arrows). Figure 49 shows one example of such a structure in which a desired RNA is provided 3' of the intramolecular stem. A specific example of such a construct is provided in Figures 50 and 51.

Example 26: Cloning of A3-5-Ribozyme Chimera Oligonucleotides encoding the S35 insert that overlap by at least nucleotides were designed GATCCACTCTGCTGTTCTGTTTTTGA 3' and 5' CGCGTCAAAAACAGAACAGCAGAGTG The oligonucleotides gM each) were denatured by boiling for 5 min in a buffer containing mM Tris.HCI, pH8.0. The oligonucleotides were allowed to anneal by snap cooling on ice for 10-15 min.

The annealed oligonucleotide mixture was converted into a doublestranded molecule using Sequenase® enzyme (US Biochemicals) in a Li,' 105 buffer containing 40 mM Tris.HCI, pH7.5, 20 mM MgCI 2 50 mM NaCI, mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 370C for 30 min. The reaction was stopped by heating to 70°C for 15 min.

The double stranded DNA was digested with appropriate restriction endonucleases (BamHI and Mlul) to generate ends that were suitable for .cloning into the A3-5 vector.

The double-stranded insert DNA was ligated to the A3-5 vector DNA by incubating at room temperature (about 20°C) for 60 min in a buffer 10 containing 66 mM Tris.HCI, pH 7.6, 6.6 mM MgCl2, 10 mM DTT, 0.066 .M ATP and 0.1U/al T4 DNA Ligase (US Biochemicals).

Competent E. coli bacterial strain was transformed with the recombinant vector DNA by mixing the cells and DNA on ice for 60 min.

The mixture was heat-shocked by heating to 37°C for 1 min. The reaction mixture was diluted with LB media and the cells were allowed to recover for min at 370C. The cells were plated on LB agar plates and incubated at 370C for 18 h.

Plasmid DNA was isolated from an overnight culture of recombinant clones using standard protocols (Ausubel et al., Curr. Protocols Mol.

Biology 1990, Wiley Sons, NY).

The identity of the clones were determined by sequencing the plasmid DNA using the Sequenase® DNA sequencing kit (US Biochemicals).

The resulting recombinant A3-5 vector contains the S35 sequence.

The HHI encoding DNA was cloned into this A3-5-S35 containing vector using Sacll and BamHI restriction sites.

Example 27: Northern analysis RNA from the transduced MT2 cells were extracted and the presence of A3-5/ribozyme chimeric transcripts were assayed by Northern analysis (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY).

Northern analysis of RNA extracted from MT2 transductants showed that chimeras of appropriate sizes were expressed (Fiq. 3536).

In addition, these results demonstrated the relative differences in accumulation among the different constructs (Figure 35.36). The pattern of S' I 106 expression seen from the A3-5/HHI ribozyme chimera was similar to 12 other ribozymes cloned into the A3-5 vector (not shown). In MT-2 cell line, ribozyme chimeras accumulated, on average, to less than 100 copies per cell.

Addition of a stem-loop onto the 3' end of A3-5/HHI did not lead to increased A3-5 levels (S3 in Fig. 35,36). The S5 construct containing both 5' and 3' stem-loop structures also did not lead to increased ribozyme levels (Fig. 35.36).

Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original A3-5/HHI vector transcripts (Fiq. This may be due to increased stability of the S35 transcript.

Example 28: Cleavage activity To assay whether ribozymes transcribed in the transduced cells o contained cleavage activity, total RNA extracted from the transduced MT2 T cells were incubated with a labeled substrate containing the HHI cleavage site (Figure 37). Ribozyme activity in all but the S35 constructs, was too low to detect. However, ribozyme activity was detectable in transduced T cell RNA. Comparison of the activity observed in the transduced MT2 RNA with that seen with MT2 RNA in which varying amounts of in vitro transcribed S5 ribozyme chimeras, indicated that between 1-3 nM of S35 ribozyme was present in S35-transduced MT2 RNA. This level of activity corresponds to an intracellular concentration of 5,000-15,000 ribozyme molecules per cell.

Example 29: Clonal variation Variation in the ribozyme expression levels among cells making up the bulk population was determined by generating several clonal cell lines from the bulk S35 transduced CEM line (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (igure 38 and 39).

All the individual clones were found to express active ribozyme. The ribozyme activity detected from each clone correlated well with the relative amounts of ribozyme observed by Northern analysis. Steady state ribozyme levels among the clones ranged from approximately 1,000 molecules per cell in clone G to 11,000 molecules per cell in clone H (Fig.

38). The mean accumulation among the clones, calculated by averaging the ribozyme levels of the clones, exactly equaled the level measured in the parent bulk population. This suggests that the individual clones are representative of the variation present in the bulk population.

The fact that all 14 clones were found to express ribozyme indicate that the percentage of cells in the bulk population expressing ribozyme is also very high. In addition, the lowest level of expression in the clones was still more than 10-fold that seen in bulk cells transduced with the original A3-5 vector. Therefore, the S35 gene unit should be much more effective 10 in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.

Example 30: Stability Finally, the bulk S35-transduced line, resistant to G418, was propogated for a period of 3 months (in the absence of G418) to determine if ribozyme expression was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme expression after 3 months. This difference probably arose from cells with varying amount of ribozyme expression and exhibiting different growth rates in the culture becoming slightly more prevalent in the culture. However, ribozyme expression is apparently stable for at least this period of time.

Example 31: Design and construction of TRZ-tRNA Chimera A transcription unit, termed TRZ, is designed that contains the motif (Figure 52). A desired RNA ribozyme) can be inserted into the indicated region of TRZ tRNA chimera. This construct might provide additional stability to the desired RNA. TRZ-A and TRZ-B are non-limiting examples of the TRZ-tRNA chimera.

Referring to Fig. 53-54, a hammerhead ribozyme targeted to site I (HHITRZ-A; Fig. 53) and a hairpin ribozyme (HPITRZ-A; Fig. 54), also targeted to site I, is cloned individually into the indicated region of TRZ tRNA chimera. The resulting ribozyme trancripts retain full RNA cleavage activity (see for example Fig. 55). Applicant has shown that efficient expression of these TRZ tRNA chimera can be achieved in mammalian cells.

Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated region of TRZtRNA chimera to achieve therapeutic levels of RNA expression in mammalian cells.

Sequences listed in Figures 40-47 and 50 54 are meant to be nonlimiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily S" 10 generated using techniques known in the art, are within the scope of the present invention.

Example 32: Ribozyme expression in T cell lines Ribozyme expression in T cell lines stably-transduced with either a retroviral-based or an Adeno-associated virus (AAV)-based ribozyme expression vector (Figure 56). The human T cell lines MT2 and CEM were transduced with either retroviral or AAV vectors encoding a neomycin slelctable marker and a ribozyme (S35/HHI) expressed from pol III met i tRNA-driven promoter. Cells stably-transduced with the vectors were selectivelyt expanded medium containing the neomycin antibiotic derivative, G418 (0.7 mg/ml). Ribozyme expression in the stable cell lines Swas then alalyzed by Northern analysis. The probe used to detect ribozyme transcripts also cross-hybridized with human met i tRNA sequences. Refering to Figure 56, S35/HHI RNA accumulates to significant levels in MT2 and CEM cells when transduced with either the retrovirus or the AAV vector.

These are meant to be non-limiting examples, those skilled in the art will recognize that other vectors such as adenovirus vector (Figure 57), plasmid DNA vector, alpha virus vectors and the other derivatives there of, can be readily generated to deliver the desired RNA, using techniques known in the art and are within the scope of this invention. Additionally, the transcription units can be expressed individually or in multiples using pol II and/or pol III promoters.

References cited herein, as well as Draper WO 93/23569, 94/02495, 94/06331, Sullenger WO 93/12657, Thompson WO 93/04573, and Sullivan WO 94/04609, and 93/11253 describe methods for use of vectors decribed herein, and are incorporated by reference herein. In particular these vectors are useful for administration of antisense and decoy RNA molecules.

Example 33: Ligated Ribozymes are catalytically active The ability of ribozymes generated by ligation methods, described in Draper et al., PCT WO 93/23569, to cleave target RNA was tested on either matched substrate RNA (Eig.) or long (622 nt) RNA (Fi. 59. 60 and 61).

Matched substrate RNAs were chemically synthesized using solidphase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res.

18, 5433-5441). Substrate RNA was 5' end-labeled using [y- 3 2 p] ATP and polynucleotide kinase (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY). Ribozyme reactions were carried out under ribozyme excess conditions (kcat/KM; Herschlag and Cech, 1990 Biochemistry 29, 10159-10171). Briefly, ribozyme and substrate RNA were denatured and renatured separately by heating to 90°C and snap cooling on ice for 10 min in a buffer containing 50 mM Tris. HCI pH 7.5 and 10 mM MgCI2.

S" Cleavage reaction was initiated by mixing the ribozyme with the substrate at 370C. Aliquots of 5 gI were taken at regular intervals of time and the reaction was stopped by mixing with equal volume of formamide gel loading buffer (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY). The samples were resolved on 20 polyacrylamide-urea gel.

Refering to Fig. 58, -AG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Supra). RPI A is a HH ribozyme with 6/6 binding arms. This ribozyme was synthesized chemically either as a one piece ribozyme or was synthesized in two fragments followed by ligation to generate a one piece ribozyme. The kcat/KM values for the two ribozymes were comparable.

A template containing T7 RNA polymerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone. The target RNA (containing HH ribozyme cleavage sites B, C and D) was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [a- 3 2 p] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1, following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. RNA was precipitated with Isopropanol and the pellet was washed two times with 70% ethanol to get rid of salt and nucleotides used in the transcription reaction. RNA is resuspended in DEPC-treated water and stored at 4°C. Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/KM) conditions [Herschlag and Cech 1990 supral. Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris.HCI, pH 7.5 and 10 mM MgCI2. The RNAs were renatured by cooling to 37 0 C for 10-20 min.

Cleavage reaction was initiated by mixing the ribozyme and target RNA at 370C. Aliquots of 5 pl were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on a sequencing gel.

Example 34: Hammerhead ribozymes with 2 base-paired stem II are catalytically active To decrease the cost of chemical synthesis of RNA, applicant was -interested in determining whether the length of stem II region of a typical hammerhead ribozyme (2 4 bp stem II) can be shortened without decreasing the catalytic efficiency of the HH ribozyme. The length of stem II was systematically shortened by one base-pair at a time. HH ribozymes with three and two base-paired stem II were chemically synthesized using solid-phase RNA phosphoramidite chemistry (Scaringe et al., 1990 suDra.

Matched and- long substrate RNAs were synthesized and ribozyme assays were carried out as described in example 33. Referring to figures 62. 63 and 64, data shows that shortening stem II of a hammerhead ribozyme does not significantly alter the catalytic efficiency. It is applicant's opinion that hammerhead ribozymes with 2 2 base-paired stem II region are catalytically active.

Example 35: Synthesis of catalytically active hairpin ribozymes RNA molecules were chemically synthesized having the nucleotide base sequence shown in Fig. 65 for both the 5' and 3' fragments. The 3' fragments are phosphorylated and ligated to the 5' fragment essentially as described in example 37. As is evident from the Figure 65, the 3' and fragments can hybridize together at helix 4 and are covalently linked via GAAA sequence. When this structure hybridizes to a substrate, a ribozymeosubstrate complex structure is formed. While helix 4 is shown as 3 base pairs it may be formed with only 1 or 2 base pairs.

nM mixtures of ligated ribozymes were incubated with 1-5 nM end-labeled matched substrates (chemically synthesized by solid-phase synthesis using RNA phosphoramidite chemistry) for different times in mM Tris/HCI pH 7.5, 10 mM MgCl2 and shown to cleave the substrate efficiently (EigU. The target and the ribozyme sequences shown in Fi.62 and 65 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using other sequences and techniques generally known in the art.

V. Constructs of Hairpin Ribozymes There follows an improved trans-cleaving hairpin ribozyme in which a new helix a sequence able to form a double-stranded region with another single-stranded nucleic acid) is provided in the ribozyme to basepair with a 5' region of a separate substrate nucleic acid. This helix is provided at the 3' end of the ribozyme after helix 3 as shown in Figure 3. In addition, at least two extra bases may be provided in helix 2 and a portion of the substrate corresponding to helix 2 may be either directly linked to the portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atleast one base. By trans-cleaving is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cleavage reaction.

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

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) has several advantages. These include improved stability of the ribozyme-target complex in vivo In addition, an increase in the recognition sequence of the hairpin ribozyme improves the specificity of the ribozyme. This also makes possible the targeting of potential hairpin 112 ribozyme sites that would otherwise be inaccessible due to neighboring secondary structure.

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) enhances trans-ligation reaction catalyzed by the ribozyme. Transligation reactions catalyzed by the regular hairpin ribozyme (4 bp helix 2) is very inefficient (Komatsu et al., 1993 Nucleic Acids Res. 21, 185). This is attributed to weak base-pairing interactions between substrate RNAs and the ribozyme. By increasing the length of helix 2 (with or without helix the rate of ligation (in vitro and in vivo) can be enhanced several fold.

10 Results of experiments suggest that the length of H2 can be 6 bp without significantly reducing the activity of the hairpin ribozyme. The H2 arm length variation does not appear to be sequence dependent. HP ribozymes with 6 bp H2 have been designed against five different target RNAs and all five ribozymes efficiently cleaved their cognate target RNA.

15 Additionally, two of these ribozymes were able to successfully inhibit gene expression TNF-a() in mammalian cells. Results of these experiments are shown below.

HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNA in a sequence-specific manner, however, the rate of the cleavage reaction is lower than those catalyzed by HP ribozymes with 6 bp H2.

Example 36: 4 and 6 base pair H2 Referring to Figures 67-72, HP ribozymes were synthesized as described above and tested for activity. Surprisingly, those with 6 base pairs in H2 were still as active as those with 4 base pairs.

VI. Chemical Modification Oligonucleotides with 5'-C-alkyl Group The introduction of an alkyl group at the 5'-position of a nucleoside or nucleotide sugar introduces an additional center of chirality into the sugar moiety. Referring to Ei 75, the general structures of belonging to the D-allose, 2, and L-talose, 3, sugar families are shown.

The family names are derived from the known sugars D-allose and L-talose

(R

1

CH

3 in 2 and 3 in Figure 75). Useful specific D-allose and L-talose 113 nucleotide derivatives are shown in Figure 76. 29-32 and Figure 77, 58- 61 respectively.

This invention relates to the use of 5'-C-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As :the term is used in this application, enzymatic nucleic acids are catalytic nucleic molecules that contain alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids S. can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

Also within the invention are 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides.

Such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 5'-C-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 5'-C-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.

Thus, in one aspect, the invention features 5'-C-alkylnucleosides, that is a nucleotide base having at the 5'-position on the sugar molecule an alkyl moiety. In a related aspect, the invention also features alkylnucleotides, and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably 114 includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above. In preferred embodiments, the sugar of the nucleoside or nucleotide is in an optically pure form, as the talose or allose sugar.

Examples of various alkyl groups useful in this invention are shown in Eiguri.S, where each R 1 group is any alkyl. These examples are not limiting in the invention. Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More 10 preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 S" carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or N(CH 3 2 amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 .carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, S. 20

NO

2 halogen,

N(CH

3 2 amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or N(CH 3 2 amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated 7 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring "i 115 atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

10 In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 5'-C-alkylnucleotides; e.g.

enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, the invention features S'-C-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 5'-C-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.

In another aspect, the invention features a method for conversion of a protected allo sugar to a protected talo sugar. In the method, the protected allo sugar is contacted with triphenyl phosphine, diethylazodicarboxylate and p-nitrobenzoic acid under inversion causing conditions to provide the protected talo sugar. While one example of such conditions is provided below, those in the art will recognize other such conditions. Applicant has found that such conversion allows for ready synthesis of all types of nucleotide bases as exemplified in the figures.

While this invention is applicable to all oligonucleotides, applicant has found that the modified molecules of this invention are particulary useful for enzymatic RNA molecules. Thus, below is provided examples of such 116 molecules. Those in the art will recognize that equivalent procedures can be used to make other molecules without such enzymatic activity.

Specifically, Figure 1 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided. This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims.

Referring to Fiure 1, the preferred sequence of a hammerhead ribozyme in a to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 5'-C-alkyl substituted nucleotides 10 that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Substitutions of any nucleotide with any of the modified nucleotides shown in Figure 75 are possible.

The following are non-limiting examples showing the synthesis of nucleic acids using 5'-C-alkyl-substituted phosphoramidites and the syntheses of the amidites.

Example 37: Synthesis of Hammerhead Ribozvmes Containing nucleotides Other Modified Nucleotides The method of synthesis would follow the procedure for normal RNA synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid. protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 26-29 and 56-59). These 5'-C-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 38: Methvl-2.3- Qsooroplidine-6-Deo -D-allranosid (4) A suspension of L-rhamnose (100 g, 0.55 mol), CuS04 (120 g) and conc. H 2

SO

4 (4.0 mL) in 1.0 L of dry acetone was mixed for 24 h at RT, then filtered. Conc. NH 4 OH (5 mL) was added to the filtrate and the newly formed precipitate was filtered. The residue was concentrated in vacuo, coevaporated with pyridine (2 x 300 mL), dissolved in pyridine (500 mL) and cooled to 0 A solution of p-toluenesufonylchloride (107 g 0.56 mmol) in dry DCE (500 mL) was added dropwise over 0.5 h. The reaction mixture was left for 16 h at RT. The reaction was quenched by adding icewater (0.5 L) and, after mixing for 0.5 h, was extracted with chloroform (0.75 The organic layer was washed with H 2 0 (2 x 500 mL), 10% H 2

SO

4 (2 x 300 mL), water (2 x 300 mL), sat. NaHCO 3 (2 x 300 mL), brine (2 x 300 mL), dried over MgSO 4 and evaporated to dryness. The residue (115 g) *was dissolved in dry MeOH (1 L) and treated with NaOMe (23.2 g, 0.42 mmol) in MeOH. The reaction mixture was left for 16 h at 20 oC, neutralized with dry CO 2 and evaporated to dryness. The residue was suspended in 10 chloroform (750 mL), filtered concentrated to 100 mL and purified by flash chromatography in CHCI 3 to yield 45 g of compound 4.

Example 39: Methyl-2.3-O-lsopropylidine-5-O-t-Butvldiphenylsilyl-6- Deoxy-B-D-Allofuranoside To solution of methylfuranoside 4 (12.5 g 62.2 mmol) and AgNO 3 15 (21.25 g, 125.0 mmol) in dry DMF (300 mL) t-butyldiphenylsilyl chloride (22.2 g 81 mmol) was added dropwise under Ar over 0.5 h. The reaction mixture was stirred for 4 h at RT, diluted with CHCI 3 (200 mL), filtered and ;evaporated to dryness (below 40 °C using a high vacuum oil pump). The residue was dissolved in CH 2 C1 2 (300 mL) washed with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL), dried over MgSO 4 and evaporated to dryness.

The residue was purified by flash chromatography in CH 2

CI

2 to yield 20.0 g of compound Example 40: Methyl-5-O-t-ButvldiDhenvlsilvl-6-Deoxv--D-Allofuranoside L6} Methylfuranoside 5 (13.5 g, 30.6 mmol) was dissolved in CF3COOH:dioxane:H 2 0 2:1:1 200 mL) and stirred at 24 OC for m. The reaction mixture was cooled to -10 oC, neutralized with conc.

NH

4 0H (140 mL) and extracted with CH 2

CI

2 (500 mL). The organic layer was separated, washed with sat. NaHCO 3 (2 x 75 mL), brine (2 x 75 mL), dried over MgSO 4 and evaporated to dryness. The product 6 was purified by flash chromatography using a 0-10% MeOH gradient in CH 2

CI

2 Yield g Example 41: Methyl-2.3-di-O-Benzovl-5-O-t-Butvydiphenvlsilyl.6-Deoxy-a D-Allofuranoside Methylfuranoside 6 (7.0 g, 17.5 mmol) was coevaporated with pyridine (2 x 100 mL) and dissolved in pyridine (100 mL). Benzoyl chloride (5.4 g, 38.5 mmol) was added and the reaction mixture was left at RT for 16 Sh. Dry EtOH (50 mL) was added and the reaction mixture was evaporated to dryness after 0.5 h. The residue was dissolved in CH 2

CI

2 (300 mL), washed with sat. NaHCO 3 (2 x 75 mL), brine (2 x 75 mL) dried over MgSO 4 and evaporated to dryness. The product was purified by flash 10 chromatography in CH 2 Cl 2 to yield 9.5 g of compound 7.

Example 42: 1-O-Acetyl-2.3-di-O-benzoyl-5-O.t-Butyldiphenylsilyl-.

Deoxy-B-D-Allofuranose Dibenzoate 7 (4.7 g, 7.7 mmol) was dissolved in a mixture of AcOH (10.0 mL), Ac20 (20.0 mL) and EtOAc (30 mL) and the reaction mixture was 15 cooled 0 o C. 98% H 2 S0 4 (0.15 mL) was then added. The reaction mixture was kept at 0 °C for 16 h, and then poured into a cold 1:1 mixture of sat.

SNaHCO 3 and EtOAc (150 mL). After 0.5 h of vigorous stirring the organic phase was separated, washed with brine (2 x 75 mL), dried over MgSO 4 evaporated to dryness and coevaporated with toluene (2 x 50 mL). The product was purified by flash chromatography using a gradient of MeOH in CH 2

CI

2 Yield: 4.0 g (82% as a mixture of a and P isomers).

Example 43: 1-(2'.3'-di-O-Benzoyl-5'-O-t-Butyldiphenvlsilyl.6'-Deoxy-.D- Allofuranosyl)uracil Uracil (1.44 g, 11.5 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT, evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of acetates 8 (6.36 g, 10.0 mmol) in dry CH 3 CN (100 mL), followed by CF 3

SO

3 SiMe 3 (2.8 g, 12.6 mmol). The reaction mixture was kept at 24 °C for 16 h, concentrated to 1/3 of its original volume, diluted with 100 mL of CH 2

CI

2 and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness. The product 9 was purified by flash chromatography using a gradient of 0-5% MeOH in CH 2

CI

2 Yield: 5.7 g 119 Example 44: NA-Benzovl- -(2'.3'-Di-O-Benzovl-5'-O-t-Butvldiphenylsilyl-6' Deoxy-B-D-Allofuranosyl)Cytosine

N

4 -benzoylcytosine (1.84 g, 8.56 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH 3 CN (100 mL), followed ."ee by CF3SO 3 SiMe 3 (4.76 g, 21.4 mmol). The reaction mixture was boiled 10 under reflux for 5 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH 2

CI

2 (100 mL) and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness.

Purification by flash chromatography using a gradient of 0-5% MeOH in

CH

2

CI

2 yielded 1.8 g of compound 15 Example 45: N6-Benzovl-9-(2'.3'-di-O-Benzovl-5'-O-t-Butvldiphenylsilyl-'- S Deoxy-3-D-Allofuranosyl)adenine (11).

I**fo N-benzoyladenine (2.86 g, 11.86 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (7 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH 3 CN (100 mL) followed by CF 3

SO

3 SiMe 3 (6.59 g, 29.7 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH 2

CI

2 (100 mL) and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness. The product 11 was purified by flash chromatography using a gradient of MeOH in CH 2

CI

2 Yield: 2.7 g Example 46: N2-lsobutvrl-9-(2',3'-di-O.Benzoyl-5'-O--Butyldiphenylsilyl 6'-Deoxv-p-D-Allofuranosvl)auanine (12).

N

2 -lsobutyrylguanine (1.47 g 11.2 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (6 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a 11 120 solution of of acetates 8 (3.4 g, 5.3 mmol) in dry CH 3 CN (100 mL) followed by CF 3

SO

3 SiMe 3 (6.22 g, 28.0 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH2C1 2 (100 mL) and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness. The product 12 was purified by flash chromatography using a gradient of 0-2% MeOH in CH 2

CI

2 Yield: 2.1g Example 47: A/-Benzoyl-9-(2'.3'-di-O-benzoyl-6'-Deoxy-p-D-Allofuranosyladenine 10 Nucleoside 11 (1.65 g, 2.0 mmol) was dissolved in THF (50 mL) and a 1 M solution of TBAF in THF (4 mL) was added. The reaction mixture was kept at RT for 4 h, evaporated to dryness and the product purified by flash chromatography using a gradient of 0-5% MeOH in CH 2

CI

2 to yield 1.0 g of compound 15 Example 48: /--Benzoyl-9-(2'.3'-di-O-Benzoyl-5'-O-Dimethoxytrityl-6 Deoxv-B-D-Allofuranosyl)-adenine (19).

Nucleoside 15 (0.55 g, 0.92 mmol) was dissolved in dry CH 2

CI

2 mL). AgNO 3 (0.34 g, 2.0 mmol), dimethoxytrityl chloride (0.68 g, 2.0 mmol) and sym-collidine (0.48 g) were added under Ar. The reaction mixture was oll 20 stirred for 2h, diluted with CH 2 C1 2 (100 mL), filtered, evaporated to dryness and coevaporated with toluene (2 x 50 mL). Purification by flash chromatography using a gradient of 0-5% MeOH in CH 2

CI

2 yielded 0.8 g of compound 19.

Example 49: N--Benzoyl-9-(-5'-O-Dimethoxvtritvl-6'-Deoxy-B-D-Allofuranosyl)adenine (23).

Nucleoside 19 (1.8 g, 2 mmol) was dissolved in dioxane (50 mL), cooled to 0 °C and 2 M NaOH (50 mL) was added. The reaction mixture was kept at 0 °C for 45 m, neutralized with Dowex 50 (Pyr+ form), filtered and the resin was washed with MeOH (2 x 50 mL). The filtrate was then evaporated to dryness. Purification by flash chromatography using a gradient of 0-10% MeOH in CH 2

CI

2 yielded 1.1 g of 23.

121 Examlole 50: Nf-e-nZovi-9-(-5'-O-Dimethoxvritvi-2'-O-t-butvldimetviilvl- 6'-Deoxv--D-Allofuranosyladenine (27).

Nucleoside 23 (1.2 g, 1.8 mmol) was dissolved in dry THF (50 mL).

Pyridine (0.50 g, 8 mmol) and AgN03 (0.4 g, 2.3 mmol) were added. After the AgNO 3 dissolved (1.5 t-butyldimethylsilyl chloride (0.35 g 2.3 mmol) was added and the reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted with CH 2

CI

2 (100 mL), filtered into sat.

NaHC03 (50 mL), extracted, the organic layer washed with brine (2 x mL), dried over MgSO 4 and evaporated to dryness. The product 27 was purified by flash chromatography using a hexanes:EtOAc 7:3 gradient.

Yield: 0.7 g Examle 51: -enzvi--( 5'--Dmethoxritv-2I uidimetsil- ~li 6'-Deoxy--D-Allofuranosvl)adenine-3'-(2-Cyan ethl N.N-diisopropy phosphoramidite) Standard phosphitylation of 27 according to Scaringe,S.A.; e* Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield.

Examcle 52: Methyl-50-o-Nitrobenzoyl-23--lsogroylidine-6-doxy-B-L- Tallofuranoside Methylfuranoside 4 (3.1 g 14.2 mmol) was dissolved in dry dioxane S.(200 mL), p-nitrobenzoic acid (10.0 g, 60 mmol) and triphenylphosphine (15.74 g, 60.0 mmol) were added followed by DEAD (10.45 g, 60.0 mmol).

The reaction mixture was left at RT for 16 h, EtOH (5 mL) was added, and after 0.5 h the reaction mixture was evaporated to dryness. The residue was dissolved in CH 2

C

2 (300 mL) washed with sat. NaHC03 (2 x 75 mL), brine (2 x 75 mL) dried over MgSO 4 and evaporated to dryness.

Purification by flash chromatography using a hexanes:EtOAc 9:1 gradient yielded 4.1 g of compound 33. Subsequent debenzoylation (NaOMe/MeOH) and silylation (see preparation of 5) led to Ltalofuranoside 34 which was converted to phosphoramidites 58-61 using the same methodology as described above for the preparation of the phosphoramidites of the D-allo-isomers 29-32.

The alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage 122 or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.

Administration of such oligonucleotides is by standard procedure. See Sullivan et al., PCT WO 94/ 02595.

The ribozymes and the target RNA containing site O were synthesized, deprotected and purified as described above. RNA cleavage assay was carried our at 37°C in the presence of 10 mM MgCI 2 as described above.

Applicant has substituted 5'-C-Me-L-talo nucleotides at positions A6, 10 A9, A9 G10, C11.1 and C11.1 G10, as shown in Figure 78 (HH-O1 to HH-O 1,2,4 and 5 showed almost wild type activity (Figure 79).

However, HH-03 demonstrated low catalytic activity. Ribozymes HH-01, 2, 3, 4 and 5 are also extremely resistant to degradation by human serum nucleases.

15 Oliponucleotides with 2 '-Deoxv-2'-Alkvlnucleotide This invention uses 2'-deoxy-2'-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or singlestranded DNA, and also as antisense oligonucleotides. As the term is used 2 in this application, 2'-deoxy-2'-alkynucleotide-containing enzymatic 20 nucleic acids are catalytic nucleic molecules that contain 2'-deoxy-2'alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

Also within the invention are 2 '-deoxy-2'-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides.

Contrary to the findings of De Mesmaeker et al. applicant has found that such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 2'-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair 123 forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 2 '-deoxy-2'-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.

Thus, in one aspect, the invention features 2'-deoxy-2'alkylnucleotides, that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the 15 invention preferably includes all those nucleotides useful for makin enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.

Examples of various alkyl groups useful in this invention are shown in :E 20 i~ where each R group is any alkyl. The term "alkyl" does not include alkoxy groups which have an "-O-alkyl" group, where "alkyl" is defined as described above, where the 0 is adjacent the 2'-position of the sugar molecule.

In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2 '-position an alkyl group. In other related aspects, the invention features 2'-deoxy-2-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 2'-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall 124 activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.

In another aspect, the invention features hammerhead motifs having enzymatic activity having ribonucleotides at locations shown in Figure 80 at 6, 8, 12, and 15.1, and having substituted ribonucleotides at other positions in the core and in the substrate binding arms if desired. (The term "core" refers to positions between bases 3 and 14 in Figure 80, and the binding arms correspond to the bases from the 3'-end to base 15.1, and .i 10 from the 5'-end to base Applicant has found that use of ribonucleotides at these five locations in the core provide a molecule having sufficient enzymatic activity even when modified nucleotides are present at other sites in the motif. Other such combinations of useful ribonucleotides can be determined as described by Usman et al. supra.

Figure 80 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided.

This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims.

SReferring to Figure 80 the preferred sequence of a hammerhead ribozyme in a to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 2'-C-alkyl substituted nucleotides •that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Although substitutions of any nucleotide with any of the modified nucleotides shown in Figure 81 are possible, and were indeed synthesized, the basic structure composed of promarily 2'-O-Me nucleotides weth selected substitutions was chosen to maintain maximal catalytic activity (Yang et al. Biochemistry 1992, 31, 5005-5009 and Paolella et EMBO J. 1992, 11, 1913-1919) and ease of synthesis, but is not limiting to this invention.

Ribozymes from Figure 80 and Table 45 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17, all of the modified ribozymes retained at lease 1/10 of the wild-type catalytic activity. From Table 45, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 55, data not shown). The order of most agressive nuclease activity was fetal bovine 125 serum, human serum >human plasma human synovial fluid. As an overall measure of the effect of these 2 '-substitutions on stability and activity, a ratio B was calculated (Table 45). This B value indicated that all modified ribozymes tested had significant, >100 >1700 fold, increases in overall stability and activity. These increases in B indicate that the lifetime of these modified ribozymes in vivo are significantly increased which should lead to a more pronounced biological effect.

More general substitutions of the 2'-modified nucleotides from Figure 81 also increased the t1/2 of the resulting modified ribozymes.

10 However the catalytic activity of these ribozymes was decreased In Figure 86 compound 37 may be used as a general intermediate to prepare derivatized 2'C-alkyl phosphoramidites, where X is CH3, or an alkyl, or other group described above.

15 The following are non-limiting examples showing the synthesis of .nucleic acids using 2'-C-alkyl substituted phosphoramidites, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance.

Example 53: Synthesis of Hammerhead Ribozymes Containing 2'-Deoxy- 2'-Alkylnucleotides Other 2'-Modified Nucleotides S. 20 The method of synthesis used generally follows the procedure for normal RNA synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 10, 12, 17, 22, 31, 18, 26, 32, 36 and 38). Other 2'-modified phosphoramidites were prepared according to: 3 4, Eckstein et al. International Publication No. WO 92/07065; and 5 Kois et al.

Nucleosides Nucleotides 1993, 12, 1093-1109. The average stepwise coupling yields were The 2'-substituted phosphoramidites were incorporated into hammerhead ribozymes as shown in Figure However, these 2'-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group II intron catalytic nucleic acids, or into antisense Cr' 126 oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 54: Ribozyme Activity Assay Purified 5'-end labeled RNA substrates (15-25-mers) and purified end labeled ribozymes (-36-mers) were both heated to 95 quenched on ice and equilibrated at 37 OC, separately. Ribozyme stock solutions were 1 mM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were 1 nM. Total reaction volumes were 50 mL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCI 2 Reactions were initiated by mixing substrate and ribozyme solutions at t 0. Aliquots of mL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each time point was quenched in formamide loading buffer and loaded onto a S"o denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

15 Example 55: Stability Assay 500 pmol of gel-purified 5'-end-labeled ribozymes were precipitated in ethanol and pelleted by centrifugation. Each pellet was resuspended in 20 mL of appropriate fluid (human serum, human plasma, human synovial fluid or fetal bovine serum) by vortexing for 20 s at room temperature. The 20 samples were placed into a 37 °C incubator and 2 mL aliquots were withdrawn after incubation for 0, 15, 30, 45, 60, 120, 240 and 480 m.

S"Aliquots were added to 20 mL of a solution containing 95% formamide and TBE (50 mM Tris, 50 mM borate, 1 mM EDTA) to quench further nuclease activity' and the samples were frozen until loading onto gels.

Ribozymes were size-fractionated by electrophoresis in acrylamide/8M urea gels. The amount of intact ribozyme at each time point was quantified by scanning the bands with a phosphorimager (Molecular Dynamics) and the half-life of each ribozyme in the fluids was determined by plotting the percent intact ribozyme vs the time of incubation and extrapolation from the graph.

Example 56: 3 '.5'-O-(Tetraisopropyl-disiloxane-1 3-diyl)-2'-O-Phenoxythiocarbonyl-Uridine (7) To a stirred solution of 3 ',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)uridine, 6, (15.1 g, 31 mmol, synthesized according to Nucleic Acid Chemistry, ed. Leroy Townsend, 1986 pp. 229-231) and dimethylaminopyridine (7.57 g, 62 mmol) a solution of phenylchlorothionoformate (5.15 mL, 37.2 mmol) in 50 mL of acetonitrile was added dropwise and the reaction stirred for 8 h. TLC (EtOAc:hexanes 1:1) showed disappearance of the starting material. The reaction mixture was evaporated, the residue dissolved in chloroform, washed with water and brine, the organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel with EtOAc:hexanes 2:1 as eluent to give 16.44 g of 7.

10 Example 57: 3'.5'-O-(TetraisoDropyl-disiloxane-1.3-diyl)-2'-C-Allvl -Uridine m. To a refluxing, under argon, solution of 3 disiloxane-l,3-diyl)-2'-O-phenoxythiocarbonyl-uridine, 7, (5 g, 8.03 mmol) S* and allyltributyltin (12.3 mL, 40.15 mmol) in dry toluene, benzoyl peroxide (0.5 g) was added portionwise during 1 h. The resulting mixture was allowed to reflux under argon for an additional 7-8 h. The reaction was then evaporated and the product 8 purified by flash chromatography on silica gel with EtOAc:hexanes 1:3 as eluent. Yield 2.82 g Example 58: 5'-O-Dimethoxvtritvl-2'-C-All-Uridine (9) 20 A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry tetrahydrofuran .o* (THF) was treated with a 1 M solution of tetrabutylammoniumfluoride in THF (3.7 mL) for 10 m at room temperature. The resulting mixture was evaporated, the residue was loaded onto a silica gel column, washed with 1 L of chloroform, and the desired deprotected compound was eluted with chloroform:methanol 9:1. Appropriate fractions were combined, solvents removed by evaporation, and the residue was dried by coevaporation with dry pyridine. The oily residue was redissolved in dry pyridine, dimethoxytritylchloride (1.2 eq) was added and the reaction mixture was left under anhydrous conditions overnight. The reaction was quenched with methanol (20 mL), evaporated, dissolved in chloroform, washed with aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel, EtOAc:hexanes 1:1 as eluent, to give 0.85 g of 9 as a white foam.

Example 59: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine 3'-(2-Cyanoethyl N.NdiisopropylDhosphoramidite) 5'-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 g, 1.12 mmol) was dissolved in dry dichloromethane under dry argon. N,N-Diisopropylethylamine (0.39 mL, 2.24 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.57 mmol) was added dropwise to the stirred reaction solution and stirring was continued for 2 h at RT. The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture 10 was concentrated in vacuo (40 and purified by flash chromatography on silica gel using a gradient of 10-60% EtOAc in hexanes containing 1% triethylamine mixture as eluent. Yield: 0.78 g white foam.

Example 60: 3'.5'-O-(Tetraisooropyl-disiloxane-1 .3-divl)-2'--Allyl-N4-- Acetvl-Cvtidine (11) 15 Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous Soxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile. To the resulting suspension a solution of 3 1,3-diyl)-2'-C-allyl uridine (2.32 g, 4.55 mmol) in 30 mL of acetonitrile was added dropwise and the reaction mixture was stirred for 4 h at room temperature. The reaction was concentrated in vacuo to a minimal volume (not to dryness). The residue was dissolved in chloroform and washed with water, saturated aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The resulting foam was dissolved in 50 mL of 1,4-dioxane and treated with 29% aq. NH 4 OH overnight at room temperature. TLC (chloroform:methanol 9:1) showed complete conversion of the starting material. The solution was evaporated, dried by coevaporation with anhydrous pyridine and acetylated with acetic anhydride (0.52 mL, 5.46 mmol) in pyridine overnight. The reaction mixture was quenched with methanol, evaporated, the residue was dissolved in chloroform, washed with sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, evaporated to dryness and purified by flash chromatography on silica gel MeOH in chloroform). Yield 2.3 g as a white foam.

Example 61: 5'-O-Dimethoxytrityl-2'-C-Allyl-4-Acetyl-Cytidine This compound was obtained analogously to the uridine derivative 9 in 55% yield.

Example 62: -Dimethoxtritl-2'--allyl-N-Acetl-tidine Cyanoethyl N, N-diisopropylDhosphoramidite) (12) 2'-O-Dimethoxytrityl-2'-C-allyl-N 4 -acetyl cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichloromethane under argon. N,N-Diisopropylethylamine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.38 mL, 1.7 mmol) 10 was added dropwise to a stirred reaction solution and stirring was continued for 2 h at room temperature. The reaction mixture was then icecooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture was concentrated in vacuo (40 and purified by flash chromatography on silica gel using chloroform:ethanol 98:2 with 2% 15 triethylamine mixture as eluent. Yield: 0.91 g white foam.

Example 63: 2'-Deoxy-2'-Methylene-Uridine Deoxy-2-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine 14 (Hansske,F.; Madej,D.; Robins,M. J. Tetrahedron 1984, 40, 125 and Matsuda,A.; Takenuki,K.; Tanaka,S.; Sasaki,T.; Ueda,T. J. Med. Chem.

1991, 34, 812) (2.2 g, 4.55 mmol dissolved in THF (20 mL) was treated 9 with 1 M TBAF in THF (10 mL) for 20 m and concentrated.in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2 '-Deoxy-2'-methylene-uridine (1.0 g, 3.3 mmol, 72.5%) was eluted with 20% MeOH in CH 2 C12.

ExamDple 64: 5'--DMT-2'-Deoxy-2'-Methylene-Uridine 2 '-Deoxy-2'-methylene-uridine (0.91 g, 3.79 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

2 (100 mL) and washed with sat. NaHC03, water and brine. The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as eluant to yield 15 (0.43 g, 0.79 mmol, 22%).

130 Ex amlle 65: 5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine 2-Cyanoethyl' N.N-diisooropylphosphoramidite) (17) 1 2-Doy2- tye mtoyrtlpDrbfurnoy) uracil (0.43 g, 0.8 mmol) dissolved in dry CH 2

CI

2 (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.25 mL, 1.12 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 mn the mixture evaporated to a syrup in vacuo (40 00). The product (0.3 g, 0.4 a: mmol, 50%) was purified by flash column chromatography over silica gel using a 25-70% EtOAc gradient iii hexanes, containing 1% triethylamine, as eluant. Rf 0.42 (CH 2 01 2 MeOH 15:1) Example 66: 2 -Deoxy-2'-Difluoromethylene:3'. S-O-(TetraiSOorODVldisilox- *03dil)Uidn 15 126 2 '-Keto-3',5'-O-(tetraisopropyldisiloxa nel ,3-diyl)uridine 14 (1.92 g, *12.6 mmol) and triphenylphosphine (2.5 g, 9.25 mmol) were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 00. A warm 00) solution of sodium chlorodifluoroacetate in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The 0, :residue was dissolved in 0H 2

CI

2 and chromnatographed over silica gel. 2'- D oy2-i rmtye VO(erispr yds *a e- 1,3-d iyl) uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.

Examgle 67: 2 '-Deoxy-2'-Difluoromethylene-Uridine 2'-Deoxy-2'-methylene-3',s O-(tetraisopropydisiloxane-.1 ,3-diyl)uridine (3.1 g, 5.9 mm ol) dissolved in THE (20 mL) was treated with 1 M TBAF in THE (10 mL) for 20 m and concentrated in vacua. The residue was triturated with petroleum ether and chromatographed on silica gel column.

2 '-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol, 68%) was eluted with 20% MeOH in 0H 2 C1 2 Examule 68: ODMT-2'-Deoxy-2'-Difluoromithyene-Uridine(16 21 -Deoxy- 2 '-difluoromethylene-uridine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (1.42 g, 4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture 131 was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

2 (100 mL) and washed with sat. NaHCO 3 water and brine. The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using 40% EtOAc:hexanes as eluant to yield 5'-O-DMT-2'-deoxy-2'-difloromethylene-uridine 16 (1.05 g, 1.8 mmol, Example 69: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-U ridine Cyanoethyl NN-diisoDropylphosDhoramidite) (18) 0 10 1-(2'-Deoxy-2'-difluoromethylene-5'- O-dimethoxytrityl-P-D-ribofuranosyl)-uracil (0.577 g, 1 mmol) dissolved in dry CH 2

C

2 (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.36 mL, 2 mmol) .was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.4 mmol). The reaction mixture :15 was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 OC). The product (0.404 g, 0.52 mmol, 52%) was purified by flash chromatography over silica gel using EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH 2

CI

2 MeOH 15:1).

**20 Examole 70: 2'-Deoxv-2'-Methylene-3'.5'-O-(Tetraisorooyldisiloxane-1 .3diyl)-4-N-Acetyl-CQytidine Triethylamine (4.8 mL, 34 mmol) was added to a solution of POC13 (0.65 mL, 6.8 mmol) and 1,2,4-triazole (2.1 g, 30.6 mmol) in acetonitrile mL) at 0 oC. A solution of 2 '-deoxy-2'-methylene-3',5'-O(tetraisopropyldisiloxane-1,3-diyl) uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h. The mixture was concentrated in vacuo, dissolved in

CH

2 01 2 (2 x 100 mL) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2

SO

4 concentrated in vacuo, dissolved in dioxane (10 mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (3 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.

NaHC03 (5 mL). The mixture was concentrated in vacuo, dissolved in

CH

2 Cl 2 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The 132 organic extracts were dried over Na 2

SO

4 concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-methylene-3',5'- (tet raiso pro pyld is iloxane-1, ,3-d iyl)-4- N-acetyl-cytidin e 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes.

Examole 71: 1 -(2'-Deoxy-2'- Methyl en e-5'--Dim eth oxytritypa:. ri bo furanosyl)-4-N-Acetyl-Cytosine 21 2'-Deoxy-2'-methylene-3',5'- O-(tetraisopropyldisiloxane- 1,3-diyl)-4-Nacetyl-cytidine 20 (1.3 g, 2.5 mmol) dissolved in THE (20 mL) was treated with 1 M TBAF in THE (3 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromnatographed on silica gel column. 21 -Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol, was eluted with 10% MeOH in CH 2

CI

2 2 '-Deoxy-2'-methylene-4-Nacetyl-cytidine (0.56 g, 1.99 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (0.81 g, 2.4 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

2 (100 mL) and washed with sat. NaHCO 3 (50 mL), water (50 mL) 'and brine (50 mL). The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, Examrole 72: l-( 2 '-Deoxy- 2 furanosyl)-4-N-Acety-Cytosine 3 2 -Cyanoethyl-N.N-diisooropylphosphoramidite) (221 l-( 2 '-Deoxy- 2 '-methylene-5'.0-dimethoxytrityl3Dribofuranosyl) 4

-N

acetyl-cytosine 21 (0.88 g, 1.5 mmol) dissolved in dry CH 2

CI

2 (10 mL) was placed in a round-bottom flask under. Ar. Diisopropylethylamine (0.8 mL, mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacua (40 00). The product 22 (0.82 g, 1.04 mmol, 69%) was purified by flash chromatography over silica gel using 50-70% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.36 (0H2C1 2 :MeOH 20:1).

Examlle 73: 2'-Deoxy-2'-Difluoromethylene-3'.5'-O.4Tetraisoprooyl disiloxane-1 .3-diyl)-4-N-Acetyl-Cvtidine (24) Et 3 N (6.9 mL, 50 mmol) was added to a solution Of PQC1 3 (0.94 mL, mmol) and 1,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 mL) at 0 00.

A solution of 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropydisiloxane-1,3-diyl)uridine 23 ([described in example 14] 2.6 g, 5 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at RT for 4 h. The mixture was concentrated in vacua, dissolved in CH 2

CI

2 (2 x 100 mL) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2

SO

4 concentrated in vacua, dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (5 mL) was added- to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.

NaHCO 3 The mixture was concentrated in vacua, dissolved in

H

2

CI

2 (2 x 100 mL) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2

SO

4 concentrated in vacua and the residue chromatographed over silica gel. 21 -Deoxy-2'-difluoromethylene.

O-(tetraisopropyldisiloxane- 1,3-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol, 78%) was eluted with 20% EtOAc in hexanes.

Examnlle 74: 1 -(2'-Deoxy-2'-Difluoromethylene-5' O-Dimeto-xtritl-B-Dribofuranosyl)-4-N-Acetyl-pytosine 2'-Deoxy-2'-difluoromethylene-3',5' O-(tetraisopropyldisiloxane- 1,3diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol) dissolved in THE (20 mL) was treated with 1 M TBAF in THE (3 mL) for 20 m and concentrated in vacua.

The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2 '-Deoxy- 2 '-difluoromethylene-4-Nacetyl.cyidine (0.89 g, 2.8 mmol, 72%) was eluted with 10% MeOH in CH 2

CI

2 2'-Deoxy-2'd if Iu orom ethyl ene-4- Nacetyl.cytidin e (0.89 g, 2.8 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (1.03 g, 3.1 mmol) in pyridine mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacua and the residue taken up in CH 2

CI

2 (100 mL) and washed with sat. NaHCO 3 (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes 60:40 as eluant to yield 25 (1.2 g, 1.9 mmol, 68%).

Example 75: l-( 2 '-Deoxy- 21 -Difluoromethylene-5'.O-DimethxritylI3D..

ribofuranosyl)-4-N-Acetylcytosine 3'-(2-cyanoethyl-N .N-diisol2rooylphosphoramidite) (26) 1- 2 '-Deoxy- 2 -difuo romethylene5' Od imethoxytrityl-p-D-ribof uran o syl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mnmol) dissolved in dry CH 2 01 2 ml-) was placed in a round-bottom flask under Ar. Diisopropylethylamnine mL, 2.9 mmol) was added, followed by the dropwise addition of 2- .110 cyanoethyl NN-diisopropylchlorophosphorarnidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mnL).

After 10 mn the mixture was evaporated to a syrup in vacuo (40 00). The product 26, a white foam (0.52 g, 0.63 mmol, 65%) was purified by flash chromatography over silica gel using 30-70% EtOAc gradient in hexanes, 15 containing 1% triethylamine, as eluant. Rj 0.48 (0H2C12:MeOH /20:1).

Example 76: 2 '-Keto-3'.5'-O-(Ttaiopyldisiloxane-1 3-divl)-6-N-(4-t- Butylbenzoyl)-Adenosine (28) Acetic anhydride (4.6 ml-) was added to a solution of propyldisiloxane-1 3 -diyl).

6 -N-(4-tbutylbenzoyl).adenosine (Brown Chitdlu Joe,. Moa,, Rees,., ianda,. Usawa A.

J. Chem .Soc. Perkin Trans. 11989, 1735) (6.2 g, 9.2 mmol) in DMVSO (37 ml-) and the resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and washed with water. The organic layer was dried over MgSO 4 and concentrated in vacuo. The residue was purified on. a silica gel column to yield 2 '-keto- 3 ',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylben..

zoyl)-adenosine 28 (4.8 g, 7.2 mmol, 78%).

Examlle 77: 2 '-Deoxy-'mehene-3' .5'-O)-(Tetraisopropyldisiloxane-.1 .3diyl)-6-N-(4-t-Butylbenzoyl)Aden sine (29) Under a pressure of argon, sec-butyllithiumn in hexanes (11.2 mL, 14.6 mmol) was added to a suspension of triphenylmethylphosphonium iodide (7.07 g,17.5 mmol) in THF (25 ml-) cooled at -78 The homogeneous *orange solution was allowed to warnm to -30 CC and a solution of 2'-keto- 3 ',5'-O-(tetraisopropyldisiloxane. 1-il-6N(--utlezy)-dnsn 51, *1 135 28 (4.87 g, 7.3 mmol) in THF (25 mL) was transferred to this mixture under argon pressure. After warming to RT, stirring was continued for 24 h. THF was evaporated and replaced by CH 2

CI

2 (250 mL), water was added mL), and the solution was neutralized with a cooled solution of 2% HCI.

The organic layer was washed with H 2 0 (20 mL), 5% aqueous NaHCO 3 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacuo to give the crude compound, which was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc 7:3 afforded pure 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 29 (3.86 g, 5.8 mmol, 79%).

Example 78: 2 '-Deoxy- 2 '-Methylene-6-N-(4-t-Butvlbenzoyl-Adenosine 2 '-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N- (4-t-butylbenzoyl)-adenosine (3.86 g, 5.8 mmol) dissolved in THF (30 mL) was treated with 1 M TBAF in THF (15 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-methylene-6-N-(4-tbutylbenzoyl)-adenosine (1.8 g, 4.3 mmol, 74%) was eluted with MeOH in CH 2 C1 2 20 Example 79: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butbenzovl- Adenosine (29) 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75 g, 1.77 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (0.66 g, 1.98 mmol) in pyridine (10 mL) was added dropwise over 15 m.

The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

2 (100 mL) and washed with sat. NaHCO 3 water and brine. The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as an eluant to yield 29 (0.81 g, 1.1 mmol, 62%).

Example 80: 5'-ODM-2-Deoxy-2'-Methylene-6-N-(4t-Blbenzoyl)- Adenosine 3'-(2-Cyanoethyl N. N-diisoprovylphosphoramid) (31 1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl--D-ribofuranosyl)-6-N- (4-t-butylbenzoyl)-adenine 29 dissolved in dry CH2CI2 (15 mL) was placed in a round bottom flask under Ar. Diisopropylethylamine was added, followed by the dropwise addition of 2-cyanoethyl N, Nd iisopropylchloroph ospho ram idite The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacua (40 00). The product was purified by flash chromatography over silica gel using 30-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant (0.7 g, 0.76 mmol, Rf 0.45 (0H 2 01 2 MeOH 20:1) :Examiole 81: 2'-Deoxy-2-Difluoromethylene-3'.5'- O-(Tetraisoprooyidisiloxane-1 .3-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine 2'-Keto-3',5'-O-(tetraisopropydisiloxane- 1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenylphosphine (2.9 g, 11 mmol were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 00. A warm (.60 00) solution of sodium *:15 chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated in vacua. The residue was dissolved in CH 2

CI

2 and chromatographed over silica gel. 2'- Deoxy-2'-difluoromethylene-3',5'- O-(tetraisopropyldisioxane- 1,3-diyl)-6-N- (4-t-butylbenzoyl)-adenosine (4.1g, 6.4 mmol, 64%) eluted with hexanes in EtOAc.

Examiole 82: 2 '-Deoxy- 2 '-Difluoromethylene-6-N(4- Butylbenzoyl).

Adenosine Deoxy-2'-d ifl u orom ethyl ene-3', O-(tetrais opropyld isilIoxane- 1,3diyI)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol) dissolved in THF mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacua. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2t -Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in 0H 2 01 2 Examole 83: r,--M-'Doy2-ilooehln--LAL& benzoyl)-Adenosine 2' ex-' fIu o ty e6 -4 tlezy)aeo n (2.3 g, 4.9 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl in pyridine (10 ml-) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 ml-) was added to quench the reaction. The mixture was concentrated in vacuc and the residue taken up in CH 2

CI

2 (100 ml-) and washed with sat. NaHCO 3 water and brine. "the organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes'as eluant to yield 30 (2.6 g, 3.41 mmol, 69%).

Example 84: 5'ODT2-em-'Dfuram-hln---4tBli benzoyl)-Adenosine 3'-(2-Cyanoethyl N. N-diisopro~ylphos~horamidite) a~ l-( 2 '-Deoxy- 2 syl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mmol) dissolved in dry

CH

2

CI

2 (25 ml-) was placed in a round bottom flask under Ar.

Diisopropylethylamine (1.2 mL, 6.8 mnmol) was added, followed by the :15 dropwise addition of 2-cyanoethyl NN-diisopropylchlorophosphoramidite (1.06 mL, 4.76 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 0 32 (2.3 g, 2.4 mmol, 70%) was purified by flash column chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.52 (CH 2

CI

2 MeCH/ 15:1).

Examole 85: 2 '-Deoxy2'-Methoxycarbonylmethylidine3I (Tetras prooyldisiloxane-1 .3-diyfl-Uridine (33) M ethyl (triphenylphosphoranylid in e)acetate (5.4 16 mmol) was added to a solution of 2 '-keto-3',5'-O-(tetraisopropyl disiloxane-1 ,3-diyl)uridine 14 in CH 2

CI

2 under argon. The mixture was left to stir at RT for h. CH 2

CI

2 (100 ml-) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCL. The organic layer was washed with H 2 0 (20 mL), 5% aq. NaHCQ 3 (20 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacuo to give crude product, that was chromatographed on a silica gel column.

Elution with light petroleum ether:EtOAc 7:3 afforded pure 2'-deoxy-2'methoxycarbonylmethylidine3stu(tetraiopopy 1 iiloan 1 ,3-diyl)uridine 33 (5.8 g, 10.8 mmol, 67.5%).

-1 138 Examlle 86: 2-Deoxy-2'-Methoxycarbonylmethylidine-1Jridine (34) Et 3 N-3 HF (3 ml-) was added to a solution of 2'-deoxy-2'-methoxy.

carboxylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 g, 9.3 mmol) dissolved in CH 2

CI

2 (20 ml-) and EI 3 N (15 mL). The resulting mixture was evaporated in vacuo after 1 h and chromatographed on a silica gel column eluting 23 -deoxy-2'-methoxycarbonylmethylidine.

uridine 34 (2.4 g, 8 mmol, 86%) with THF:CH 2

CI

2 4:1.

Example 87: 5'-O-DMT- 21 -Deoxy-2'-MethoxycarbonylmethylidineUridine 21 -Deoxy-2'-methoxycarbonylmethylidine-uridine 34 (1.2 g, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-CI (1.5 g, 4.42 mmol) in pyridine (10 ml-) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 ml-) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken :15 up in CH 2

CI

2 (100 ml-) and washed with sat. NaHCO 3 water and brine.

The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using 2-5% MeOH in 0H 2 C1 2 as an eluant yield 5'-O-DMT- 2 -deoxy-2'-methoxycarbonylmethylidine-urid in e (2.03 g, 3.46 mmol, 86%).

Examlle 88: '-O-DMT- 2 '-Deoxy-2'-Methoxycarbonylmethylidine..Uridine 3'-(2-cyanoethyl-N. N-diisooropylrphosphoramidite) (361 l-( 2 '-Deoxy- 23 2 1 D-ribofuranosyl)-Liridine 3 5 (2.0 g, 3.4 mnmol) dissolved in dry 0H 2 C1 2 ml-) was placed in a round-bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2cyanoethyl NN-diisopropylchlorophosphoramidite (0.91 mL, 4.08 mmol).

The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 00).

5'ODT2-ex-'mtoyabnlehldn-rdn cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1.8 g, 2.3 mmol, 67%) was purified by flash column chromatography over silica gel using a EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.44 (CH 2

CI

2 :MeOH 9.5:0.5).

139 Examole 89: 2 '-Deoxv- 2 siloxane-1.3-diyl-Uridine 37 2 '-Deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 33 (5.0 g, 10.8 mmol) was dissolved in MeOH (50 mL) and 1 N NaOH solution (50 mL) was added to the stirred solution at RT. The mixture was stirred for 2 h and MeOH removed in vacuo. The pH of the aqueous layer was adjusted to 4.5 with 1N HCI solution, extracted with EtOAc (2 x 100 mL), washed with brine, dried over MgSO 4 and concentrated in vacuo to yield the crude acid. 2'-Deoxy-2'- 10 carboxymethylidine-3',5'-O-(tetraisopropyidisiloxane-1, 3 -diyl)-uridine 37 (4.2 g, 7.8 mmol, 73%) was purified on a silica gel column using a gradient of 10-15% MeOH in CH 2

CI

2 The alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.

Administration of such oligonucleotides is by standard procedure. See Sullivan et al. PCT WO 94/02595.

Oliqonucleotides with 3' and/or 5' DihaloDhosDhonat 20 This invention synthesis and uses 3' and/or 5' dihalophosphonate-, 3' or 5'-CF 2 -phosphonate-, substituted nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule.

As the term is used in this application, and/or 3'dihalophosphonate nucleotide containing ribozymes, deoxyribozymes (see Usman et al., PCT/US94/11649, incorporated by reference herein), and chimeras. of nucleotides, are catalytic nucleic molecules that contain and/or 3'-dihalophosphonate nucleotide components replacing, but not limited to, double-stranded stems, single-stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA or DNA transcript. This invention concerns 140 nucleic acids formed of standard nucleotides or modified nucleotides, which also contain at least one 5'-dihalophosphonate and/or one 3'dihalophosphonate group.

The synthesis of 1-O-Ac-2,3-di-O-Bz-D-ribofuranose 5+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-Oisopropylidene-B-D-ribofuranose 5-deoxy-5.dihaomethylphosphonate is described for the difluoro, in Figure 87). Condensation of this suitably derivatized sugar with silylated pyrimidines and purines affords novel nucleoside 5'-deoxy-5'-dihalomethylphosphoates. These intermediates may be incorporated into catalytic or antisense nucleic acids by either chemical (conversion of the nucleoside 'dihalomethylphosphonates into suitably protected phosphoramidites 12a or solid supports 12b, Figure 88) or enzymatic means (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into their 15 triphosphates, 14 Figure 89, for T7 transcription).

Thus, in one aspect the invention features 5' and/or 3'dihalonucleotides and nucleic acids containing such 5' and/or 3'dihalonucleotides. The general structure of such molecules is shown below.

II

((30)2RX2

(R

3 0) 2

PCX

2 2

B

(R

3 0) 2 P 0

(R

3 0) 2

P=

where

R

1 is H, OH, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, or carbonate; each

R

2 is separately H, OH, or R; each

R

3 is separately a phosphate protecting group, methyl, ethyl, cyanoethyl, pnitrophenyl, or chlorophenyl; each X is separately any halogen; and each B •is any nucleotide base.

The invention in particular features nucleic acid molecules having such modified nucleotides and enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleside dihalo and/or 3 '-deoxy-3-dihalophosphonates by condensing a dihalophosphonate-containing sugar with a pyrimidine or a purine under conditions suitable to form a nucleoside and/or a 3'-deoxy-3'-dihalophosphonate.

Phosphonic acids may exhibit important biological properties because of their similarity to phosphates (Engel, Chem. Rev. 1977, 77, 349-367). Blackburn and Kent Chem. Soc., Perkin Trans. 1986, 913- 917) indicate that based on electronic and steric considerations _-fluoro and _,_-difluoromethylphosphonates might mimic phosphate esters better than the corresponding phosphonates. Analogues of pyro- and f 10 triphosphates 1, where the bridging oxygen atoms are replaced by a difluoromethylene group, have been employed as substrates in enzymatic processes (Blackburn et Nucleosides Nucleotides 1985, 4, 165-167; Blackburn et al., Chem. Scr. 1986, 26, 21-24). 9-(5,5-Difluoro-5phosphonopentyl)guanine has been utilized as a multisubstrate analogue inhibitor of purine nucleoside phosphorylase (Halazy et al., J.

Am. Chem. Soc. 1991, 113, 315-317). Oligonucleotides containing methylene groups in place of phosphodiester 5'-oxygens are resistant toward nucleases that cleave phosphodiester linkages between phosphorus and the 5'-oxygen (Breaker et al., Biochemistry 1993, 32, 9125-9128), but can still form stable complexes with complementary sequences. Heinemann etal. (Nucleic Acids Res. 1991, 19, 427-433) found that a single 3 '-methylenephosphonate linkage had a minor influence on the conformation of a DNA octamer double helix.

NH

2 O O O N -o-P-X-P-O-P-0

NI

N3N 0 0- 0-

N

o-- OH OH 1 0

H

N N

S(HO)

2 0PCF2 N NH 2 2 2

(ETO)

2

POCF

2 Li 3 0 One common synthetic approach to c,a-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive substrate by diethyl (lithiodifluoromethyl)phosphonate (Obayashi et al., Tetrahedron Lett. 1982, 23, 2323-2326). However, our attempts to synthesize nucleoside 5'-deoxy-5'-difluoro-methylphosphonates from using 3 were unsuccessful, i.e. starting compounds were quantitatively recovered. The reaction of nucleoside aldehydes with 3, according to the procedure of Martin et al. (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842), led to a complex mixture of products. Recently, the synthesis of sugar a,cc-difluoroalkylphosphonates from primary sugar triflates using 3 was described (Berkowitz et al., J. Org.

Chem. 1993, 58, 6174-6176). Unfortunately, our experience is that nucleoside 5'-triflates are too unstable to be used in these syntheses.

The following are non-limiting examples showing the synthesis of nucleoside 5'-deoxy-5'-difluoromethyl-phosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon these examples. These examples demonstrate that it is possible to achieve synthesis of 5'-deoxy-5'-difluoro derivatives in good yield and thus guide those in the art to such equivalent methods. The examples also indicate utility of such synthesis to provide useful oligonucleotides as described above.

Those in the art will recognize that useful modified enzymatic nucleic acids can now be designed, much as described by Draper et al., PCT/US94/13129 hereby incorporated by reference herein (including drawings).

10 Example 90: Synthesis of Nucleoside difluoromethylphosphonates Referring to Fig. 87, we synthesized a suitable glycosylating agent from the known D-ribose c,a-difluoromethylphosphonate (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842) which served as a key 15 intermediate for the synthesis of nucleoside difluoromethylphosphonates.

Methyl 2 3 -O-isopropylidene-13-D-ribofuranose a,adifluoromethylphosphonate was synthesized from the according to the procedure of Martin et al. (Tetrahedron Lett. 1992, 33, 20 1839-1842) (Figure 87). Removal of the isopropylidene group was accomplished under mild conditions (1 2 -MeOH, reflux, 18 h (Szarek et al., Tetrahedron Lett. 1986, 27, 3827) or Dowex 50 WX8 MeOH, RT (about 20-25*C), 3 days) in 72% yield. The anomeric mixture thus obtained was benzoylated with benzoyl chloride/pyridine to afford the 2,3di-O-benzoyl derivative, which was subjected to mild acetolysis conditions (Walczak et al., Synthesis, 1993, 790-792) (Ac20, AcOH, H 2

SO

4 EtOAc, 0°C. The desired 1-O-acetyl-2,3-di-O-benzoyl-D-ribofuranose difluoromethylphosphonate was obtained in quantitative yield as an anomeric mixture. These derivatives were used for selective glycosylation of silylated uracil and N 4 -acetylcytosine under Vorbr0ggen conditions (VorbrOggen, Nucleoside Analogs. Chemistry, Biology and Medical Applications, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F 3

CSO

2 OSi(CH 3 3 as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron

S.

144 Lett. 1987, 28, .3623-3626 and references cited therein) (SnCl 4 as a catalyst, boiling acetonitrile) to yield P-nucleosides (62% 6a, 75% 6b).

Glycosylation of silylated N 6 -benzoyladenine under the same conditions yielded a mixture of N-9 isomer 6c and N-7 isomer 7 in 34% and yield, respectively. The above nucleotides were successfully deprotected using trimethylsilylbromide for the cleavage of the ethyl groups, followed by treatment with ammonia-methanol to remove the acyl protecting groups.

Nucleoside 5'-deoxy-5'-difluoromethylphosphonates 8 were finally purified on a DEAE Sephadex A-25 (HC0 3 column using a 0.01-0.25 M 10 TEAB gradient for elution and obtained as their sodium salts (82% 8a; 87% 8b; 82% 8c).

.Selected analytical data: 3 1 P-NMR (31P) and 1 H-NMR (1H) were "o recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H 3

PO

4 o and TMS, respectively. Solvent was CDCI 3 unless otherwise noted. 5: 1

H

5 8.07-7.28 Bz), 6.66 J1,2 4.5, aH1), 6.42 PH1), 5.74 J2,3 4.9, PH2), 5.67 (dd, J3,2 4.9, J3,4 6.6, 0H3), 5.63 (dd, J3,2 6.7, J3,4 3.6, aH3), 5.57 (dd, J 2 1 4.5, J2, 3 6.7, aH2), 4.91 H4), 4.30 CH 2

CH

3 2.64 (m,

CH

2

CF

2 2.18 PAc), 2.12 aAc), 1.39 CH 2

CH

3 3 1 P 5 7.82 (t, JP,F 105.2), 7.67 JP,F 106.5). 6a: 1 H 5 9.11 1H, NH), 8.01 11H, Bz, H6), 5.94 J1',2. 4.1, 1H, 5.83 (dd, J 5 ,6 8.1, 1H, H5), 5.79 (dd, J2',1' 4.1, J 2 6.5, 1H, 5.71 (dd, J3', 2 6.5, J3', 4 6.4, 1H, 4.79 (dd, J4',3 6.4, J4',F 11.6, 1H, 4.31 4H, CH 2

CH

3 2.75 (tq, JH,F 19.6, 2H, CH 2

CF

2 1.40 6H, CH 2

CH

3 3 1 p 8 7.77 JP,F 104.0). 8c: 31p (vs DSS) (D 2 0) 5 5.71 Jp,F 87.9).

Compound 7 was deacylated with methanolic ammonia yielding the product that showed Xmax (H 2 0) 271 nm and Xmin 233 nm, confirming that the site of glycosylation was N-7.

Example 91:Synthesis of Nucleic Acids Containing Modified Nucleotide Containing Cores The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (Figure 88 and Janda et al., Science 1989, 244:437-440.). These 145 nucleoside 5'-deoxy-5'-difluoromethylphosphonates may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 introns, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 92: Synthesis of Modified TriDhosphate The triphosphate derivatives of the above nucleotides can be formed as shown in Ei989, according to known procedures. Nucleic Acid Chem., Leroy B. Townsend, John Wiley Sons, New York 1991, pp. 337-340; Nucleotide Analogs, Karl Heinz Scheit; John Wiley Sons New York 1980, 10 pp. 211-218.

Equivalent synthetic schemes for 3' dihalophosphonates are shown in Fiures 90 and 91 using art recognized nomenclature. The conditions can be optimized by standard procedures.

*4 The nucleoside dihalophosphonates described herein are 15 advantageous as modified nucleotides in any nucleic acid structure, e.g., catalytic or antisense, since they are resistant to exo- and endonucleases that normally degrade unmodified nucleic acids in vivo. They also do not perturb the normal structure of the nucleic acid in which they are incorporated thereby maintaining any activity associated with that structure.

These compounds may also be of use as monomers as antiviral and/or antitumor drugs.

Oliaonucleotides with Amido or Petido Modification This invention replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and portions of the sugar of a nucleotide may be substituted, or the phosphate group may be substituted with amido or peptido moieties. Generally, such a nucleotide has the general structure shown in Formula I below: 146 0 hl R2 H. RI R 3

P--"O

I 0 FORMULA I The base is any one of the standard bases or is a modified nucleotide base known to those in the art, or can be a hydrogen group. In 5 addition, either R 1 or R 2 is H or an alkyl, alkene or alkyne group containing between 2 and 10 carbon atoms, or hydrogen, an amine (primary, secondary or tertiary, ea, R 3

NR

4 where each R 3 and R 4 independently is hydrogen or an alkyl, alkene or alkyne having between 2 and 10 carbon atoms, or is a residue of an amino acid, ie., an amide), an alkyl group, or 10 an amino acid (D or L forms) or peptide containing between 2 and 5 amino acids. The zigzag lines represent hydrogen, or a bond to another base or other chemical moiety known in the art. Preferably, one of R 1 R2 and R 3 is an H, and the other is an amino acid or peptide.

Applicant has recognized that RNA can assume a much more complex structural form than DNA because of the presence of the 2'hydroxyl group in RNA. This group is able to provide additional hydrogen bonding with other hydrogen donors, acceptors and metal ions within the RNA molecule. Applicant now provides molecules which have a modified amine group at the 2' position, such that significantly more complex structures can be formed by the modified oligonucleotide. Such modification with a 2'-amido or peptido group leads to expansion and enrichment of the side-chain hydrogen bonding network. The amide and peptide moieties are responsible for complex structural formation of the oligonucleotide and can form strong complexes with other bases, and interfere with standard base pairing interactions. Such interference will allow the formation of a complex nucleic acid and protein conglomerate.

147 Oligonucleotides of this invention are significantly more stable than existing oligonucleotides and can potentially form biologically active bioconjugates not previously possible for oligonucleotides. They may also be used for in vitro selection of unique aptamers, that is, randomly generated oligonucleotides which can be folded into an effective ligand for a target protein, nucleic acid or polysaccharide.

Thus, in one aspect, the invention features an oligonucleotide containing the modified base shown in Formula I, above.

o In other aspects, the oligonucleotide may include a 3' or 5' nucleotide 10 having a 3' or 5' located amino acid or aminoacyl group. In all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard modifications can be made. For example, an may be replaced with an S, the sugar may lack a base abasic) and the S° phosphate moiety may be modified to include other substitutions (see Sproat, supra).

Example 93: General procedure for the preparation of 2'-aminoacyl-2'deoxy-2'-aminonucleoside conjugates.

O" Referring to Fig 92, to the solution of 2 '-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or amino acid (1 mmol) in methanol [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1- S" ethoxycarbonyl-2-ethoxy-1, 2 -dihydroquinoline (EEDQ) [or 1isobutyloxycarbonyl.2-isobutyloxy-1, 2 -dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to *C from 3-48 hours. Solvents are removed under reduced pressure and the residual syrup is chromatographed on the column of silica-gel using 1methanol in dichloromethane. Fractions containing the product are concentrated yielding a white foam with yields ranging from 85 to 95 Structures are confirmed by 1 H NMR spectra of conjugates which show correct chemical shifts for nucleoside and aminoacyl part of the molecule.

Further proofs of the structures are obtained by cleaving the aminoacyl protecting groups under appropriate conditions and assigning 1 H NMR resonances for the fully deprotected conjugate.

Partially protected conjugates described above are converted into their 5'-O-dimethoxytrityl derivatives and into 3'-phosphoramidites using standard procedures (Oligonucleotide Synthesis: A Practical Approach, 148 M.J. Gait ed.; IRL Press, Oxford, 1984). Incorporation of these phosphoramidites into RNA was performed using standard protocols (Usman et al., 1987 supra).

A general deprotection protocol for oligonucleotides of the present invention is described in Fig. 93.

The scheme shows synthesis of conjugate of 2 '-d-2'-aminouridine.

This is meant to be a non-limiting example, and those skilled in the art will recognize that, variations to the synthesis protocol can be readily generated to synthesize other nucelotides adenosine, cytidine, 10 guanosine) and/or abasic moieties.

Example 94: RNA cleavage by hammerhead ribozymes containing 2'aminoacyl modifications.

Hammerhead ribozymes targeted to site N (see Fig. 94) are synthesized using solid-phase synthesis, as described above. U4 and U7 15 positions are modified, individually or in combination, with either 2'-NHalanine or 2'-NH-lysine.

RNA cleavage assay in vitro: Substrate RNA is 5' end-labeled using

[Y-

3 2 P] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace 20 amount 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37°C for 10 min in a buffer containing 50 mM Tris-HCI and 10 mM MgCl2. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 4l are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X formamide stop mix. The samples are resolved on 20 denaturing polyacrylamide gels. The results are quantified and percentage of target RNA cleaved is plotted as a function of time.

Referring to Fig. 95, hammerhead ribozymes containing 2'-NHalanine or 2'-NH-lysine modifications at U4 and U7 positions cleave the target RNA efficiently.

Sequences listed in Figure 94 and the modifications described in Figure 95 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (base-substitutions, deletions, insertions, mutations, chemical modifications) of the ribozyme and RNA containing other 2'-hydroxyl group modifications, including but not limited to amino acids, peptides and cholesterol, can be readily generated using techniques known in the art, and are within the scope of the present invention.

Example 95: Aminoacylation of 3'-ends of RNA 1 I. Referring to Fig. 96, 3'-OH group of the nucleotide is converted to 10 succinate as described by Gait, supra. This can be linked with amino-alkyl solid support (for example: CpG). Zig-zag line indicates linkage of 3'OH group with the solid support.

1. Preparation of aminoacyl-derivatized solid support A) Synthesis of 0-Dimethoxytrityl (O-DMT) amino acids Referring to Fig. 97, to a solution of L- (or serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4,4'-dimethoxytrityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25*C) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was partitioned between 5% aq.

NaHCO 3 and dichloromethane, organic layer was washed with brine, dried (Na 2

SO

4 and concentrated in vacuo. The residue is purified by flash silicagel colum.n chromatography using 2-10% methanol in dichloromethane (containing 0.5 pyridine). Fractions containing product are combined and concentrated in vacuo to yield white foam (75-85 yield).

B) Preparation of the solid support and its derivatization with amino acids Referring to Fig. 97, the modified solid support (has an OH group instead of the standard NH 2 end group) was prepared according to Haralambidis et al., Tetrahedron Lett. 1987, 28, 5199, (P denotes aminopropyl CPG or polystyrene type support). O-DMT or NHmonomethoxytrityl (NH-MMT amino acid was attached to the above solid support using standard procedures for derivatization of the solid support (Gait, 1984, supra) creating a base-labile ester bond between amino acids 150 and the support. This support is suitable for the construction of RNA/DNA chain using suitably protected nucleoside phosphoramidites.

Example 96: Aminoacylation of 5'-ends of RNA I. Referring to Fig, 98. 5'-amino-containing sugar moiety was synthesized as described (Mag and Engels, 1989 Nucleic Acids Res. 17, 5973). Aminoacylation of the 5'-end of the monomer was achieved as described above and RNA phosphoramidite of the monomer was prepared as described by Usman et al., 1987 supra. The phosphoramidite was then incorporated at the 5'-end of the oligonucleotide 10 using standard solid-phase synthesis protocols described above.

II. Referring to EigJ., aminoacyl group(s) is attached to the phosphate group at the 5'-end of the RNA using standard procedures described above.

VII. Reversing Genetic Mutations 15 Modification of existing nucleic acid sequences can be achieved by homologous recombination. In this process a transfected sequence recombines with homologous chromosomal sequences and can replace *the endogenous cellular sequence. Boggs, 8 International J. Cell Cloning 1990, describes targeted gene modification. It reviews the .use of 20 homologous DNA recombination to correct genetic defects. Banga and Boyd, 89 Proc. Natl. Acad. Sci. U.S.A. 1735, 1992, describe a specific example of in vivo site-directed mutagenesis using a 50 base oligonucleotide. In this methodology a gene or gene segment is essentially replaced by the oligonucleotide used.

This invention uses a complementary oligonucleotide to position a nucleotide base changing activity at a particular site on a gene (RNA or genomic DNA), such that the nucleotide modifying activity will change (or revert) a mutation to wild-type, or its equivalent. By reversion or change of a mutation, we refer to reversion in a broad sense, such as when a mutation at a second site which leads to functional reversion to a wild type phenotype. Also, due to the degeneracy of the genetic code, a revertant may be achieved by changing any one of the three codon positions.

Additionally, creation of a stop codon in a deleterious gene (or transcript) is defined here as reverting a mutant phenotype to wild-type. An example of this type of reversion is creating a stop codon in a critical HIV proviral gene in a human.

Referring to Figures 100 and 101, broadly there are two approaches to causing a site directed change in order to revert a mutation to wild-type.

In one (Fig. 100) the oligonucleotide is used to target RNA specifically.

RNA is provided with a complementary (Watson-crick) oligonucleotide sequence to that in the target molecule. In this case the sequence modifying oligonucleotide would (analogously to an antisense oligonucleotide or ribozyme) have to be continuously present to revert the RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying ooo oligonucleotide. The transient nature of this approach is an advantage, in that treatment could be stopped by simply removing the sequence modifying oligonucleotide (as with a traditional drug).

15 A second approach targets DNA (Fig. 101) and has the advantage that changes may be permanently encoded in the target cell's genetic code. Thus, a single course (or several courses) of treatment may lead to permanent reversion of the genetic disease. If inadvertent chromosomal mutations are introduced this may cause cancer, mutate other genes, or 20 cause genetic changes in the germ-line (in patients of reproductive age).

S. However, if the base changing activity is a specific methylation that may S" modulate gene expression it would not necessarily lead to germ-line transmission. See Lewin, Genes,1983 John Wilely Sons, Inc. NY pp 493-496.

Complementary base pairing to single-stranded DNA or RNA is one method of directing an oligonucleotide to a particular site of DNA. This could occur by a strand displacement mechanism or by targeting DNA when it is single-stranded (such as during replication, or transcription).

Another method is using triple-strand binding (triplex formation) to doublestranded DNA, which is an established technique for binding polypyrimidine tracts, and can be extended to recognize all 4 nucleotides. See Povsic, Strobel, Dervan, P. (1992). Sequence-specific doublestrand alkylation and cleavage of DNA mediated by triple-helix formation.

J. Am. Chem. Soc. 114, 5934-5944 (1992). Knorre, Valentin, V.V., Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 152 1993) describe conjugation of reactive groups or enzyme to oligonucleotides and can be used in the methods described herein.

Recently, antisense oligonucleotides have been used to redirect an incorrect splice into order to obtain correct splicing of a splice mutant globin gene in vitro. Dominski Z; Kole R (1993) Restoration of correct splicing in thalassemia pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci LSA 90:8673-7. Analogously, in one preferred embodiment of this invention a complementary oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by 10 antisense.

In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence.

This can be accomplished by activating an endogenous enzyme (sj Fiure 102), by appropriate positioning of an enzyme (or ribozyme) conjugated (or activated by the duplex) to the oligonucleotide, or by .appropriate positioning of a chemical mutagen. Specific mutagens, such as nitrous acid which deaminates C to U, are most useful, but others can also be used if inactivation of a harmful RNA is desired.

RNA editing is an naturally occurring event in mammalian cells in which a sequence modifying activity edits a RNA to its proper sequence post-transcriptionally. Higuchi, Single, Kohler, Sommer, and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GluR-B:

A

base-paired intron-exon structure determines position and efficiency Cell 75:1361-1370. The machinery involved in RNA editing can be co-opted by a suitable oligonucleotide in order to promote chemical modification.

The changes in the base created by the methods of this invention cause a change in the nucleotide sequence, either directly, or after DNA repair by normal cellular mechanisms. These changes functionally correct a genetic defect or introduce a stop codon. Thus, the invention is distinct from techniques in which an active chemical group an alkylator) is attached to an antisense or triple strand oligonucleotide in order to chemically inactivate the target RNA or DNA.

Thus, this invention creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base.

1_.

153 This includes correcting a sequence instead of inactivating a gene but can also include inactivating a deleterious gene.

Thus, in one aspect, the invention features a method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule. The method includes contacting the nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After formation of the duplex or triplex molecule a base modifying activity chemically or enzyrnatically alters the targeted 10 base directly, or after nucleic acid repair in vivo. This results in the functional alteration of the nucleic acid sequence.

By "alter", as it is used in this context, is meant that one or more chemical moieties in a targeted base, or bases, is altered so that the mutant nucleic acid will be functionally different. Thus, this is distinct from prior methods of correcting defects in DNA, such as homologous recombination, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the transfected nucleic acid. This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.

By "functionally alter" is meant that the ability of the target nucleic acid to perform its normal function transcription or translation control) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.

By "mutant" it is meant a nucleic acid molecule which is altered in some way compared to equivalent molecules present in a normal individual. Such mutants may be well known in the art, and include, molecules present in individuals with known genetic deficiencies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal expression of a gene, such as cancer, thalassemia's and sickle cell anemia, and cystic 154 fibrosis. It allows modulation of lipid metabolism to reduce artery disease, treatment of integrated AIDS genomes, and AIDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a "wild type" phenotype and/or genotype. For deleterious conditions this involves altering a base to allow expression or prevent expression as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant non-human gene) to a wild type no production of a non-human protein). Such modification is performed in trans rather than in cis as in prior methods.

In preferred embodiments, the oligonucleotide is of a length (at least 12 bases, preferably 17 22) sufficient to activate dsRNA deaminase in .vivo to cause conversion of an adenine base to inosine; the oligonucleotide is an enzymatic nucleic acid molecule that is active to chemically modify a base (see below); the nucleic acid molecule is DNA or RNA; the oligonucleotide includes a chemical mutagen, the mutagen is nitrous acid; and the oligonucleotide causes deamination of methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to In a most preferred embodiment, the invention features correction of a mutation, rather than inactivation of a target by causing a mutation.

i Using in vitro directed evolution, it is possible to screen for ribozymes with catalytic activities different than RNA cleavage. Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences. Science 261:1411-1418. Using these methods of in vitro directed evolution, an enzymatic nucleic acid molecule, or ribozyme that mutates bases, instead of cleaving the phosphodiester backbone can be selected. This is a convenient method of obtaining an enzyme with the appropriate base sequence modifying activities for use in the present invention.

Sequence modifying activities can change one nucleotide to another (or modify a nucleotide so that it will be repaired by the cellular machinery to another nucleotide). Sequence modifying activities could also delete or add one or more nucleotides to a sequence. A specific embodiment of adding sequences is described by Sullenger and Cech, PCT/US94/12976 155 hereby incorporated by reference herein), in which entire exons with wildtype sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 3).

Thus, in another aspect, the invention features ribozymes or enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule. Applicant is the first to determine that such molecules would be useful, and to provide a description of how such molecules might be isolated.

Molecules used to achieve in situ reversion can be delivered using :i 10 the existing means employed for delivering antisense molecules and ribozymes, including liposomes and cationic lipid complexes. If the in situ reverting molecule is composed only of RNA, then expression vectors can :be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes expression vectors.

15 There are several advantages of using such an expression vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that already express that gene. Furthermore, the corrected gene would be o. properly regulated by its natural transcriptional promoter. Lastly, reversion can be used when the mutant RNA creates a dominant gain of function protein in sickle cell anemia), where correction of the mutant RNA is 0. necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.

Endogenous Mammalian RA Editing Syster It was observed in the mid-1980s that the sequence of certain cellular RNAs were different from the DNA sequence that encodes them. By a process called RNA editing, cellular RNA are post-transcriptionally modified to a) create a translation initiation and termination codons, b) enable tRNA and rRNA to fold into a functional conformation (for a review see Bass, B. L. (1993) In The RNA World, R. Gesteland, R. and Atkins,

J.

eds. (Cold Spring Harbor, New York; CSH Lab. Press) pp. 383-418). The process of RNA editing includes base modification, deletion and insertion of nucleotides.

Although, the RNA editing. process is widespread among lower eukaryotes, very few RNAs (four) have been reported to undergo editing in I 156 mammals (Bass; supra). The predominant mode of RNA editing in mammalian system is base modification (C -4 U and A The mechanism of RNA editing in the mammalian system is postulated to be that C--U conversion is catalyzed by cytidine deaminase. The mechanism of conversion of A--G has recently been reported for glutamate receptor

B

subunit (gluR-B) in rat PC12 cells (Higuchi, M. et al. (1993) Cell 75, 1361- 1370). According to Higuchi gluR-B mRNA precursor attains a structure such that intron 11 and exon 11 can form a stable stem-loop structure. This stem-loop structure is a substrate for a nuclear double strand-specific adenosine deaminase enzyme. The deamination will result in the S.conversion of Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.

In the present invention, the endogenous deaminase activity or other such activities can be utilized to achieve targeted base modification.

15 The following are examples of the invention to illustrate different methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific embodiments of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims.

Example 97: xoiti cul RNA dpnent dein to nosne An endogenous activity in most mammalian cells and Xenopus oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. Weintraub, H. (1988). An unwinding activity that covalently modifies it double-stranded RNA substrate..C., 55, 1089- 1098.). This activity can be used to cause an in situ reversion of a mutation at the RNA level. Referring to Figures 102 and 104, for demonstration purposes a stop codon is incorporated into the coding region of dystrophin, which is fused to the reporter gene luciferase. This stop codon can be reverted by targeting an antisense RNA which is long enough to activate the dsRNA deaminase, which converts Adenines to Inosines. The A to I transition will be read by the ribosome as an A to G transition in some cases and will thereby functionally revert the stop codon.

While other A's in this region may be converted to I's and read as G, converting an A to I cannot create a stop codon. The A to I transitions

I

I 157 in the region surrounding the target mutation will create some point mutations, however, the function of the dystrophin protein is rarely inactivated by point mutations.

The reverted mRNA was then translated in a cell lysate and assayed for luciferase activity. As evidenced by the dramatic increase in luciferase counts in the graph in figure 103, the A to I transition was read by the ribosome as an A to G transition and the stop codon has successfully been reverted with the lysate treated complex. As a control, an irrelevant non- S. complementary RNA oligonucleotide was added to the 10 dystrophin/luciferase mRNA. As expected, in this case no translation (luciferase activity) is observed because of the stop codon. As an additional control, the hybrid was not treated with extract, and again no translation (luciferase activity) is observed (Figure 103).

While other A's in the targeted region may have been converted to I's 15 and read as G, converting an A to I cannot create a stop codon, so the ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a dystrophin protein made from an mRNA reverted by this method should retain full activity.

20 The following detail specifics of the methodology:

RNA

oligonucleotides were synthesized on a 394 (ABI) synthesizer using phosphoramidite chemistry. The sequence of the synthetic complementary RNA that binds to the mutant dystrophin sequence is as follows to

CCCGCGGTAGATCTTTCTGGAGGCTTACAGTTTTCTACAAACCTCC

CTTCAAA (Seq. ID No. 1) Referring to Figure 104. fifty-nine base pairs of a human dystrophin mutant sequence containing a stop codon was fused in frame to the luciferase coding region using standard cloning technology, into the Hind III and Not I sites of pRC-CMV (Invitrogen, San Diego, CA). The AUG of luciferase was deleted. The sequences of the insert from the Hind III site to the start of the luciferase coding region is to

GCCCCTGAGGAGCGATGGAGGCCTTGAAGGGAGGTTTGTGGAAAA

CTGTAAGCCTCCAGAAAGATCTACCGCGG (Seq ID No. 2) 158 This corresponds to base pairs 3649-3708 of normal dystrophin (Entrez ID 311627) with a Sac II site at the 3' end. This plasmid was used as a template for in vitro transcription of mRNA using T7 polymerase with the manufacturers protocol (Promega, Madison, WI).

Xenopus nuclear extracts were prepared in 0.5X TGKED buffer Tris (pH 12.5% glycerol, 25 mM KCI, 0.25mM DTT and 0.05mM EDTA), by vortexing nuclei and resuspended in a volume of 0.5X TGKED equal to total cytoplasm volume of the oocytes. Bass, B.L. Weintraub, H.

Cell 55, 1089-1098 (1988).

10 The target mRNA at 500ng/ul was pre-annealed to 1 micromolar complementary or irrelevant RNA oligonucleotide by heating to 70°C, and allowing it to slowly cool to 37°C over 30 minutes. Fifty nanograms of mRNA pre-annealed to the RNA oligonucleotides was added to 7ul of nuclear extracts containing 1mM ATP, 15mM EDTA, 1600un/ml RNasin 15 and 12.5mM Tris pH 8 to a total volume of 12ul. Bass, B.L. Weintraub, H.

o supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 25°C. Next, 1.5ul of this mixture was added to a rabbit reticulocyte lysate in vitro translation mixture and translated for two hours according to the manufacturers protocol (Life Technologies, 20 Gaithersberg, MD), except that an additional 1.3 mM magnesium acetate was added to compensate for the EDTA carried through from the nuclear extract mixture. Luciferase assays were performed on 15ul of extract with the Promega luciferase assay system (Promega, Madison, WI), and luminescence was detected with a 96 well luminometer, and the results are displayed in the graph in figure 102.

Example 98: Base changing activities The chemical synthesis of antisense and triple-strand forming oligomers conjugated to reactive groups is well studied and characterized (Knorre, Valentin, Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993) and Povsic, Strobel, S. Dervan, P.

Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation J. Am. Chem. Soc. 114, 5934-5944 (1992). Reactive groups such as alkylators that can modify nucleotide bases in targeted RNA or DNA have been conjugated to oligonucleotides.

**r 159 Additionally enzymes that modify nucleic acids have been conjugated to oligonucleotides. (Knorre, Valentin, Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993). In the past these conjugated chemical groups or enzymes have been used to inactivate DNA or RNA that is specifically targeted by antisense or triple-strand interactions. Below is a list of useful base changing activities that could be used to change the sequence of DNA or RNA targeted by antisense or triple strand interactions, in order to achieve in situ reversion of mutations, :10 as described herein (see figure 100-104).

1. Deamination of 5-methylcytosine to create thymidine (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

Also, nitrous acid or related compounds promote oxidative deamination of C to be read at T(Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.). Additionally hydroxylamine or related compounds can transform C to be read at T (Microbial Genetics, SDavid Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226- 230.) 20 2. Deamination of cytosine to create uracil (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993) or by chemical groups similar to nitrous acid that promote oxidative deamination (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.) 3. Deamination of Adenine to be read like G (Inosine) (as done by the adenosine deaminase, AMP deaminase or the dsRNA deaminating activity Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

4. Methylation of cytosine to Transforming thymidine (or uracil) to 0 2 -methyl thymidine (or 0 2 -methyl uracil), to be read as cytosine by alkynitrosoureas (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

160 6. Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521;770-778 (1978)-which can be done with the mutagen ethyl methane sulfonate (EMS) Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.

7. Amination of uracil to cytosine (as performed by the cellular enzyme CTP synthetase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

The following are examples of useful chemical modifications that can 10 be utilized in the present invention. There are a few preferred straightforward chemical modifications that can change one base to another base. Appropriate mutagenic chemicals are placed on the targetting oligonucleotide, nitrous acid, or a suitable protein with such activity. Such chemicals and proteins can be attatched by standard 15 procedures. These include molecules which introduce fundamental chemical changes, that would be useful independent of the particular technical approach. See Lewin, Genes.1983 John Wilely Sons, Inc. NY pp 42-48.

The following matrix shows that the chemical modifications noted can 'o 20 cause transversion reversions (pyrimidine to pyrimidine, or purine to purine) in RNA or DNA. The transversions (pyrimidine to purine, or purine to pyrimidine) are not preferred because these are more difficult chemical transformations. The footnotes refer to the specific desired chemical transformations. The bold footnotes refer to the reaction on the opposite DNA strand. For example, if one desires to change an A to a G, this can be accomplished at the DNA level by using reaction #5 to change a T to a C in the opposing strand. In this example an A/T base pair goes to A/C, then when the DNA is replicated, or mismatch repair occurs this can become G/C, thus the original A has been converted to a G.

ISR matrix Reverted Base Mutant base A T(U) C

G

i.

161 A I sTransversion ransversion DNA'3/RNA3 T(U) ITransversion

NARNA

7 Transversion C Transversion

RNA

2

/DNA

6 TransversionI G

DNA

6

/RNA

6 Transversion

T

ransversion 1 Deamination of 5-methylcytosine to create thymidine.

2 Deamination of cytosine to create uracil.

3 Deamination of Adenine to be read like G (Inosine).

5 4 Methylation of cytosine to Transforming thymidine (or uracil) to 0 2 -methyl thymidine (or 0 2 -methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron S* Letters 35:303-306 (1994)).

6 Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978)).

7. Amination of uracil to cytosine. Bass supra. fig. 6c.

In Vitro Selection Strateav SReferring to Figure 105, there is provided a schematic describing an approach to selecting for a ribozyme with such base changing activity. An RNA is designed that folds back on itself (this is similar to approaches already used to select for RNA ligases, Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences.

Science 261:1411-1418). A degenerate loop opposing the base to be modified provides for diversity. After incubating this library of molecules in a buffer, the RNA is reverse transcribed into DNA (that is, using standard in vitro evolution protocol. Tuerk and Gold, 249 Science 505, 1990) and then the DNA is selected for having a base change. A restriction enzyme cleavage and size selection or its equivalent is used to isolate the fraction of DNAs with the appropriate base change. The cycle could then be repeated many times.

162 The in vitro selection (evolution) strategy is similar to approaches developed by Joyce (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635-641; Joyce, G. F. (1992) Scientific American 267, 90-97) and Szostak (Bartel, D. and Szostak, J. (1993) Science 261:1411-1418; Szostak, J. W.

(1993) ILB~ 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein, each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence.

The known nucleotide sequence domain enables: 1) the nucleic acid to 10 bind to its target (the region flanking the mutant nucleotide) 2) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their base modifying activity, 3) introduction of restriction Sendonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry, A. A. and Joyce, G.F. (1992) Sciance 257, 635- .i 641). In this invention, the degenerate domain is flanked by regions containing known sequences (see Figure 105), such that the degenerate domain is placed across from the mutant base (the base that is targeted for modification). This random library of nucleic acids is incubated under conditions that ensure folding of the nucleic acids into conformations that facilitate the catalysis of base modification (the reaction protocol may also i include certain cofactors like ATP or GTP or an S-adenosyl-methionine (if 25 methylation is desired) in. order to make the selection more stringent).

Following incubation, nucleic acids are converted into complimentary

DNA

(if the starting pool of nucleic acids is RNA). Nucleic acids with base modification (at the mutant base position) can be separated from rest of the population of nucleic acids by using a variety of methods. For example, a restriction endonuclease cleavage site can either be created or abolished as a result of base modification, If a restriction endonuclease site is created as a result of base modification, then the library can be digested with the restriction endonuclease The fraction of the population that is cleaved by the RE is the population that has been able to catalyze the base modification reaction (active pool). A new piece of DNA (containing oligonucleotide primer binding sites for PCR and RE sites for cloning) is ligated to the termini of the active pool to facilitate PCR amplification and subsequent cycles (if necessary) of selection. The final pool of nucleic acids with the best base modifying activity is cloned in to a plasmid vector 3S Z^ S 163 and transformed into bacterial hosts. Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques.

Base modifying enzymatic nucleic acids (identified via in vitro selection) can be used to cause the chemical modification in vivo.

In addition, the ribozyme could be evolved to specifically bind a protein having an enzymatic base changing acitivity.

Such ribozymes can be used to cause the above chemical modifications in vivo. The ribozymes or above noted antisense-type molecules can be administered by methods discussed in the above referenced art.

VIIl Administration of Nucleic Acids Applicant has determined that double-stranded nucleic acid lacking a transcription termination signal can be used for continuous expression of 15 the encoded RNA. This is achieved by use of an R-loop, an RNA molecule non-covalently associated with the double-stranded nucleic acid and which causes localized denaturation ("bubble" formation) within the double stranded nucleic acid (Thomas et al., 1976 Proc. Natl. Acad. Sci.

.USA 73, 2294). In addition, applicant has determined that that the RNA 20 portion of the R-loop can be used to target the whole R-loop complex to a desirable intracellular or cellular site, and aid in cellular uptake of the complex. Further, applicant indicates that expression of enzymatically active RNA or ribozymes can be significantly enhanced by use of such Rloop complexes.

Thus, in one aspect, the invention features a method for introduction of enzymatic nucleic acid into a cell or tissue. A complex of a first nucleic acid encoding the enzymatic nucleic acid and a second nucleic acid molecule is provided. The second nucleic acid molecule has sufficient complementarity with the first nucleic acid to be able to form an R-loop base pair structure under physiological conditions. The R-loop is formed in a region of the first nucleic acid molecule which promotes expression of RNA from the first nucleic acid under physiological conditions. The method further includes contacting the complex with a cell or tissue under 164 conditions in which the enzymatic nucleic acid is produced within the cell or tissue.

By "complex" is simply meant that the two nucleic acid molecules interact by intermolecular bond formation (such as by hydrogen bonding) between two complementary base-paired sequences. The complex will generally be stable under physiological condition such that it is able to cause initiation of transcription from the first nucleic acid molecule.

The first and second nucleic acid molecules may be formed from any desired nucleotide bases, either those naturally occurring (such as o.:o.

adenine, guanine, thymine and cytosine), or other bases well known in the art, or may have modifications at the sugar or phosphate moieties to allow "greater stability or greater complex formation to be achieved. In addition, such molecules may contain non-nucleotides in place of nucleotides.

Such modifications are well known in the art, see Eckstein et al., 15 International Publication No. WO 92/07065; Perrault et al., 1990 Nature ~344, 565; Pieken et al., 1991 Science, 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Sproat,B. European Patent Application 20 92110298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these i publications are hereby incorporated by reference herein.

By "sufficient complementarity" is meant that sufficient base pairing occurs so that the R-loop base pair structure can be formed under the appropriate conditions to cause transcription of the enzymatic nucleic acid.

Those in the art will recognize routine tests by which such sufficient base pairs can be determined. In general, between about 15 80 bases is sufficient in this invention.

By "physiological condition" is meant the condition in the cell or tissue to be targeted by the first nucleic acid molecule, although the R-loop complex may be formed under many other conditions. One example is use of a standard physiological saline at 370C, but it is simply desirable in this invention that the R-loop structure exists to some extent at the site of action so that the expression of the desired nucleic acid will be achieved at that site of action. While it is preferred that the R-loop structure be stable under 165 those conditions, even a minimal amount of formation of the R-loop structure to cause expression will be sufficient. Those in the art will recognize that measurement of such expression is readily achieved, especially in the absence of any promoter or leader sequence on the first nucleic acid molecule (Daube and von Hippel, 1992 Science 258, 1320).

Such expression can thus only be achieved if an R-loop structure is truly formed with the second nucleic acid. If a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that transcription is enhanced.

In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by forming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or transcription termination signal such that once expression is initiated it will continue until the first nucleic acid is degraded.

15 In another related aspect, the invention features a method in which the second nucleic acid is provided with a localization factor, such as a protein, an antibody, transferin, a nuclear localization peptide, or folate, or other such compounds well known in the art, which will aid in targeting the R-loop complex to a desired cell or tissue.

20 In preferred embodiments, the first nucleic acid is a plasmid, e.g., one without a promoter or a transcription termination signal the second nucleic acid is of length between about 40-200 bases and is formed of ribonucleotides at a majority of positions; and the second nucleic is covalently bonded with a ligand such as a nucleic acid, protein, peptide, lipid, carbohydrate, cellular receptor, nuclear localization factor, or is attached to maleimide or a thiol group: the first nucleic acid is an expression plasmid lacking a promoter able to express a desired gene, it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNA/DNA heteroduplex; no promoter or leader region is provided in the first nucleic acid; and the Rloop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular transcription machinery.

In other preferred embodiments, the first nucleic acid encodes one or more enzymatic nucleic acids, it is formed with a plurality of T66 intramolecular and intermolecular cleaving enzymatic nucleic acids to allow release of therapeutic enzymatic nucleic acid in vivo.

SIn a further related aspect, the invention features a complex of the above first nucleic acid molecules and second nucleic acid molecules.

R-loop complex An R-loop complex is designed to provide a non-integrating plasmid so that, when an RNA polymerase binds to the plasmid, transcription is continuous until the plasmid is degraded. This is achieved by hybridizing an RNA molecule, 40 to 200 nucleotides in length, to a DNA expression 10 plasmid resulting in an R-loop structure (see figure 106. This RNA, when conjugated with a ligand that binds to a cell surface receptor, triggers internalization of the plasmid/RNA-ligand complex. Formation of R-loops in q: general is described by DeWet, 1987 Methods in Enzymol. 145, 235; Neuwald et al., 1977 J. Vio. 21,1019; and Meyer et al., 1986 J. Ult. Mol.

15 Str. Res. 96, 187. Thus, those in the art can readily design complexes of this invention following the teachings of the art.

Promoters placed in retroviral genomes have not always behaved as planned in that the additional promoter will serve as a stop signal or reverses the direction of the polymerase. Applicant was told that creation 20 of an R-loop between the promoter and the reporter gene increased the j transfection efficiency. Incubation of an RNA molecule with a doublestranded DNA molecule, containing a region of complementarity with the RNA will result in the formation of a stable RNA-DNA hetroduplex and the DNA strand that has a sequence identical to the RNA will be displaced into a loop-like structure called the R-loop. This displacement of DNA strand occurs because an RNA-DNA duplex is more stable compared to a DNA- DNA duplex. Applicant was also told that an 80 nt long RNA was used to generate a R-loop structure in a plasmid encoding the B-galactosidase gene. The R-loop was initiated either in the promoter region or in the leader sequence. Plasmids containing an R-loop structure were microinjected into the cytoplasm of COS cells and the gene expression was assayed. R-loop formation in the promoter region of the plasmid inhibited expression of the gene. RNA that hybridized to the leader sequence between the promoter and the gene, or directly to the first nucleotides of the mRNA increased the expression levels 8-10 fold. The

I

167 proposed mechanism is that R-loop formation prevents nucleosome assembly, thus making the DNA more accessible for transcription.

Altematively, the R-loop may resemble a RNA primer promoting either DNA replication or transcription (Daube and von Hippel, 1992, sugra).

One of the salient features of this invention is to generate R-loops in expression vectors of choice and introduce them into cells to achieve enhanced expression from the expression vector. The presence of an Rloop may aid in the recruitment of cellular transcription machinery. Once an RNA polymerase binds to the plasmid and initiates transcription, the 10 process will continue until a termination signal is reached, or the plasmid is degraded.

This invention will increase the expression of ribozymes inside a cell. The idea is to construct a plasmid with no transcription termination signal, such that a transcript-containing multiple ribozyme units can be generated. In order to liberate unit length ribozymes, self-processing ribozymes can be cloned downstream of each therapeutic ribozyme (see figure 107) as described by Draper supra.

Lioand Targetin Another salient feature of this invention is that the RNA used to 20 generate R-loop structures can be covalently linked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, etc.). Specific ligands can be chosen such that the ligand can bind selectively to a desired cell surface receptor. This ligand-receptor interaction will help internalize a plasmid containing an R-loop. Thus, RNA is used to attach the ligand to the DNA such that localization of the gene to certain regions of the cell is achieved. One of several methods can be used to attach a ligand to RNA.

This includes the incorporation of deoxythymidine containing a 6 carbon spacer having a terminal primary amine into the RNA (see fiure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 L.Big.l_QhaJm 269, 3198-3204). The RNA containing a 6 carbon spacer with a terminal amine group is mixed with folate and the mixture is reacted with activators like 1-( 3 -Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC). This reaction should be carried out in the presence of 1-Hydroxybenzotriazole hydrate (HOBT) to prevent any undesirable side reactions.

168 The RNA can also be derivatized with a heterobifuctional crosslinking agent (or linker) like succinimidyl 4-(pmaleimidophenyl)butyrate (SMPB). The SMPB introduces a maleimide into the RNA. This maleimide can then react with a thiol moiety either in a peptide or in a protein. Thiols can also be introduced into proteins or peptides that lack naturally occurring thiols using succinylacetylthioacetate.

The amino linker can be attached at the 5' end or 3' end of the RNA. The RNA can also contain a series of nucleotides that do not hybridize to the DNA and extend the linker away from the RNA/DNA complex, thus 10 increasing the accessibility of the ligand for its receptor and not interfering with the hybridization. These techniques can be used to link peptides such as nuclear localization signal (NLS) peptides (Lanford et al., 1984 Cell 37, 801-813; Kalderon et al., 1984 Cell 39, 499-509; Goldfarb et al., 1986 Nature 322, 641-644)and/or proteins like the transferrin (Curiel et al., 1991 Proc. Natl. Acad. Sci. USA 88, 8850-8854; Wagner et al., 1992 Proc. Natl.

Acad. Sci. USA 89, 6099-6103; Giulio et al., 1994 Cell. Signal. 6, 83-90) to the ends of R-loop forming RNA in order to facilitate the uptake and localization of the R-loop-DNA complex. To link a protein to the ends of Rloop forming RNA, an intrinsic thiol can be used to react with the maleimide or the thiols can be introduced into the protein itself using either iminothiolate or succinimidyl acetyl thioacetate (SATA; Duncan et al., 1983 .Anal. Biochem 132, 68). The SATA requires an additional deprotection step using 0.5 M hydroxylamine.

In addition liposomes can be used to cause an R-loop complex to be delivered to an appropriate intracellular cite by techniques well known in the art. For example, pH-sensitive liposomes (Connor and Huang, 1986 Cancer Res. 46, 3431-3435) can be used to facilitate DNA transfection.

Calcium phosphate mediated or electroporation-mediated delivery of the R-loop complex in to desired cells can also be readily acomplished.

In vitro Selection In vitro selection strategies can be used to select nucleic acids that a) can form stable R-loops b) selectively bind to specific cell surface receptors. These nucleic acids can then be covalently linked to each other.

This will help internalize the R-loop-containing plasmid efficiently using receptor-mediated endocytosis. The in vitro selection (evolution) strategy is 169 similar to approaches developed by Joyce (Beaudry and Joyce, 1992 Science 257, 635-641; Joyce, 1992 Scientific American 267, 90-97) and Szostak (Bartel and Szostak, 1993 Science 261:1411-1418; Szostak, 1993 TIBS 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence.

The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (a specific region of the double strand DNA), 2) 10 complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their affinity to form R-loop and/or their ability to bind to a specific receptor, 3) introduction of a restriction endonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides represented at every 0 15 position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 Science 257, 635-641). In this invention, the S•degenerate domain is flanked by regions containing known sequences.

This random library of nucleic acids is incubated under conditions that ensure equilibrium binding to either double-stranded DNA or cell surface receptor. Following incubation, nucleic acids are converted into complementary DNA (if the starting pool of nucleic acids is RNA). Nucleic acids with desired characteristics can be separated from the rest of the "i population of nucleic acids by using a variety of methods (Joyce, 1992 sura). The desired pool of nucleic acids can then be carried through subsequent rounds of selection to enrich the population with the most desired traits. These molecules are then cloned in to appropriate vectors.

Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques.

Other embodiments are within the following claims.

Page(s) 2 1 K- are claims pages they appear after the sequence listing 170 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, bluegreen 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 all are bacterial in origin.

Hammerhead Ribozyme Size: -13 to 40 nucleotides.

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

S.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 (Figures 1 oand 2) Hairpin Ribozyme Size: -50 nucleotides.

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) Ribozyme 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. Found in human HDV (Figure 4).

Neurospora VS RNA Ribozyme Size: -144 nucleotides (at present) 171 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 172 Table 2 Human lOAM HH Target sequence nt. Position Target Sequences CCCCAG C GCwXCtJ-G CUGAGCU C ctJCUGCE nt. Position Target Sequences lo...

00* oo 9 .00.

U1 23 26 31 34 48 54 58 64 96 102 108 115 11-9 1-20 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 AG-tTCC CtCUGC-U

UGCUACT

UCAGAGU

C-CAACCU

UCAGCCU

CCU~CGU

UAUGGCU

CCGCACtJ

UCCU

t7CCUGCU

CGGGGCIJ

GCUCUGU

CtTCtGUU

CAGACAU

UCUGUGU

UCCCCCU

CAAAAGU,

AAGUCAU

GGAGGCUi

AGCACCU(

CCCAAGU I

AAGUUGUI

UGGGCAU

ACCCCGUJI

GUUGCCU I

AAGGAGUI

AGUUGCEJ C AAGGUGtJ

AGAAGAU

AUGUGCUJ.

GUGCUAU L UGCEUAUU c GGGCAGtJ c AACAGCU A AAAACCtJ U AAACCUU C CCTUCCU C C L'GCUACU A CUCAGAG C AGAGUUG U GCAACCU C AGCCTJCG C GcahuGG A UGGCUCC C CCAGCAG C CUGIGUCC C CUGCUCG C GGGGCC C UGUUCCC U CCCAGA C CCAGGAC C UGUGUCC C CC CCCA C AA.AAGUC C AUCCUGC

CCUGCCCC

CCGUGCUG

CCUGUGAC

3 GUUGGGC 3 GGGCAfLA k GAGACCC I GccaAAA

LAAAAGGA

I GCUCCUG

CUGCCUG

UGAACUG

LGCCAACC

6UUCAAAC rCAAACUG

AAACUGC

AACAGCU

AAACCUU

CCUCAkCC CE3CACCG

ACCGUGU

386 394 420 425 427 450 45i 456 495 510 564 592 607 608 609 6211 6=:6 6=-7 668 6-7 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851

ACCGUGU

CUGGACU

CACCC

AGAA =tJ

GAACCUU

CCAACCU

UGCUGCU

CUGAGGU

GAGAGU

AGCCAAtJ

GCCAAUU

CCAAUUUj AAUUt7CU GAGCUEtJ 1 AGCrUGJU I GCCCCCtJ

ANCCAGCEI

CAGACCu i AGACCUU t CCUrjUGU AGCGACU c CACAACt3 L AACUUGT C CCCGG~T c CGGEJCCU CCGUGGTJ C GGUCUGu T) GtYCUGtJU C GG-UG~u u GGCtJGUU C UCCCAGU C CCAGt3CU C CCCAGGU C CAGAGTJU Z CCAC.XGU C GUC;LCCU A A, CUGGACU

CAGALACG

3 GGC-CC 3 ACCCUAC :L C=CUACG

ACCGUGG

CG=-C-GG

ACGACCA

ACCAUGG

TUCUCGt7G

TCUCGUGC

UCGUGCC

GUGCCGC

UGAGAAC

GAGAACA

GGCCCCC

CCAC-CUC

CAGACCU

UGUCCUG

GUCCUGC

CUGCCAG

CCCCA6CA

GUCAGCC

AGCCCCC

CUAGAGG

GAC-GUGG

UGUUCCC

CCCUGGA

CCIGGAC

CCCAGUC

CCAGUCU

UCGGAGG

C-GAGGCC

CACCtJGG

GAACCCC

ACCUAUG

UC-CCAAC

173 o 0* 0 0* *0 0* 863 866 867 869 881 885 933 936 978 980 966 987 988 1005 1006 1023 1025 1066 1092 1093 1125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 1334 1344 1351 1353 1366 1367 1368 1380 1388 1398 1402 AACGACU C CUtUMCG C-ACJCCU U CtJCGGCC ACUCCUU C UCGGCCA UCCDUCTJ C GGCCAAUG AAGGCCU C AGUCAGtJ CCtJCAGU C AGGGA GtJGcAGU A AflACUGG CAGUAAU A C~GGGGA UGACCAIJ C UAC-AGCU ACCMJJC A r-CAGCUU UACAGC-U U UCCGGCG, ACAGCUU U CC,-GGC CAGCUUU c CGGC-GCC_ ACGUGAU U CUGACGA CGUGAUU C UGACGA.A CAGAGGU C UCAGAAG GAGGUCUJ C AGAAGGG CCAC--CU A GAGCCAA AUGGGcGU U CCAGCC UGGGGUU C CAGCCCA CCC-AGCU C CUG%-tJGA CGCAGCU U CUCCUGC GCAGCUU C UCCUGCU AGCUUCTJ C CUOCUCE) UtCGCEJ C UGCAACC C-CCAGCU U AUWACA CC-AG-CU A UACACAA AGCEJUAU A CACAAGA C-C-GAGCU U CGUGUCC GG-AGCUEJ C GUGUCCEJ UUCGUGU C CUGLUG GUJCC=GE A UGGCCCC GAGGGAU U GUCCGGG GCGAUUGU C CGGGAAA AGAADA U CCCAGCA G-AAAAUjU C CCAGCAG GCAGACU C CAAUGUG CCAGGCU U GGGGGAA AACCCAU U GCCCGAG CCGAGCrJ C AAGUGUC CAAGUGU C UAAAGGA AGUGUCU A AAGGAUG UGGCACU U UCCCACU GGCACUU U CCCACUG GCACrUUU C CCACUGC tJGCCCAU C GGGGAAu C-C-GGAAU C AGUGACE UG-ACUGE) C ACUCGAG UGUCACU C GAGAuUE 1408 1410 1421 1425 1429 1444 1455 1482 1484 1493 1500 1.503 1506 1509 1518 1530 1533 1551 15959 1563 1565 1567 1584 1592 i599 1651 1661 1663 1678 1680 1681.

1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802 1812 1813 1825 1837 1845 U'CAC-AU C UUGAGGG GAGAUTCE U cGCA GGCACCEJ A CCUCUjGU CCTJACCU c uGuCGGG CCEJCUGUj C GGGCCALG GAGCACU C AAGGGC-A GGGAG-.U c ALCCCGC:G AUGUCU C UCCCCCC GUOGC--UcE C CCCCC CCCCG-GU A UGkAGtU AUGAGAU U GUCAUCA 2LGAEJUGrJ C AUC;LtJCk UUGUCAU C AUCACUG Ur-UrAE) C ACUGrJGG CUGUE A GCAGCCG CCGCAG;U C AUAAUGG CAGUCAU A AUGGGCAz CAGGC-CU C AGCACCGU AGCACGEJ A CCUCUAU CGLTACCU c UTAzCC UACCUCU AL tAACCGC CCUCEMtJ A ACCGCCA GGAAGArj C AAGAAAU AAGAAAU A CAGLCEJA ACAGACTJ A CAACAGG CACGCCEJ C CCUGAAC UGAACCU A UCCCGGG, XACCEJAU C CCGGGAC AGGGCCU c uucCUCG GGCC-UCtJU ccucGGC- GCCUCUU c CUCGGCC UJCUUCCU C GGCCUUC UCGGCCTJ U CCCAUJAU CGGCCUU C CCAUJATUJ UJUCCCAU A UUGGUGG CCCAUTAU U GGUGGCA AAGACAU A UGCCAEJG UGCAGCU A CACCUAC UACACCEJ A CCGGCC~c AGGGCALu u GuJCECA GCAUUGU C CUCAGUC UUGUCCU C AGUCAGA CCUCAGU C AGAUACA GUCAGAU A CAACAGC ACAGCAU U UGGGGCC CAGCAUU U GGGGCCA CCAUGGU A CCUGCC CACACCEJ A AAACACU AAAC-ACU A GGCCACG 174 1856 1861 1865 1868 1877 1901 1912 1-922 1923 1928 1930 1964 1983 1996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2115 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173 2174 2175 2176 2183 2185 2186 2187 tTGGAUGU GGAL7GUU

UUAAAGU

AAAGLTCU

GAGACAU

AGGACAU

GGGAAAU

UGAAACU'

C-C-UGCC-U.

UGCCU

ACAGACO I

CAGACUU

UGG%-CCO(

CCUCCAU;

CAUGUGU;

GtTAGCAUC CCACACU t CACACUU C GCCAGCU L CUG%=J-J C GC-UG7JCJ A CAACCC TJ UGAUGAU A G;AL'tGtJ A UAUGUAU U At3GtUtU u tJGt.W3UU A UALUCAU UJ AUU!MUU C tUUCAU U AtJUCAUrJ U CALUUrJGU U AUULUGU A UUUA U t7GU!AUU U GUUAUUU U UUAULrXt A ACCAGCEJ A CAGCMJU U AGCUAUU U G%-tVATUUU

A

U AAAGUC A AACCJA C UAGCCOG A C-C--UCAU A GC=CAC A CAACUGG A CUGAAAC UY G C UG'-U A~ UUGGGUA Ly GGGUAUG ;L UGCUGAG J3 ACAGAAG k~ CAGNPAGA

CAUGU

GCAtTCAA

AAAACALC

ICCLGACG

CJGA-;CGG

TGGGCACU

UACTJGmC

*CUG-ACCC

GUGATlUU

UUGAUUC

UALUUCAU

AUrJ-C AIJU UUCAmU

CAUUUGU

AUUUGUU

UGEXVLT-U

GUUAUUU

AUUUUAC

UUULVACC

UUACCAG

UACCAGC

ACCAGCUj

CCAGCUA

UUUAUUG

UAUE3GAC; AUUGAGtJ

UUGAGUG

CACCAUW C UGiflCUG AUCUGAU C U=tAGUC G-AUCUGU A GUCACAU CtJGCAhGU C ACAUGAC CAUGACU A AGCCAAG CAAGACU C AAGACAU ACALUAU U GAUGGAU 2189 2196 2198 2199 2200 2201 2205 2210 2220 2224 2226 22-3 224 2 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 UAUUAU U GAGUUC MUG C UUUUG AGVGUCEJ U UMVJGUA, GUGUCUU U UAXJGUA UGCUUU U AUGLAGG GtJCUUUU A UG-3AGGC uLUUAxGu AL c-GMAA GtIAGGC-U A AAUGAAC UGAACAU A GGCjC CAUAGGtj C UCUC-GC UAGGLUcj c LrG-_,jc CUICGCUt C :LCGG;LGC CGGAGC.T C CZ-AGUCC UCCCAGU C CAUGEJCA UCCAMlU C ACAUUCA GUCACAU U CAAGGUC UCMcAU C AAGGDCA UCAAGGrJ C ACCAGGEJ ACCAGGU A CAGUUGU GU:;AG U GUACAGG CAGUUGUJ A C-kCGUUG UACAGGEJ U GEUhC~cr AGG-UQGU A CACUGCAk AAAAGAU C AAAI7GGG UGGGCACU u CUC~uG GGG-ACUU C UCAUUGG GACtJUCU C AUUGGCC UUCUCAU U GGCCAAC CCUGCCU U UCCCCAG CUGCCUU U CCCCAGA UGCCUUU C CCCAGAA GAGUGAU U UUUCU AGUGAUU U uucaAUC GUGAUJUU U UCLUflCG UGAUUUU U CUAUCGG GAUUUUU C UAUCGGC UUUUtJCE A UCGGCAC UUUCEJAU C GGCACAA AAGCACU A tUGGAC GCACUu A UGGACtIG GACtJGGU A AUGGUUC UAAUGG;U U CACAGGtJ AAUGGUTU C ACAGGUU CACAGGU U CAGAGAU ACAGGTJU C AGAGAUE) CALGAGAU U ACCCAGU AGAGAUJU A CCCAGUG GAGGCCU U AUUJCCUC AGGCCUU A UUCCUCC 175

'I

K

2451 2452 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2545 2568 2579 2585 2588 2591 2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2691 2700 2704 2711 2712 2721 2724 2744 GCCUUAtJU CCUCCCU kccu c CCUCCC CCUCCCU U CCCCCCA CUCC=U C CCCCCAA GACAC=t U UGUUAGC ACACCUt) U GuEahGCC CCUUUGU U AGCCACC CtJUUGUU A GCC-ACC-U GCCACCU C CCCACCC CCCALCAU A CAUUUCU CAUACAU U UCUGCcA AUACAtJU U C=GCAG UACAUUEJ C UGCCACU CCAGUGU U CACAAVG CAGUGUU c AcAAuGA UGACACE) c AGCGGuc CAGCGGU C AUGUCUG GUCAtUD C UGGACAU AsGGGAAIJ A UGCCCAA CCAAGCU A UGCCtJUG UAUGCCtJ U GUCCtJCU GCCUUGU C CUCtJUGU UUGUCC C u uGCCU GUCCUCt) U GUCCUGU CUCUtJGt C CUG7UUG GUCCtJGU U UGCAJJEJ UCCUGtjU T GCAtuuuc UtJUGCAUJ U UCACUGG UUGCAUU u cAcuGCG UGCAUUU c AcuGGrA GGGAGCU U GcACuU UUGCACEJ A UEJGCAGC GCACEAU U GCAGCUC UGCAGCU c cAGuuuc CUCCAGU U UCCUGCA UCCAGCrJU U CCUGCAG CCAG~tU C CUGCAGU CACUIGAt) c AGGUcc UCAGGGtJ c cuGcA CCAAGGU A UUGGAGG AAGGLrhIJ U GGAGGAC GAGGACt) C CCUCCCA ACUCCCU C CCAGCrU CCCAGCU Ui UGGAAG CCAGtJU U GGAAGGc GAAGGu c AUCCGCG GGGTJCAU C CGC-GtJ UGUGUGU A UGUGUJAG 2750 2759 2761 2765 2769 2797 2803 2804 281-3 2815 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 293 2914 29315 2916 2917 2918 291.9 2931 2933 2941.

2951 2952 2955 2956 2961 2962 2965 UAUGUJGU A GACAAGC ACAAGCrJ C UCGCtJCU AAGCUJrJ C CUCEJGT UCUCGICU C UGUCACC GCUCUGU C ACCCAGG GUG-CAAU C AUGGUUC UCAU'GWr U C-kCGCk CAUGGt) C ACUGGAG '-CCAWj C UG-ACCU GCAGUCU UJ GACCtUU UUGACCj u utGGGC-u UGAC--UU U UGGGCtJC GACCUUU U GGGCUCA UUGGGCU C AAGUGAU AAGUGAU

CUCCCAC

U-GAUCI-V C CCACCUC CCCACCU C AGCC-UCC UCAGCCU C CtJGAGUA CCUGAGj A GCUGGGA GGACCAU A GGC-UCAC ALMGGCU C ACAACAC GGCAAAUy U UGAUUUtj GCAAAUtj U GA.UUUU AUIuA U UUUUUtUU UUUGAUU U UUUUUU UUG-AmUU u UUUrJU UGAUuuu u urUUUUU GAUEUUU U UUUXU~ AUrUUUU U UUrjUUUU UUEJUUUU U UUUUUUU UUUUUUty U UUUUUUU ULUUUU U UUUUUUC UUUuu U UUUUUCA, UUUtUUU .U UuuucALG UUUUUJU U tUUCAG-A UUUUUUU U UCA-GAG UUUUU~rU U UJCAGAGA UUUUUUU U CAGAGAC tUUUUEUtJ C AGAGACG ACGGG-U C UCGC-AAC GGGGUCt) C GCAACAU C-CAACAU U GCCCAGA CCAGACtJ U ccuuuGU CAGACUU c UUGUG ACUUCCtx U UGGUA CtJUCCUU U GUGUUAG UUUGUGU U AGUUAAU TUUGUt A GUTJAAUA UGUUAGTJ U AAUAAAG

S

S

176 2966 GULUhGUU A AUAAAGC 2969 AGUtUA A AAGCU 2975 tkkAA-CU U UCUCAAC 2976 AAAG--C U CUCMACU 2977 AAGCUUU C LUCAACEIG 2979 G-t7UUCU C AACEUGCC 'Table 3 Mouse lOAM HH Target Sequence nt. Position Target Sequence nt. Position Target Sequence 0* 0 00..

4, 11 23 26 31 34 40 48 54 58 64 96 102 108 115 1219 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 785 786 792 794 807 833 846 851 CCCugGt CaGuGgU uGgE~uCrJ

CJCUGCU

UUCUcatJ gCAC*-?CU aggACCU CaUgc-tJ c-AcccCU CLucugcu UgCcaGU cuCVGCU uGGuuCt7 C-gaaUCtJ CDCUGcUC UCUGLJGUJ

C

CAgAAWr u AAGcCuU c GGuGGgU

C

gcCCUt C CagAAGU

U

AAGUUGu u UGUIGCuU u AaCCCaU c ccUGCCU

A

AgGGuuU c AGggGCrJ

C

AAGct3GUu C C CIGUG u CTCtJCU C LMGCUCCU c CUCcaca a AC-GUcG, U GuAgCC-3 C -AG-CgG C GuAUGG U UaGCUCC C CCAGCAG C CVGGcCC EL CVGCE~gG :CUGGCcC

UGCUJCCU

aCCGA CugGccC cG"CUUCC: agCCaCu LAAAAaC gUuuUGC

CUGCCCC

CGUGCaG CUCUGgC GUuuGC UUGCucc GAGAaCu uCCO~AA AggAaGA UCUaCOTG CUGCCUa

UGAG

367 374 375 37 8 386 394 420 425 427 450 451 456 495 510 564 592 607 608 609 E :-I 656 65 7 668 677 684 692 693 696 709 720 723 735 738 765 769 770 1353 1366 1367 1368 1380 1388 1398 1402 AAugGCrj u c-AACCcg gAAgCcU U CCUgccC AAgCCUU C CL~gCCCC CuacCaU C ACCCGU ACCGUGtJ A UcGuUU CCGGr-ACr U ucG-AUOLu CACaCuU C CCCcCcg CaCCCCU C CCaGCAG CagCucu c aGCAGug AGgACCU c ACCCUgC GAAaCcU u uccruuUG UUACCCU c aGCcaCu CUAcCaU C ACCGUGu UGCUGCrJc

C-,JGC,-,

CEUCAGGrJL a uCcAuCc GA;aAGAU C ACaugGG AGCCAAu U UCUCaUG GCCAALUt U CUCaUGC C CA AU U C UCaUG-C AAUUUCU C allGCCGc &AGC-UGU UI U-AGcug AGC-UGUU U GAGcugA cgagCcu a GGCCaCC GaCCUCU A CCAGCcU UUCAG,-rJ C CgGuCCU C9GACUU U cGauCt~u AGgaCcu c acCCUGc CCtUgtUU C CUGCCuc gGCGgCU C CaCCUCA uACAACEJ U uUCAGCu AACUUUEI C AGCuCCg aCCaGaU C CUgGAGa I-GC-gCcu c GuGaLTG CaGUcG;U C cGcUuCc GG-CCUGU U U.CCUGcc UUr-TcU C CCrJGGAa AGUC-ggU c gAaGgtJG UaaCAgU c UaCaACtJ aGCACcU c CCCACcu GuACugU a CCACt~cu UGCCC-AU C GGGGugg GGaG-AcU C AGUGgCEJ UGgcUGtJ C ACaga.Ac UGUgcU-U u GAC-AaCUj cUGUGCU GT~cCaAU aGCUgU GuGCAGtJ GGcCTJGU GcCT3GUU UggagGU CugGgCU CUCgGaU CAaAGcU CCCUgGUI GagACCU UgagAAC

CACACUG

gAgCrJGa gUCcGCrJ UCCUGcC CCuGcCU UJCGG-AaG GGAGaCu

UACCTIGG

GAcaCCC ACCguuG UacCAgC 178

N

S

863 866 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 1092 1093 1125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 1334 1344 1351 1793 1797 1802 1812 1813 1825 1837 1845 AgCCACU GAagCCUT AkuGGCuU CUgGu cUauAaU uAaUcAU tUaACagU ACagTCU UACAaCU ACAaCUU CAaCUL7 ACcaGAU uGaGAgU3 ugG-AG-U

GAGGUCU

CCACuCTJ AcuGGaU.

UGGacct3 CCCAaCtJ i CGaAGCU .1 GaAGCtU ;.GCDTJCt UCCtGUU A cCuCU( gCuGC-UU I AcuUUuU i GGUAcaU a GaAGC3U C tJUCGUUU C GAaGGgY c uGAgaGtJ C AGgAgAU a G;ggggtj C GCAGACU c gaAGGCY c AACCCAUc auGAGCtJ C ugAaUGU a UgGUrCCU C CacCAGU C acCAGAU c ACuGgAU c CAGCAtJU U CCACGcU A u ccucugG U CCUGcCC u CCagA C augCAAG C AacCc(tJ a gagGUJGA c AUuC3GG u CUGGuGc A CAaCUUU U UuCaGCu U uCaGCuC u CaGC-uCc c CtJGgaGA C UGggGAA C UCgGAAG C gGAAGG a.AkauAA uCAGgCC *i CAflCCaA J CUuuUGC UuuUGCtJ a1 UGCUCU i aaaAACCcrcCACA I UgaACAg I CACcAGu LCGtJGUgC CgGagaG t7GGuCCu Gt~gCaaG uGGGgAA CugAGc

UCAGCAG

ugAaaUG aGGaGgA uCCuaAa gAGaGUg UAAguuA gGotigGA AcAflAaA.

CuggAGa UcaGGCC acccucA CCUctigc 1408 1410 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 1518 1530 1533 1551 1559 1563 1565 1567 1584 1592 1599 1651 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 2173 2174 2175 2176 2183 2185 2186 2187 gCGAGAU C ggGgaGG GAC-gU~t c GgaaGgg CCCACC!J A CuUuUGU aCUgcctJ u gGUaGaG UCEJCtaU u GccCCuG GAaggCU C AgGaGGA GGaAuGU C ACCaGga AgUUIGU~U u Ug ccc cUGutjCr u CCuCauG CLIguGCU u UGAGAac AXJGAaATJ c allggUCc gG-ActaU a AUCAUuc UUaUguU u AUaACcG cuAcCAU C AccGtGu ucar-GG-U c CCAGgCG CuauAaU C AUUCUGG ugGUCAn u gUGGGCc CAuGCCEJ u AGCAgcU AGCACCLY c CCcaccT CUUAugU u UALAhACC U~ugUuU A LMAAfCGC ugtuUWO A ACCGCCA GaAAG~tj c AgGAuAU AgGAUAU A CAaguUA ACAaguU A CAgaAGG CcCaCCTJ C CCUGAgC gaAACCUu UCCuuuG AACCUuEJ C CuuuGAa AGGaCCU C agCCUgG aGCCaCEJ U CCEJCuGg GCCaCUJE C CUCuGgC aCOjUCCU c uGgCtjgu cCGGaCEJ U uCgAUcu CGGaCt2U u CgAUcUEJ UgCCCAU c ggCGGUGG CggAUjAU a ccUGGag gAGACcU c UaCCAgc gGCgGCU c CACCca gAagCCU u CCuGCC gaGaCAU U GUCCcCA GCAUUGu u CtCuaau UUagagU U UUACCAG UagagUU U UACC-AGC agagUUU U AcCAGCU gagUUUtJ A CCAGCEJA ACCAGCrJ A UUUAUUG CAGCUTAU U UAUtJGAG AGCUAtJU U AUUGAGEJ GCUAUUU A UUGAGt~a cgAgcCtJ A GGCCACc 179 i856 1861 1865 1868 1877 1901 1912 1922 1923 1928 1930 1964 1983 i996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2 115-- 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2417 2418 2425 2426 2433 2434 2448 2449 CggaCuU AcaUGAU cAcuUGE7 CaccAGU

CAUGCCU

uAAaACU AuAI~agU tTGaPJJGU uGAUGCU UrJAgAGrJ AgAGt~uU

GAACAU

AG-AuAU.

aG-GAgAXJ TGgAgCU G%-tUauutj LrGC-CcAU gguGGuU gCuGgCt3 CuGACcU MicuCCU CuaCCAUc CAcuUGU GtJAGCctJ C CaACuCt7 L CACACUEJ C GCCAGCTJ c CaGCUaU u cCU.Gt~uU c CAACuCY U tUauUaAU u uugAUJGU A gAUUU U AUGUAUU U UGUA~UUU A LUfUUAU U AUgtrAUU u acUUCAtJ u AkUguAUU U UUUaU U AgUUGtJU u gAAUIGWt a AcUGGau c CAUgGGU c AkuuaaUU u uAG-AGutJ u AG-AGuUU u G-AaGCCU u AaGCCUUr c u CGAUJCLu a UccAGt~a A CUCAg C ACAUaAa u AGCagau C AAGggAc a GAUcagU a uAAGUua c AgGUaUc u UuaCCaG u aCCaGcU u GuCCCca A CAAgt~ua h CDCGAgcC a GCgGaCc ;L UUGaGUA :GGGgugG

:UUCUGAG

L gCAGAgG CuGgAGg CAcAucacCgUGU LGCcCCAg rCUM.AuG Cccc,-c-G GGaggaU UrAuUr.Ag Ctr c C-1C cuXCAfg UagAqUU UUtCh.Ua UAUUaAU AUUaAEUU UUaADUU aAIUUag AUUaaUUr CCUcfU allUaUUJ AaUUU~hg UgcUcCC CAuAcGU UCAGGcc gAGgGuU AGAGuUU uaCCAGc aCCAGcu cugcC cugcCC 2189 2196 21.98 21-99 2200 2201 2205 222.0 2220 2224 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 22 91 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2691 2700 2704 2711 2712 2721 2724 2744 UJAUUUAU U G-AGUacC caAcUcU u cUUgAUG gcaGccu c uTvjG-u GCCUCtUU a UgUuUku UCtUUCCU c AUGcAaG aagUUtj AL UGUcGGC UTJUAUGU c GGCCugA GgAGaCU c AgUGgcu-, c'-g9CAU uUGE;CrjCUj C-jcAG-tj a UCcauCC UgGaUCtJ C &aGGCCgC Ct;GaC::j c cuGG-AGg UC-GAr,-U a gCgGaCC tjauCcatj c CAUccA- UCCAauU C ACAcUgA allCACAU U CAcGGUg UCACAULI C AcGGUgc ggAAUGU C ACCAG~a ACCAGaU c CuGgaGa GaAggW u C UgCAaG aAGCUGU u ugaGcuG UAuAaGEJ U allggcCu caGJgGU u CUCVGCu gAAAGAU C AcAUGGG UGaGACU c CUgccUiG GaaACcU u u-CCUI~uG GACcCUE a ccaGccu UUucgAU c uuCCAgC CCcagCU c UCagCAkG CUGCuUU U gaaCAGA aaCCEUtU C CUuuGA6A agGUGgU U CUUCUga gGUGgUU c UUCt~gag agGgUEUu c UCUTACUG U]GcUUU c ucAI~aaG aAgUUUUj a ugTJcGGc alUtCUCUi A UuGCCC allcCagu a GaCACAA AAaCACU A UgUGGAC aagCtigt u -uGagCUG uACtr.GU c AgGaUgC AAuG3cU c cCGAGCcC GAaGcCU u CCt~gCCc gaCUCU a CCAGCCU CCCAGCU c tUcagcaG gagGucti c GCAAGGG GAAGGr-tu C gtlgcaaG GGuaC-AU a CGuGUGc gGUGgGU c cGUrcAG 180 2451 2452 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2545 2568 2579 2585 2588 2591 2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2941 2951 2952 2955 2956 2961 2962 2965 CCUguu gAagCC CCaCaC CaCaCU GAgACC uCACCgl CCaaUGI cttuuuu agCACCI CC-CACcl uAUcCA= ut3LAggt LlAgAgUl

CUUUUGE

CA~caUE tJGAUgCt CAXGCaGE Gt~gcUMt guGaAgt auAA~uL cugGCat GCaU=G UgGuuC cuCutu CUuUUGU acCgrUGO UCCaGcU ct~cGgAU caGCAgU gGaATJgU iiGCAcCU UUuCgaU GCACact7 UuCAGCU ggCCuGu cCCAGcU CCuGUUrJ uAcUGgU gaAGGGU CuAAuGU GagACAU CCAcgCU CAGcagt7 AgUgaCti uuucctIT UcUGUGEJ allGUaUUJ UuUgAaU u C CLugCCc U u CCrjgCCC LT U CCCCCCc U C CCCCCcg U3 c UaccAGC 3 U GUgAnCC J c AGCCACC J c aCCAguc J C CCCACCU J A CuUUUgU J c caUcCCA 3 U uUaCCAG I u UaCCAGc I U CcCAAUG 7 u ACccUcA IC AGguaUC 7C cgcigUG Ta UGGuCcU Tc U~uCaAA rA U~gCcUG rU uCUCU ru CtTCraaU rC Ugcucct7 U GcCCUGc u CccaaUG a UuCgUtU a cCAE~ccC a UacCU)GG c CgCUGuG C ACcaGGA aCcCtigc

UUCCAGC

U GuAGCcu C CgGUccu U UCCU)GCc c uCaGCAG C CUGCcuc C AGGaUgC C gUGCAAG c UccGAGG U GUCCccA a CCUcL-,, C CgcJGUG c UGUIGUcA UJ GaatjcAa AGccActJ aUlUAAEUU

AAUAAAG

GCCt~gUU U CCUg=C 2750 2759 2761 2765 2769 2797 2803 2804 2 8:13 282-5 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 291-2 2913 2914 2915 2916 2917 2918 2919 2931 2933 UAtUt.,tat cCggaCtj AgGacCti

UUUGC-U

agJCU~TJ allGaAAU UCATiOC%-U ggUG3gtJ Cccgc-u aCAG;UCtJ ccGACtJ gGwhgct ugCCUUtJ cLUGGaCtJ AgGUGgU UGAgaCU CCaAugtJ gCAGCCtJ gCcaAGU GGACCutJ UUcCCG-tJ cGgAcuUI UUAAuUU ACUE~cAUI cUUCATUJ UUGAUgU UGuaUrU-j a GAagctij- c AgcUjtcU L tUgUat3DU a UgUatitit a UtigUtict c UUUCcU a UgcUtitjt c aUlUUaUUT a UaUUcgU u aUlUcgUu u jucgULOJU c tiUctjcaU a ugGaGGU C GaGGucEJ c u GAguAcc u UC-%aE3Ct aCcCUGc :uGcCgCu AaaCAGG AUGGtJC CcagGCg cgCG CUGACCc LcAaCUtU ,ct7Gagg CGGaCtiu LuCC.C LuAat~cAU LCtu~U0ga CugCCtug AGCCaCC utiauGtiu aC;GuG-A aGCcaAg cCAuCAC cG-AUcUU GAgUUUU~ cUct~aUU Uct~aUjUg tUtrJaUTa UrjaaUUU UEUUMgctJ tmgcucu UE.aaUUU UUaaUUU UaaUgtiC CLTggUCA, UcaUa.AG a~Tj-uAGA UcCgGAG cCgGAGA CgGAGAg AG9GU.CG UCGgAAg GgAAggg 2966 GcUgGcU A gcAgAGg 2969 Aa~cAAU A AAGLuUUUr 2975 UAgA~uU U UacCAgC 2976 gAgGgUU U CUCUACrJ 2977 AAGCUgU U .gAgcUiG 2979 uCaUUCY C uAxuGcC 182 Table 4 Human IOAM HH Ribozyme Sequences nt. Position Ribozyme Sequence a a a. *5

S

a.

aSS.

11 23 26 3, 34 48 54 64 96 102 108 115 1U9 1.20 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 394 420 425 CAGCGt3C CUGAVGAGGCCGAAAGCCGA ACrCG AGCAGAG CUGAGGCC,-,-kz CAA AGCt7CAG AGUAGCA CUD AUGAG.GCC-,AAACCCGA

AGGAGCJ

CUCUGAG CUUGG-,-C-,CGA A~ AGCAnG CACCU CUGAUrGG-CC-AAG-CCG;A~

AGUAGCA.

AGG-UUC-C CUGAt GAGGCC ACUC-,-Ca CGAGGCtJ C~rJGAtGAGGCr~-AAGCGAA AGGa~ CCAAGC CTCA;AGCCAA,,CA AC GCu GGAGCCA CGAMG-CGA--CA

AGCGAGG

CrJGCJGG CUGAt3GAGGCCGAAGGc=-AA ;rAA GGACCAG CGGArCCGAGCC,-.A AGtMCGG CG-AGCAG CAGAGCCGAAAGOCC

ACCAGGA

GAGCCCC Cr-AUAGCCAG--CA

AGCAGGA

GGGAAC CUaDGAGCC 'A CGAA AGCCCCG DJCCUGGG CLC-AUGAG7GCCrG-AAG~CCGAA

ACAGAGC

GTJCCUGG CDG-AUGAGGCC-AGCGA

AACAGAG

GGACACA. C=GUGAGGCC-,-AGOCCAA AUGUCrJG tJGAC,2G-,GA =GGC. G ACACAC.A GACUUtTU CGA GAG C-,CAAG-,C

AGGGGGA

G'CAGGU CLGAVGAGGCCGAAAGGCCG.A ACUUrUUG GGCAG CUGAUGAGGCC =GAA AUGACUE) CAGCACG CUGAUGAGGCCGA=GAA~

AGCCTJCC

GUCACAG CUAGGCCAA--CA

AGGUGCU

GCCCAAC CUGAUGAGGCCGAA GGCCGAA

AC'ULIG

UArJGCC C~AGG-C-ACGCA

ACAA=U

GGGTj~ct7c Ct=UA~C-AAAGCCCGAA

AUGCCCA

UUUAGGC CUGAUGAGGCCGA CCA;. ACGGGGrJ UCCUUD CUGAkUGAGCCCAGI,-CCAA

AGGCAAC

CAGGAGC CUGACGAGGCCCQ AC-CA ACUCCtU CAGGCAG CM3AtAGCCCAAGCCk

AGCAACU

CAGtJUCA CUGAUGAGGC CA ACACCUUr GGUUGGC CUGAUGAGGCCGAAGGCG;L

AT.CUUCTJ

GUEUtGAA CUr.AU -GGCCGA AGCCCAA

AGCACAU

CAGUUUG CUC-AUGAGGCCGAAGCCG AtMhGCAC GCAGUUU CUGAGCAAGGCCGAMGt AGCUGUU CUGAIUGAGGCCGAA rCCGAA

ACUGCCC

AAGGUUt7 CUGAUGAGCCCAAGGCGA AGCr.TGWE GGUGAGG CUGAUr.GGCAGGCCGAA AGCtUUj CGGUGAG CUGAUGAGGCCGAAAGCCAA AAG~Irjr ACACG-GU CUGATMAGGCC -AAGGCCCAA

AIGGAAGG

AGTJCCAG CUCAUGAGGCCGAAGCCGAA ACACGGrJ CGtUtCUG CUG-AUGAGGCCGAA rCCA

AGTUCCAG

AAGAGGG CUGAUGAGrCCGAAAGCCGA-

AGGGGUG

CUGCCAA C'GALGAGGCCGA AGrGCCCGAA

AGG.

a. a.

183 2.

a.

a 427 450 451 456 495 510 564 592 607 608 609 611 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 867 869 881 885 933 936 978 980 986 987 988 GGCUGCC CEIGAUGAGGCCGAAAGGCCGAA AGAGGGG GMGrGU CUAUAG CGAAAGGCCGAA AGGUUCrJ CGUAGGG CLIGAtJGAGGCCGAAAGGCCGAA AAGGUjUC G7GCAGCG CGAGA CCGPAAGGCCGAA AGGEtMA CCACGGU CGGACCGAAAGGCCGAA AGGUUGG CCCCACG CUG AGCCGAAAGCCGAA AGCAGCz, 0UCG CUAGAGCCGAAAGGCCAA ACCUCAG CC-AUGGO-. CUGAUGAC-CGAAAGCCG-AA AUCUCUC CACGAA CGUA-CGAAAGGCCG,)AA AflUGGCU GCACGAG CGAUGAGGCCGAAAGG-CG-AA AAUrJGGC C-GCACG-A CAGAGGCCCAAAC,-c,-

AAALUUGG

GCG-GCAC CUGALGAGCCGAAAGCCGAA AGAAAUU GUUCrCA CliGAtJGAGGCCGAAAGGCCGAA

ACPAGCUJC

TGOUCEC CUGAUGAGGCCGAAAGGcCGA AArAGCrj GGGGCC CUGAUAGCCGAAAGGCCCGAA AGGLTG=r GAGCUGG UGGACCGAAAGCCGAA AGGGG;GC AGGUCETG CUGAUGAGCCCGAAAGGccC.A AGlCUGGrJ CAGGACA CUAG GGC~AAGG-CGAA AGCUG GCAGGAC CUAUAGGCCGAAGGccGA AAGUtCU CUGGCAG CUAGGCGAAAGGCCGAA ACAAG UGUGGGG cuUGAGGCCGAAAGGCCGAA

AGUCGCU

GGC-UGAC CUGAGGCGAAAGGCCGAA AGUUGUG GGGGGCU CUGAUGAGGCCGAAAGGCCGAA ACAArGrJ CCUCMG CUGAI3GAGGCCGAAAGGCCGAA

ACCCGGG

CCACCUC CUGAUGAGGCCGAAAGGCCGAA

AGGACCC

GGGAACA CUX'AG-CAAGCA

ACCACGG

UCCAGGG CtJGAUAGGCCGAAAGGCCGAA ACAflACC GUCCAGG Ct3GAUG2AGGCCGAAAGGCcGAA

AACAGAC

GACU=G CtL AM CCAAGCCGAA ACAGCc AGACUGG CUAGGCGAAAGGCCGAA

AACAGCC

CCUCCGA CUGADGaAGGCCGAAAGGCCA

ACUGGG;A

GGCCUCC CUGAUAGCCGAAAGGCCA

AGACUGG

CCAGGUG CUGAMJGGCG .GGC ACCUGG GGGGUC CUGALTGAGGCCGAAAGGCQAA

ACCUCUG

CAUAGG;U LUG zCCGAAAGGCCGAA

ACUG

GUUGCCA CUAGGcGAAAGGCcCGAA

AGA

CGAGAAG CGATJGAGGCCGAAAGGcCGA

AGUCGUU

GGCCGAG CtJGAt AGGCCGAAAGGCCGAA

AGGAGUC,

UGGCCCGA CUGAU .AGGCCGAAGGCCGAA

AAGGAGTJ

CUUGGCC CUArGcGAAAGGCCGAA

AGAGG

ACUGACU CUGAUGaAGGCCGAAAGcGC

AGGCCUJ

UCACACtJ CUGAUGAGGCCGAAAGrCCGAA

ACEJGAGG

CCAGUAU CUGAUGGCGAAAGGCCGAA

ACTJGCAC

UCCCCAG CUGAUGAGGCcGAAAGGCCA AUtjACUG AGCtJA CUGAUGAGGCCGAAAGGCCCA

AUGG;UCA

AAAGCUJG CUr-AUGAGGCCGAAGCCA

AGAUGGTJ

CGCCGCGA CUGAtJGAGGCCGAAAGGCCGAA

AGCUGUA

GCGCCGG CUG-AUGAGGCCGAAAGGCCGAA

AAGCUGEJ

GGCGCCG CUr-AUGAGGCCGAAAGGCCGA.A

AAAGCUG

a. a a a a.

184

S

a a C a a.

a a.

1005 1006 1023 1025 1066 1092 1093 1125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 1334 1344 1351 1353 1366 1367 1368 1380 1388 1398 1402 1408 1410 1421 1425 1429 1444 1455 1482 1484 1493 1500 03 1506 UCGtJCAG CUGAUAGGccGAAAGGCCpaA

AUCALCGU

UUCGUCA CUGAUGAGCCGAAGCC

AAUCAC-,

CUUCMGA ~COU CGA.-CG;

ACCEC=

CCCUUCU Ct 7 GAtGAGGCCGAAAGGCCGAA AC-%CCr7C UWGzCTJ CUGAflGAGGCCGAAGCGA~,AGUG GGGCOGG CUGAL7GAGGCCGA aCC-CcAU UGGGCLUG CW-kVAGGCC-AAGC3CCGAA

AACCCCA

UCAGCAG CUAGGCGAkGCA

AC-CUG

GCAGGA CUG AGGAW= GAA ALC-JrCG AGCAGGA CUAUAGCCGACCG AACUGc AGAGCAG CUGAGAGGAcc,-rGA

AAA

GGUUGCAk CUGAUGAGGCCGAAGCCGA AGcakGfGt7GUGAU CEUGAUGAGGCCGA kG'CGAA. ArGc UUGUGA CVAGGCGA~r-C

AAGCUVGG

UCUuGUG C GAUGAGGCCGXV--CCG;A AtUhAG,--J GGACACG CUGAGAGGCGAAAGCCGAL -C-Cc-.

AGGACAC CUGAMXAW4W-UAAGG AAGCLr-UCc CALULCA CUGAUGAGG GAAGCGAA ACACG6 GGGGCCA CUGAX AGGCCGA yGGCCGA

ACAGGAC

CCCGGAC CUG DAGGCCGAA GCCGAA~ AUCCCLc ULt3CCC CUG AGAGCCGAMWCCGA

ACAAUCC

UTGCUGGG CUGAUGAGWGCCQ GCGAA AUUEIEJCU CUGCUGG CUGAUGAGGCCGAAACCAA AAUGU~c CACATUG CUGAUGAGGCCGAA =CGAA AGtiCUG UUCCCCC CUGtr-GGCCAAGCCA

AG-CCUGG

CEJCGGGC CUGAUGAGCCGAAGGCAA

AUC-CGUU

GACACUU CUGAUQAGCCGAAAG~C

AGCUCGG

UJCCUA CUGAUGAGGCCGAA=CGAA

ACACE=G

CAt7CCUU CUGAGAGGCCAAAGGCCA

AGACACU

AGUGGGA CUAGGCGAAGCCA

AGUGCCA

CAGMaGG CUGAX GAGGCCGAAGGCCGAA

AAGUGCC

GCAGUGG CEAGGCGAG.CA

AAAGUGC

AUUCCCC CUGAUGAGGCCGAAA--GAA

AUGGGCA

AGUCACU aM ACC AtJTCCCC CTJCGAGU CUJGAUGAGGCCGAAAM 1 G.A ACAGUCA AGAUTCUC CUGAUGG GAAM

AGUGACA

CCCUCAA C"UGAtGAGGCCGAAACCGA

ADUCGA

tJGCCCUC CUGAtr-%GGCC AAGCGAA

AGAUCUC

ACAGAGG CUGA6UGAGGCC AACL

AGGUGCC

CCCGACA CUG-At -GGCCAAGGCGAA

AGCGUAGG

CUGGCCC CUGAUGAGGCCGAAGCUCGA

ACAGAC,-

UCCCCUU CUGAUGA-GGCCGAAAGG;CCGAA

AGGCUC

CGCGGGU CUGAUGAGGCCAAGGCGA

ACCUCCC

GGGGGGA CUGAUG.AGGCCGAA CGAA AdJCACAXJ CCGGGGG CUG-AUGAGGCCGAA~,CGAA

AGAGCAC

AAUCUCA CUGAUGAGGCCGAA GrCCGAA~

ACCGGGG

UGAUGAC CUGAr-GGCCGAAGCCA AE7CECAUJ UGAUGAU CUGAUGAGGCCGAAG CGAA ACAAUCtJ CAGUGAU CUGAUGAGGCCGAAAGGCCGAA AtGACA a. a a Ca 185

"I

1509 Isla8 1530 1533 1s51 i55-;9 1563 1565 1567 1584 1592 i599 1651 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802 1812 1813 1825 1837 1845 1856 1861 1865 1868 1877 1901 1.912 1922 1923 1.928 1930 1964 1983 CCACAGU CUGATJGAGGCCGAAAGGC-CGAA AUG;tG CCCGU AGGCCGAAAjGCCGA ACCACAG CCAULULU CUG7&XGAGGCCu-AAAGGCcGA

ACUGCCG

UGCCCALT CGDAGCCGAAAG3GccGAA AflGACLTG ACGUGCtJ LUGUAGCCGAAAGGCCGA

AGGCCUG

AUAGAGG CtUGAtXUG cGMGcc3J ACGUC-U GGU~ktUk CUGAUGAfGCCGrAAAGGCcGAA ACgjAG GCGGUtA CGAUGAGCCGAAAGGccG-,A AGaruM CIGAU AGGCCGAAAfGCCGA

AUAAW

AIULMCU CtJGAUAGGCCGAAAGG--CGM At~UUCC UAGUCJG CCGAE GXGCCGAAAGGCCGAA AUUUCruUT CCUGUUG CtJG AGC-CGAAAGCCGAA AGUC-Grj GUUCAGG CUA GCCGAAAGGccG

AGGU

CCCGGGA CUGAUGcccAC kG=UCA GUCCCGG C tLUAGGCCGAAAGCCCAA AUAGGaUu CGAGGAA CUAt GCCGAAGCCCA

AGGCCCU

GCCGAGG CUAU GCCGAAGCccL AC3AGC, GGCCGAG CUAGAGCGGCCGAAc

AAGAGGC

GAAGGCC CUGA~r.AGGCCCGLCAA~

ACGAA

AUAUlGGG CUGAGAGGCCGAAC-CCG

AGGCCGA

AAUGG CUG UAGCCGA CCC-, AAGGCCG CCACCAA CUGAUGAGGCCGAAAGGCCGjA

AUGGA

UGC-CACC CGUAGCCGAAAGGCCGA ArJfG CAUGGCA CUA~GCGAAGCCA kUGUCUU GtU.GGM GA rACCGAAGCCGAA AGcUGCA GCGCCGG CUrAD GCCGAAAGCGCCGAA AGGUGUjA L'GGGA CUGAXXAW~XGGCCG cc AUGCCCU GACUGAG CU~rGCGAAGCA

ACAAEJGC

UCLJGACU CUGAUGAGGCCGAAGGCCtG,,

AGGACA

u R~ucu CUGAUAGCCGAAGCCA

ACUGAG

GCXUUG C~zUaGCAAG~.A

AUCUGAC

GGCCCA CUGAUAGGCCGAACCA

AUGCEJGU

UGGCCC CUGAOACGCCGAAGC-C AAjG-U GUGCAGG CUGAU GGCGAAAGGCCCU

ACCAIJGG

AGUGUUU CUrGAAGGCCGAAGCCGAA

AGGUGUG

CGUGGCC CUGAGAGCCGAA G AGUGUUUr CA4WrCA CUGAUGAGGCCGAAAGGCCtGJA

AUGCGTJG

GACUACA CUGAUGAGGCCGAAAGCCA

AUJCAGAU

AtJGUGAC C GAUGAGG--CGAAAGGCCGA

ACAGAUC

GUTCAUGU CUGA rACCGAAAG CGA ACUACAG CUTUGGCU CUAGGCGAAGCA

AGUCAUG

AUGUCUU CUGACAGCCG GCGAA AGUCUUG AUCCAIJC CUGAGAGGCCGAAGCCGAA

AUCAIU

AGACUUU aW=AW4AGGCGICCQ ACiUCCA UAGACUU CUGAU -AGGCGMAGCCGAA

AACAUC

cAGGCuA CuA AGGCGAAAcc ACutJA AUCAGGC CUGAGAGGCCAACC-AA AGAcrajr GtrjGGGC CUGAUG-AGGCCGAAGGCCGAA

AUGUCUC

CCAGUUG CMAGfGCMWQ CCA AUGUCCtJ 9 .9 1996 GULtCALG CUGAUG-AGCGAAAGGcA

AUUJUCCC

2005 AGGCAGC C GLCt--',-AAG-,A AGUUUCa 2013 MCCCAA CtUGAGAC-CGAAAG~cc-,, -CGCj= 2015 CAIkCCC CUr3~AGGC-CGAAAGGccU AtUAGGCA 2020 CUCAGCA CUGAmaGcAAC-GCGCCGA;

ACCCALAU

2039 ;UUCDG CUGAUGGG-C,-AA.G-CC

AGUCUGU

2040 t~3~~CUGAMAG="-CGAAAGGCCGAX

AAGUCUG

2057 GUCETAUG CUGAG,--CcGAAc,-CC

AGGCCA

2061 ACAUT3GC C-;tA~~G-,CuiAGCCCL-

AMGAG

2071 tJUGAUGC COGAUGCGCC,-,AA-CcGAA

ACACAUG

2076 G.AGu uU CUGA C-CCGAAAGGCCA

AUGCUAC

2097 CWCAGG C~r-UADG,-GCCGr-AAAGC-,CGA A3GG **:2098 C-GUCAG CCAGGCAA.,C-L

AALGLG

*2115 AGcCCC CLMMUGAGGCCG ccG AGCUGCC **2128 GtCAU COGAUGAGC-C-CG AcG,-

ACAGCAG

2130 GGGjUCAG Ct3GAGAGCCGAAAGCGAA

AGACAGC

2145 UTCAXJC CUGAUGAGG-CGA GCCAA AGGGUUG- 2152 AAAflACA CGAGA-G CC-cr AUCAUA .:2156 GAAMLAA CUGAIGAGGC- "CrC,-A ACj=Aj,; 2158 ALMAIUA COGAUGACG-CGAA CGA AUACAUA 2159 AAX3GAAU CMUAGGCC-_.AC,-CCA AAtACAU ~.**2160 AAAI3GAA CUGAfLGAG--A;G,-GA

AAAUACAA

*2162 ACAAAUG C GuXUGAG-ccGAAG CCGAA AU~AAUA 2163 AACAAAU CUAr-G~,;AGCA AAt~hAAU 2166 AAIaAkCA CUC-AUAGGCC-,AAr-CGAA

AJGAATUA

2167 AAAU3AAC C~r-AUGAC-G-CGAc CCGAA AAUGAUU *2170 G71AAAU CrUGAGGACC aLGC-L-A ACAAAtUG 2171 GGOAAA CUGAUGGCCa -L3C--GAA

AACAAAU

2173 CT5GGUAA CUC-AUGGC,-'CAGCCG-AA

ATIAACA

2174 AGCDI~GU CUGAUGAGGCCG ACCGAA

AAUAC

2176 tUC2CUGG CU-UAGC-,AG;C-A AAAALTmA 2183 CAAUAAA CUGAAGC-CCGc GGCC-;

ACUGGU

2185 CUCAADJA CUGAtrxAGGcc-C,-CG- AThAGCUj 2186 ACUCAAU CUGAL.A~r-C rkAGGCG AALMC~ 2187 CACUCAA cuGAflGArGGCCGAAGGcrAA AAAImhGC 2189 GAZACACU ;AC-CAAG-rC

AJTAAAUA

2196 CALVJAAA CUAU~c~~ C AACCCA 2198 tUhCALULA CLTGAT,-AG,AAA CCGAA AGACACU 2199 CEIACAiiA CUC-AVGAmGCC a.GCGAA A.ArACAC 2200 CCUIACAU CTJGAI GAGG-CGAACGCCGAA

AAAGACA

2201 GCCUACA CUGADGAGGCCC-AAC-GAA

AAAAGAC

2205 UUtUAGCC CUAGGC-GACCG.

ACAAAA

2210 GUUCAUUE CUrAUGAGGCCC '2CCCGA

AGCCTJAC

2220 AGAGACC CE 3 GAtJ.GnGCC .,AAGGCCGALA AUGtJUCA 2224 G-GCCAGA CUJGAtG-AGGCAC-GCCGc

ACCUAUG

2226 CGGCCA CtJGAUG AC-GCCGAAGGC-GAA

AGACCUA~

2233 C-CUCCGU CUGALA~GC GAACCC-A

AGGCCAG

2242 GGACUGG CUGAt=AGGCCG A-GCC-AA~

AGCEUCCG

187 2248 UGACAIG c -G GA C ACUGGQA 2254 UAAUGU CMUAI CA ACAflGGA 2259 GACCUTJG CUGA GCCGAAAGGCCCAA

AU(GUCAC

2260 UIGACCUU CUGAIdmAGrcCGAAA GCCGAA AAflGUGA 2266 ACCUGGtJ CUAGGCGAAGC ACC3UGA 2274 ACAACUG CUGAUGAGGCCGAA GA. ACCJGGT 2279 CCUGUC CUGAL1GAGGCCGAAGCCGAAj ACtJGUC 2282 CAACCUG CUC-AWAGGCCGAAAGCCGA

ACAACUJG

2288 AGUGCAC CL-A -G-,CZriGGCCGA

ACCUGEIJA

2291 UGCAGUG CUGAUXGCGA

ACAACCU

2321 CCCAUtjt C GAflGAGGCCGAAAGGCCA

AUCULUW

2338 CAAflGAG CUGAUG GGCC-GA AGCGAA AGUCccA 23- 9 CCAA, CU-UAG-CAAOCA AAGEJCCC 241 GGCCAAU CU-UA.--CAAGCA

AGAAGUC

2344 GUUTGGCC CUAGGCGAAGCA

AUGAA

2358 MGGGACUGA-UGAGC,CGAAAGGCCGMA

AGCG

2359 uactJGGG COGAUGAGGCCGAAGGCCGA

AAGGCAG

2360 UUCUGGO CUCAUGAGCCGAAGGCGAA

AAGC

2376 A~kAAA C 3GAUGAGGCCGAGGCGAA AUCACUC *2377 GAXkGAA cwGAMlACZ2C AAuC-Acrj 2378 CGA.UAGA COGAuGAGcr-cGAA CGOCGAA AAAXJCAC :*2379 CCGAk cuGAu GccGAA~c-A

AAC

*2380 CCCG-AUA CUGAI GAGGCCGAAGCCA

AAAAAUC

2382 GUGCCG-A CUGAUGAGGCCGAA GCGAG ACa ZAJkA 2384 tJUGUGCC CTJGAGAGGCCGACUGA

AUAGAAA

2399 GUCAU CUJGAUGAGCCGAAGCGAA AGUGCtUU *2401 CAGU=cA Ct~UGAGGCCGAA GCG AtGUGC 2411 GAACCAXJ CUGAUGAGGCCAAAGCGAA ACCAGUc 2417 ACCUGUG CUGAI GAfGGCCGAAC-

ACCAUEJA

2418 AACCUGUCUGAIJGAGGCCGAAAGGCCGAA

ACU

2425 AUCUCUG

CUGAUGAGGCCAAGCCGAAACCEJGUG

2426 AADCUCuJ CUAGGCGAAGCA

AAC

2433 ACUGGGU CUGAflGAGGCCGAAGGCGA

AUCUCUG

2434 CACUGGG CUAr AGGCCr-lGGCGAA~

AAUCUCU

%2448 GAGGAAu CUGAI AGGCCGAAC CGAA AGGCCUC 2449 GGAGGAA CMUAGCA~GCA

AAGGCCU

2451 AGGGAGG CUGAI GAGGCCGAGGCGA

AIAAGGC:

2452 AA66GGAG CUJGAtGGCCGAAA,-CGAA~

AAUAAGG

2455 GGGAAGG cuGAUrGfGCCGAAACGcAA

AGGAAUA

2459 UGGGGGG CUAUGAGGCCGAAGGCCGA

AGGGAGG

2460 UtJGGGG CUGAtrG CGAAG CAA AAGGGAG 2479 GCEJAACA CTJAUAGGCCGACGA AGG-UCtJC 2480 GGCU~AC CtJGAUGAGGCCGAAAG~c-GAA AAGG~uGu 2483 GGEJGGCU CUGAUGAGGCCGAAGGCCA ACAAAccG 2484 AGGUGGC CUrGAUGAGGCCGAA GCrA

AACAAAG

2492 GGGUGGG CUrGAUGAGr-CCG AGGCCGAA

AGGUGGC

2504 AGAAAUG CUGAUGAGGCCGACGA

AUGUGGG

2508 UGGCAGA CE7GAUGAGGCCGAA ,-CGAA,

AUGUAUG

2509 CUGOCAG CUGALMGG GAGGCA

AAUGUAU

188

S.

S.

S. S S S

S.

S

*5

S

S 2510 2520 2521 2533 2540 2545 2568 2579 25835 2588 2591.

2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2 642.

2642 2653 2659 2689 2691 2700 2704 2711 2712 2721 2724 27144 2750 2759 2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 ACUIGGCA CNIMUAGGCCGA AACCGaM

;LAM

CAUUGUG CUGAUGAGGCCGAAGGCCMk

ACXMWG

UCAUU CUG GAGM1GcGCGA

AACA=~

GACCGCU CUAGGCCGGC-CA

AGGC

CAGACAU CUGAAC-GICC GCGAA

AC-G

AtJGUCCA~ CUrGAG CGAAAGzGCCGA

ACUGA

UUGGGCA CUUGAGCGAAGC ADtUCC CAAGGCA CEGLr -GGCCGAA C-CGA AGM=uG AGAGGAC CTMArCGAAAGCC

AC-GCU

ACAAGAG CU-UGccGAAAc,

ACAAGGC

AGGACAA LVC-C-A,-GAc-r,

AC~

AC-AGGAC COGAUGAGGCCGAA CGCAA AZGAGGCic CAAAC-AG C-rGCCGAAAGCMU

ACAGA

AAAUGCA CUGAD AGGCCGAAAC,-CGAA

ACAGGAC

GAAAUGC CUGAUAGGCGAAA-C-

AGG

CCAGUGA C LrXGAGGCCGAAAGGCCAA

AUGCAAA

CCCAGUG CUGAfGAGGCCGAC--GA AAtGC;A UCCCAGU CUGADGG cGCAACA

AAC

AXMGGC. CCAUA GrAAGGccXM

AG-CUCCC

C-UGCAA CUGCGXr--C

AGUGCA

GAGCUGC CUG CCGAAAGG CC AMVGUrGC GAAACW CUAGAGCGAAAGGCGCA

AGCUGCA,

UGCAGGA CUAGGCGXC--CA

ACUGGAG

CUGCAGG CUAGGCCAAGL

AAC=GA

ACUGCAG CUAMGCGAaCCA

AAACUG

GGACCCU CUGAM~AGGCCGAAAGCCr

AUOACUG

CUUGCAG CUAGGCCXAGCA ACcCCJGA CCUCCAA CLAUAGCC~kr G- GA ACCUUGG GJCCUCC CUG~trGGGCC CG~AA AM==CU UGGGAGG COGAUGAGGcCCrAGGC

AGCU

AAGCUGG CCUGGCCGAAAGGCC;L

AGGGAGU

CCUUCCA CUGAflGAGGCCGAXG,-rA

AGUG

CCC-UUC CUGAGGCCGXX-CGAA

AAGCOGG

CGCGGAU CUGAUAGGCCGAAG CGALA ACCcuUC ACACGCG arUAGCXMM

AXJGACCC

CUACACA CU AMGCCGAWG

ACACC

GCUUGEJC CUGAUGAflCCG uCGAA

ACACAUA

AGAGCG.A CUAGGCGAGCrA

AGCTJUGU

ACAGAGC CUGADIXAGCCG AG-CGAA

AGAGCUU

GGUGACA Cr.UGACGGCCG

AGCGAG

CCEXGGGU CUGAUGAGGCCG AGCL

ACACAGC

GAACCAU CUGAUGAGGCCGA AGC, GAA~ AUUGc-Ac UG-CAGUG CUGAUGAGCC AAGCGAA

ACCAUGA

CUGCAGU CUG-AUGAGGCCGAA GGCCGAA

AACCAD

AGCtJCA.A CUGAUGAGGCM- CCAA

ACUGCAG

AAAGGtJc CUAUGAGGCCGAA,-tA

ACACG

AGCCCAA CUGAA GG CGAA~ AGGrJCAA GAGCCCA curAuGAGGAGccGiA AAGWuCA UGAGCCC curAIJ GcrCGAAAGCcGA;.

AAAGGUC

189 9* 4.

2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 2975 2976 2977 2979 AUCAhDU CUO=AGAGGCCGAAAGGCCGA;A

AGCCCAA

GWGGAG CGUA GrAAG-CcA ;CACrUu GAGGUGG CU CGAAGGCc-,t

ACGUC

GGAGGCTJ COAUACCGAA~GC

AGGUG

!ThCUCAG CUlr GGCAAAGCGAA.

AGGMCA

UCCCAGC CO GAGGCC

ACUCAC

GtMAGCC CVGA~rGCGLAACCC,

AUGGUCC

GOUUGU COUGUZCCCGAAACGGCCAA AG3CCUD AAAAUCA CUGfGAGGCCG CcU ;fUM-rC AAAAWC CUG AGC--ACGAcA

-AXUXC

AAAAAAA Ct3G GAGG =AUCA AAAAAAA CUAGAGCaAAAGGC-X

AAUCAA

AAAAAAA CGV-JG-CGAAAC

AAAIJCAA

AAAAAAA CUAGGCGAAAGCA AAAAUAc~ AAAAAAA CUAGGCGXG-CA AAAA7 AAAAAAA CUGAUAGCCMAGCCr.JA

AAAAAAU

AAAAAAA CUAU GCCMLGGAAA

AAAAAA

AAAAAAA CUAWC<CCGAGCC

AAAAA

GAAAAAA CUAUA CCGAMGCCGA

AAAAAAA

UGAAAAA CW-UGA~r-CGAAAGGCCA

AUAAAAW

CUGAAAA CGUGAGCCGAAAGGC.A

AAAAAAA

UCUGAAA. CMA GCCGAAAGGCCC-A

AAAAAAA

CUCUGAA CUAG GCCGAAACCA

AAAAAAA

UCUCUGA COGAtGAGGCCGA AGCCCGAA

AAAAA

UCCUGA r GCCGAAAGGCCG;L

AAAAA

CGU]CU C"Grc A

AAAAAAA

GUUGCGA CUGAtAGGccGArGGGA Acccu At~UGC CUG;t3 AGGCCG AAGGCCGAM AGAC~cC UCUGGGC COG rIGCCGAGCG AIJGtUGc ACAAAGG CcAGGCA AGGCA AGUCUGG CACAAAG CUGA]JGAGGCCGALAGGCCAA

AAGUCUG

UAACACA ~CUGUACGGGCCA

AGGAU

CtLILCAC CUGAI UAGGCCGAAGGCCA

AAGGAAG

AtUAACU CUGG AGGCccGAA, AcACAAA, ~UECAC CMUAG~a-A,,CA

AACAA

CUUUAUU CUGAUGAr-AAGGCC GAA ACOAACA6 GCUUAU CO UGGGCCGCGAG;A AAcm~ AAAGCU CUGA GAGGCCGAAAGCGA AAcu AGUE3GAG COGAUGAGGCCGAAGCCXA

AAGCUEU

CAGOUGA CUGAUGAGGCCGAAGCCGAA

AAAGCUU

GGCAGOJ CUr- AGCAGGCC--M

AGAAAGC

Table Mouse ICAM HH Ribozymne Sequence nt. Position Ribozyme Sequence 11I 23 26 32.

34 48 54 58 64 96 102 108 115 1U9 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 394 420 425 C-NACGGU CUGA.UGAGG-CCGAAAGGCCGa2A

ACCAGGG

AGCAGAG CUDGAUGAGCC-CAAAG-,--GAA ACCACtUG AGCGAGCA CEGATJGAGGCC-GAAAGGCCGAA

AGAACCA

UGUGGAG CUGAUGAGG---GAAAGGCC-G-A

AGCAA

CGACC CUGAflGAGGCCGAAAGGCCAA

AUGAGAA

AGGCCAC COGAUGAGCCGAAAGGCCA AGU C CCAGGCU COAU GCCGAAGCCGA

AGGUCCU

CCAUCAC CUAUAGCCGAAGC-CG

AGGCCCA

GGAGCE77 CUAGGCGAAGCA

AGCU

CUGCUGG COGUGAGCCGAAAGGCCGA~

AGGGGUG

GGGCCAG CUGcUGAZ AAG cC .AGCAGAG CCAGCAG CUAGGCGAAGCA'Cr GGGCCAG CVGGAGGCC rL AGCAGAG AGGAGCAN CUAGGCCA~

AGLA

UCCUGGU CtJGAUGAGGCCGAAGGCGA

ACAUUCC

C-GGCCAG CUGAMG CCGAAGGCC AfCG GGAAGC-G CUGATJGAGGC-CGAAAGGCCGAA

ACGACUG

AGUGGCU CUGAX1GAGGCCGAAAGGCGL

ACACAGA

GGUUUUU CUMNGCC occAMCG GCAAAAC CGAGAGGCCGAAAGGCCA

ACUOCUG

GGGGCAG ~CUGUACGGG~rA

AAGGCUU

CUGCACG CUAGoCCGAGCAA

ACCCACC

GCCAGAG CGUGGCCGAAAGoGA

AAGUGGC

GCAAAAC CUGAIJGAGGCCG AGCGA

ACUUCUG

GGAGCAA COAUAGCCGAAGCCG

ACACUU

AGOUCUC arUAGAGGCCCAGCtGAA (kGCAcA UUUAGGA CUGAUGAGGCCGAGCtJA

AUGGGUU

UCUUCCtUG~lAGC

AGGCAGG

CAGUAGA CUGAUGAG.GCCGAAAGG;CCGAA AAACCCtj UAGGCAG CUrGAUGAGGCC ~AGCCA

AGCCCCU

CAGCUJCA aCAUGAGGCCGAAGGCGGA ACAGCUrJ GGCUCAG CLTGAUGAGG;CCGAAAGGCCGAA AUCUCCu GUUCUCA CUGAUGAG-GCCGAA £GCGAA

AGCACAG

CAGUGUG CUGAUVGGCAAGGCC C-cAA AUJUGGAC tJCAGCUC CUJGAUGAGG;CCG .AGGCCGAA AACA .GCU AGCGGAC CUG3AUGAGG-CCGACGCGAC

ACUGC-AC

CGGGUIUG cucGAGCCAGGCCGAA~C

AGCCAUEJ

GGGCAGG cuJGAuGAGG-cCCAA ~crA AGGcuuc GGGGCAG CrGUAGCAAGCA

AAGGCUU

ACACGGtJ CLTGUAGCCCZZGO~CU

AU==UA

AAACGAA CUGAuGAGGCCGAA GCCA

AMCGT

A'GAUCCAl CUGAUGAGGCCGAAAGCCG

AGUCCGG

CGGGGGG Cj;;-C-CCAACGCA

AA'GUGUG

CUGCUGG CUGAUIGAGCGCCGA -GGCCGAA

AGGGGUG

427 CACUGCU CMJAUGAGGCCGAAAGGCGAA

AGCUG

450 GCAGGU CUGAGAGGc-CGAAAGGCCGAA

AGG-U=C

451 CAAGGA CGAGAUC.GCGAAGGCCGAA

AC---UUC

456 ATJGGCU CUGAUC-AC,-CGaAGGcG

;LGC-UA

495 ACA.CGGrJ CUGAUCNXGGCCGAAAGGCC3AA, ALG,-rj 510 CCCCACG CUGAUC-AG.GC-CGAAAGrCC,-M

_AGCAGI

=64 GGAXUGGA CUGAMGCGCGAGGCCGM~

ACL-,VAG

392 CCC.nGU CLrG-UGAG~CCG A AG~Cc_-A., Ar-C-uuc 607 CAUGAGA CUGAflGAC-G-CakAAGGcCGAA AXU-GGCrj :608 GCAUGAG CUGAUGAGGC-CGAAAGCCGtAA

AADUIGGC

609 GGCAflGA CUGAUGAGCCGAAAGGc-CG-AA

AAUUGG

611 GCGGCALU CUGAAG--GAGGcCGAA AC.GAUt ~636 CAGCUCA CUGAL-GAG~CCGAAAGGCCGAA

ACAGCUU

657 UCAG-CUC CVUAUGAGGCG G-c AACAsGCU 668 GGUGGCC CUGAUGACCCCGAAAGGCCC.A AGGCt7G 677 AGrGCUGG CUGAUGAGSCCCG'AAGGCGA

ACAGGUC

684 AGGACCG CUGAUGAGGCCG-AAAGGCGAA

AGCDGAA

692 AAC-AIC%- CUr-AUGAGGCCGAAAGCC .A AXc, S.693 GCAGGGU CUGAtJGAGGCCGAAG~ccGAA AGGTJCCrJ *696 GAGGCAG CUGAUGAGGccGAAAGCCA.

AAAC.A.G

709 LM-AGGUJG CU M-UC-AGGC-CGA flGCrCGAA AGCCG;CC 720 AGCUGAA CJGALUGAGGCCGAAAGGCCGLA

AGUUGUA

723 CGGA-U CGAUGAGGCCGAAAGGCCGA

AAAAGUUT

735 UCUCCAG CUGAUGAGGCCGAAGCCA AUCTrG 738 L'CAUJCAC CUGAUGAGCCG.AGGCCGA AGXcCCA 765 GGAAGCG CrGAUCAGGCCGAAAGGCCGA ACCACEjG 769 GGCAGGA CUGAUGAGGCG G ACAGGCC .770 UUCCAGG CUGAUC-AGC-CCGAAGGCC-A

AGCZAAAA

785 GGCAGGA CUGAUGAG-GCCA AGGCCGA ACXGGCC 786 AGGCAGG CUGAIGAGGCCGa.AGGA

AACAGGC

792 CUUCCGA CUGAUGAGGCCGAAAGGCCG.A ACCrJCCA, 794 AGUCUCc CUGAUGAGGCCGAAAGGCCA

AGCCCAG

807 CCAGGUA CUr-AUC-CG-AAGGCCG AA AUCCGAG 833 GGGUGUC CUGAUGACGCCGAAGCA

AGCIJUUG

846 CAACGGU CUGAuGAGGCCGAAGGCCA

ACCAGG

851 GCUGGUA CUGAIJGAGGCCGAA GGCGAA AGGIJCrJC 863 CCAGAGG CUGAUG.AGGCCGAAGGCCGAA

AGJGGCU

866 GGGCAGG CUGAUGAGGCCGAAAGrAA AGGcuJTC 87UCUCCC-G CUGAUGAGGCCGAA GGCCGAA AACGAAU 869 CtJUGCAU CU .UCAGGCCG AGGCCGAA

AG-GAAGA

881 ACGGGEjM CUGAUjGCCAGGCCGAAAAG rAj 885 UCACCtJC CEJGAUGAC-GCCGAG~CcG AccAAG 933 CCAGAAU CUG-AGAGGCCGAGC. AUTJAtAG 936 GCACCAG CUjGAUGAGGCcGAAAGGCA

AUGADUA

978 AGUUGUA CtJGALTCAGGC-.CGAA .GCCAA AcutjLrj 980 AAAGUUG CUGAUGAGGCCGAAGGCGAA AG-AcUGtU 986 AGCUGAA CUr.AUC-A-GCCGAAGGCA AZtVUGt 987 G-AC-CUGA CUGAUC-G CC-A.GCWA A.A (7,jCJG Z88 GGC-:CtG CUGAUG.AC-CCCGAA6GC AAGLTUrG 1005 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCTJGGU 1006 UUCCCCA~ CTJGAUGAGGCCGA.AAG~cCGA AcucuCA 1023 CUUCCGA CGAUAGCCGAAAGGCCGAA

ACCUCCA

1025 C^CUUCC CGTAGCAAGcAAAGACCUC 1066 UUJAUUUU ctAGGcGA~cGAAGAGUGG 1092 GGCCUGA CtJGAXGGCCGAAAGGCC,-.A AU~CcAGU 1093 uUGGCUG CUGAUGAGGCCGAAAGGCCGAA AG,-tiCCAz 112)5 UC-AAGAA CU~'AGCAAGCA.AGUUGGG *0 *1163 G-CAAAAG CUGAUGAGGCC~AAGGCGA A~cULTC^ *1164 AGCAAAA Ct~UGAGCCGAAGGCcG AAflCUUC 1166 AGAGCAA CUGAtJGAGGCCGAAAG~ccGAA AGAACCU S*,,1172 GG-UUJUUU CUGAUGAGGCCGA-aAGCCGAA AACAGG;A 1.200 OCGOGGAG CLMWGAGCCGAAt3GCGA AGCA=a 1201 CUGUUCA CtJGAUGAGGCCGAA-AGGCCGAA AAGCAGC *.:1203 ACUGGUG CUGAflGAGCCGAAAGGCCGAA AAAAAGU 1.227 GCAr-ACG CUGAUG-AGGC-CGAAAGGCCGAA AUGUACC 1228 AGCAAAA CUGAUGAGGC-CGAAAGGCCGAA AAGCUUtC *.*:1233 CUCUCG CLTGAUGAGGCCGAAA53GCCGAA AAACGAA *sees, 1238 AGGACCA CUGAIJGAGGCCGAAACGCCGAA ACAGCAC @to1264 CUUGCAC CUGAUGA GGCCGAAAGGCCGAA

ACCCUUC

1-267 ULTCCCCA CUGAIWGAGGCCGAAAGGCCG-AA ACUCUCA .1294 GGCt7CAG CUIGAUGAGGCCGAAAGCCGAA AUCUCCU *1295 CUGCUGA CUGAUG.AGGCCGAAAGGCCGAA ACCCCUC 1306 CAfUUCA CUGAUGACGCCGa-aAGGCCGAA AG;UCUGC 1321 UCCUCCU CUGAUGAGGCCrGAAGCCGAA ArCCUUC 1344 CACUCUC CUGAflGAGGCCGAAAGGCCGAA AGCUCAU 1351 UAACUUA Ct3GAUGAGGCCGAAAGGCCGAA

ACAUJUCA

1353 CACCUUJC CUG-AUGAGGCCGAAAG.GCCGAA ACCCACUj 1366 AGUUGUA CUGAUGAGGCCGAAAGGCCGAA ACUGUUA 1367 AGGUGGG CUGAUGAGGCCGaAAXGcCGAA AGGtJGCU 1368 AGAGUGG CUGAl~iJAGGCCGAAAGGCCGAA ACAGUAC 1380 CCACCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA 1388 AGCCACU CUGAUG.ACGCCGAAAGGCCGAA AGEJCUCC 1398 GUUCUGU CUGAUGAGGCCGAAAGGCCGAA

ACAGCCA

1402 AGUUCUC CUGAUGAGGCCGAAAGGCGAA AAGCACA 1408 CCUCCC CUGAUG.AGGCCGAAAGGCCGAA AUCtJCGC 1410 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA

AGAC

1421 ACAAAAG CtJGAUGAGGccGAA GCrCGAA AGGrJGGG 1425 CUCUACC CUGAUGAGGCCGAAAGGCCGAA

AGGCAGU

1429 CAGGGGC CGAUGAGGCCGAAAGGCCGAA

AUAGAGA

1444 UCCtJCCtJ CUGAUGAGGCCGAAGGCCGAA

AGCCUUC

1455 UCCUGG;U CUGAkUGAGGCCGAAAGCCGA

ACAUUCC

1482 GGGAGCA CUGAUGAGGCCGAAAGCCcA

AACAACU

1484 CAUGAGG CtGMMA GC XGAA AGACAG i493 GUUCUCA CUGAUGAAGGCCGAAAGGCCGAA

AGCACAG

1500 GGACCAU Ct3GAUGAGGCCGc GGCCGAA ATUUCAU 1503 GA-UGAU CUGAUr=AGGCCGA-AGGCCGAA AUAGucc 1506 CC-%-zUAU CUGAUGAGGCCGAAAGGCGA

AACAUAA

193 00 0 0 00000 :0Oa 0@ *0SOS 5 1.509 1518 1530 1533 1551 i559 1563 1565 156-7 1584 1592 1599 1651 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802 1812 1813 1825 1837 1845 1856 1861 1865 1868 1877 1901 1912 1922 1923 1928 1930 2.964 2.983 ACACGGU CUGAAUGAGGCCGAAAGGCAA

AUGGU]AG

CGCCrJGG CGUGCCGAAAGGCCGAA ACajjGA CCAGAAU CUGAflGAGGCC,--AAc,-ccG;A

_MMUAG

GGCCCALC C GAfl GCCGAAAGGCCGAA AUGACCA AG,-CGCT CUC-AUGAGGCCG AAGGcA AGGcAIJG AGGUGGG CUvU-CG--AAAGCCGAA kG,-GUGCU G.G-tVUA CUG-AL GCAGGCC-x-A AraCAAGf GCGGUtUA CEG~ G-CAAAs2GCCGAA AAACAMA UGGCGGU CUGAGALGCCAAAGGCcGLA

AUAAACA

AUAUCCU C~rG AtGCCGAAAG~ccG AI7CUUUC UAACUUG CUAGCGrAAAGGCC,-AA

AMCCU

CCUUCUG COGAUGAGGCGAAAGGCCGAA

AACTUGU

GCUCAGG CUGANUGAGCCGAAAGCCGAA

AGGUGGG

CAAAGGAL CUG? GCCGAA ~CC-A AGULUC UUCAAAG CUAGAGCCGAA -GCCGAA AAAGGUTJ CCAGGCU CUGAUrGACGCCGAAAGGCCGALA AGGrJCCU CCAGAGG CUGAvUGAGGCCGAAAGCCAA AGUGGCtJ GCCAGAG CGJAGCAAAGCCGAA

AAGQGGC

ACAGCCA CUGAUGAGCCAA ~cCA AGGAAGtJ AGAUCGA CUAG GAAAG-GCCGAA AGUCCGG AAGAUCG CUGAUGAGGCCGAAAGGCCAA AAGUCCG CC-ACCC CL-GAflGAGGCCGAAAGCCAA

AUGGGCA

CUCCAflG CLMG AGGCCGa-AAGCCGAA AUADCC GCUGGUA CUGAUGAGccGAAAfGGccc

XGGUCUC

UGAGGUG UAGCCrGAAAGGccGAA,

ACCGCC

GGGCAGG Ctr=XUAGGCCGAAAGccGA

AGCUUC

UGGGGAC CUGAAGCCGAAGCC;L

AUGUCUC

AUUAAG CUGAUGAGGCCGAAAGGccGA.A

ACAAUGC

tJCCAGCC CUGaAraGCCGAAAGGCCCAA

AGGACCA

UULVWW GUGAGA CGAAAGGCCGAA ACGU UCUCCAG CUG-ALIGAGGCCGAAGCCGA AUCUGGU GGCCUGA CUATACAAAGc-CCGAA

AUCCAGU

UGAGGGU CUGJAUG GAAAcICCGAA AAXJGCUG GCAGAGG CUAUGCCGAAAGCCGAA -AGCGUGG G-GAGCUA CUGAUGAGGCCGAAAG.GCCGAA

AGGCAUG

GGUGvGCc CUGAUGAGGCCGAAAGGCCGAA

AGGCUCG

.AAGAUCG CGGACCGAAAGGCCGAA AAGUCC UACUGGA CUGAUGAGC-CCGAAAGGCCGA

AUCAUGU

CUGAGGC CUGACXGCGAAAGGCCGAA

ACAAGUG

UUUAUGU CUGAUAGGCCGAGC-

ACUGG;UG

AGCUGCU CUGAUGAGGCCGACCLGA

AGGCAUG

GUCCCEJU CUGAUGAZGCCG AGCCGAA AGUJUUA ACtJGAUC CUG AGCCG GCGAA AzCtJATAU UAACUUA CUGAUGAGCCGAAAGGCA

ACAUUCA

GAUJACC CUGAUAGCCAAGCCA

AGCAIJCA

CUGGUAA CUGAUGAGC-CCG GCCGAA ACUCUAA AGCUGGU CUGALMAGGCCAA AGCC A.AAC'JCU UGGGGAC CUG-AC-CCC-r-GC-CCCGA AtcauICC UAACUG CUGAUGAGGCCGAC.CC-GA AUA2 UCCU 194 1996 GGCUCAG CUGAUGAGGCCAA GCC-,AA AUCtJCCTJ 2005 GG-.UCCGC CUGAUGAGGCCG-AAG-'CCAA

AGCUCA

2013 tUhCUCAA CUGALAGGCC--AA CCA AAAtiA 2015 CCACCCC CUGAUGA CGGCC-,AAG- .AL, AUGGCA 2020 CM3AGAA CUGAZMAGGCC: AGC--A

AACCAC

2039 CCUCUGC CUGAUGAGGC-

AGCC;GC

2040 CCUCCAG Ct3GAtJGAGGCC, A -GC--AA

ALG.-,JCAG

2057 GGAUGtJG CUGAUGAGC--ACC AA ACGAG'CA 2061 ACACGGU CUGAXJGAGC-C 'CAA G-,,rA AUGGtTAG :*:2071 CUGAGGC CUAGG:-GAAGCA

ACXAG

2076 UGCUCUCUGAUAGGCGAAAG'CcA

AGGCUAC

*2097 CAIJCAAG CUGAUGAGGCC-AAXM

AC.

*2098 CGGGGGG CMGAUGAGGCC- ,Z AAGUGUJG 2115 AUCCUCC Ct3GAXGAGG-CGXAG C,-A Acrj,'-GC 2128 CUCAAUA CUGAUGAGOCCGX"AGGCC.AA~

AUAGCLTG

*.:2130 GAGGCAG CL7GAIGAGGCCGXXAGGCG

AAACAGG

2145 CAUCAAG CUGAUGAGCCGAA GCA .AGAGUU 2152 AACUCUAaCUauGGc ,I AtJUAAflA .:2156 tMVAAAA CUGAUGAGCC,-kAGlC--A

ACAUCAA

2158 AUr3AAUA CUGAUGAGCCAAGCC.-

AUACATTC

2159 AAUUAAiJ CUGAXJGAGCCC 'GC-CGA; AAUACAtJ 2160 AAAUUAA CUGAUGAGGCC-AACG C- AAAUAC.-.

2162 CUAAAUU CUGAUGAGG-AGCc CGA AtUhAAtAh *2163 AUUAAU CUGAUGAGGCCGAGC.I

AUAU

2166 AAtWAGAG CUGAtGAGCCC-AGCC- i UGAAGU 2167 AAUEMAU CUGAUG G-CC- ,'G-CGAA~ AAUACAU *2170 CUAAAUEJ CUGAUC-AGGC~aA GA A AUAAAUA ***2171 GGGAGCA UArGC-AA---,ACAU 2173 CUGGUAACt3AUGAGGCCCGAAAGGCC,,..A AaC'A 2173 CUGGmA CUGUGt1GG~CCGAGCA, ACUCmA 2175 AGCUGGEJ CUGhUAGGCAAGGc GCCC-AA AAACUCU 2176 UAGCUGG CLGAt GGCGcAAa-CC-;.A

AAAA~CUC

2 183 CAA C~aUG AGGCCGL CG-A AGCtIGGU 2185 CtJckiUA CUGAUGAwcc-GA -CGAA A!hGCLTG 2186 ACUCAAU CUGAUGAGGCGGCCGA AAMJGCrJ 2187 UACUCAA CUGAUGGCGGcc G CCGA; AAAUAGC 2189 GGt3ACtJC CUGAGAGGCC- ACGCGA

AUAAAUA

2196 CAUCA.AG CUGAUGAG-Cc~GAA.CG- AGAGtJUG 2198 AACAUJAA CUG-AUGAGCC-G-CC-L

AGGCEJGC

2199 AtJAAACA CUGAuG-AGGCCGAA GGCCC AGAG 2200 CUUGCAU CUGAUGAGGCCC-AAGGCCC-

AZS&AGA

2201 GCCGACA CUGAUJGArGGCI~,C~ rA AA 2205 UCAGGCC CUr-AUGAGGCCGAA GCCC.aA

ACAUAAA

2210 AGCCAC CUGAUGAGGCCG GA AGUCUCC 2220 AGAGAACaUAUAGGCCGAAAG.C AtJGCCAG 2224 GGAUGGA CUr-AUG-AGGCCCG AGCGAA

ACCUGAG

2226 GCGGCCU CUGALTMGGCC az C-CCC;

_GAUCC.

2233 ccuccAG cuG3AU-Ac-GccrG

AGGUC=-

2242 GGUCCGC CUGAUGAc-GCCGA;GGCa;, ;CtJ-CC.: 195 2248 tJGGGAUG CU~GGc,-AAGCA

AUGGAUA

2254 UCAGUGU CU~. cAAGCA

AAUUGGA

2259 CACCGUQ CUtAG AGGCCGAAMGGCC-.-AA

AUGJGAU

2260 GCACCGU Ct;GAIG-AGGCCGAAACrA

AAUGUGA,

2266 UCCUC-cGu CCGAUGAGGCCGAAC---%

ACAUUCC

2274 UCUCCAG CUGAUGAGGCC-GA AGCCGAA

AIJGUU

2279 CUUGCAXC CUGAUGAGC-CGA~c,-cG-,

ACCCUUC

2282 CAXGCXCA CUGAI CA,-GA CCCAA ACAGCU 2288 AGGCCAU CUGAX UGAGrGCCC AAAGGCCGAA rrj :2291. -AGaG CUGAX3GAGGCCG-AAA GCCGAA ACCACU *2321 CCCAflGU CUGAUGAGGCCGAAA CCGAA AUCUUUC 2338 CAGGCAG CUGAU AGGC-CGAA GGCGAA AG=Cry 2339 CAAAGGA CUGAUGAGGCCAAAGCCt-A AGGUUc 2341 AGGCUGG CUGAUGAGGCCGAAGCAA

AGAGU

2344 GCUGGAA, CUG UGAGGCCGAAGGCGA AUCGAzAA .:2358 CUGCUGA COGAflGAGG-CGCCCG AA AGVG 2359 UCUGUt3C CUAGGCGAG-LA

AAAGCAG

2360 UUCAAAG CTJG AzGGAA GCG; AAAGGUU 2376 UCAGAAG CUGMflGAGGCCGAA CGA ACCACCU 2377 CUCAGAA CUarGA CGAAAGGCCGAA

AACCACC

2378 CAGt~kGA CUAGGCGAGC-A

AAACCCU

2379 C~nZJUGA CUGAUGAGGccGAAAGCCGAA

AA.AAGCA

:..2380 GCCGACA CtUGAGCCCGAA~CCG

AAAACUU

2382 GGGGCAA CUGAUGAIGGCCGAAGCCA AGAGAAtJ 2384 UUGUGUC CUGAUflGcAAGGCC GCG,) ACUGr.AU 2399 GUCCACA CUGAUGAGGCCGAAAGGCrAA ATjUrjUy 2401 CACCUCA CUGAUGAGGCCGAA G~CG1A ACAGCU *2418 GGACCU CUGAU-GGCAGGCCGAAr

ACCAGU

2417 ACUC CUGAUGAGG-CCG AGGCG ACCAUGr 2418 GCCUA CUcLA3GAGGCCGAAAGGCMrAh

AAUA

2425 AACUC~J CUGAflGAGGCCGaAGCA

CUA

2426 AACUCU CUGAI3GAGGCCGAAGC;L AAAC UCU 2433 GGCGG~ CUGAtJGAGC-GGCC GAA AUCUAC 2449 AGCG CUG-AUGAGGCCGAA GCGL A.AGGCUU 2448 GGCAGG CUGAUGAGGCCc A AGGC:CUAAACC 2449 GGGGCAG CUGAUGAGCGAAGCGA

AACG

2451 AGGCAGG CUGATGA CGAAGA;CG

AAAGGC

2452 GGGCAG CQGUAGGCCGAGGCC-

AGGG

2455 CGGGGG CUGAUGAGGCCGAAGCCAA

AAGGU

2459 GCGGGG CUALGAGGcCGAGUrCccA

AGGUGG

2460 CGGGGG CUGAUGAflGCCGA AGCCGA

ACGGUG

2479 GCUGGU CUGAUGAGGCCG AGGCCA CU~Uc 2480 GAAUCAC CUGAUGAGCCGAGL

ACGGUGA

2483 GGUGGU CrJGAUGAGGCCGAaCCA

ACAGUGU

2484 ACUGGU CUG-AUGAXGGCCCA- 'CG"'A AAAAGG 2508 UGGGAUG CEJ -AU GC-CCcLAAGGCCC AuGGAUA 2509 CUCGrUAM. CUC.AUGAGCC;C-C z. CUtA 196 2510 GCUGGUA CUC-AUGAGGCCG-AAAGG-CCGAA AACUCtEh, 2520 CAUUGGG CGAGACCAGCGMACAAAAG 2521 UGAGGGU CUC-ATGAGCCGAAAGGCcGAA -AuIGCL-G 2533 GAUACCt3 CUGALUGAGGCCCGAAAGcGAA AG_-fAU 2540 CACAGC-% CUGAUG~CZCGA A-GA~A AC~r-tG 2545 AGGACCA CUGAflGAGG--CAAAGCCGAA ACAGCAC 2568 tUUCA CUGAU A-GCCGAAAG,-CGAA AiCUUCAC 2579 CAGGCCA CUG~AGGC-GAAGGcc,-GAA AACUUAU :2585 AGAGAAC CUGAUGAGCCGAAC-C-CcGAA AUGCc.= 2588 AUUtCAG CUCA GGIZCCGAAGCCCAA ACX3ZW 2591 AGGAGCA CrGAU-AGCCGAAG--GAAG AGLACC:A :2593 GCAC-AGC CUGALGAGCCGAAAGGCCGAA AAAGAAG 2596 CAX3UGG CUGAUCAGGCCCGAAAflGCcGAA AAAAAJG 2601 AAACGAA CU XJAGACGCCGAAZG~CCGAA ACACG= *.:2602 GGGAflGG CU;UAGLIAA~CA AGCCUGGA *2607 CAGGUA CUGAIJGAGGC-CGAAAGGCCG-AA

AUCCGAG

2608 CACAGC-G CUG--tJGACGx-CC-AA GCGAA ACUGC-UG *.:2609 UJCCUGGtJ CUGAUGAGGCCGA-AAC.GcCGAA ACAUUCC 2620 GCAGGG3U CUGAUGAGSGCCGAAAGGC--CGA AG-UCCU *2626 GCLUGGAA CUG UAGGCCGAAAC-GCCGAA

AUCGAA

2628 AC-GCTJAC CUGAUGAGGCC -CCAA AGUGUGC 2635 AGGACCG CUC-AUGAGGCCCGAA.c CCGAA AC-UGA *2640 GGCAGGA CUG;LUGAGCCGAAAGGCCGAA

ACAGGCC

2641 CUJGCUCGA CUGAUGAGGCcAA,,A--GAAc AGCUGG 2642 GAGGCAG C -A=GGCC flAZGCCGAA AAACAGC; *2653 GCAUCCU CUGAIr=AGGCCGAAAG,-_-CAA ACCAGUA *:2659 CUUGCAC CUGAUGAGGCCGAAAGGccrGA

ACCCUUC

2689 CCrCGCGA CUGAUG-AGGGAA~r_0GCCGAA ACAUUAG 2691 GGCCUC%' CUGAUGAC-GCCGAACGCCA AC-ACAUU 2700 GGGCAGZG CUGAUGAGGCCGAAA 3 I-CCGAA AGGCUUC 2704 AGGCUGG CUGAUGAG~CCCAAAGG-CGA AGaGCrjC 2711 CUGCUC-A CUC-AUGAGlCGAAA-c-CGAA AGC-UGGG 2712 CCCtjaCC CUGAUGAGCCCGAAAGGCCGMA AGACCtUC 2721 CUUGCAC CUGAUG.AGG-CCGAAALC-CCCGAA, ACCCUTJC 2724 GCAC-ACG CUGAUGAGGCCGAAAGGCCGAA AUGtACC 2744 CUGCACG CUGAUGAG=CGAAAGGC-CGAA

ACCCACC

2750 GGUACUC CGAUGAGGCCGAAAGGCCGAA

AUAAAUJA

2759 AG-AUCGA CUGAUGACGCCGA rr,-CA AGUCCGG 2761 GCAGG-tJ CUGAUGAGGCCC-AGGCCGAA

AGGUCCU

2765 AGCGGC:A CUGAUGAGGCCGAAAGGCCGAA

;GCAAAA

2769 CCUTGUUtJ CUGAUGAGCCAAGGCGAA DCAGACtU 2797 GGACCAU CUC-AUGAGGCCC-. £-GCCGAA AUUUCA 2803 CGCCUGG CUGAUGAGGCCGAAAGGCCGA

ACCAUGA

2804 CUGCACG CUGAUGAGGCCGAAAGGCCGAA

ACCCACC

2813 GGGUCAG CUGAUGAGGCCGAAAGCA

ACGA

2815 AAAGUUG CUrGAUGAGC.-C .A~zGGAA~ AGACUjGU 2821 CCUCCAG CUC-AUGAGCC-:-AGGCCGAA

AC,-UCAG

2822 e-kCUCC:3 CUC--GUC-.'C-CCGG;kC-CGAA

AGC-;CC

2823 CCCUGAUGA. CGCCC--X 'C-CC AA).C-C-C 197 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 2975 2976 2977 2979 AUGAUUA, CUGAUGAGr.CCWAA GCC AGUCCAG UCAGAAG CUGAUGAGGCCCGAkkGGCCGAA ACCACCtJ CAGGrCAG CDGAtyGAGGCCGAA CC AGEJCUCA GGUGGCU Ct3G~uGAGGCCGAACGA

ACALIUGG

AACAUAA Ct7GAUGAGCCGAAGGC,-

AC-GCGC

UCACAGU Ct3GAUGAGGCCcAAGGCCGA

ACUUGGC

aC-MGc CrJGAUGAGGCAAkGCCCGA AAGGtJCC GUGAUGG UAuGCCGAAGc,-cGAA

AG,-:A

AAGAUCG CUGAUTGAGCCGAAAGGCCA

AAGUCCG

JAAAACUC CUGAGACGCCGu'AACGGCU

AAAXUA

AAUA.GAG CUGAUGAGGccnAAAGGCCA

AUGAAGU

CAALTAGA CUGATGAGG-GAGcc ACGA

AAUGAAG

UAAlAA CUGAUGAGCCG CGGCC

ACALTCAA

AAAtUAA CUAGLGCCG,-ZAAA=C c AAAIjACA AGCAAAA CUGAUGAGG-CGAAGGCCGA AAGCUUtC AGAGCAA C3UAGAGCCGAAGGCCC-A

AAAGCU

AAAUU1AA CVAUAGCGAAAGCCALA AAAUjACA AAAUUAA CUG~rAGCCAAAGGc AAAL-g= GACA.UUA CUGAUGAGCC AAGCGAA

AGAACAA

UGACCAG CUAGMGAGGCGA

AGAGWA

CUCAUGA C~.UAGCAAGCA

AMAGC:A

UCUAAAtJ CUGAtJGAGGCCGAAAGCGAA

AAUAAAU

CUCCGGA C JGAUGAGGCCGAA GCCA ACGAAUA UCtJCCGG CUCUGAGGAAGGCCGA~A

AACGAAU

CtYCUCCG CUGAUGAGGCCGAG,-CGA

AAACGAA

CGACCCTJ CUGAUGAGGCCGAAGCGA

AUJGACAA

CUUCCGA CUGAUGAGGCCGAAGCCAA

ACCUCCA

CCCUUCC CUGAUJGAGGCCGAAAGGCCGAA

AGACCEIC

UGGC-GAC C GAUGAGGCCGAAGGCCrA

AUGUCEUC

GCAGAGG CGAGAGCCGAAAGGCCGA

AGCGUGG

CACAGCG CUGAUGAGGCcGAAAGGCCAA

ACUGCUG

UGACACA CUAGGCGAAGCA

AGUCACU

UUGAUUC CUGAUGAGGCCAGCCGA

AAGGAAA

AGtGGCU CUAUGAGGCCGCCA

ACACAGA

AAUUlAAU CUGAUGAGGCCG GGA AAtUhCAU CUUE)AUU CUGAUGAGGCCGAA GCCGAA

AUUCAAA

.CCUCUGC CUGAtMAGGCCGAA GGCCGAA AGCCAGC AAAACUU CUr-AUGAGGCCGAAGCCGAA

AUUGATU

GCUJGGUA CUGAUGAGGCCGAGCCGA

AACEJCUA

AGUAGAG CUGAUGAGGCc GGCCGAA

AACCCUC

CAGCUCA CEJGAUGAGGCCGAAAGGC CGA ACAGCEUU GGCAAUA CVG4UGAGGCCGA CGCCGAA AGAAUGA T Substrate Trable 6 Human ICAM Hairpin Ribozyme/Substrale Sequences nt.Hari ioyeSqec PositionHariRioyeSqnc 86 343 635 782 920 1301 1373 1521 1594 2008 2034 2125 2132 2276 2810

GGGCCGGG

GGAGUGCC)

CCCAUCAG

GCCCUUGG

UGUUCUCA

AGACUGGG

CUGCACAC

ACAUUGGA

CCCCGAUG

AUGACUGC

CUGUJGIJA

ACCCAALJA

UUCLJGUAA

GGUCAGUA

GGGUUGGG

ACCIJGUAC

AAGGUCAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAM

AGAA

AGAA

AGAA

AGAA

AGAA

GCUG ACCAAGAAACACACGUUGUGGUACAUU1ACCUGGUA CCC ACCAGAGAAACACACGIJIJ)GGk

ACAIJIJACCUGGIJA

GUUIJ ACCAGAGAAMCACACGUUGUGCJUACAUUACCUGGUA GCAG ACCACAGAAACACACGUUGUGUACAUTACa-JGUA GCUC ACCAAACACACGGUACAcc yUAJCCUGGUA GCCC ACCAGAGAMCACACGUUG!JGGUACAUUACCIJGUA GCCG ACCAGAMCAACGUJGUACAUACCzTGUA GCUG ACCAGAGAAACCACGUUUGUACIJTJACCJGGUA G.UGG ACCAC.AGAAACA

CGUUGUGGUACAUUTACCUGGUA

CCUA ACCAGAAMCACAcGUGUGUAUUACCUGGUA GUAU ACCAGAGACCACGUUGGUGACAUJACCUGGUA GCAA ACCAGAGAACACACGJIGUACAUUACCIJGUA GUGG ACAAAAAAGUGGUCUACGU GCAG ACAAAAAAGUUGAAUCIGU GUAG ACCAGAGAAACACCGUTGUGGUAJUACCTJGTJA GUAC ACCAGAGAAACACACGuUGUGGUACAUUIACC!JIGJA GCAG ACAAAAAAGUGGUCUACGU

CACCA

GCGCU

MAACLI

CUJGCG

GAGCU

GGGCLJ

CGGCU

CAGCA

CCACU

UAGCA

AUACA

UUGCU

CCACA

CUGCU

CUACU

GUACA

CUGCA

GCC

CC

CC

CC

GCUU

GUU

GAC

GAC

CC

CC

GAC

GCC

CAC

GC

GAC

GUU

GUC

CCCGGCCC

CGCACUCC

CUGAUGGG

CCAAGGGC

UGAGAACA

CCCAGUCU

GUGUGCAG

UCCAAUGU

CAUCGGGC

GCAGUCAU

UACAACAG

UAUUGGGU

UUACACAA

UACUCACC

CCCAACCC

GUACAGCO

ULIGACCULI

9 9 9 9 9t 9** 9 9* 9 >9 9 .9 2999 9 9 9 *9* Table 7 Mouse ICAM nt.

Position 76 164 252 284 318 447 804 847 913 946 1234 1275 1325 1350 1534 1851 1880 Hairpin R ibozyMe/S ubst rate Sequences Hairpin Ribozyme Sequence GGGAUCAC AGAA GUGA ACCAGAGAAACACACGuLJJGUGGUACUUACCJGGUA UGAGGAAG AGAA GUUC ACCAGAGAMCACACGUGUGGUAAUUACCUGGUA TJCAGCUCA AGAA GCUUJ ACCAGAGAAACACACGUUGUGGUACAJTJACCUGGUA GCACAGCG AGAA GCUG ACCAGAGAAACACACGUUGUGUATJUACCUGGUA AAGCGGAc AGAA GCAC ACCAGAGAAMCACAcGuJGuGOAcWJACCUGGUA AGAGCUGG AGAA GCGG ACCAGAGAACACACGUGGUAJJACCUGGUA UCUCCUGAGAA GCAU ACCAGAGAAACAccGuuGuGGAJTACCJfGGUA UCUACCAA AGAA GUGG ACCGAGAAACACACGUUGUGUAAUACCUGGUA AGGAUCUG AGAA GCUA ACCAGAGAAACACACGUlUGUGGU ACuCcJ~uA AAGUUGUA AGAA GUUA ACCAGAGAAACACACGUUGUGGUAAUACCJGGUA CCCAAGCA AGAA GUCU ACCAGAGAAACACACGtJGUGUAA1AcC![jGGA AUUUCAGA AGAA OCUG ACCAGAGAACACACGUUGUGUACAUACCJGGUA UGCCUUCC AGAA GCAG ACCAGAGAAACACACGUU!GUG(UAAUACLIJUA CCCCGAUG AGAA GCAG ACCAGAGAAMCACACGUIJGUGJACLJIJACCUGGUIA ACAUAAGA AG-AA GCCA ACCAGAGAAACACACGTJUCGUGGAAUUACOJQUA GUCCACCG AGAA GUAG ACCAGAGAACACACGUGGGUACJJACCGGUA AGAAUGAA AGAA GCGU ACCAGAGAAACACACGUUTGUGGTAAUACCJGGUA Substrate UCACc GUll GUGAUCCC GAACU GUU CUUCCUCA AAGCU GUll UGAGCUGA CAGC2A G!JC COCUGUGC GUGCA GIJC GUCCGCUU CCGCG GAC CCAGCUCU AUGCC GAC CCAGGAGA CCACU GCC LJUGGUAGA UAGCG GAC CAGAUCCU UAACA GUC LJACAACUU AGACG GAC UGCUUGGG CAGCA GAC UCUGAAAU CUGCA GAC GGAAGGCA CUGCU GCC CAUCGGGG UGGCA CC UCUUAUGU CUACA GCC CGGUGGAC ACGCU GAC UIJCAUUCU

S..

0 0 0 0 0 0 0 S 0 5 9..

0** 0 0 0 *50 0 0 g S .0 Se 0 Substrate Table 8 Rat ICAM Hairpin Ribozyme/S ubst rate Sequences n t.Hari ioyeSqec PositionHarnRioyeSqnc 59 84 295 329 433 626 806 849 915 1182 1307 1357 1382 1858 1887 2012 2303 2539

AAAGUGCA

GGAGCAGA

GGGAUCAC

GCACAGUG

AAGCCGAG

UUCCACCA

CAUUCUUG.

UCUCCAGG

UCCACUGA

AGGGUCUG

ACCUCCAA

AUGUAAGA

UGCUUUCC

UCCCGAUA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCAG

GCAU

GCGA

GCUG

GCGU

GQGc

GUGA

GCAU

GUGG

OCCA

GCAG

GCUG

GCAG

GCGG

GUAG

GCCU

GUGU

ACCAGAGAAACACACGUUGUGUACAUUACCUGGUA

ACCAGAGAMCACACGUUGUGUAAUUACCGGUA

ACCAGAGAAACACACGUGUUAUACCUGGUA

ACCAGAGAAACACACGUUGUGUACQArJACCUGGUA

ACCAGAGAAACACACGUUGUGUAAUUACCUGGUA

ACCAGAGAMCACACGUUGUGGUAAUUACCUGGUA

ACCAGAGAAACACACGUTGUGGAAUUACC1JGIJA ACCAGAGAAACACACGUUGUGUACUACCzTJUA

ACCAGAGAACACACGUGUGUACUACCUGGUA

ACCAGAGAAACACACGUUGGGAAUACCUGGUA

ACCAGAGAACACACGGUGGACJUACCIJGTJA

ACCAGAGAAACACGUGGUACAUACCUUA

ACCAGAGAAACACGUGUAUACCUGGUA

ACCAGAGAACACACGUUGGUACAUACCUGGU1A

ACCAGAGAAACACACGUUGUGGUACAUUACUGGUA

ACCAGAGAAACACACGUUUGGUTAJAUUACCUUA

ACAAAAAAGUGGUCUA~Gu

CUGCIJ

AUGCU

UCGCC

CAGCA

ACGCA

GCGCU

UCACU

AUGCU

CCACU

UGGCG

CUGCG

CAGCA

CUGCA

CCGCU

CUACA

AGGCU

ACACU

CCACA

AAGCU

GCC

GCC

GULJ

GAC

GUC

GCC

GULJ

GAC

GC

GAC

GCC

GAC

GCC

GCC

GCC

GAC

GUC

GCC

GUrJ

UGCACUUU

UCUGCUCC

GUGAUCCC

CACUG;UGC

CUCGGCUtJ

UGGUGGAA

CAAGAAUG

CCUGGAGA

UCAGUGGA

CAGACCCU

IJUGGAGGU

UCUUACAU

GGAAAGCA

LJAUCGGGA

UGGUGGGC

UIJCCUUCUj

CCCAACUC

UGGAGUCU

GUGGGAGG

GCCCACCA AGAA AGAAGGAA AGAA GAGUUGGG AGAA AGACUCCA AGAA CCUCCCAC AGAA GCUU 201 Table 9: Rat 10AIM IM Ribozyme Target Sequence ~s.

nt.

Position U1 23 26 31 34 48 54 58 64 96 102 108 15 I1i9 120 i46 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386

GAXUCCAAU

GCUGA=D

GAACUGCU

CCUCU=C

CUGAAGCT

CTJCAAGGV

GAGAACCET

CCCCGCCU

CCGUG=U

CAAUGGC

CCUC~U

CDCCU=G

GGACUG-CU

UCCUMcc GACACtUG

GUUGUGAU

CCAGA=C

ACCCGGCU

AUt1UCUU

UGAACAGU.

GAAGCCTU

GGGUGGALU

CAGCCCCU

GACCAAGU

CAAGCU

CtJGAAGC

GGCCCCCU

CACUGcc= GAGCCAAU

TC

G-AAGCCL7U C GAAGCtJCU TU CGGAGGAU

C

ACUGUGCU

U

t3GUGCAU A AAGCUCULT C CACGCAGU C CAAUGGCU

U

UUACCCCT C AGAAGCCU

U

ACCCAC=t C U CACACUGA c cuumcaCA C UUCCUCE7U C CUGuCC C AGAMUM~hC A CAAGCCC C G-GCCU~GG C CCU1GAGCC U UwGCCCC C CUGGOCCrJ C CUGGUCGC U GGGGAACtT U UGUUCCCA C CCCAACrJC C CCCGGGCC U GGAACUCC C CACCUCAA C ACGAGUA A CtJUCCCCc C CGCCUCG :cGUGcAGG kAUCUGACC kACUGUGAA 7GUGGGAGG

GACACCCC

CCVUAGGA

AGUGGAGG

UCUCAUC

*CUGCCUCG

CAAGCUGA

ACAAACGA

UGAGAACU

UGGUCCrUC

AAGCUGAG

CUCGGCUU

CAACCCGU

ACCCACCrJ cCTCCTC HM Target sequence Po0sition 394 420 425 427 450 451 456 495 510 564 592 607 608 609 611 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 GCAC-CCu

AAGAACCU

CUCGG-,-rj-U GCCACCpAU

GAAAAUU

GGGAGU

C-ACCAAr-

AGCCAAUU

CAAUUUCU

GOCACOUU1

UCACUGJUI

GAACUGCUI

GCACCCU

AGGCAGCU

CCAGC.XU

CGGACEUU

GCCUGUU

CAGCUuJww CUACAACU IC CAACUUUU C CUCCUOGur C UCCLIGCC C AC"UGU U CUUGUGU U AGGCCUGU U GGCCUGUJU

U

CU7CCLIGGU C UCCUCCU C GCUTCAGAU A CCUTGGt U CUC-ACAGU U GCUCACCU

U

CAAUG.GCU

U

CCAUGCUrJ c UJ CMAACAG C CCAGCGCCA U CCCACC U AAAAACCA C AUCcV--, C CCmCCc C UGCCACA C ACUGUGuM C CGU1GGGQM UJ CCAACCAC C AccAG U UCLcAUGC U C~CAtGC-U C UCAfGUU C AUC-CUrJ U CAAGAAxjG CUUCCUCvu :cGGACUrU

ICGACUCC

L CC-CUMC r AGCUC

CUCGGUCC

GCGGGA

UGAGAACU

CCCUGGAA

CCUGGAAG

UCCUGCCU

CCCCMC

CUCGUCGC

U2-hCCUGGA AUUUAUxyo

UAGCAGCU

CAACCCGtI

L'CCGACA

HETarget Sequience CGCUGUGU U UrGGCU 202 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 1092 1093 115 2.163 1164 1166 1172 220 1201 2.203 2227 1228 1233 1238 1264 1267 1.294 1295 1306 1321 '334 2.344 2.351 1353 2.366 2.367 1368 1380 1388 1398 1402 1408 142.0 GACCkCC

CCUCU

AAUGGCEUU

G-ACICAAGUT

GCAGAGAU

TJUGAGAAU

GAGAAUCU,

C~CAACU

Ar-AACUUU i GUGGGAGU A UCACCAWG CCGGAGGU C UCAGAAGG GGAGGCU C AGAAG=G CCLccuu U GOCCA AGAGG=G C UCGA AGGGGAAUJ C CAGCCcCt7 CCC~ACm C UtUUMl ACGACGCU U cOuuCuu CACGCU C UUUCuC= ACGCUOC~r U VUGCUCU CUUUUGCU c UGCG=C AUCCAA13U C ACACUAA TUGGGCUU c T3crCAAW UUGGAACU C CAnUtGUG GCGGGCUU C GUGAU CUCCGGU C CUGGEJCGC UGUGCEPhD A UGGUcCL-C GGAAAGAU c Amu==G~ GUCACUGU u cAAGAAUG CAGAGADU U UGUGUCAG AGAGGGGU C UCAGCAGA AGCAGACU c UakcA=,G AACAG=G c uaGGGAAA G~U -t CCCAGAGC UCGGUGCU C. AGGU=C UCAGGcc A AGAGGACU UAGCAGCU C AACAAXJGG AGGGUACU U CCCcGG GGGLACU c CCrCAGGC GAUGGW)U C CCCCUGCC CUGCCEWhD C GG.GAt.GU UGGAGACU A ACUGGAUG CUGGCUGU C ACAG.GACA~ CUGUGCEIU U GAGAACUG UUCGUGkU C GCGCrUC CC-AACUALU C GAGUGGAC

CCCACO

UU~CGACa.G

AACCCG

ACUGUGAA

GUUC~CAo

MCAACU

CAACUUUU

UUCAGCC

UCAGCOCC

CAGCUCC

1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 1518 1530 1533 L951 1559 1563 1565 1567 1584 1592 1599 1651 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802.

181 1813 1825 1837 1845 1856 1861 GGGUACU3 C CCCCAGC ACCCACCU C CtJCUGGCU AUCUUGU A GCCtUCA~r AGAAGGCU C AGGAGG~A GGGACUw C ACCAGGGA AGGGOaCU u CCCCCAGG ACUGCUCU~ U C-CUCUU= CCOGG=G U GGAGACUjA CG GAAAU U ALTGVCA GAAAAUGU U CCAACC.kC UGGGUCU A ALTUGUC, GCCACCAU C ACtUGUGuA.

Gr3CCUGGU C GCCGUUGU ACCUG=G C AaAUGU CUGAUAU U GCGC.G=U GUGGcCCE C UGCVCGUJA UGGGAU C CCUGUUUA UCCMVcu U UGIUCCCA UUACACCU A UUakCCGCc ACACCrUA U ACCGCCAG AGGAAGAU C AGGAIJUA CAC-C-MW A CAAkGuUAC tUhAAGUU A CAGAAGC CCC~CG= C CCUGGCCC MCtGCUU U GCLcU GAACAGAU~ C AAMMODC GAGAACCU C GGCCUGGG GGGCDDU C CACAGGUC GGCCUGU U CCtGC CUGCUCGU A GAcCUCEC CCCACCU A cCAcU CCGGAC=r U CGAUCUUC CUCCUGG C COGGUCGC UCAGA&U A CCUGaGA GAUCACAU U CACGGUGC GUCCWUU A CACCUaL= CCUCUGCU C CUGGUCCU GAGAACCU C GGCCUGG GACAxrj c CCCAACUC AUGGUccr C ACCUGGAC UCC~UGU U AAAAACCA GCUCAGAU A UACcL1GGA AACAGAGU C UGGGGAAA GCGGGCU C GUGAUCGU GCCACC= C ACUGUGUJA ACCCcCCU C ACAGGGUA AGAGGACU C GGAGGGGC CCCCUMAU C UGACCUGC CAUGUGCU A UATUGucc 203 a. a.

a.

1865 1868 1877 1901 1912 1922 1923 1928 1930 1964 1983 1996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2115 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173- 2174 2175 2176 2183 2185 2186 2187 2189 2196 MiUMGCMCJ A GACACAAG UCACG.-GU C AUAUAAAI3 ACAGUACU U CCCCCAGG CZAAAACU C AACGG-UAc, G-AACAU C AAUGGAC-A AtUGUkAGU U AUCCUtAh LCGCCU C ACCUUG Ct-w-CAU A UAC G tXaGACU A ACUGG=~ A~uAL-u-U U GMGUOAG G-AG-ACCU C GGCCCGGG L-GCAAGCU-T C UUCAAGCU'r AUGUAAGU U AVUGCCUA CS-UMCCU A UCGG I CUG-CCM.U C GC-CJJU-x UWUUGAGU A CCCOGThC CGGAGGALU C ACAAA=G CCUGAXCCU C CT3GGAGGU CCCIGUC7 C CAADGGCU AUACUUGU A GCCUCAGG LGUACC C AGGCCLCA CCXACDLt U GUtGAY.U CUGAC-U C CUGGAGGU UUCCGACU A G,-UCCE;G -AGUG-CCGU A CCAtJGAUC GCCEJGUUY C CUGCCUCE CCAAUC U GUU-AU UUGAGAAU C MCAACLU tGCAGUU A UUUA CGAUGUAU U MflU~AUU C-AUGMA=t U ALUAAC AUGMfU= A UaLUAA ACAUUCCU A CCUUUGUU TJAUUMUU A AUUCAGAG UGAUGtUUu UaZmou G-AUGMhUE U .AJJOkAIUC GUAUUt;rU U AAWtCAGA CAG~tahuu u AuuGAGUA UUGCOAU A UGGUCCUC UCUCOAUU A CCCCUGCU AUUUCUUU C ACGAGUCA G-AAAAX=~ U CCAACCAC tJGACAGtJU A UUUAUUGA ACAGUUAU U UALUUGAW CAGUtUt3 U AflUMGUAt AGUOMxUu A tJUGAGAC UUAUUUAU U GAGUACCC CLMGACAGU U AULTUAUG 2198 2199 2200 2201 2205 2210 2220 2224 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338.

2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 GAA=lCU C CGAG-UCA AGACVCU A CAflGccAG, GGG~CUU C CCCCAGC-C GGGCO-UCU C CACAGGUC UUUGU C AGCCA=t MGGA= A ACL'GGAUG GAGAACC C GGC-C,Gc AC~ACf U CC',DCr-,U Cu GA=~r C ALC,,CAMA UCAUGCUU C ACAGAACEJ ACAC;L-CU C UcAGMGu CUCCL'GMt C CCUCuC AUCCAAUU C ACACUC-A GAUCACAU U CACGGtC AUCACA.DU C ACGGC-U AI3CAGGAU A UkCAA=~ GAGCAGU U AACAUGU GGAAAGAU C AUACGC,-M ACAGUUAU U MAOUGAGU GCCCU,,GGU C CUCCAAUG CAGGAMlU A CAAUOc GGAAAGAU C ADRCCGG-U UUJGGCUu C UC=CcAG GG.CtU- C CCC,-AGC-C GGGCCUGU C GGUGC-UCA.

Ct'G-UCCGU A GACCU~CC CCCUC-CCU C CUCCcCAZ CCAUCCAU C CCACAGAA CDUGUGU C CCLX;GAG GAACUGCUT C UUCCUCU GACUtJCc-U U coca.UAu GCUGAIUU

C'UUU~CAC.:

CCUCJU C CUCULUG UGAIUMUCU U UCACGAGU AUUUCOUu C ACGAGUCA M~UCCCGGU A GACACAAG MAAMXAMr A UGUGC.G UGVGCrIAU A UGGUCCUC CAAUUUCu C AUGCUUCA AXJCAGGAU A UACA.AGU UCAUOG=U C ACAGAACU UUOAfl~Ar U CAGAG=~ CCUGGGGU U GGAGACcJA UCAGAGUU C UGACAGU CGQAGGAU C ACAAACGA UGAACAGrj A CUUCCCCC GAAGCCtju C CUGCCUCG GGCCUGUU U CCUCCUC GCC3GU C CjC-CCL7CU 204 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2545 2568 2579 2585 2588 2591 2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2691 2700 2704 2711 2712 2721 2724 2744 2750 2759 ACAUUCCU A CCUUUU CCCUG=C C CUCCCA~CA CC~kCCrJU U GDUCCrAA U~CACCU A Ut1k~CCM GUGCCGU U GUGAUCC= ACCUUUGU U CCCAAUGU CCtUUU= C CCA=C G-ACCAcc C CCCACCUA AC~tak=f A CAUUCCUA AC-AU~cA Uj CC~kC=t ~rCAMCA C CrUACC=u GUCCA=U A CACCfL-U ACCULUU U CCCAAUGU CCUOUUU C CCAAXUC ACAGCXtIU U ACCCCOCA VcGG;Gc C AGGMUCC AG~cGCU c CGGACUU CAGAGAUT U GUGUCAG CCUGCACU u uGcca=G CUGCUMU A GACCUCO VCCJCCU C CCACAGCC CUCUUCCU C UUGCW.AC ucucaDuu A ccccuccu CUCCUGG C CUGGU~C UGCr,--U A UGGUCCuc GUCCUGGU C GCCGUUG GUGGGAGU A UCACCAGG CUUAGCU C CCGOGGGA UGGAGACU A ACUGGAUG OVAGAGOrj C UGACAGOIJ CDCMOA A GUGCCU MkAckc U UCX= UCACAGAu C CAAUUC?~ GCCAGM A UCCAUCC CCCCMcCU A CAtlca=r GCCULGUU C CUGCCUC CCACAG=r C -AGGUrGCr AGAAGGGU C CUGCAAGC ACOAGGGU C CUGAAGCU UCAGGCCU A AGAGGAcU AGGUAC U CCCCCAGG GACCACC C CCCACCUA CCCUACC U AGAArjL CCrJACCULT A GGAAGG GGAAAGAU C AUACGGGU AAGAUCAU A CGGGTYit3G GGGUGGAU C CGUGCAGG GUCCCUGU U UAAAAACC GACGAACU A TJCGAGUGG 2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 2829 2837 2840 2847 2853 2560 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2931 2912 2913 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 CGACUUU C GAtCUUCC CUUUGCU c UGCCGCCrJ UtCUCMU U ACC-CoC CGUGAAAXJ U AU,-UA CUCAXX3'cU U CAcAGAAC UCALDG=U C AC-AAACr GCUCCCAu C CUGACCj =cGGACuU c GAUCUUCC CCUGACCU C CrrGAGM UACAC r u t;CAG-rUc CAACOU=r C ACUCCCA UCG-GUGCU C AGGUAUCC CACAGGCGU A CVUCCC- GCACCCCrJ C CCAGCGCAL UUACCCCU C ACCCACCrj UUCGAUDCU U CCGACaG tU~UrGrx Lu CCCUGGAA GGCrG C GGUCC3CA UGGAWrCU C CCAGCAc AGGCAGCU c CGGACUUU GGCUGAC!J U CCUUCJCT GAACUGCUT c UUCCUCUU~ GC-CUGACU U- CCUUCUCTJ GVUGAUGTJ A UUUUA CUGCUCLUF C jrjv=( UGAUGtJAU U UIAUU]AAUU GAACUGCU c UUCCUCUty ACUUCCUrj C UC~UAuC UUC=X"U~t C UAUUACCC AGrkou A UUAAUUCA UUGUAhUU C GUCCCA GauAuA U AAUUCAA VUAUU~r A AUC~z COCUUCCU C UUGCGAG CUUCCUTCU U GCGAAG).C ALUUcUUU C ACCGUCA UUtUGuJGU c A~cczCUG GAUGG=~ C CCGCEJGCC UGGAGUCU C CCAGCAC CAGACT= C CCCCACGC ACCAUGCCu U CCUCtjGAC CCGGAC!Jrj U CCAt3CUUc UGCUUicCU C UGACAtjGG CUUUCCUUr U- GAAUCAAU UUTUUGUGIJ C AGCCACUG UGUGUATJU C GUUCCCAG CUUUG.AAU C AAUAAAGU UGGAAGCU C UUCAAGCrJ GAAUCAAU A AAGULUUA 205 2975 UG-GAAGCU C uucAAGCU 2976 tWAGGf *C cucACCOG 2977 GZAAGCL3CU U cAAGcuG 206 Table 10: Rat I0AM H[H Ribozyne Sequences nt.

Position 1 23 26 31 34 48 54 58 64 96 102 108 115 119 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 WJ~ U&AJ I Li

GAV

UAGAGAAG C~JW GCC--AAACGXGCC.,

AAGUCGC

AAGAGGAA CJrAG-C--wjA~

ACCAGUTC

AGGACCAG CGUACCGAAAGC-,,?A

AGCAAG

GtUhUAUCU C rNUAGCCAL;r,-CCA AGC=uCA GGGCUUG CUGAVGAC-GCCGAcCCC,

ACCUG

CCCAGCC c AGGcccGAGGCCA

AGGUUCC

G7GCUCAGG GccGAUG AGccC;

AGGCGGC;

GG3GAGCA CUArGCGAAGGcCw

AGC.-

ACGGGUUG CUACGCCA1G~,i AGCCA=u AGGACCAG CU wNGCUAAAGCCCA AG-a.GAGG GCGACCAG CGUG ~AGGCCG,-C;

XCCAG

AGUUCCCC CGUAGCCAGCC.A

AGCAGUCC-

UGG-GAACA. CUA GCCGAAAGCCC- AGGUkGGA GAGOUGG CUAGGCCAAGCA A~kG GGCCCGGG CUCUGAAGGCCGGCCW.A

ACA~C

GGAGUCC CUG GGCCGAAkfGCCA AGMrCUGG TJTJGAGGOCUGAG CGAAACCC;

AGCCGCGGU

UGACUCGU CUGAUGAGGCAGCcr AAAGAAAxj G-GGGGAAG CUGAflGAGGCCC-AC- LA ACUG,-ucM CGAGGCAG CUAGACC-AGG--G

AAGCUUCV

CCUGCA.CG aMUZ CAAGIA

AUCCACCC

GGCAGAU CUAGAGGCGAAGc=

AG=G=

UUCACAGU CUAGCG

ACUUGGUC

CCUCCAC CMGGOAAGGC CCQA ACOUG- GGGGUGUC COAGGcAGGa=

AGCUUM

UCCUAAGG CtJG&UGAC r r AGGGGGC CCUCCACU CUGAUGAGGCCGG.AA

AGGC=

GCAGAGA CUGGXJGAGGCCGAAAGCCCA

AUUG-,-UC

CGAGGCA L

AAGGCUTJC

UCAGCUUG CUGAUGAGGCCGAAGC .A AGAGCUUC UCGUUUGU CGGAGCCGUGGCA

AUCCUCCG

AGDUCUCA CUGAGAGCCGXUZGGAc

AGCAAG

GAGGACCA CUAGGCCAGCGAA~

AUAGCA

CUAGCUU CUGAGGCAGCCCA AAGAGCUU3 AAGCCGAG UAGGCGVZG

AGIW

ACGGGUUG CUGAUGAGCctAGCCAA ArCCAUrJG AGGUGGG-J CUGAUGAGCCGAAGC

AGOMI

GAGGCAGG CUAGGr AGcCUUcu UACCCUGTJ CUGAUGAUGCAr.cccrA Ac.GE u AGCtICCAA CUGAtJGAGCCAAGGCAA

A=~AG

Rat E Ribozyme Sequience 207 394 420 425 427 450 451 456 495 510 564 592 607 608 609 611 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 852.

863 866 867 869 88i 885 933 936 978 980 CUGUUCAG CGAGcGCAAMGccGA& AGcACCAC UGCGCUGG CAGCCGAAAG~CCGA

AGGCGUGC

GGUGCAG CVAGCCCJACGCA ACCGrAG UGGUUEUU CUAGGCCGAA~c.C~r

ACAGG

CGCAGGAU CUACGCtAAAGcc%

AGGUUCUU

GCCUGMG CVAGGCGGC~CGci XbAGACC UGGtOGGCA CGt-AGCCGAAAGCCC

AAGCCCAG

MXACAGU CUAGGCCGAAAGGCCGAA AflGGVWGC UOCCCACG CUAGGCGAAGOA

AGCAGCAC

GGGGU=~ CGU G-CGAAAGGCcGAA ACAnUUUrC UCCC=GUGGU GCCGAAGCCGA

A~CC,

GCAMAGA CUAGG^CAGCCGAAc AuUGGCUCx AGCAUJGAG CGUGGCCGAAGCCGA

AAUUC.GC

AAGCAVGA CAGCCGAAAGCCG AAAUGf,- UGAAGCkAU CA G c acc AAnUM CAUCoC CU &GGGCGZC-AA A~kGAC ACAVUCUU CUM GCMUV Cr.A AACC AAGAGGMA CUAUGCCGCA

ACAG

UGCGCDGG CGUAGGAAGGAA~

AGGGC,-U,-

AAAGOC CGAVG cc-uG~CCC A~CaCWc GGAGIGU C UGGCAGGCGAA

AGGUCUWG

GGAAGArJC CAUkGCGAAAGrA

A&GCC

AGAGGCAG CAUkGCCGAAAGGCcr.A AAkCAGC GUGAGGGG CAVVGCCGAAAGM=

AMIUGCUG

GAGCVGAA CAGCCGAAGXCcM AGUUGt~hG UGGCGAGCU CUXAGCC~aZA AAAAGUuc GCGACCAG ~COGDAC G

ACCAGA

UCCACCCC CWGGCcGAAG cc AGGCAGGA AGUUCUCA. CWUAGCGAGX

AGCACAGUJ

UCCCAGGG CUAGGC~kGCA

ACA

CUUCCAGG COAAGXAGCrA

AACAM

AGGCAGGA CMMGGCCXGCGA

ACW.~CU=

GAGGO= O3GG GCCMXG

AACAGGCC

GCGCCAG CM WGCGAACr ACcAGGAG C-AGCUUCA, CUDAGCCGAAGCC

AGOW.

UCCAGGU CUGJAGGcctAACrA AU GAG UGUCUCC CUNmgccG c AcCCCAGG CAA~kAAU CWAUGAOGXAGCCt;A

ACUUC

AGCUGCU CMGOCAGC3A

AGGG

ACGGUG MUAGMAMXA

CAIG

UGUCAGAG CMMGCXAGCA

AM

UAGGUGG CMrGCCGAAGCC AGUG~c CUUCGCAACAGGa=-A

AGGAAGA:G

CACGGGU CAIGCCGXGCXcc

AAGCCAXI

UUCACAGU CEGAIGAGCCG AAG ACUUGtJC

CUGGGAACVGUAGCAAG~-AA~C

VGACACAA CUAUAGCCGAMC~rA AUCXUc AAGUUGUA CUGAUGGCGAAGG CG AUUCUCAA AAAAGUU CUGAMAGGCCG AACCG.A AGAUjUCUc 208 986 GAGCOUGAA CMGUGAGClGAr.CCAGCr-

AGUUG

987 GQAGCOGA Lut-lC~~ CGAA=L AAGUGA 988 GGGCUG C~r GA -ACGC-,GAAAG.-,C AAA UIGU= 1005 G-ACGCCAC CUC~.XG~aAcGC,%

AUAC;L

1006 CCCGG CLtAULAC-CCGAAArC-

ACUCCAC

1023 CCUCA CUGAUGAGGcCGA CCGAA ACCUCcM17 1025 ccCCUCV CLGUACCGAA,-C--

A~C,,C

1066 UCGGGPLC CMUAGCAAC-C;-

AAGGUAGG

1092 LCUGCUGA Ct4GTAGGCCGAAAGCC,X ACcCC-, 1093 AGGGCUG CUGflGACC-CGAAAc.GC; ,j~tCCU 11=5 AtCAACAA COAGAGGCC".X GCG-

AUUG=

1163 AGAAAA~ CIUGlAGGCGcGC;_ A AGCCGrjG 1166 CAAGA CMAUACG,CrG =CA ACA 1172 AGGCCuCAL COAGGCGA~.-C

AGCAAAAG

1201 CCUUGA, CUGAGAGCCGAAGCW

AACCA

1203 GCcrLGUG CUAGAGLCcG- CCGAA AGAAGCC 1.227 AGCAAU COGAUC-AG-CCGAAAG-CCGAA,

AGUCCAJ,

1228 ACCAtJCAC COG G.floc=.GCa C; LCCG ***1233 GCGACCAG CMUGtGAGGCCGA AG--CGALA ACCAG=A 128CAGA* COGAUGAGGCCGA-AAGGC-CGAA Ak=C 1264 ACCCGETh.U CUGAUAGGCCG ACC..-A AUCUUUC *.:1267 CAMUCUUG CUAGGC~AA-r,-C,.

ACAGUGA.C

*1294 Ctr-ACACAL CUAGGCGA~----A

AAM

**1295 UCLGCUC-A CUUACCGAGCCA AcCCCrJ 1306 GCUGA CUAUAGGCCG XGGCGL

ACVGCU

1321 UUUCCCCA CUGAflGAGGCCGAA GCCGA;A ACDCUGruU *1.334 GC-UCUGGG CrxGAUGAGGCCGAG rxr-AA ACAM 1.344 G-GAM.CC C7GAZGGGCCJUZ 6=rAA AG-C i151 AGCC13CU CCGMtMGGCCGAAGGCCGAA

AGCC

1.353 CCAflUGUU COGAtTGAGGCCMA GCGL AGCUG=U *.1366 CCTUGGGGG CUGAX3GAGGCCG A QG~rA A~apccc 1367 G-CCGG CUGkflGAGGCC GGCCMIL

AAULC

1368 GGA G LUll

ACACCAC

1380 ACCAUCCC CUADAG-C

AMGCAG

1388 Lrr;Trk

AGUCUCCA

1358 UGUCC=G CUGWUGAGGCC-,XAGGCCG

ACAGCCAG

1402 CAG=UCUC COGADGAGOCGAAAG-CW

AAGC=

1408 GACGCCAC CUGAGAGGCCGXGCCA AU CG 1410 GUCCACUC CUG~AM~rCGA

A~GU

1421. GCCUGGGG CUGAUXJGGGXaAG CGA AAGtJ cc 1425 AGCCrAGAG CUGAUAGG C CIC j AGOLG=r 1429 CCLMAGGC ClrGAUGAG~CC)OAAA--CG

ACAG

1444 CUC3CUCU CUGADJGAGrcc C, Acccr.CCAjcrj 145;= tCCCUGGU Ct3GAGAGGcCGa CCL~UCUc 1482 CCUGGGGG CUGAGAGGccGA- AG~kC 1484 GCAAG-AC, CUG UGAGCGU CC-k AC-.rAGU~ 2.493 UAGUCUCC CUGAAGGCCA-;CC-A p=C 209 1500 UMACAU CUGAUGAGGCCGAAAGGCCGA AflUUCACG 1503 GUGGOUGG CUGAt1GAGGCCGAAAGGCCr=-A

ACAUUUC

1506 CCAACAAU CUG3AXrAGCCGAA GGrraA, AflACCCA 1509 tACACAGU COGAX AGG -CGAAG~cC, A3UGGUCGC 1518 ACAACGG CUt;AUCAGGCCCGAMGCCGA

ACCAGGAC

1530 ACA.AMAU C XAUAGGCCCAAC, CA .ACC-CAGGU 1533 AAGCCCCGC COUGAGGGAAAGCC.A

AUA

1551 UACGA C CWUAGAGGCCCIG cc AGC-GCC-AC 1559 UACAGG COGAflGAGGCCGAAAGG--CCAA

ACVUCCCA

1563 UGGGAACA CUGVGAG=CC3AAG.-C. Aa AC- 1565 GCGM~A CUAGGCG, M cG ;UG A 1567 CUGGCGGU C:CGAGCCGAAGCA

AVGG

1584 UDAMUCCU COGAUGGG AAAGGCGA AflCUU=C 1592 GEUACMa CUGAUGAC,-GAA CGA AMW=cG 1599 GCCUU=CGA AGCGAGGCA -ACUr.Gj .:1651 GGCOUAGG COGAAGCCG~ AAA CMA AGGG= ,-\1661 ACCAGGGC CUMAGGCc MUZMA

AAGUCA=

*1663 UMCCADU CGGAXGAGGCCC-U=flGAA AflCUM= 1678 CCCAGGCC CUWMfGAGGCCGAAAGtGryG AaMUuc 1680 GACCOGUG CUGAUGAGGccGaAC GCG

AGAAGCCC

1681 GAGGCAGG CUGAGAGGCCGA~ c AAGGCCA AW.,CC :1684 GAGAQ=U COGUGVAGCCGAAG ACGrrGCAG 1690 AAUGtU<fG CUAGG-CAAG-rA

AGGUGGG

.:1691 GAAGAflCG CGAUGAGG~CGAAAGGCCr.A

AAGCGG

1698 GCUICCAG CUGAXJGAGGCCGAAAC-CGAA AUCrJG 1737 GCAccGU CVGAUGAGCGAAAGCGAA AflGUGAT-TC 1750 AAUAGGUG CUGAIJGAGGCCGXGCCA

AAAUGGAC

*1756 AGGACCAG CUGAIXGAGGCC AGCCUAA

AGCAGAGG

1787 CCCAGGCc CUMVAGGAAGG;AA

AGGUUCUC

1790 GAGUO= G CT3GGCCGGC CGAA ACZUM 179 GUM CUGAM~AGCC

AGGACCAU

1797.I, UGGUoUUrJCMUAXCAAGCA A.ACAGGGA 1802 VCCkGGUA C ThCAGCri CGGAA AflCjA 1812 ULMCC CUGAWGAGCcr- ACUCUGrj 1813 ACGAUCAC COGAUGAGC~CGAAGCCA AAGdCCGC 1825 tWC=c~ Ct GUGAGC<.Cc

AUC-GUG

1837 UkCCCUGU CtXAUGGCGAAAGGCMj A TGrGW 1845 GCCCCUCC CMUAGCGAG-C AcjcCUCL 1856 GCAGGUM CMUGAGCCGAAAGMA

AVUAGGG

1861 GMICCA~k CUWWUGAGGCGGCCGA

AGCCA

1865 CUUGUGETC CUGAUGUl AGAG C~J r zv ACCGGCuz, 1868 AXUUD COGUGAGCCGAAAGCC Ak=CGUGA 1877 CCOGGQ CUGAGAGCC GCCA AGUACrJG 1901 UGUACCUU CUGATJGAGGCCGAA CCGAA

AGUULTMAG

1912 UGCCATJU CUGAUGAGCCGAAWCGAA AuCUGEJc 1922 UAGGCAAU CUGAUGAGCCAAAGGCCGA

AUUAAI

1923 CUAAAGGU CUGAflGAGGCCCAAG CGA

AGCGUCCA.

1928 UCCAGGUA CUGAUGCGAA(CCCAA

AUCUGAW

210 1930 CAUCCAGU COGAUGAGGCCsAAAGGCCGAA A~CUCOC, 1964 GCUG-A=C CU UGA~NGCCGAAAGGCCGAA AAAuC=C 1983 CCCAGGCC CUGAGGCtSAAAZcCcGAA AG~uUCUC 1996 ACCUUGALA CUNVAG= ACCUUCCA 2005 MGGCAAI7 CUG~~~caA~~~ aco 2013 CADCCCGA CVGAUGAGGCCGAAAGGCCGAA AGGcAcC,- 2015 ACCAUC CCAUvGCGAAGc AakGGCA 2020 GUACAGGG CUGC-AvGGCCAAAGGCCGAA AcucA~ 2039 VUUU~uu COGAVGAGGCCG-AAAGCGA AucCu=c 2040 ACCDCCAG CUACAGCCAAGCA AGGuCAGG 2057 ACCAUUG CCAGC-CAAGCA AcaACCAG 2061 UAGM LMUGAGGGCGAAAGCcaAA AuGAGC 2071 CCUGAGGC COGP GAG-GCCGAAAGGCCGAA AcAAGUU 2076 ULGGC.^U CUGAUGA-GCCAAGGCcGAAL AG~cu~r 2097 ACATJCAAC CrJGWGAG GAA cGA AGAGUUGG 2098 ACCUCCAG C-ARAGOAAG~A GUA I2115 CA.GGACCC ACGCAAG~ol GCC *2128 GAv'CAUFGG CUGAUGAGGCCGAAAGGccGAA, ACAGCACU 2Z30 AGAG CUGAUGAG~CCGAAAGGCCCAA. AXCAGGC 2145 ACAUCAAC CUGCMA GGcGA cGA AGAGU=G 2152 AAGUUGM! UA GOCAAGCC AUUC~A 2156 UCAXA CGAUGGGCUGAaaGccj AACC 2i58 AAUAAL CUGAUtGAGCCGAAAGGCCrAA AtCVCA ~:22159 GAAfUAAU CUGCflAXKCCGAAAGKGcCGAA. AADACAUjC 2 150. UGAUA CUGAIaAGcCG AGcMAA AAAUCAU 2162 AACAAAGG CUGAGAGGCCOAAAGGCCGA AGGaM= 2163 CUCM3AAD CUGAflGAGGCCGxa-aGGcCGA AAAAA .2166 AAUAAtm. CUC flGAG-CcGAAAGccCGAA AckCA *2167 GAAM3AU CUG~AGGCCGAA ccA AAACy 2170 UCUGAAU 'CUUGGGC :AAGGccCAA AMLV 2171 UACUAAU CUGAAGCCGAA GGcCCAA AtM.ACUG 217 GAGGA~CCk CGUa3GCCGAAAGGCUGA~ hfAGCACkL 2175 VGACUCGU Ct flTGAGCAG~~GCCCAA AAAGAAAU 2176 GUGUUG COUG~AGrGAAAcCG ACflUUUC 2183 UCAMAA CUC% GAGGCCGAAGCCGA AACUGUM 2185 ACUCAAk CUACGCGAAGC AACUGU 2186 UACUCAAU CUAGGCGAAGC

ALVLX

2187 GCMCUCA CUGAUGAGCCGAAGCCGA AAAcuj 2189 GGUCUC CUGAUGAGGCCGAAAGGC~GA

AAAL

2196 CA.AMAATJ CUGWAUGcca

ACUUCA

21.98 UGACCUCG CUGAXGAGGCCGAGCCGA A~kCA=U 2199 CUJGGCAUG CUGaALGAGGCCGAA CCG; AAGauc 2200 GCCUGGGG CUG~lrAGC-~rcGCCGAA

AAGLTACCC

2201 GACCUGUG CUGAUGAGGCCGAAGGA

AGAAGCCC

2205 CAGUGGCU CUGAX7AGGccAA~cGA

A~CA

222.0 CAIJCCAGU CUGAUGAGGCCGAA GCGA A~uaCoCc 2220 CCCAGGCC CUGAUGAGGCCGAA GCUr. AGGUUCUC 2224 AAGGUAGG CUGAUGcAAGGCCG A~C~~A AUGUAUGU 211 'I go 00 *005 0 0* Ot 0 0 go 0*e 0 *Pgg 0 0 .000 00 0 00 0* 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 2455 2459 2460 2479 2480 2483 2484 2492 UGGGCMCUG GGGCG ,A AGGUCCA AGUOCGJCOGAVM c -CGAA

AAGCU-

ACWCDG CMGGGMAccA

AGCUGMU

GCGCCAG CUAG c=,AGC~CQc

ACCAGGAG

UUMAG=G CVMNDUGCAGGccW

AA-C,,

GCACCGTJG CVAGGCGXA--CA ALGUGA~c AGCACwr CUGJ -CGAGG-,-A

AAUGUGAU

A)ACUUGLU, CUGAW=M W ACCUGAU UACAUGUU CUGAGC,-Gcc GCCGA ACcUGcrj ACCCG= COUG G-CGUGAAA

AV=UMU-

ACUCAAA CUG -GCC.--V-LGGCC; aL~Cw CAUUGGAG CGUAGCCGAAAW=CC ACco Gt~hACUUG CUGAMG'-MkAGC AMccU ACCCGaAU COGAUGAGG-CGAACA =Uc CCUGUGGA CCXGAVXCC =CC AAGC=cA GC =CCOGMGGLA A M GC GAOAXGGUC L; AGAGCGAAC GA ACQGAG UGUGGGAG C. XflGGCCG~-kGCG.ZA

GCG

UUCUGUGG CUACCGCMACCtG

AW-AU,

CUUCCAGG COMGU GCGGCCGA AAkCUG AAGAGGAA CA GCGGGCCGAA

AGCAU

UAAUAGAG CUG IJGAGCCtaGCCA AGrJC T'CGUGAAA CCGAGAGGArcc~C-

AAUCG

CCAAGA CUGAX GAJGGCCC G;A A;GiGC ACUCGUGA COAGGCGAGCGL AGkMAUCA UC-ACUCGU CUGA~rAGCr ACC AA 2kAMA.

CUUG=C CtJGAUGAGGCCGXGe- ACCGGCu CGUCCACA CUAAG'MAGCrA

GUU

GAGGA=CWAGAGCMAGCCA AflACXM UGAAGAU COMAAGOCAGGc rCUA AGAM=j AACUUGUA CMUGGG cct AUCCrJMu AGUUCUCU CUAAGCMAGCA AAGkCA GAACUCOG CVAGGCCXC-= AU~aM U~AUCUCC COAGGCGUGCrA

ACCCAC,,

AACUGUCA CUGAGGCCGGCGA

AAC~CCVG

UCGUUTUGU CUGAUGAGGCC~ GLCIC

AUC-UCCG

GGGGGAAG CUAGGCGAAUCA ACUCAu CGAGGCAG CMUAGCAAGCA AAGGCUjUC GAGGCAGG CUGAUMW GCLt CUA

AACAGGCC

AGAGGCAG CUAGGCGAAGCA

AAACAGGQC

AACAAAGG

CWUAGCXGCGAAGUG

UGVGGGAG CUAUGG- GA

C-GCCGAAG--

UUGGGAAC CEGAfGAGCCAGGCGAA AAGZtJW= GGCGGA t- GAAAC AC,GUAtA GGGAUCAC =GAUAGGCCAGGCcA

AC-GCGA~C

ACAUUGGG CGUAGCAAGCA

CMG

GACAUUGG =UG GGCCGAMDCI-C

AACAAG

UAGGUGG GAUAGC-AAGCA AGG-Uc- 212 2504 UGGAAUG CUGAUGAC-GCGAAAG,-CCcA

AUGM=

2508 AAGGUAGG CUGAflGAC-GCC a-aaGCCA AUUU 2509 AAAGGMG COGAI GAGGCCMG GGCCGAA~ AWJGM=D 2510 AAZGGM GAU GCGAGCC, AAAflGGAC 2520 ACADUGG CUGAI AGCCGAAAGcCC AcMAA(= 2521 GACAMW CU~GAGC7CGAAAGCa.

AACAAAGC

2533 UGAGGGGU CLGA;MG~CLCGAAAGG-Cr=A XNGCUrU 2540 GGAM=CUGAGG MAA~Cwj

AGC.ACCGA,

2545 AAAGUCCG CUGAGAGGCCGAAAGCmAA

AGCUGCU

2568 CJGA.~k CUAM CGA AAUCUCtM 2579 CCGGGCA CUGAUAGGCCC-AAGOCGA

AGGCAGG

2585 GAGAGGUC COAGC-CAAGCA ACMAGcAG 2588 GGCGUG C ~kUGAGCCGAAG

AGGAGGCA,

2591 CUCGCAA CUXGAUGAGGCCGAA GCGA AGAAGAG Sce 2593 AGOLGGGG CUGAJGAGGCCrG cCJA MAAA e2596 G~~GC~UGAGCCM GCCA ACCWAG 2601 GAGGACA CUGAVGAGcGMa. AkGA 2602 ALACGGC CUGAGAG-CGAc GCA ACCAGMAC 2607 CCUGGUGA CUGAGAGC AC CCA 2608 UcC~CCGG CWZAGCAA4CA AGCUaA.AG *2609 CAUCCAGU CCMX~GCGAAAGGCGA

AGUCUCCA

2620 AACVGUA COGUGVAGcCAAGGCG

ACUCGA

2626 AGCAGCAC COGAUGAGGCCGAAAGC .A ACOGAGAG 0*2628 GGAGCUGA. COGAUGAGGCAAGCrA

AAGUUGM

**,*2635 GUGAAUUG COGAUGAGGCCGAA GC.A AUCUGGA 2640 UGGAWGGA CUGA~UGAGCCGAAA4- ACCUGAGAa 2641 AAGGAG Ct3GAtGAGGCCG eArGGCCA AACA 2653 AGCACCCU CUGWUGAGGCAA G ACCGu= 2659 GCUUGCAG CUMVXAGCC LAoa ACCCoUc 0002689 AGCUUAG CUWZMUXaGAAA GGCcc=A A~CC=C *2691 AGUCOCU CUAGGI AGCcCA 2700 CCUGGGGG CUA~GCCAG= A A CCU 2704 UAGGUGGG CUGATJGAGGCCMQ AAGGU G=C 2711 ACCUU=C COMWGAGGCCMU ~GA

AGGUAGGG

2712 CACCUUCC C GfAGGCCG.G;

AAGM=

2721 ACCCGUAU CUGAUAG c AUCUrUC 2724 CAAACCCG CGAGAGGCCGAk ML AUMwcUu 2744 CCUGCACG COGGMAGCCGAAGCC;

AXUCCA~CC

2750 GGUUUUA CUIGkUlGGGcckGCA ACAGGG

C

2759 CakCUCGA CUGAUAc.C j

AGUUCGEX

2761 GGAAfC CUGWGA GCCMAAGC

AAAGUCCG

2765 AGGCMGCA CL1GAXXGAGGCGGC CA AGMAA 2769 GCAGGGU COGAUGAGCCXAGGC AA AGA 2797 UUGALCCA CUGAUG-AGCC~kUZfc

AUMCACG

2803 GOUCUGUG CUGAfGGCGAGCrCA AGC G 2804 AGUUCUGU CUGAWGAWC AArG MM 2813 AGGGOCAG CUGAflGAGCCGAAGC

AUGGAC

-2815 GGAAMAUC CUGAUGAGWCcGAAGcG

AAAGUCCG

213

V.)

2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 292.3 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2962.

2962 2965 2966 2969 2975 2976 29777 ACCCAG CUGAUGAGGCCGAAAGGccGAA AGCCi GGAGCOGA CUAU AGGCA G~;AAGU l TJGGGAGCU CGUAGCGAAAW-CcGAA AAAG-j GGAUCCU CUGAUGAGGCCGAAAGGrC,-,;

AGCACCGA

GGGGGAAG VGr.AGCCGAAAGbcGrAA ACCCU3G UGCGCUG UAG ccAAAcCC AGG,- A,,GGTGGU CUA~GCCAAGGCu-

AGG.C

CtTAGUCGG L.'v4GCG;CAAAGG-CCGAA -C,;UCr.-A UUCCAIGGG CUGA GCCGAGCcr. ACaAC;-C UGAGCACC CVUGACXfl jAG G cCr-r AOCGCCC GGC,^G C=O flGAGz~uAGGC ACACYA AAAGUCCQ CUAG CGAAAGcC.,AA AGCUGCCU AGAGAAGG CUGAr3C-CGAAA-GCcr.AA AGtUCAG-C AAGAGA CZAGGCGAAAGCCC-

AC,G~UEC

AGAGAAGG CUuJCG-%GAACGM

AGUCACC

UAhAAA CrAwGCCGAAAGGCC~ ACrA CGMcAMXG CUAGGGA G: AAGAGCAG AAfUMAV CUccGA ~AAGGCCXa~ AOkAUCAn AAGAGGAA CO A GcGA:uAAGGCCGAA AiGCXGMX GLTAAGA CGATGCCGAAAG~CCG.A AACGAGj~ GGGA= CUGAG GCCCGAAAGCC%:- AG3aAGGAA TJGAAC17A C~rAUAGGCGAAAG~CCGA2L AAACa CtJGGA CCAtjACCCGAAA-C'CGAA AAflAC UCUGAADJU COGA GC CGCGG AUAAAUAC CLUCUGAAU CUGAUGAGCC,-uG -CCGAA AAMLhAUA CTJUCGCAA CUATAGC-AGGLCAAC'k G%'OCUU=G CO AMGCGGGL

AGAGGAG

U'GACUCGU CU~7GGCCGAAAfGCCCGA

AAAMAAAD

CAGUGGCU CGrAGCCGAAAGc, U ,,ACAAAA GGCAGCGG CUA GCCaA-JaGcCCM ACCwC GOGCEOG CGUGcGC- AAAGGC(!U AGAC'Uc G-CCUGGG GA -AGCCGAAGaCCGA

AAGUA=O

GUCAGAGG% CG~rAGCCGAc-MU ,iGCAD= GAAM CUG GCcAA~GcCr., U AUCC= CCAUGUCA CUGO ,GCCGAAccC

AGCAAGCA

AUDGAIJUC CUGAflGAGGCGckGc~r

AAIGMAG.

CAGUGCU= CUC-AUGAC-CCAAGCOG

A~CAAA,

CUCGAAC CGGcUGAGoCcc AAflACACA ACUULIUUU CUCOGAGCGCr.A

AUUCAAAG

AGCO-UGAA CtJGAUGAGGCCGAA GGCGA AG-CUUCCA UAAAACUU GCCAAAacc

AUUGM=U

AGCUUGAA CUGAGAGCAGCcA

AGCUUCCA.

CAGGUGAG CUAGGCGAA-CCA

ACCAVADA

UCAGCUUG COGALMAGGcc AA,--W

AGAGCUUC

214 Table 11: Humazin IL-5 EH Target Sequence nt. EM Target Sequence ut.

Position E Target Sequence 8 9 12 13 36 37 38 56 57 63 64 69 70 74 78 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 1-92 200 201 206 207 212 216 222 AUGCACU U UCACUU U GCACUUU C AcC= U CUUU U AGAACGU U G-AACGUU U AACGUUU C GGAUGC U GAUCGCUU C UCUGAU U CUGCAtJU UJ VCOUGCw CUUGc

UUUGCCA

UGCCAAA

GCCAAAG

VCAGAGC

CAQAGCC

AGAGCCA

COCCUU

UGCA=U

UGAGOUU

GAGUtrG UUUGGU U UGCCAGC UUGAGUUT U GcaACU GUUU A GCUAMGCU C MGCUCU U GCUGCCUT A UAG A AUGCCAUJ C CAGAAAU U AGAAAUU C AGUG=U U GAGACCU U CAcUGCU U ACUGCUjU U CUGCUUL C GCUOUCU= A UUCM=C C UACUCAU .C UCGAACU C tMCUGAU A UGAGACU C tJGAGGATU U GAGGAUU C UUCCUGU U UCCUGUrJ C UUCCUGU A UGUACAU A TJAAAAAU C

GCDCCEUG

UUGGAGC

GGAGCO3G

CGUGU

UGCCAUC

CCCACAG

CCCACAA

CCACAAG

GGUGAAA

GGCACUG

ucaACUC

UACUCA

CUAUCG

AUCGAAC

GAACUCU

UGCUGAU

GCCAAUG

UGAGGAU

CUGUUCC

CUGIOACA

CAMAAA

AAAAUCA.

ACCAACU

245 247 248 249 257 273 291 305 307 308 316 319 322 323 326 334 338 380 388 389 392 397 409 410 411 413 419, 437 440 447 454 462 463 466 479.

480 481 497 498 4-99

AAGAAAU

GAAAUCU

AAAUCUU

AAUCUUru

AGGGAAU

GGAGAGU

AGGGGGU

AAAGACU

AGAcaAU

GACUU

AAAAACU

AACUUG

UUGUCC

UGUCCDU.

CLCDAAU

AAGAAAU

AATACAU 1

GGAGAGUA

ACCAATJU

AAUUCCU 2 caAGACU* AAGAGUU AGAG= C AGUUUCUt UOGGUIGU AGUGGAU P GGAUJAAU A AGAAAGU 1L UGAGACU A AAC~U TU ACqG=U lo GGUUUU U CAAAGAU U AAAGAUJU U JAAAUU U AGGACAU U GGACAJU U GACAUUU U C UUucaGG :1 UCAGGGA J CAGGGAA :L GGCACCAC :AAAC=Jt k. CUGUGGA kUUCAAAA I CAAAAC

"AAAAACU

7GUCCUtIA

TAAUAAAG

AUUAGA

*AACCAAU

CCUAGAC

CUAGACU

GACUACC

CCUGCAA6

UCUUGG;U

CUUGG

UUGGUG

GGOUtA

AUGAACAL

AMUGAA

GAAAGUU

GZAGACUA

AACUGG-U

UGUUGCA

GUUGCAG

GCAGCCA

UMGGAGG

UGGAGGAL

GGAGGAG

UUJACUGC

UACUGCA

ACUGCAG

215 0 500 531 538 539 542 543 544 545 549 551 554 556 560 561 573.

577 579 580 581 588 597 598 61.1 616 517 6i9 620 625 627 629 630 631 636 638 644 647 633 655 656 657 558 661 672 676 678 581 682 UAA=U U UAAU=U C UUUCAAU A UCAtAhD A ADUW U AUAUU A UULACU U GGAAAGU A AGU~AAW

A

CAAM

AAUUM

~AUUCA

AACOUC

ACOUAG

CAGAGGG

AGAGGGA

AAUM=O

ACAVUU A CU AAAGAGL7 C AGGCCCU CAGC4= U AADUUC= .ACGCCUU A AUUUUM CCUraAAU Uj UUCAA FM AAU U UCAAt;U ULAU U UCAQ3CA AAXG= U CAGGCAU AAM=UU C AGGCXA CAGGCAU A COUA= UCACAcu U UGCCAGA GCACcUci U GccmAGA AAAGAU A AAAfUU*U AUAAAAU U cLuaAAA ULAAJLWU C UALAAU AAAU=C U AAAAMW AAVUCUUJ A AAAMDA UUAAAXU A WUflUUcA AAAAA A UUUXGA AAMMU U UCAGAWL AMUAU U CAGAXIAU MUM=D C AGAEV=IC UUCAGAU A UCAGAAU CAGAMU c AGA~flck UCAGAAU C AfltM.A GAAUCAU U GAAGA UUQAAGU A UUCU GAAGr.J.U u uuccurcc AAGUAUU U 1JCCu=A AGMflU u ccoccAG Ga.mmr c CUCCAGG DUUUCCU C CAGGcMA GCAAAAUT U GAT.T.UC AAUMGAU A UACUOU UUGAMU A CUUUUU AUUA Uuuco ATICUU U UUU~uAk 684 685 686 688 689 691 692 693 697 698 703 704 708 715 719 720 724 725 728 731 733 734 735 745 746 752 753 757 761 762 765 767 768 769 771 772 773 778 779 783 788 789 791 794 805 M=OU U UCDUAlu ACOUU rU CDurU CUU C Ut~hDUuA UUUCU U AfULAC UUUU= A UUAAzCrj UUCUM W U UAUtYA UCU= U AACUUAA CUMV=u A ACuuAAC UUMACU U AACAX=u UMACUU A ACAXJUCty UMACAU U CUUAA UAAC=~ c LvathAAA AflUCU~u A AAAflGUC AAAAFlGLT C UGUUAAC UGUCUU U AACOIA GOCGU A ACu~am3 GUUAC U AAUAGUA UU=Ut A ADaGOW ACTUtMAU A GaWMM UM=U A UUau~fGA AtThILA U UNafGAAA MUM U AUGAAAU AGMfU= A UGAAAUC; AAATUGM U AAGAAUU AAVGGUU A ACA~DuU tMhAGAAU u tJGGaAAA AAGAADJU U GGUAUW AIUtJWG A AAfUAGU GGUAA U AGM=U GaAAAUU A G~UUUA AAUaM= A UDMWMU uaumu u u~ua;L UAGMWIU UJ AUUUAAU AGOAUuU A uruaAX UAUUUAu U. m~vA~uu AUUMflU U AAUaGUL UUrMUou A AflGOuah UUAG UT AXJUG TLAU A UGUtU= GUMUWu U GUGUUCU GtUUGtJ U CMUAA UuGuruu C UAVMA ~GUuCUr A AtTAAAAc UCM A AAACAAA CAAAAAUJ A GACAAcu 216 Table 12: Humnan IL-5 HIR Ribozyme Sequences Ut.

Position E Ribozyme Sequence *0 0* 0 8 9 12 13 36 37 38 56 57 63 64 69 74 78 80 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 192 200 201 206 207 212 216 222 245 GCAAAGA cG A GCcc~

AGM~CAU

GGCAAAG CW-UGr-'.AACCG

AAGGCA

UGGCAAA CCAUCr-, CMA AAAG-,GC UUEJGGCA CGUAGCCGAAGGC-A

ACGAA-U

GGOUGC CUGaAX3GGCC GCCGAAGA- kGot U GCU C GAGC--AG~GCCM-

AAACGU

AAAUGO.G CMUGAGC3UAc CG;'A AAGC.= AAACWCUGAUGGccs--GAC,( L AUC=CA~ CAAACUC CMUAGCGAAAGGCGAA AArMCAG GC MGMA CUGGCAGCC-;

ACUCA

AGCUAGC CUGAUGGC CGcL AACUA CAAGQAGC CUAWGGCC-GCL--

AGCAAC

GCUCCAA CUGMCC-AAGG C-C- AGCrJAC-C CAGCUCC CGUAGCCCGAAAGLCC AGAflCUA AUACACG CUGAUGAG=-CGAC,-CCAA

ACGCAGC

GADGGCA CGAUGAGGCCGAAGCCA

ACACGUA~

CUGOUGGG CUGAUGA=GGCCQ A~rZCILU ULKGUGGG CUGAI AGG-CG AGCW A. UTTJCT fUU UG UAGCGAGG=A

AAUUUCU

tUU CACC CUGAUGGGCCG~.A

AUGCACU

CAGUGCC COGAUGAGGCCA rGC=-.

AGMCUC

GAGZCGM CV GCGGCCG

AGCAGUG

UGAGMG CUCCGArA cc; AAGCAGU ATJGAGUA CGUAGCCGXUZGCCC-

AAAGCAG

CGX~UGAG CGGAGCCGAAGCC

AGAAMGC

UUCGU CUGAGA~rC-GC CCYA AGt3AGAA AGAGUUC CU;UAG-CAAGCA AfGAGUA MAbCA CUAGGCC-,AGC-;

AGUUCGAL

CAUXUGGC CU A CGAAGC~CGM

AUCAC

AUCCUCA CMUGAGAGCCXkXGyCCGA,

AG

7

JCUCA

GAACAGG CUAGGCGXZCG;

AUCCUCA

GGAACAG CUGAMAG-XAGGC Ccr AAU=Cc GUACAGQ CUAGG-CAAGCA

ACAGGA

t3GtACAG CUAGGC-XGCM-

AACAGGA

UUUUAUG CUGAUGAGGC-C-;GGCCGc AcAGCGAA UGAUULTU CUGAUGAGGCCGAAGCCA

AUGMA.A

AGLt3GGU CUG UGGGCCAWCCGAA c AUOUUT1A CCUGAAA MrGAM7AGGCr-AAAAC

AUUUCU

217

C

247 248 249 257 273 291 305 307 308 316 319 322 323 326 334 338 380 388 389 392 397 409 410 411 413 419 437 440 447 454 462 463 466 479 480 481 497 498 499 500 531 538 539 542 543 544 545 549 551 UCCCOGCUGtGAGcGAGGC'

AGAUDUC

UUCCCUG CUGU~GGCCAAAGCGAA

AAGA=U

AM=cc C~zAG-CCG;a.CCG AAAGACj= GUM=C COM GAGGCCAGM=CAr

AUUCCCUT

ACAGOU CAUGGCCGAAGCCG

ACUCUCC

UCCACAG CMDAGGGGC CGAA ACCCCCU UUUUGAA CUAGGCAAGCA

AGUCUUU

GUUUUUG CUGAUGA=-GCCAA GCG AUAGtICU AGOUUULJ CLUG AGGC,-GAAGGCCCA

AAUAGUC

UkGGAC CGUAGCCGGGCCA AGUUtUU UAUUtAAG COAGGCAGOCCAA~

ACAGJQ

MUM=U LGUGGCCGAAA-GAAc

AGGACA

UCUUUAU CAGGGAAACGAA

AAGGACA

AUUU CMUMGASCCGAAAG--UA

AMA=

GUCAAUG CUGAUGAGGCCAACCL

AUUUC=

GGCCGUC CUGAxGAGGCCr -AGcc.A AuGrmu AUUGUU CUM'AG ccG

ACUC

GUCUAG UA GCCkGAA~

AAUU=

c-G&=COGUMGCCGAGM AGGA fl UUCAGG CUAAGC~A=rA AGUCtChG ACCAAGA CTX3 fGAGGAGCCGAAr

ACUCUUG

CACCAAG Lcc

AACU=U

ACCA C Gc }cc

AAACUCU

Utcc NO OAGGCGAGCrA

AGAAACU

XUUI CMAGGG GCC~J r.Ar~A ACACCAA UUUCUAU CUGAXMGA~C4GC AGC

AUCCACU

AACUUUC CtMXA ~CZGAAA cc AUMUCc aAGUCUC ~CUcUW GAGCGA

ACUUUCU

ACCAGUU CGMc1r cc AGUCUCA W-CAA CAMGCCMAGGC-AA

ACCPAGU

CUGXAAC COAGAGCCGAGGAA

AACCAGU

UGCOGC CUCAUAGGCC kA GGAA ACAAACC CC;UCCAA CMAUGAGCCGAAGCCG AT7CUUUG U CCucA CU~rG;'C2AjGCA

AAUCUUU

r;UCC C UG~ CZGAAGC,-CC" AAAU=tJ G-CAGaAA CMUAXGCC~kU,-CC;

AUGUCCU

UGCAGUA C VGCCGGCcM

AAIGUCC

C rCAGU CoaAIXAGGccGAAAG~rmjA

AAAIGUC

ACUGCAG CMXG GCCGraAA AAAAlGU AAGGCCU CMU~CGAXcGAGC~r

ACUCUUU

GAAAAUJU CMAcMrr AA

AGGCCUG

UAA CVAGGCGAGCA

AAGG=C

UAUUGAA CUAMAGC CM AflTJAGG ALUUUGA CrUAGCC-kCCGA

AATAA

UATUAuuG a UGGCGZCA

AUA

UMTUAUU CM~AGCXAGCAA

AAEXU

UAAuumaMAGGCAACC

AXUMAAA

UL AAAU CUGAIJGAGkAG G AUAIUG 218

C

554 555 556 560 561 573 5-7 579 580 581 588 :Z97 598 611 616 617 619 620 625 627 629 630 631 636 638 644 647 653 655 656 657 658 661 672 676 678 681 682 683 684 685 686 688 689 691 692 693 697 698 GAAGOUA CUAGAG AAG--Cz AUpU u GV= OU DAGGCCGMAAA3G-CCGAA

AA~U

u AM~ CO GCCCGACC~

AAU=

CUV.LCU AGCGAccGGCcr.

AGWA

ut,;nnCCUrA]t;CCC(AAGGCCCA ;LaUu~ AAAMUU CO CGCCGAAAGC=

ACUUUCC

7XCmGAAA CGUAGCCGAAAGCCGA AUMUAcu UGCC,.UGA CUGAUCAGGCCGXAGCCA AUAUlUA AUGCCUG CUAGC-CGAGCCG

AM=

a=GCCU AGGCCGAAAGGC;, AAAfl7nu GUGUCAG CGUAGCCGAAAGCCCA AUGCCua UCUGG-=L CUG UGAGGCCGAGCCGA

AG=

UUCUGG COGAXJGAGGCCGAAAGGCCG.A

AAGUGUC

AGAAUUM CUG GCCMGAcGCCM AnMcoUu ~u tMAG CUG AGGCCUGCCCUA AfUUU AUUUUAA COGAAGAGGccGA G~crA AmxJuUA AUAfUUU CUA-G-CGCCGA AGaUU UM CGUAGCCcGAAAGGc AAG;a=tI UGAAAML CUGACVGCCGAA AflMuA 0COGAAA COAGCCCGXUaCCG AaDUUU UUCUGAL CGUAGCCGAM-CGAAc

AUU

ALVLCUJG COUGZGCCUGG C GA AAIUATAU GAXMUCU UAGCr-

AXM

AUUCUGA CMUAGCCGAAGCCG

AUCVGA

UC-AUUCU CUAGGCcacc AMlUXUG CUUCAAU COGAUGAGGCCGXGCM f A pCrx At.U=C CUGAXJGAGGCCGAAGCc

AUGIJU

AGGAAAA CVAGGCCGAGCCGA

ACVUCA

GGAGCAA CUAGGCCGAAr3CGAA AUIACUUc UGGAGGA CGUAGCCG cc4= AAMLCUU CUGGAGG CUGACGCCGAAGCCGA

AAM=

CCUGGAG COAMGCC~kGCCG

AAA

UUGCCUG CUAGCCGGCCC

AGA

GakU=CM GCCGXUZCCG AflUUUGC_ AAAAGtIA CUA GGCCGACA

AUAAD

AAAAAAG CVAAGCGAGCM AUAflCAA AAGAAAAa UAGCGAGC

AGUALULU

UAAAAA CUAGG-CGAGCA

AACUM

AUAAG CUAGCCGAGCW AAAGaATJ AAU~aAGA CUAM4CAAOCA

AAAGU

AAAMAG VAGGCMk

AAU

tG7NM W W C G U A G C A G C G A A AAAA GUAAA COUG G G AGCCA AAG A UAAGUUA CUGAGAGCGAGCCGA

AMAA

UtU.AGUU CUAGGCGAAGCA

AAUAAGA

GUEDLAGU CUGA U~r-GAGCCA AAAtMG CuAAUGUrJ CAGGGAAAC AGuaAAM AGAAUIGU CUGAUGAGCGAGCC GA AAGUM 219 a. L a 703 704 708 715 719 720 724 725 728 731 733 734 735 745 746 752 753 757 761 762 765 767 768 769 771 772 773 778 779 783 788 789 791 794 805 UUUACAG COGGMAGGCCG-CA AU~aM UUOUACA CUGAUGGCCGAAAGGCcGAA AAtXGUU GACAUUU CGUGGCCGMAAGGCA

ACAGAAIJ

GVC~a.CA CUAUAGCCGAAAGCCGAA

AMM

LV,=XD COAGGCCGkULGGk

ACAGACA

AuaAG CCGAMrCCcAA~aA

AACAGAC

utcahm C GUAGGCCGAGCCA

AGUUA.C

ALMCLIU CUGAMAGG-LrAAGGCGA

AAGUA

UAAAmkcCOGUAGCCGAAGGCCGA

AUM=AU

UCAMAA CGUAGCCMAGMCcA ACuMWu UUCAUA CGUA'-CAG-c-,

AUA

AUCUCAU CU~-GAAAGGCCGA AA~k= CAUUCA C-jAGGCC-AGCA AAAMCu AAUU=COUC GCGAAGCM

ACCAUUU

AAADUCU CGUAGCCGAC-CCGA AAcC=fU UUJACCA CGAGGCCGC

AIUCUUA

AUUA C GGCGUGCUAU AA U CGUGCMG= AA AAU AAAMCU COAMGCCGAAAGM AfUCc AAAMAA CUAMC<I- ACL~hAU ULD6AMA CCAUZCCGAAAGGCcGM AcA AuaAAAU C UMGCCGGCCGAAcc

AA~CDA

CAUUAAA COAUAGCCGAAGCC.A

AA

AACUML C~AGC,-cGC~CM At1AML UA.ACAU CUGAflGAGCGccGOC ry AADAAAU ALMhACAu CCGAGAGC-r AAAflAA ACAACAU CAOAGCCGAAAGCCGA

ACULA

CACAAC;L ~COMUM CCGAAGC-

AAM

AGXAMkC CUAGGCGAGCrA

ACAMAC

DUDUAG _UAGGCGAGCCA CAA uD gum COAGCCGAXGc-M

AM=U~

GUUCU COAGCGGCCGAA

AGACA

UUUGGUUU GcAGCCC

AUTJGAA

AGUGUC ~CGAAGG-CGAcr.A AfuuUUG 220 Table 13: Mouse IL-5 B ibozyme, Target Sequence nt.

Positi±on

S

*5 8 11 12 36 36 37 43 58 59 59 66 82 91 112 2.13 141 141 158 167 196 197 197 202 202 206 212 212 218 218 218 232 241 241 241 241 243 243 244 245 HfM Target Sequience cGCUCOU C CUOUU UCUUc-CU U UGCULAA Ct7UCCUU U GCUgAAG GAAgacU U CAGAGuC GaAgAcU u cAgAG~c AAga=1 C AGA~uCA.

UcaGaGU c AUG%3gaA GGAfU U CUG;cU GAXUGCrJU C UC-C~cU gADGcUU c uGcAcUU CCGCAcU U GAG~gUu UgAcucTJ c aGcUG GcUgUGU c uggGCCA.

ugGAgAD U CCCAugA gGAgAIJ C CCAL~gAG GAGACCU U GaCACaG GAgACcU U GaCAcAg gUCcgCU C AcCGAgC cCGAgCU C UGuGM UGAGGcU U CCUGrcC GAGGctUU C CUGUcCC gAGGCuU c COGuCcC UUCCCU c CCUacuC UUCCUGU c CCUACUC UGUccU a cuCaAA tMCUCAU a aAAaUCa UacuCAU A AAAAUCA uaamaaU c accArcu UAAAAAU C ACCAgCU UAAAAXJ c acCAgCrJ uaUGCAU U GGaGAAA gAC-AAAY C UUUCAGG gAgAaAU c UUucAGG gagAAAU c UUUCAGG gAg-APAU c UUUCAGg gaAAucU U UCAGgGg G-AAAUC U UCAAGGGg AAAUCUU U CAGGGgc AAUCUUU C AGGGgcU Ut.

Posi-tion 253 259 269 269 269 287 301 301 303 303 304 315 318 319 322 330 334 334 384 385 393 405 406 409 481 482 483 483 495 553 557 564 564 565 565 569 569 613 614 AGGGgc'J UP-9ACAU GaAC-Aaty GaAGAau GAAgaAU

UGGGGG;U

AA~ugCU AAAugaU AUCuA AugCJAU u6gcafuu AACcU(GU cCGUCaU1 LM-CaUU CaLTUAAII AAGAAAtJ AAUACAUt AAUaCaU i.

AggCAgu 1: 9gCAgUU C CLTgGAuU A CAAGAGU U AAGAGUU c AGUCCU U TUcaCAAXJ

U

C-kAAU Uj AzAAUUU A AcAATuU a AA.AUUgU c GC-UGuuU c UUUCCAU U UUaUA~UU U tUtuaUtJ u uaJAUUU a UAUU a UutAtGU c U UUAUG= c AAAGuGU u AAgUr~ut

U

A GaC-XuAc a CtUC-aAgA C kzAAMGU AkAaCugU aAAcUgU A CUGuGGA A tUCAA a uUCCaaA u CCaAaAc U7 CcAAAAC C cAAAACc Caltl.ArUA .3 AAUAAG k. AUAAAGA kAAGAAATJ

CUUGAC

7 GACcGCC I GACcgCC CCt~gGAu *Ct~gGAutJ

CC=UGC:AA

cLIGG-U CUUGGrJG *C-WJUgA UAA9UU7A AAgUUaA AgUUaAa aGUtUhAa AAcAgAU CaUuVAU UauaUUU allgUCCuT AugUcC7 ugUCCuG ugUcug cUGUaG~u cUGUagU uaaCCUUt ea~CcJUU HE Target Sequence 221 UUAACcU u uuuGMhU 793 816 818 825 825 839 840 863 864 864 913 917 957 960 960 962 975 987 990 1000 i027 1034 1037 1039 1039 1041 1051 1148 1213 1213 1214 1215 1234 1236 1275 1276 1280 1298 2.310 1310 1310 1350 1358 1370 1375 1377 1383 1405 caAC~gCU CuGagtUu GAguU ACLcCcU aiicucU uCcucU ucugta A-UgUAU

AAGU

gAaCUCU Ucuuggu UUagcAU GCAnCcU GcaUcCU AflcCuuU gccccDU I aGaOU lAuov tGACUCLT CgggGCt3I LUCCUGcU C UgcUJCU cUccuAU c cur-ckU~ c cDmcu UUcAAuUtJ UGAcUUU u GCUgGaU u gcuGAX u cugGAIlU u ugGAfl uU gGGAAU c GACAUcU c UgGGCCU U gGGCCO A CUUACUU c UgAACUU a gCAAAGtJ a GCAAAgU a GcaAAgU a AAAGCAU A AAAUtGGU U UgUuaUU C t1UCAGgU A CA.GgtMhU C UCAGggU C cccCAgU U u UGuGcAU a UACU=cc a cUCCcuC c CccCLTCA c CcCcUCa U cGUWGCA c GUUGccu U cCAGGCu CAGGCug caggCuig U G%-ucCaG CAGAuGG *cCuuucUc UcUcCuA Li UCUCcUa c. VCcUaGC ai AgA~kgA k cuuAAUG a1 AAugacU UugCuGA J C~gCUC 'CEaUcuA LUcUAACU

:UAACUUC

UAACUUc LACUCcAa FAAuAccc cUuaUGU *UUGGAaa uUgGAAA *UGGAaaA4 GGAaaAG UccuGcc

CUUGCA

ACUUCUC

cuucucc UCcgUqu AGAaGcA aAuACcA a.Acca AAUAccA AAAUggU ggGAugU AGgUAUc UCAGgqU AGggUC-A ACUGgAG

UALCUCCA

1407 1407 1410 1434 14.34 1434 1435 1435 1438 1438 1439 1443 1447 1458 1458 1460 1461 1463 1475 1479 1483 1483 1484 1487 1487 1489 1489 1489 1490 1490 1490 1491 1491 1491 1491 1494 1502 1502 1507 1509 1509 1510 1510 1510 1510 1512 1515 cCAgUtuU A Ct~cCA~g ccAgUrJU a CuCAG gOUU~aCt C CAGGaAA AXgCUUU U alluUaAU allgcUuU U AUUUAAu allgcuu u AUUAAU UgCU=u a UuuaArUt ugcjUUUU a uUUjkaUU UuUUAUU U AAuUcug UUU=I U AATucUg UL"UALTUU A AUucUgU UUUaAuU c JGuaAGa AflUCUaU A AgAIJGu UgtJrcac a tUMUUUA ug1UUcAu~ A uUUUA OucAflkU u AfUtLIhug UCAUAUU A UUaflGA Au~o U aMGAug AuCGg=f c aGL1AAgU AIUcaGU A AgUUAaU aGuAAGU u AAUMJ aGUAAgU U AaUU GUAAgULT A aUAZUuA agUEIAAU a UUuAuUiA AgUUAat A uuaMhUa UtAAUaU U u.AuUAcAN UUAAuAU u UAUUaCA UUAaUAU U UAUUacA UAAUaUJU u AUUAcAc UAaM=fl U AUuAcAc UMaUAIU U AIJUacAc AAMDU= a uuaCkcg AAXMhXuU a UuAcAcg AaUU A UuAr-AcG AaMU= A tJUacAcG AUUUAUU a CAcgUu cCGUaU A UaauAUu cAcgUA a UAAUaUU AtIADAaU a TUUcUaaU AUAuaAU u CUaAuAA allaaUaU U C'UAAIAA UAAUAUty C UaAuAAa UAAuAUL C UaauAAA UAAuAU c UaaUTAAA UaaUaUUr c UAAUAA allaUUcu A AUAAAgC UUCUPhA'U A AAgCAgA

S

S

S

S S S

S

SS* C S S C S S rn-C> S S S. Tabnle 14: Human 1I- airpiRihozyrne Sequences nt.

Position 86 151 172 203 Hairpin Ribozyme Sequence UACL~ AGAA Ga=lA A~AAMCAanaApLG GAGM PA3AA GJGXCA AMWGtA~mn3nwmm Lmau= AAA GN~aW AOWGAA~xunwuom UJaWN 3 AGAA GGAAW. ACAWACAaX~WAlcm Substrate UGGGC GCUACL UGOAU GCU UUACUC GAU OCU GUPLM GALUXU GUU CCUGUACA

S

S

S S S S

S..

S

S S S~ 5 S S Table 15: Molise 1L-A Hairpin Ribozyrne Sequences nt.

Position 83 147 150 154 168 199 274 381 454 499 548 701 710 870 919 1030 1170 1205 1402 1421 Hairpin Ribozyme Sequence AGCUCArA AM GAACAC GCL-AC A GLX3A GOUSAC AGAM GC K GC03LU AGAA GACG UGLX AGAA G~A= UAACCA AGAA GCCL)U G GQXXXXIGC ?LAA GAANJLJ LMAAUOA AGAA 03W3AUA GWCAGAG NAM GCCA C N3hrW UGAACIAW AGAA GSAG AGUUCA AGAA GCCUGG CMMCUM AGAA GfltC- C~LWWLUA PAA GGAAGC C CA GLZAU A AA G JUUW ALAT~ Substrate GfLXU GAC LXt)Cua APUXL)Ch GCU GUGULX WGACACA GC 6 CCL CACGCU G6W CGCUAC2 GCURM GCU CACCGAO GAGC~UC GU GACAN~ GCU1XU GUC 6UAQUA WAAACU G6W COU33 O3N3XA GUU QTC3AIU CMAGCU OC CUM GUtAAA GUI Q2AAAAAC ALWXGDu GW LUALI AAUUfLzU GAU OCLXmUG LXIOU

OWVUXLI

OAOU GAC UUMAM UGiUOCA GAU Q3ACOCA GCWXU C CXIUCIA WrAAU~h GAC UGxUzCCA, UG WGA a OGA~UWX LICtflCA GUU UAUOCAG ~AAACA GAIJ GUIJ1flt i

A

Table 16 :Mouse 11.-5 Hairpin Ribozyine Seqttences nt:.

position 83 154 168 199 274 381 454 499 548 701 710 870 91-9 1030 1170 1205 1402 14211 Hlairpin Ribozyme sequence AGCUGAGpj AGAA GAACAC

A-G

CCAGAGCAC ?L3AA G N3XWN-m rmA Ga3C3G AGAA GUCAX QXCUGan PAGAA GAG=AN3Z UGAUG AGAA GC-Tfl CCCCCmC AGAA GUUA ANC AGAA GOCU03 CALCC[EX AGAA OCCA GJUUZ AGAA GJ~r.AC OGCflbM3 NAM GAAAW aGU tCAA AGA Q3C A 3 N A jrM CALGOU3 AGAA QGACAtG~ PUflLPM AGMA @ffAAC CANJ AGAA GMLUA CWNGA AGA1A G39 AAGAUAC AGAA QUUXXI Substrate GfLUX)C GC LUyUMGcU ACEUIZA 0CU GU2 UGACAICA OC LUO oau= GCr Coar GDLIU GJ GAMANh UZAAALj GU CGGM CGP3A@X GWX CUW3Au CrACUu ou CCmjnGUW GrAACh GAu acAAAAAC AUUG GWU LtUAEXA AANLU GAu ccttai3 WQCWW QC ucalx= CCUGGC GAC UUUAP~u UOXEQM GAU GaAA3CA 0WLUOC GU Ctapmmu WMALWA GAC UGLnrCM W3TO O0U GGAXnM UOCttCA GUUECCA AAACA GAU GJLwja 225 Table 17 Mouse re/ A nt. Position

I

C

C.

19 22 26 93 94 i00 103 105 106 129 138 148 151 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 795 796 797 798 829 HH Target sequence HH Target Sequence AAUGGCU a caCaGgA aGCt7CcU a cGUgGUG CcUCc-aU u GcGgACa GAuC3GU U UCCCCuC AuCUGUU u CCCCVCA.

UUCCCCU C AflCUuC CCml. C UuCCCuI C.UCAI=C U uICCCUCA UCAUC U CCCucAG CAGCuU C UGGgCCu GGgCCuL7 A UGUaGG UGGAGAU C AUCGAaC AGAXJCAU c GAaCAGC AUGGAU U CCGCCI;n UGCGaUU C CGC~huA UUCCGCU A uAAaUGC GGGCGCU C aGCGGGC GCAGuAU u C~GGCG CACAGALT A CCACCAA CICCAU C AAGALTCA tJCAAGAU C AAMIGCU AAUGGCU A CACAGGA UUC~aAU C UJCCCUGG CGaAUTCU C CCUGGUC CCC'UGG C ACCAAGG GGcCCCU C CUCcuga uCCaCCU C ACCGGCC CCGGCCU C AxiCCaCA AuGAaCU U GUgGGgA AGAUcaU c GaAchGc G-AUGGCU a CtUflGAG AUG~ucU c UCCGgaG GGCUaCU A UGAGGcu CEJGAcCUT C UGCCrCaG GCaGuAU C CAuAGcU CCgCAGU a UCCjuAg CAuAr~cU U CCAGAAC AuAGCUU C CAGAACC UGGGgAU C CAGUG GC-UCCU U UUCUCAA GCUCCEJU U UCUCAAG CUCCUUrj U CuCAAGC UCCDUUU C uCAAGCrj UGGCCAU U GUUUCC 467 469 473 481 50i 502 S08 509 514 534 556 561 562 585 598 613 61.6 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 1167 I16 1169 "1182 1183

CCAGGC-

AaGCcAfl UUgAGU AC-CGa.AU

AACCCCU

AC-CUU=

UUCAc-,U IC-XcGUU

CGUMCCU

UUCCtJAU

GC-OG-CACU

UGICGCCU

CUjCoGcu

UCUGCU

aAgCCAU

GGCCCCU

CCCCUGU

CL'GUCCU

gucCCUU cctjL-WCC UCC'UgcU AUCCgAU1 Cg2UuU CgAUuUrjT UGgCcAU 1 CCGAGjCCU C

TJCAAGAUC

CGgAACUC G-u'GCCU c AUGAGAUT C GAGAUCU U AC-AUCUU C UuUCrCU c AaGAC~u U GAGGUGU A GGUGEIAU

U

GUGU=U U 'UGUALUUu C CGAGGCE) C GAUGAGU

U

AUGAGU U T'jCAGtUU u AUGCtJGu u UrGCLJGU a C CUgUUCg u AGCCAGC C AGaucAg C CA;GACCA U uCACGUU U CACGUUC U CCUAG C CUAUAGA A JAGAgGA A GAgGAC A uGACuUG C UGCDUjCc U CcAGGUTG C CAGGUGA u AGCCAGc C CLUCCOGa C CUcuCaC c uCaCAUJC C CUcAgCC- SA4CCaug

-CC:AUC

IUUUGAuA 3 UGAu±AAc I G-AuAAC IG-uGUUCC

AAGIAUCU

U GCGAG UGGgAGC

GGCCUGG

UUCuUgC C0.UgCUG uE~gCUGu CauUGCG

G-AZGUGU

UUUCACG

UCACGGG

CACGGGA

ACGGGAC

CrjUUUCU

ULUCCCCC

UCCCCCA

CCC-CCAU

aCCaUCa CCaUCaG nt. Position HH Target Sequence 226 a a a a.

834 835 845 849 872 883 885 905 906 9i9 936 937 942 953 962 965 973 986 996 1005 1006 1015 -1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 1098 1102 115.

11.27 1131 1132 1133 1137 1140 1153 1158 1680 1681 16583 1686 1690 AUUGUGU U UUGUc c GACUlCCU C CCOC~gU A CCAGGCLT C UuCGaGU C C~aGGCU C GC-GGCCU U CGGCC= C GcGAGCU C AUGG~gU U UGG~gQU C UUC"=E) A GCCucAU c AGAuGXU C CagUacUu ACCGGAU U GAgACcU u( AGGACcU A GAGACC U AGACCDU C3 AGAGuAU C3 GAAGAGU CC GAGUCCU U AGUCCUU U GuCC C CCGGCCU CC UaCACC uG GgCGuAU UG UUCUaC aaGCCUU cC CGaAacu cA CUCAaCU U C UCAacuu c U C;UUCrJGU C C CAGCCCU A c GCCaUjAU a gc CAUCCCU ca AcaCCUU c c UCCaUcU c C UUaACuU u A cCagCAU C C GCACCAU C A AUCAACU u U GAAGACU U C

CCGACU

CGGACuC CgL1ACGC

CGCCGAC

CUGUUCG

UCCA=G

CAUGCAG

CUGAUCG

UGAuCGc

AGUAGC

CCAUC

CUUGCO,.A

CCAUG&

GCCACCG

gCCaGAc lAaGAGA :AAGagu :AAGAGu LAGAGuA

ML-AACA

-OUUAa PCauGG MaMGGA

AUGGAC

AaCcCG AucCha CCGaAa CGaAGu 3UCCC

'CAAGC

:agCAU LgCuuc rcgCgc "UcAGC

JCUUUG

~UA

ccucc

CUCCA

CCAUU

OOGCG

GGACA

1184 1187 1188 1198 1209 1215 1229 1237 1250 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 3.525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 GGccccu Gucccuu Ur~aCCaU GGgAGuU

CAGCCCU

cuGGCCU GGuCCCU CCCAgcU CCAGcCU CCCaGCL7 CCAUGu gOGGgcU AUgA~uU CuCCUGU cCCCAGU CAGr~uCU gGGuCCUT CuUUETCU

ACGCUGU

CtUGu

UG=GUU

GGGcU

CCUUG~CU

GgaGUGU gaGUGTUU C CUGGCAU c CuUCgGU a GACAACUc UCaGAGU U CaGAGUU U aGAGUOTU c gGuGCAU c AUGGAWu A UGAaGCU A AaGCtULU A UAhUAACU c CUCUCCU A CCCAGCU c UCCtrCu u CGGGGCU u cU~aCCU c cucugCt u UCUgCruU c CtJCgcutJ u C CUcCUGa c CUcaGCc C aGGGCAG u AGuCuGa a caCCtUc U aGCaCCG u CCucAGC C CUGCCCC C CAS~gCuC C CuGCr-cc c CCUUCCU C AGCUgcG

UCCCCCA

CgA<GUCu U C"UAaCCC- A aCCCCgG C CCCAGUC C AaGCUGa C gGzAaGC%^ LT UGAUGcU 31 GALUGCUG J GCUUGGC 3 GGCAACA I C-ACAGAC

ACAGACC

UGUgGAC GggAACU *aGAGUUEJ

UCAGCAG

*CAGCAGC

AGCAGCU

CCUGUGU

CCCUGAa

UAACUCG

ACL1CGCC GCCUgGU GaGAggG CUGCcCC CggUaGG

CCCAAUG

ugccCAG cCAGGuG CAGGuGA cGGAGgU

AAGACUU

G-ACUUCU

UUCUCCU

CCUCCAU

UC

CU

CA

GO

227 1704 AUGGACU U CUOjGCu 1705 3;=~uU C UCuGCuC 1707 GCUUCEJ= C UGCUCUU 1721 UUUGAGU C AGAVCAG '1726 GVCACAU C AGCU=~ 1731 AUCAGCU C CMAGGu 1734 AGC-UCC A AGGuGcU 1754 CaGugC3 C CCaAGAG 228 Table 18 Human re/ A nt. Position HH Target Sequences HH Target Sequence nt. Position HH Target Sequence 9t** 9 *9 *9 9 00 *9 9 *999 9 9 9* 1-9 22 26 93 94 100 103 105 106 129 138 148 151 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 795 796 797 798 829 834

AATJGGCUI

GGCGU

CGOCUGU

GAACUGUI

AACUGUU

UcccccUC CCCUCAU C

COCA=CI

UCA=cuu

CAGGCCUC

GGC, C7 U U GGAUC AGAUCAU I AUGCC X W7 M 13U C

UCCCGCU.A

GGCGCU C CAGCPJJ C CA.CAGAU A CCACCAU C UCAAGAU C AAtJGGC A UGCGCAU C CGCAUCU C CCCUGGU C GGACCCU C CcCCC C CGC C A :GCU U AGCUUGU A GAflGGCT U AUGGCU C GGUUCU A CUGAGCU C GCUGCAU C CCACAGU U CACAGUU U ACAGUUU C UGGGAAU c GGCUCCU U GCrJCCUU U CUCCEU; U UCCUUUTJ C UGGCCAU U AUUGUGU U

:UGUG

!L GUGCACG J CCCCCt;C

CCCCUCA

AUCUUCC

UOCCCGG

ICCCGGCA

UGGCCCC

LUGOGGAG

:AUUGAGC

TGAGCAGC

T CCGC~kC

CAAGOGC

*CGCGGGC

*CCAGGCG

*CCACCAA

*AAGAUCA

AAUGGCU

CACAGGA

UCCCUGG

CCEJGGUC

ACCAAGG

CUCACC

ACCGGCC

ACCCCCA

GGAAAGG

CUAUGAG

UGAGG

UGCCGG

CACAGUU

UCCAGAA

CCAGAAC

CAGAACC

CAGUG

UJUCGCAA

UCGCAAG

CGCAAGC

G7CAAGCU

GUGUUCC

CCGGACC

467 469 473 481 501 502 508 509 512 514 534 556 561 562 585 598 63 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 1167 1168 1169 1182 1183 1184

GCAGGC-U

~UCAGU

AGC-GCAU

AACCCC-,U

ACCCCEU

UCCAAWJ

CCA.AGC"u AGUUCaT.

UUCCU

GGGGACU

LTGCGGCUi Ct;CCU-

UCEJGCE

G-AC=CU

GGCCCCU

C C C C G-,

CUGUCCUZ

UGt;CCjuU c CCUUCCU C CCCu c AUCCCAu c CCCAUCU Uj CCAU0CUU U UGACAAU C CCGCU C UCAAGAtJ c CG-AAACu c CUGCOCU C AUGAGAD c GAGAUCU U AGAUCUJt C UCUUCCU A -AGGACAU U GAGGUGU A GGUGUAU U G'UGWAUU U UGOAUU C CAGGC C GAMJAGt; U AUC-AGUUt U UG-AGUUEJ C AUG-.GU U IUGU-GUU U GGLU7UU C A UCAGUCA C AGOCAGC C AGCGCAU C CAGACCA- U CCAAGULTJ C CAAGtJUC U CCLIAUG C CUALULGA 2L UAGAAUGA PL. CAAC-AC SCGACCrUG :UGCtjucc 3 CCAGGUG-w

CAGGUGA

CGCC*,UGC

TCCUCAUC

CCAUCT-jrJ U0E'GACk rUGACAAU

G-ACAAUC

GUJGCCCC

AAGAUCU

UGCCGALG

VGGCAGC

tJUCCUh*C

CCEIACUG

CMUGUG

GAGG;UGU

UUCALCG

UCACGG

C-ACGGC-A

ACGGC

CLUUUCG

UCCCACC

CCCA

CCACCATJ

UCCMJU-t CCtJUCUG CrUUCLUGG 229 S. S S0 0e

S

SS*O

5,*9 S. S *9 .5 S. o @6

S.

S

S. 56 5 S. S 9 @5 835 845 849 872 883 885 905 906 919 936 937 942 95S3 962 965 973 986 996 1005 1006 1015 1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 1098 1102 1U25 1127 1131 1132 1133 1137 1140 1153 1158 1680 i681 1683 1686 1690 1704

UUGUU

GCAGGCU

VGCGCJGU

CGUGUCUI

GCGGCCU I

CGGCCUU(

GG~uGAGC

AUGGAAUI

UGGA6AUUC

UUCCAGUI

GCCAGAU AGA6CGAUC CGAtJCGU c ACCGGAU t

GAAACGUA.

AGGACAU 21

GAGACCUIC

AGACCUU C AGAGCA.U C GAAGAGxU C GAGUCCU U AGUCCUU U GCUuU C CCGGCCU C UJCCACCU C C-ACGCAU U UG 3GCCU U GUGCCUU C CGCAGCU C CUCAGCU U 'UCAGCUU C CUUCUGU C CAGCCCU A G--CCCAu c UAUCCCU U cC GGAC= A, CGCAGAC I CCGACC

CGACCGG

AGUGAGC

7CCAGEIAC

LCCDGCCA

LCA-ACG-A

GUCACCG

ACCGGAU

rGAGGAGA

LAAAGGAC

6UGAC.A= r AAGGOA

COUUCAG

CAGCCGGA

AGCGGAC

CACCDCG

GACCAU

GCCG=G

CCCGCAG

CCGCAGC

AGCUUCU

CUGCCC

UGtJCCCC

CCCAAGC

CCUUUA

CDUC

1.187 1188 1198 1.209 1215 1229 1237 1250 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1.577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 GtJUUCCU u CLGGGC-A UUUCCUU C UGGGCAG GGC-AG= C AGCCAGG CAGGCCU C GGCCDUG UCGGCCU U GGCCCG GGCCCCU C CCCAAGU CcoA=G c CUG-CCCC CCAGGCU C CAGCCCC CCCUGjC'J C CAGCCAU CCAUGGU A UCAGCUTC AMC=UA C AGCUCLUG AUCAGCU C UGCGCCCA CCCCUGU= C CCAGUJCC UCCCAGU C COAGCCC CAGUCC-U A GCCCCCAG AGG.CCC-U C CUCAGGC CCCUCC C AGGCUGU ACGCUGU C AGAGGCC CUGCAGU U UGfLTGAU UGCAGUU U GAUGAUG GGGGCCLT u GCUUGGC CCUUGC-U U GGCAACA GCUGUGU U CACAGAC COGUGUrJ C ACAGACC CUGGCAr. C CGUJCGAC CAUCCGU C GACAA<Ct GACAAcU C CGAGUUU UCCGAGU U UCAGCAG CCGAGUU U CAG-CAGC CGAGUUU C AGCAGCU AGGGCAU A CCUGUJGG AtJGGAGEJ A cCCrGAG UGAGGCU A ACCG AGGCtWJ A ACUCGCC UM=AC c GCCaAGU CUCGCCU A GUGACAG CCCAGCU C CUG;CuCC UCCUGCU C CACUGUG CGGGGCU C CCCAAUG ALIGGCCU C CUUUCAG GCCOCC U UCAGGAG CCUCCUU U CAGGAGA CEJCCtUUTE C AGGAGAU AUCCCUU U Aa;UCAU UCCCDUU A CGUCAUC UM=G C AUCCCUG ACGUCAU c ccoaAGC GCACCAU C AACtUiD AUCAACU A UGAUGAG GAAGACtJ U CUCCUcc AAGACUU C UCCUCCA CGA~CUU c CUCCAUU JUCUCCrj C CAIJUGCG CCUCCAU U GCGGACA AUGGACtJ U cucAGCC 230 1705 LM'GACU C UCAGCC 1707 G-ACOUCU C AGcCTU 1721 GUAGU C AGAUCAG 1726 GCAGAU C AGCUcc= 1731. ADCAG-CU C CUAAGGG 1734 AGCCCC A ACCGGG 1754 C;U C CCCAGAG 0@ 0 0000 0 231 Table 1.9 Mouse rel A HH -Ribozyme Sequences nt. HH Ribozyme Sequence Sequence *j

K.)

0* 19 22 26 93 94 100 103 105 106 129 3.38 148 151 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 467 469 473 481 UCCUGOG CMDAC-GAGcrA AGCCAzuu CACCACG CUGUAG-CcGAAAGCGAA

AGGAGCU

UGUCCGC CUAGGCCGAACCG;L

AUGGG

GNAGGGGA COGAflGAGG--..AGGCGA ACAGWuC UGAGGGG CUGAflGAGG c CGAA AACAGAU GAAAGAU COAC;G-CAGCCGAA~

AGGGGAA

AGGGAAA CUGAflGAG-CGAAAGCCGAA

AUGAGG

UGAGGGA CUCArG~ GAAACGAAc

AGAUG

kCUGAGGG CGGLr-GGGC CGc AAGAUGA AGGCCCA CUAGGCCCGGCGA AAGC=u CU3CCACA CU AUGA GCrG cccc AA=Cc GUUCGAU COGUG GAaUC<V- AflCUCCA, GCWGUUC CUG=AGCCGAAGCrA

AUGAUC

AUhGCGG C~aAVMGGCC CAAAGCGA

AUCGCAU

TJAUAGCG CUGAGCGAAcc

AATJCGCA,

GCAtUUA COA GAGCGkZCCGA

AGCGGAA

GCCCGCU C~-7ArCCGAGCCGA

AGCG=C

CGCCAGG CUGAflGAGGCCGAAGGCGA

AMAC=G

0 XOZTGG cUAUA GCCXZGCCG

AUCUGUG

UGAUCU

CUAUAGCCGAAAGGCCAOGUGG

AGCCAUU CUGAGAGCGAAAGGCCA

AUCUUGA

UCCUG CUGAUAGCGAGGC

AGCCAUU

CCAGGGA COGAUXJGCAGGCCGA A AU

T

JCGAA

GACCAGG CUAGGCCAAGM

AGAUUCG

"CUUGU CMUAGCM~GCG

ACCAGGG

TJCAGGAG CUAGGCCGAAGCC

AGGGGCC

GGCCGGU aXUAGCG ACC AGGUGGA UGUGGAUGAGG A AGCGAA

AUGCCGG

UCCCCAC CM~AI3GAGGCCCAr CCA AGUUCAU GCCIGUC- GCCkWZAA"

AIUCU

CUCAUJAG CAGGCGAMGC

AGCCAUC

CUCCGGA CUAGC4CAAGCA

AGACCAU

AGCCUCA CMUAGCAAGCA

AGUAGCC

CUGGGCA aAUGAGGCCGAGGCG

AGGUCAG

AGca3AUr. COAGGCC~AcaCCr.

AVACUGC

CWWAGGA COAGCCGAAGCA

ACUGCGG

GUUCUGG CUG UGGCCGACW

ACUAUG

GG~uUCUG CUGAX3GAGGCCMA ccr AAGCMTu CAMMCU CUGA GGCAGGCCr.A

AUJCCCCA

CGAACAG CUAGGAGCCGAA

AGCCUGG

GCUGGCtJ LUGATUGAGGCCGAAAGGCCGAA AtJGGCEUU CUGAUCtJ CUGAUGAGCCGArAA

ACUCAAA.

UGGUCUG CUAGG-CCAAGCA

AUJUCGCU

232 501 AACGUG& CUC7AI3GAGGCCCGAAACCCGAA

ACCGGGUU

502 GACGUG CVGAUGAGCCGAAGCCGtrAA

AAGGGGU

508 CMUMM CO~GAUCCCG XNxaCCC tV G CGGA 509 UCAUG C~ flCAGCGAAGGCGA

AACGUGA

512 UCCUCrC, UU AUAG-CAAGC-;A

AAACG

514 GCUCCUIC CUGAUflAGGCCG A.AGCGA

AAGGAA

534 LGOC CUGAflGAGGCCGA GC AGUCCCC 556 GCAAGCA CUGflGAXGCCGAACGCCGA

ACGCGCA

561 CACCUGG CUGANUGAGGCGAAACG AC7CAGAG 562 UCACCUG CUArAGCAAGCA

AAGAA

585 GCUGGC-U CUAGGccAAGCA

AUGG=U

598 UCAGGAG CUG UAGGCCGaCCCCAA pCGGGc 613 GGAG CUA~XC-CAA~CA ACAnGG 6i6 CAUG~h CUAGGCGAAGC

ACGACG

617 GGCUGAG CUGAUGAGGCCC-G,-G

A.GC

620 CrAxGGCu COGAtJGAGGCCGAXG CGA AGGAAGG 623 GAGA3G COGAVGAGGCCGAAAGCCGA

AGCAW-A

~.*628 MUXMAA CUMUAGAGCCG AGCCGA

AUCGGAU

630 GUAC CUMVrGAGGCCkG

GCGAAAUG

V 631 G-GUtADC CMWAGCAGGAA AAArjG 638 GGAACAC CUGAUGAGCCG)krGGCCGA AflGGCCA 661 AGAUCUU CUGAUGAGGCkGGC C AJ AGCUCGG :667 CUCGGA CMUfGAGGCCMUAGGrY

AUCUUGA

687 GCUCCCA CVOGAG AGGCU~ ~AAUrCC 700 CCCCACC COG flGAGGCCGAAGCCA Arc~ -CC- *715 GCAAGAA CUGAUGACGCCGAA CGCCGAA

AUCUCAU

717 CAGCAAG CVGAUGAGGCCGA-UGCG;L AGAUCuc 718 ACAGCA CUGAUGAGGCCG AGCGA

AAGAUCU

721 CGCAAUG CUGAGAGGCCGAAC<CCA

AGGAGAA

751 A~C=C C O G CCA UGCU 7S9 CCGUGAA CWGAUGAGGCCGAAGCCQCJA

AACACC

762 CCCGUGL COAGG<VAAGCA

.AUCAC

:763 GUCCCGU CUAMGCG~AGCA

AAAUVAA

*792 AGAAAAG COGAXGACCCGAflrGCCCrm

AGCU

795 U0GAGAA CUGA3JGAGGCC r ArGrAjC 796 CUUGAGA CUGAXGAGGCCAAGCAA

AAGG

797 GCUUGAG CUGAXJGAGGCL)M rCCGAA AAAGGAG 798 AGCUUGA CUGAflGAGGCCGA.

AAAAGGA

829 GGAACAC CtXGAfGAGGCCGA CCA AUGGCCAL 834 AGUCCGG CUGAUGAGCC~k GC AA ACACAAU 835 GAGUCCG COGAUGAGGCGGCQGAA

AACAC.AA

845 GCGUAC CUGAt1GAGGCC

AGAU

849 G;UCGGCG CUGAGAGC~Cr AGCC

ACGGAGG

872 CGAACAG CUGAfLrGCGAAA GCGA AGCCUGG 883 GCAUGGA CUGALUGAGCCGLrCGAA ACtJCGAA 885 CUGCAUG CUGAUGAGGCL r AGACt3CG 905 CGAUCAG CUGAXJGAGGCC akGCCA

AGGCC=

906 GCGAUCA CUGAUGAGCCGAGCGA

AGC

233

K

9i9 936 937 942 953 962 965 973 986 996 1005 1006 101.5 1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 1098 1102 1125 1.127 1131 1132 1133 1137 1140 1153 1158 1167 U168 1169 1182 1183 1184 1187 1188 1198 1.209 1215 1.229 1237 1250 GCUCACU CUGAOCAGGCCMAMGCCGaA

AGM=CG

GUAOCDGGAAGGAAG-CGA ACUC=w AGCCUG CMrGGALUCCCGA

AACUCCA

UGGCAAG COVGCCMGAGCM

ACUGGCA

UCWUGOG CUGUlG~GCCMLGC~. AflGAMC CGGOGGC COAGGCC-AGCA AUCAlUM GUCUGGC CUAGGCGAAGCA

AGMZM

ucucrauc COGAUGAGCoCGAA CA AUCCGGU ACUCUG C GU~CCGA AGG CUC GGUCUCW-NGGCCGAAAGC

AGU=

ACUC=UG CMNAGGCCGAGCG

AGUCUC

U.CUCUU GccGrGcc

AAGGUCU

LMCUUCAU CMUArCC--AGCCIG AXhca UUGAAA CUGAMAGCGAAAGGCCGA ACUC~U~c CCAUUGP. CAGGCCGcAGG-CG

AGGACUC

UCCADUMGAWAGCCGAGCCr.

AAGGACU

GVCCAUU CVUAGOAAGOA

AGA

CGGGXkuUG C- kUAGCMLJLGCA AGGCc= uUGGw= ccmxaWCCW

AGM

GCACAGC CWMGCMUGCA

AUMCGC

UUUCGGG CUWGCCWAGCCM

AGGCAA

ACUUCGG CMGCCMMGCCA

AAGGMU

AAGGOiU CGMGCMAGCA

GUC

GGMXACAG AGCc

AGUA

GGGCWA CrMAMGGCCMAGCA AM GCDU UGGAVGAGC~CMUG

ACAGAAG

GAAGGM UGAUAW AA-CA

AGGGCUG

GEUAGC COAAG-GAAGCA AOflArc UGGOG CUG AGCGAGGC

ACGGG

AUGC-UGG CUAWGCGAG-L

AAGGUG

GAAGCM MUAGG

GUG

X= cCGU GAGCCGUGCC

AAGMAA

GCEJMWG CAGAGCOMUC

AUGOG

CkAAGU UAUkGXAGMA

GOC

CUaCA VkGGaAAOCA

AGUGT

CGGAGammCCAAGGCGAA ACUcOc UGGGGA CGMGCCMA

AACUCAU

AUGMWGGAUGVCCMAGCCM AAACUcA UGAUGGU CUAGGCM-

AACA

CDGAUGGAWAGC<=MAGCG

AAAC

UCAGGAG CWUAGCtAGCA

AGGGGCC

GGt7aMG CAMGGCCGAGG~CCM

AAGGGAC

LDGvCCUGAOMcc c AUGGOA UCAGACU CUGAMGAGCCMUGCr A~axc GAAGGUG MGAGGcAMAGccCA

AGGGCUG

CGGtJGCU CEJGAUGAGGCCC kGCCA

AGGCCAG

GCt7GAGG CUAJA~c~AGGGACC GGGGCAG CUAGGCGAAGCA

AGCUG

GAGCCUG CUAMGGGAAGG-CGAA

AGGCUG

234 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 i566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 1680 1681 1683 1686 1690 1704 1705 1707 1721 1726 1731 1734 1754 GGCG,= C~xVGcGAAcc

ACCUCGG

AGGAAGG C~aUwtG-tGAAAGGCcM AC, ALGG CGAGCU CO GrAGGCcGAAAGGCCGrA

AGCCCXC

UGGGGA CCUGAGGCGAAc_CG AACLTCAt AGACUCG CUGAGAGCCG C,--Ca ACaGG3AG GGGUtUhG CrUGGCCGAAGCCG

ACUGGCG

CCGGGGU CGMGGCCaAAGCc,. A~acu GACtGGG CMAUGAGGCCGAaC-Ccc AGM;cC UCWJU CUGAflGAGGC-C AGccG-,AA A53AAA GGC-UUCC CGU -C,-,GAAG-Crc;

ACAGCGU

AGCADCA C~3~=G-CGAAAGcc

XMCLG

CAGCAUC COAGCG---;AC-)GL

AAC,-'CA

GCCAAGC COGAflGAGGCCG,'ACSC;A

;L=,CC~

TJGUUGCC GGAC,,cc-AAAGCc AGf~akGG GUCUGUG CUPGAMGC-CG AACGACA

ACACUCC-

GCGU CUAGG~rAA ,CGAA AACACLTC GUCCkCAL CUcAVG AGG-CCCGA

AIGCCAG

AAACUCU CUAGGG

AGUUGUC

CUGCUGA COAUAGCCGAAG,-rA AC rJG GCUGCUG CUGAAr Gcc-AAGGcc-G;

AACMTCG

AGCUGCU CUAGGC~CGAC,--CA

AAACUCU

ACACAGG CUGAXUGAGCaA~ GCGALA AL.GC;LCtJUCAGGG COGGAMAGCCGA--c

ACTUCCAU

CGAGUUIA ctUG A UGAG-,- M Lx.A L Anr.,,c GGC~uAGU COGAUGAG-GAAAC GCC~AA

AUAGCEJ

ACCAGGC CUAGGcCGA=CCC AGU3UA CCCD=CU GAUGAGGC-CGA GCCGAA

AGGAGAG

GGGGCAG CrXGGC-,r-AG,-

AGUG

CCUACC CUGAI GAGGCCa GAAC&

AGCAGGA

CAUUGGG CUGAGGCAAAGCC C,--AA AGCCCCG CVCGGGCA CUACGGCAA-CC X AGGUCAG CACCUGG CUGAXUGAGGCCGA G,CGAA

AGCAGAG

UCACCUG CGUAOGGGCCGAA

AAGCAGA

ACCUCCG CDGAGGCCGAAG-,CGAA

AAGCGAG

GGAGGAG cuGUGAGGCCGAAAGAA AGucuuc VGGAGGA CUGAGCCC-AGICGA A AGrjr AALIG=A COGAXJGAGGCCG AG-,CGAA

AGAAGUC

CGCAAUG C~rUGAZCGAccG,-,CGAA

AGGAGAA

UGUCCGC CUGGAGGCCA-G

AUGAG

AGCAGAG CG lG GCGAA G AGUCCAU GAGCAGA aAUWM CWAWM

AAGUCCA

AAGAGCA CUGAt3GAGGCCGaAG,-C=M

AGAAGUC

r; GATJCU Ct GAl AGGCC-.a aCGM--r-A ACtJCAA AGGAGCU CUGAUGAGGCCGA AGGCGAA

AXJ

T

TGAC

ACCUUAG CUGjGA-GGCC A CGAAC AGCUrA AGCACCtJ cEGAuGAGCCGAA GGCGM AGAU CUCU GG I AGGC G~ AA AGCACU 235 Table Human re/A HH Ribozyme Sequences nt. Position HH Ribozymne Sequences 19 UACAGAZCUUAD-IrUAGCMA

AGCCAUU

22 CACOACA CVGAt7GAGGCCGAAAGCG.

ACGAXGCC

26 CGUGCAC CMGGGGCCGAA.GQGCCrGJL ArZXGACG 93 GAGGGGG CUGACAGGCCAAAGCCA ACAGUrJC 94 UGAGGGG ;AMGCGAAG-GL AACA~u 100 GGCAAGAU C UM5-AGCC -GccA

AGGGGGA

103 ctGGAA CMUGIGAGCcGA GC

UAG.

105 TJGCCGG CUGAVOAGGCCCG AG,-CCGAA

AGAUCG

106 Ct3GCCGG CGUAccJ AAGAtJG.

129 GGGGCC CM MAACCGAAGG

AGGCCUG

138 CUCCACA C MWL-'WAGCGAAGG 148 GCUCAAU CUWAGCMGCA

AUC

T

JCCA

151 GCUGCUC C MDMUGAGccJL)r

AUGAUJCU

~180 G M .AGC~cAU 181 UGUAGCG CMUMAfGAGGCG.,, 186 GCACUUGCGXGGMG GC.

GGA

204 GCCCGCG CVMGIGGCGA CC AGCG-CCC *.:217 CGCCUG CUGAJGG--GCC WC AX3GCUG,- 239 UUGGUGG CMUMWZCGAGG Cc G At7CUG 262 UCALTCU CUGAt3GAGCCQ _;C-r 26 AGCCATJU COAX GAGGCCG A -CC ;A AUCUUTGA 276 UJCCUGUG CUGXJACCCG r-CCU 301 CCAGGGA CUGAUGAGGCCGG-CCGA AUrC~CCA 303 GACCAGG CVUGAAGMGAAGG

AGAUGCG

**310 CCUUGGU

CUGCCAGG

*323 CG33GAG CUGAUCkG~G~ccrGJCJr# A AG3GUCC 326 GGCCGGU CcJGZWAGCcJUF

AGGAG=

335 UGGGGWu CMMG~ UGC.

AGGCCGG

349 *UCM =UIG AGCUCGu 35 CCUC al"

.ACAAGCU

375 CUCAUAG CM7AXJGAGGCC J

AGCCALTC

376 UGACAG axm AAG.CCAt 378 AGCCUCA CUGAGCCMAj

AGAAGCC

391 CCGGGCA CtGIJeji.~AGCUCAG 409 AACUGUG CUGAUGAGCCAAGCCG

AUGCAGC-

416 UJUCUGGA Ct~IGGCQ~AC=G- 417 GUUCUGG CMUGAAWCC C A AUG 418 GGUUCUG CWU~GGCCGAAG^CGAA

AAACTJGT

433 CACACUG CUGAUGAGGCCrAAr"rCGA6

AUUCC

467 UGACUGA

A.GCCUGC

469 GCUGACU CUGAUGAW.C LCA

AJGI

473 AUGCGCU CUGALTGACC ,,AGCGA

ACUGAUA

481 UGGCUG CUGA C CGA

AUGCGCEJ

501 AACUUGG CUGMflAIGrr rm GAA AGGGGuU 236 9* .1 502 508 509 512 514 534 556 561 562 585 598 613 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 795 796 797 798 829 834 835 845 849 872 883 885 905 906 919 GAACUM JG wCCGA~-~A

AAC

CUAMZG CtArCG -,--rC~hAGCCA ACUcGGA UCLUI-UW COA(AGCCAAGCA

AACUJUGG

OCOUC CUGAUGAGGCCGAAAGGCCGAA

AGGAACU

GC-UCUUC CtJGAT AGGCCGAAAGCCCGAA AflAGM CAGGUCG CUAGGGCGAAAGGCCGM

AGUCCCC

GGAAGC;L CArGGCCGAAAGGC

AGCCC

CACCEGG CUAJGGCCGAAAGr,-CGA

AGCAA

UCACCCG CMUAGCGAAGCA

AAGCG

CCUGCCU C MWAGCCGAAGCCG AflGG~c GCAGGCG Ct3GAfGAGGCC .,aGCCGAA

AGCC

GAGGAAG CU CC aAcc ACAGc GAUGAGG c~-UAGCCGAAAGC.-cA

;GGAAG

GGAUGAG CUAUAGCCcAAG-.A--CcA

AAGGACA

AUGGGAU CUGrGaC-Car CA AAGAUGG CUGC-'f AAGClAAGGGA VGUCAAA CW-UMr- cAAGCCA AflGG= AUGOCA COAGGG'

AGA=

GAUUGUC CMUfGArG CMAc CGAAGM GGGGCAC CUAr.C-CA-a,-GL *,jaDGC AGAUCUU CAG=CGAAAGGCCA

AGCL,=

CUCGIGCA CGAGG- -CAAtGCCGAAm AtUCUUGA GGCCWCA CUAG CCGkAZGCCGA

AGUUUCG

CCCCACC CAUG AGGCCGAA crA AGacAGC- GtJAGr-AA CUAUG GAXUGcc AUCW-TT CAGtTh.GG CUGUGA=GCCGAA.GCCA AGAflCUC ACAGUAG CUGAIGA-C G GCCGAA AAGAUCU C-ACACAG CUGAU =AGGCC r.GAA AGGAAGA ACACCEJC CVGAMGxDAGGCC r.cAA .AIGU=C CGUGAAA CUGAfl ArGGCGAGGCCA

ACACCU'C

CCCUG COAT GCCGAAAGccM

AUCACC

UCCCGUG CUG GAGCCGUGCMt

A

GUCCCGU COA GCCGAC-CU' AAAflMc CGAMAAG COAGGCCGMXGCr

AGCCUTCG

TUUGCGAA CMUGGCLcaAkkoCCGA

AAGC

CTJUGCGA L cuAGGcG ccr-A AAGGAGC GCUUGCG. CGUA,-CcAAGCCA

AAAWA.G

ACUUGC CUAGGCCWGCA

AAAAGGA

C-GAACAC CUGAGAGGCCGAkkG'CGAA

AUGGCCA

GGUCCGG CUAGGCGAGCrA

ACACAAU

GGGUCCG CtAGAGCCGAAAGGCCGA6

AACACAA

GCGCAGG CUA

AGGGGUC

Gt3CUGCG CUAGGCGAAGCA

AGGMG

CGCACAG alAUOCGAAAGGCCA

AGCCUGC

GCAUGGA CUr-AUGAGGCCAAA G--CrAA

JACACGCA

CtGCAUG MCULGGAG CCC-AA AGACACG CGUCGG Ct3GUGAGGCCGAAAG-CCAA

AGCCG

CCGGUCG CUGAUGAGGCCGA GCG; AAGGCCG GCUCACU CrJG UGAGGCCGAWCCGAA

AGCUCCC

237 Ye

CCC.

*CC.

C

C.

C

CC..

C

C.

C .C

C.

936 937 942 953 962 965 973 986 996 1005 1006 1015 1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 1098 1102 1125 1127 1131 1132 1133 1137 1140 1153 1158 1167 1168 1169 1182 1183 1184 1187 1188 1198 1209 1215 1229 1237 1250 1268 GUaCUGG. CUGAUGAGGCCGAAAGGCCGAA AflUCCAtJ GGtmkcm3 CUG GAGGCCGAAAGGCG AA-cr; UGGCAGG CcGAUGAGccGAA ACUGCA UCGUCUG CUM CMAGG AA-G cGAA AUCUGC-C CGGUGA MGAUtGAGCCG AG-cGA AjCGU AUCCGGU CUGAXJGAGGC.CGAAAGG-cCGAA

ACGAXCG

UcCUCC TC CUAUGAGCr~kUGGCAA

AUC-C,-U

GUCCUUU CDG1*X3GAGCCCGAAAGG=CGAA ALCGuuuC GGUCUMk CUGACGAG AGGCCGA UGCCU GCUCUUG CUGPXJGAGCCGAAAGGCCGAA AGtjCVjC UGCUCUU CUUAZ-CGAAAGG,CGA

AAGGUCU

UCUUCAU C~.A~'CC-CGAAAGGC-CGAA AL-GCUCU CUGAAAG CUGAUGAGGCOGAAGCCG AC-JC-,UC CCGCUGA, C1GAflGAGGCCGAAAGGCC,-A AG.ACc TJCCGCUG CUAACGG--CGAAG=CrA AAGGACU GUCCGCU CUGAflGAGG-cCAAAGGcC,-- A;LXGGC CGAGGUG CUAGG-CGAAAGcCC~AA AGCCGG AUGCGUC CuNuX CGAAAGGcCAA AC,-%rrA GCACAGC CGUAGCCGAAAGGCcGA AV1GCGUC CUGCGGG UAGGGCCGAAAGGCcGA

ACC,-,C

GCUGCGG CUGAUAGGCCGAAAGG,-c-,- AAC.CAC AGAAGCEJ COGAUGAGGCCCGAACGCCCA AG,-=,C7 GGGACAG CUGAUGAGCCGMAGGccc-

;LGCUTGAG

*GCACA CUAUGGCCCGAAAGGCCAAAAGUG GCUUGGG CUAGGcGAAAGGCCGAA

ACXGAAG

AAAGGGA. CUGAnGAGGCC-GAAAGGCCG--,A AGGGCE7G GUAAAGG CUC-AGAGGCCGAA~cCCG AUOAGGG TJGACGrUA CUG GA-CCAAC-CCG AGSGAUjA AVGACGLI CUGAGAcGCCGuAA CCWA. AACGC_;L GAUGACG COGAfGAGGCCGAAGA

AAAGGGA

CAGGGAU CUGAUGAGCCGAAGCCGA

ACGCZAA

GCUCAGG CUArGGAACGCCC.

AUGACGU

CAURGU COAGGCCGAAAGGCCIGA;L

AXGGUGC

CUCAVCA COGAUGAGCCAAc.CCA

AGUUGAU

GGUGGGA CUGALUGAGGCCGAAAGG,-CCAA

A-UCC

UGGUJGGO CUGAUAGCGAAGGCCA AtACUCAU AUGGUGO CUGAGGccAAccCGA

AAACUCA

AGAAGGA CUGAUGGGC-CrAAG~C

ACACCAU

CAGAAGG CUCAUGAZGCCGAAG,-CCGaA -tACACrk CCAGA.AG CUGA AGGCCGA C-CGAiA AAACACC UGCCCAG CGAflGAGGCCG AAGGCGA

AGGAAAC

CUGCCCA rJArAGCGAAAGGCLC-aA

UAGGAAA

CCUGGCU CVGAflGAGCCGA GC AUCUG=c CAAGGCC C GAUGAGGCCGAAG~cCGLA

AGGCCUG

CGGGGCC CUGAUGAGGCCGAAG C-A. A-GCCCA A=tUGGG CUGAt GAC-G!CCG AGGCCGAA

AGGGGCC

GGGGCAG CUJGAUGAGWCCC-AA GAr-A AcOuGGG GGGGCt7G COGAUGAGGCCGAAAGGCCA AGCCUcG AtIGGCUJG CUGAUGAGGCCGAGGCCCgA

AGCAGGG

238 1279 GAGCUGA CUGAUGAGGCCGAAAGCCGAA _kccA=G 1281 CAACU CU~GAGGCCGAAAGGCCGAA Auacc.~u 1286 UGGGCCA C GAflAGCCGCAAAGCCGAA ACUGAU 1309 GGACUGG CUGAflGAGGCCG~lAAGCCGAA ACAGGGG 1315 GGGCUAG CO0GlUGAGCCGAAAGGCCGAA ACtJGGGA 1318 CUGGGGC CtJG AUGCCGaAAAGGCCGAA AGGACUG 1331 GCCUGAG COGAXJGAGGCCGAAAGCcGA AGGCCU,- 1334 ACACCU CUCAflaGGCAG t~r.CCG- %CC-iG 1389 GGCCUCtJ CUGAL-GAGGCCGAAAGCCGc AcAGCGU 1413 )AICA COGAUGAGGCCGAAAGGCcGAA. AcuGCAG 1414 CAUCAUfC C LGAUGGCCrGAAAGCCCA AAcUCA 1437 CCCAAGC COGAUGAGCGAAGGCCGAA AGGcCC- 1441 UGUUGCC CUGAUAGGCCGAAA-ccGA

AGCAAC-G

1467 GUCUGUG CUGA UGAGGCCGAAAGGC-CGAA. AcACAGC 1468 GGUCUGU CUCflGAC-GCCGANAAGGC-CGAA

AACACAG

1482 GUCGACG CUCAXGAGGCCGAAAGGCCGAA AUCCAG 146AGGUGUC CGLGAGGCCGAAAGGCCGAA

ACGGAUG

*1500 CUGCGA CUGALIGAGGCCGAMGGCCG;

ACUCG-A,

1501 GCUGCUG CUGAUGAGGCCGuAAAGGCcC.A AACtJCGG *1502 AGCUGCU CVGAXP3AGGCCGDJaMGGCCGA;.

AAACDCG,

1525 CCACAGG COGAUGAGGCCGAAAGGCC=A~

A=GCCU

.:1566 CUCAGGG CUGAIUGAGGCCGAAGCCrA

ACUCCAU

.1577 CGAGU. CUGAUGAGGCCG AAGGcC=~ AGCCUCAL 1579 GGC-GAM~ CUGALUGAGGCCGAAAGGCCGAA

AUAGC

*CC1583 ACUAGGC C fGAUCAGCCGAAGcGAC AGUA 1588 CUGUCAC CUUGAGCC -CCGA AGGCGAG 1622 GGAGCAG CUGA UGAGGCCGAA GGCG AGCUGGG 1628 CCCAGUG CUAUACCCGAAAC-GU

AGCAGGA

Veto*, 1648 CAVUGGG CUGAUaGAGGCCGAAGC

.AGCCCCG

1660 C 3AAAG CUGWUG~cGAGGCCG G A~ AGGCCAUj 1663 CUCCUG C GALIGAGGCCGAAGGCCGAA jaGGAGC; 1664 UCUCCU CUGAt1GAGGCGAAAGGCCA

AAGGAG

1680 GGAGGAG CUGGflGAGGCCGAAA cGA AGUCUUC 1681 3GAGGA CUGCflGAGGCCG AGGCCAA AAGUCUU 1683 AAUGGAG CUGAUGAGGCCXDAZcGCAJ

AGAAGEJ

1686 CGCAAG CUGAGAGGCCGAAAG-cCGAA

AGGAGAA

1690 UGUCCGC CUGA GAGCr,-CGAAAG-CAccL

AUGGAGG

1704 GGC~r=%G CLUGAUGAGCGAGAGCCA

AGUCCAU

1705 GGGCUGA, CUGUGAGGCL"c GGCGA AAGUCCA 1707 CAGGGCU CUGATUGAGGCUrAAG GA AGAAGU 1721 CUGAUCU CMUGXAGGCCGA AGCCC. ACtJCAGC 1726 AGGAGCU CUGAUGAGGCCGAAGGCCGAAL

AIJGAC

1731 CCCUEIAG cGIA cG cG AGCUGAU 1734 ACCCU CMUtGAGCGAAGGCCG-A

AGGAGCU

1754 CUCUGGG CLUGADGAGCCr zAGCG GGA C *o C C C C t0 C. CCC C C C C C C OC C bC* C C

CC.

Table 21 Human re/ A ft. Position Hairpin RibozymefTarget Sequences Hairpin Ribozyme sequence Substrate 156 362 413 606 652 695 853 900 955 1037 1045 1410 1453 1471

UGAGGGGG

GCUGCUUG

GCCAUCCC

GrJUCUGGA

GAAGGACA

UUGAGCUC

CCCACCGA

AGGCUGGG

GGUCGGAA

UGACGAUC

GUCCGGJOG

GGCCGG

CAUCAUCA

ACAGCUGG

GAUGCCAG

AGMA GUUC ACCAGAGMAACAACGUUGUGOACAUUACCJGGUA AGAA GCUC ACCAGAGAAACACACGUUGUGUAAUUACCJGGUA AGAA GUCC ACCAGAGAAAccACGu1J1JGGOuAcJTACCTJGGUA AGAA GUGG ACCAGAGAAAcACAC0u1GuGACAUUTACC1IGGUA AGAA GCAG ACAAAAAAGULGUCUACGU AGAA GIGU ACCAGAGAA~cAcAcGuuGuGTJACAUJACCIIJUA AGAA GCUG ACCAGAGACACACGUTJGGUACJUACCGGUA AGAA GCGU ACCAGAGAAMCACACGuEruGuGUAAUUJACJGGUA AGAA OCCG ACCAGAGAAACACACGUUGAUACCUGGUA AGAA GUAU ACCAGAGAAACAC~cGuJrJGGutAcAUUACCTJGGUA AGAA GCUG ACCAGAGAAACACACGUUGGGUTAAUJACCJGGUA AGAA GUGG ACCAGAGAAACACACGUUGGGuVAAUJACCJGGUA AGAA OCAG ACCAGAGAAACACACGtJ!JGJ(,JGUACAUUACCIGGJA AGAA GUGC ACCAGAGAAACACACGUTJGUGOTJACAUUACCIJGGUA AGAA GUGA ACCAGA AAACACACGUUGUGG!AJTJACCUGGUA GAACIJ G011 GAGCA GCC GGACU 0CC CCACA Guu CUGCC GCC ACACU 0CC CAGCIU 0CC ACGCA* GAC CGGCG 0CC AUACA GAC CAGCG GhC CCACC GAC CUOCA GUU GCACA GAC UCACA

GAC

CCCCCUCA

CAAGCAGC

GGGAUGGC

UCCAGAAC

UGUCCUOC

GAGCUCAA

UCGGUGOG

CCCAGCCU

UUCCGACC

GAUCGUCA

CCACCG2AC

CCCCGGCC

UGAUGAUG

CCAGCUG3U

CUGGCAUC

V N Table 22 Mouse rel A ft. Position Hairpin RibozymefrTarget Sequences Hairpin Ribozyme sequence Substrate 137 273 343 366 633 676 834 881 1100 1205 1361 1385 1431 1449 1802 2009 2124 2233 2354

GUUJGCUUC

GAGAULJCG

GCCAUCCC

GGGCAGAG

UUGAGCUC

CCCACCGA

AGGCUGGG

GATJCAGAA

AGGUGUAG

GGGCAGAG

GGGCUUCC

CAGCAUCA

ACUCCUGG

GAUGCCAG

AAGUCGGG

UGGCUCCA

UGGUIGUCG

AUUCU)GAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GUuC

GUCC

GCCU

GUGU

GCUC

GCGLJ

GCCG

GCGG

GUGC

GCGLJ

GCAG

GUOGC

GUIGA

GCUG

GUCC

GCAC

GCCA

ACCAGAGAAACACACUUGUGGUACAACGGUA

ACCAGAGAAMCACACGUUGUGGUAAUUACCGUA

ACCAGAGAACAACGUUGUGUAUACCJJGGUA

ACAAAAkAGUGGUCUACGU

ACCAGAGAAACACACGUUGGGUACAUACCGGUA

ACCAGAGAAACACACGUUG1J-GOUACAUUAI-CJJGGUA

ACAGAGAACACACGUUGUGUAAUUACCGUA

ACCAGAG.AArACACCGUUGUGAATJACCJQ.GUA

ACCAGAGAAACACACGUUGUGGJJUACCJGGUA

ACCAGAGAAACACACGUUGUGGUAAACGGJA

ACCAGAGAACACGUJGAAEACGJA

ACAAAAAAGUGGUCUACGU

ACCAGAGAAACACACUUGUGGJACAACCJGGUA

ACCAGAGAAACACACGUUGUJCGUACUUACCJGGUA

ACCAGAGAAACACACGUUGGGUACAACJGGUA

ACCAGAGAAACACACGUUJUGG(UACAUUACCJGGJA.

ACCAGAGAAACACACGUUGUGGUACJAuCCUGGUA ACCAGAGAAACACAcCU1JGUG(UTJAUU!ACIWCUGA

ACCAGAGAAACACACGUUGUGGUAAACCUUA

GAACA

GAACA

GGACU

AGGCU

ACACLJ

GAGCU

ACGCC

CGGCG

CCGCA

GCACC

ACGCU

CUGCA

GCACA

UCACA

CAGCU

GGACA

GUGCUJ

UGGCC

AGACA

GCC

GUU

GCC

GAC

GCC

GCC

GAC

GCC

GCC

GUC

GUC

GUU

GAC

GAC

GCC

GAAGCNAC

CGAAUCEUC

GGGAUGGC

CUCUGCCC

GAGCUCAh

UCGGUGGG

CCCAGCCU

UUCUIGAUC

CUACACCU

CUCUGCCC

GGAAGCCC

UGAUGCUIG

CCAGGAGU

CUGGCAUC

CCCGACUU

GCC C2GACACCA GCC UUCABGAAU GCC

UUUACUGA

UCAGUAAA AGAA GUCU 241 Table 23: Human TN:F-a HE Ribozyme Target Sequence Ut.

Position E Target Sequence Pont.

PoIti on HR Tzrget Sequence 28 29 31 33 34 37 39 44 58 67 69 106 136 165 177 180 181 184 190 192 193 195 198 199 205 226 228 229 243 244 253 273 286 288 290 292 295 302

GGCAGGU

GCAGGtU

AGGUCU

UECUCUU

lU~c~CU

CACGOCU

CCAcccU=

ACCCUCU

CCUCUCU,

GCAUGAU

AGCGCU

CAGGCU

CGUGCU 1 UGCEJVGt I

GCUUGUU

UGDU=C

UCAGCCU

AG'-C= It GCCD cD CUM= C uucoccut UcucuUC UCCUAU C CCACGC-u c ACGCUCU L cGcujuU c CU.GC.AcU U UGCACU U GAGUGAU C GAAGAGU c GGGACCU C GACCUCU c cCcuctJ C tJCtCUCU A CUCL'AU c *AC-CCCU C

:CUCUUCC

ucuucc UUCC=c i CCUCUCA

CUCDCAC

:UCACXI

:ACAACU

LCUGACCC

CACCCUC

UCU~CCI_

uccccuG

CCCUGGA

CGG-GACG

CCCAA

CAGGCG

GUUC

CCUCAGC

CUCAGCC

AGCCUCU

uucUccrj cuccuoC

UCCUUCC

CUUCCEIG

CCL'GAU

CUGAUCG

GUGGCAG

UUCOCICC

CUGCCUG

UGCCOGC

UGGAGUG

GGAGTJGA

CICCCC

CCCCAGG

UCUCUA

UCUAAUJC

UAAUCAG

AUCAGCC

AGCCCUC

UGGCCCA

321 324 326 327 329 352 361 364 374 391 421 449 468 480 484 487 489 492 499 502 504 505 525 538 541 553 562 568 570 573 586 592 595' 597 604 657 667 669

GJUC-AGAU

AGAUCAU

XUCAUCU

UCAUMU~t AUCUtJCU

AGCCUGU

CCC~UM

A13oGUUG

AAACCCU

GGCAGCU

AUGCCCU

GAGAGAU.

Liu CCAU

GGCCUGU

UGUACCU

ACCUCAU

crCAUCU At7CEICU

CCCAGU

AGGL'CCUT

GUCCUCUt

UCCUCUU

tGCCCC AUGUGCLI C UGCUCCLT C ACACCAU C GCCGCAu C UCC-CG C GCCGtJCU C GUCUCCU A CCAAGGU C UCAACCU C ACCUCCU C CtUCCUCU C CUGCCAU C CCCT.JGG

A

AC-CCAU C CCCAt~cU

A

C AUCL7cU C tLMcucGA U CUCGAAC C OCG-AACC C GAACCCC A GCCCAnG U GUAGCAA A GCAAACC C AAGCUGA C C-AGJGGC C CUGGCCA A. ACCAGCU

CAGAGGGC

AL CCUrCAUC

CAUCLTACU

UACUCCC

k. CUCCCAG

CCAGUC

CUCUUCA.

UUCAAGG

CAAGGGC

AAGGGCC

*CACCCAIJ

*CUCACCC

ACCCACA

*AGCCGCA

GCCGUCU

UCCUIACC

CUACCAG

CCAGACC

AACCUCC

CUCtJCUG

UCUC-CCA

UGCCAUC

AAGCAGCC

UGAGCCC

.UAUCEIGG

UCUGGGA

242 *5

S

S 682 684 685 709 721 725 735 737 739 744 745 753 763 765 768 769 775 778 801 808 809 820 833 837 838 839 841 842 849 852 853 863 869 871 872 878 890 898 899 904 917 918 924 925 926 945 946 959

I

C

C

C

C

C

A

A

U

C

C

A

C

C

C-flCXU C UGGGAG cGGG C UUCOGC GGGGUCJ U CCAGCUG GGGUCCU C CAGCUG ACCGACU C AGCGCUG CUGAGALT C AAIUCGGC GAflCAAU C GGCCCGA CCCGACLT A UCUCGAC CGACtI.U C UCGACUU ACtUCU C GACUUU CtJCGACEJ U UGC-%r.AG UCGACUU U GCCGAGU GCCGAGU C UGGGC.AG GGCAGGU C UACUUUG CAGGUCU A CUUUGG GUCUACU U UGGGAUC JCEMhCU U GGGAUCA JUGGGAU C AtUUGCCC G;c~aU3 U GcCCOGU raAA~CAU C CA.ACCUU CrAACCU U CCCAAAC :AACCO C CCAAACG LACGCCU C CCCUGCC .CCAu c ccuuUAu.

AujcCCU U tUhfltAC .UCCCUt7 U AUCC CCCUUU A UUaCCCC CUOUAU U ACCCCCU UUUAUU A CCCCCLUC CCCCCU C CUUA ccuccrU U CACGACAC CUCCUU C AGACACC 960 1001.

1007 1008 1021.

1029 1040 1046 1047 1051 1060 1067 1085 1086 1090 1091 il11A 1124 112-9 1135 3.151 I1152 U.58 1159 1162 1164 1166 1174 1175 1176 1183 1184 1187 1208 1224 1228 1230 1232 1233 1.234 1238 1239 1245 1251 1252 1254 3-255 1256 1258 UGC-C-UU C AGGAAUG AACCACU A AGAAUTUC tLIAACAAu u cAAACUG AAGAAUU c AAACUGG GGC-GCCJ C CAGAACTJ CAGMACU C ACMGGG GGGGCCU A CAGCUjuU UACAGCU u UGAXJCC~C ACACUrJ v G-AUCCCU- CUUUGAU C C-,UGLC-A C'GACAU C LrGGAAZLC C UGGAAU C UGGAGCU, GGAGCCu U UG=Uu GAGCCUU U GGUUCUG CUtUGU u CrJGGCCAr: tUUMAGMU C UGGC-CAG CAGGACEJ U GAGA- AAGACCU C ACCUAGA CUCACCU A GAAAUUG UAG-AAAU U GACACAA UGGACC U AGC-CCUU GGACCUE3 A GCCUUC UAkGGCU LT cCUCUCU AGGCCUU C cUCUCU.C CCUUCCU C UCCCAG UUrCCUCU C 'UCCAGAU CCUCUCIj C CAGAUGU CAGAUGU U U'CCAGAC AC-AL'GWr U CC.AIGACU GAUGUU C CAGACUU CCAGACu U CCUUGAG CAGACU C CUUGAGA ACECCUT U GAGACAC CAGCC-t C CCCAXJGG GCCAGC-u C CCUCU;tJ GCUCCCU C UAUUUAIJ UCCCUCU A UUTUAUG CCUCUAUT U UAtJGUrU CL'CUAUJU U AUJGUUUG UJCEUU A L-GUUGc UUUAUGU U UGCACU uOaUGUU U C-CACUU UUGCACJ U GUGAIJUA OUGUGAU U AUUUAriUU UGUG-ATU A UUUA UGAUUMTJ u U.AMULJUJ GAUMAUU U ALUUAUUU AUUUUU A TUUAUUUA UAUUMty U AUUUALUU ACACCCUT C UCAACCUT cU AACCUCU UC ACCUCUU c UCDGGCU CA AGAGAAU uG GGdC& UA

GGGGCUAG

UUAGGUCG

CCAAGCU U A CAAGCUU A G UAGAACU U AGAACUU U A GAACUUEJ A A CPLCCACU U C ACCACUJU CG CUGGGAU U C

ACCUCU

VCD=C

UGGCUC

GGC=C

AAAAG&

GGGGCU

GGGUCG

~GUCGG

3AACCC 3AACUU

ULGCAA

kGCAAC

;CAACA

AIAACC

LAACCU

C7GAAU 243 1259 1261 1262 2.263 1265 12 66 1267 1269 1270 1272 1273 1274 1276 1277 1278 1280 1281 1282 1294 1296 2.297 1298 1300 1301 1315 '1317 1334 1345 1350 1359 1360 1361 1362 1386 1393 1394 1401 1414 1422 1423 1425 1426 1427 1431 ,432 1436 1437 1438 MAMU~ A AUUAUUU A UAUOCAU U AUL1MUU U UDUAfUU A AfUUUU A utUU U A.V3U A tLVflUAU U AUUAU U UUMlU A AUUM=l U UUMfUU A

UAUU=U

AUuakuu

AUUU

AUMU

UUUA=UU

AUUCA

AUL~haku

UCAGALU

CAGAUG

1440 1441.

1446 1448 1449 1451.

1456 1457 1461.

1464 1466 1479 1480 1494 1498 1501 1512 1517 1528 1S33 1537 1540 1546 1549 1551 1552 1566 1572 1576 1577 LUG-U U'

GUUUUUU.

UUUAAI

AAAAUJAU

I

-AANUAUU

AUkUAU

AUCM~AZ

IUCUGAUU;

AMIAAGUI

AAGOUGUC

GML.-cu UG-tGA C-CAUU- L CAACUCu c UGU c CACM~AU U GAGGCCU C CUCUGCU c AGGGAGU u GUUG=~ c VGUCUGU A CUGtAAU C UCG~CCU :A GCCUMAMU A CCUA=u u UACMTJU C GAGAXAAU A UA,A G t; UU GG-UUGCEJ

U

GUTUGCTJU

AL

UGX%=U A UUM.UU AAUD U M~UM=G AUGM=l U AUUGGG UGWUflOU A UUDGGGA tPLuumTh U UGGGAGAL AUUMUAt U GGGAGAC CCGGGGU A UJCCWGG GGGGU.U C CUGG CCAAU A GGAGCUG GCOUGCC-U U GGCtJCAG CUUGGCU C AGAAUG GACA3u u uuc)=G ACAUGULT U UCCGGA CAUU U ccGM~AA AUGUUOU c CGuG;AA GAACAAU A GGCUGUU AGCGCUU U CCCAUGU GGCUGru C ccAuG1A CCCAUGU A GCCCCCU CUGGCCU C UGUGCCU LIGUCCU U CUUUUGA GUGCCUU C UUCUAU GCCDUCU U DUGAMLT CCUUCUTU U tIGAUU CUUCUUU U GAUUAU UUUtrG-AT U AUGVUU UUUGA.UU A UGUUUU ALR-MTGU u TUUOULA UMUGUU U UUAAA LUUUU U ULAAAAU U AAAAUJAU ;L AAAUAUU h~ UUAUCUG J AUUGA k. UCUGAU :UGAUtEhA 7AAGUUGU L Ac;tJUGC

IGUJAA

LAAACAA

LAACAAUG

ruGMCAC rGG-UGA;CC

ACUNMU

AflGMG

TGCCCC

UG~kAUC

AUCGGCC

GGCCt;LC

CUAUUCA

UUCAGU

CAGUGGC

AG=GGCG-

AAC-GUUGm

GCUUAGG

AGGAAAG

GGAAAGA

244 Table 24: Hulm-an TNF-a Hammerhead Ribozyme Sequences Int.

Positi±on H Ribazyme Sequeonce

I

28 29 31 33 34 37.

39 44 58 63 67 69 106 136 165 177 180 181 184 190 192 193 195 1.98 199 205 226 228 229 243 244 253 273 286 288 290 292 295 302

C-CAAGA

AGGAAG

AGAGG~

UGAGAGC;

GUGAGA

UAUG=G

AGLChUGE

GGGUCAC

GAGGGM.

GGGGArI

CAGGG

UCCAGGG

CGUCCC

GAGGAAC

GCUGAGG

GGCUGAG

AGAGGCU

AGGAGAA

GAAGGAG

GGAAGGA

CAGGAAG

GAUCAGG

CGAUCAG

CUGCCAC

GGCAGAA

CAGGCAG

GCAGGCA

CACUCCA

UCACLUCC

GGGGGCC

CCUGGGG

UtP.GAGA

GAUEMGA

CUGAULA

G-UGAtJ GAGGGCtJ

LTGGGCCA

CUGAUGAGGCCGAAAGGCCcGAA

SCUGAEC-ACGCCGAAAGGCCG;A

CCC-AUGAC-CCCGAAAGCCGAA

CCGATJGAG-GCCGAAAGGCC-.AA

CUGAU2AGGCCGAAAGGCCGAA CUGAUGAGCCGAAA~cCGAA

CUC.AUGAGGCCGAAAGGCC,-AA

CGAUGAGGCCGAAAGGCCGA

CUGAUGAGG~rGAAAGGCCGA

LCGAUGAGGCCGAAAGGCCGAA

CUGAtJGAGCCGAAAGGCCGAA

CUGG-CCAAAGGCCGAA

,LJLArnArCCGAAAGG,-C~CGAA

CUGAUGAGGCCGAAAGCGCCGAA

CDGAUAGGCCGAAAGGCCGAA

CUGALIGAGC-CC.AAAGGCCGAA

CtJGAUGAGGCCGAAAGCCGAA

CUGAUGAGGCCGAAAGCGA

CUGAUGAGGCCGAAAGGCCG--AA.

CUGATJGAGGCCGAAAGGCCGAA

CUGAtLAGG CG AGCGA

CUGAUGAGGCCGXAAGGCG;LA

CUGAGGCCGAAAGGCCGAA

COGAIGAGGCCGAXGGCCGAAL

CUGAT2GAGGCCGAAAGGCCGAA

COGAUGAGGCCGAAAGGCCGAAJ

CLCAAGGCCAAAGGCCGAA3

CUGAUGAGCCGAAAGCGAA

COGAUGAGGCCGAAAGGCCAA;

CUGAW.AGG--CGAAAGGCCCAA CrUGGCCGAAAGGCCGA CMUGfGAGG,-CGAAAGCCGAA

A

CIJGAIJGAGGCCGAAAGGCCGAA

CUGAUGAGGCCGAAAGGCCGAA

COGAUGAGGCCGAAGGCCGAAA

CEIGAL3GAG-GCCGAAAGGCCGA.A

CUGAUJGAGG-CCGAAAGGCCGC-A

CUGWl-AGGCCGAAGCC

A

CUGAUGAGGCCGAAAGGCCUGA

A

ACCLIGCCC

AACCUGC

AGAACCU

AGAGAAC

AAGAGAA

AGGAAGA

AGAGGAA

AUGUGAG

AGCCGUG

AGOGUGG

AGAGGGrJ

AGAGAGG

AUCAUGC

AGCGCCLT

AGCCCEG

AGCACCG

ACAAGCA

AACAAGC

A.GGAACA

w2GGCUGA

P.GAGGCUT

LAGAGGC

kGAAGAG kGGAGAA

LAGGAGA

UCAGGA

kGCG=G

LGAGCGU

LACAGCG

LGUGCAG

LAGUGCA

LUCACUJC

CUCUEJC

.GGUCCC

CAZGUC

CAGAGG

GAZ~GAA

UUAGAG

GGGCUG

245

A

321 324 326 327 329 352 361 364 374 391 421 449 468 480 484 487 489 492 499 502 504 505 525 538 541 553 562 568 570 573 586 592 595 597 604 657 667 669 672.

682 684 685 709 721 725 735 737 739 744

ACAAGA

UCGAGA

GUUCGA

GGOUCG

GGGGUL,

CAUGGG

UUGCtM

GGUUUG

UCAGCU

GCCACU(

UGGCC.=

AGCtUW

GCCCUC

GALUGA

AZAGA

GGGAGUW

CUGGMA

GNACCUM.

UGAAGMC

CCUUGA;

GCCCCUUG

GGCCCUM

AUGG=U

GGGUGAG

UGGGGU

LrxcWGGu

AGACGGC

GGtJAGGA CLGG%-UkG GGUCuG

GGAGGUU

CGAGAG

C GCAGA

G-AUGGCX

CYGCOCUU

GG-UCAI

CAGAU

UCCCAGA,

cuccc,'

GCUGGA

CAGCOGG

CCAGCrUG

CAGCGCTJ

GCCGAUU

UCCGGGCC

GOCGAGA

AAGrJCGA CAAAGt7C

CUCGGCA

UCUAUAGGCGMAGCCGA

ACUGAGAGCCGAAAGC,.

GCUMMAGOCCGAAAGCCGA

SCtJGAUGAGGCC-AAAGGCCG-;A

CUCGAGAGGCCGAACGCCG

CUGAUGAGGCC-AABGaCCAA

CUGAUXGAGGCCGAAAGGCCGA

7CUGALr.AGr3CCGAAAGGCrC-AA SCUAVMAGGCCI!Ar.--r.-AA

SCJGAUGAG-GCC'.AAAGG-CCGAA

SCOAUAGGCCGAAAGGCCG-A

ICUAUAGGCCAAAGCC--AA:

CCUTGAC-GCCGAAAGGC,-,AA

CUGAUGAGGCCGAAAGCCGAA

CDGAUGAGGC GA.AAGG,-CGAA CUGAUGAGGCCwAAAGGCCGAA iCGUZCGAU;CCGA vCUGAWXAGGCCGaAAGGCCGXZL

LCGAUAGGCGAGGCCGA

CDGAUGAGGCCGAAAGGCCGrAA CUGAUGAGGCCGAAAGCCGAA1 CUGAUGAGGCCGXU~GG~aA *CUGAMlAGG--CGAAAGGCCGA CtUGALMAGGCCGAaGccA~x

CM-AUGAGGCCCGAAAGGCG.AA

CUGAAGGCCGAAAGCCGA*C.-U

CLJGAIJGAGGCCGXAAGGCCGAA

CtJGAUGAGGCCGAAAG~CCrAAA CUGAUGAGGCCGAAAGGCCG.A

A

COGAUGAGOCCGAXZM~CC.-AA

CUGAGAGGCCGAAGCCGA

CU~hUGAGCCGAAAGGccr

A

CUGAUGAGGCCGAAAGGCCGA

A

CUCAIJGAGGCCGAAAGGCCGMAA

COGAUGAGGCCGAAAGGCCGAA

A

CUCGG-CC-AAACCCGA.A,

CUGAVGAGGCCGAAGCGA

CUGAUGAGGCCGAAAGGCCGAA

CtJGAUWZAGCCGAXAGGCCGAA COAGG~CCGAAGGCCGA

AC

CVGAUGAGGCCGAAAGGCCCGAA

A

CAUGAGGCCGAAAGGCCGAA

CUC-AUGAG.GCCCGAAAGC;-CC-A

AC

CUAGGCCAAGCAt CUGAUGAGGCCG~aAGGCCGAA

A

CUGAUGAGGCCGAAA=CC-AA

A

AUCUGAC

AUGAUCU

AGAUGAU

AAGNUC-A

AGAAGAtJ

ACAGC

ACAUGG

ACAACAU

AGGGUUU

AGCUGCC

AGGCAU

AIJCUCUjC kUGGcAxc AGr.C- AGGUaCA-

AUGAGGU

AGAUGAG

PAGUAGAU

kccuGGG kGGACCU kGAGGAC LkAGAGGA

UGGGGCA

LGCACALT

LGGAGCAL

LIGGUGU

aUGCG-c

JCGGCGA;

rACGGC

ZGAGAC

CCUUGG

GGUUGA

GGA=G

GAGGAG

OGGCAG

CCAGGG

UGGGCU

3AUGGG

MGAUG

crccuc 3ACCCC

GACC

7JCGGU

ICUCAG

~AUC

tJCGGG

MMGCG

;AUAGU

UCGrAG 246 745 753 763 765 768 769 775 778 801 808 809 820 833 837 838 839 841 842 849 852 853 863 869 871 872 878 890 898 899 90Q4 917 918 924 925 926 945 946 959 960 1001 1007 1008 1021 1029 1040 1046 1047 1051 1060 ACUCGGC CCCAAAGG CCGAA AAGJCGA CUGCCCA CUAGCCvuAaAG~cc,-A AcCCGC CAAAM CUAUAGGCCG-AAAGGCCGCAA

ACCUGCC

CCCZAAAG CUG~AGCCCGahAG=ccGAA AGACCUG GAUCCk COUGAACGCCGAcA A~mkAC UGAUcc cOGUGAGGCCGAAAGcGAA AAGaThGA GGGCAAfl CUGUAGGCCGuAAAGGCG

AL-CCC.;

ACAGGGC C GAUAGGCCGAlAAGG-CcGAA AfLCaC AAGUG CLAGGCCGAAGXCC.AA ATJUrJ'c GUUGGG CGUGGCCGAAAWGC,;

AG,-UUGG

CGUUG COAUAGCCGAAAGGCC3,;,

ACM-

GGCAGGG CtGAUGAGCCGAAACGGCC-,aA

AGC,!-GUU

AT.TAAAGG C GAACCrAAG--CAA ALL-L-CG GGUAhAUA CUGAUGGCCGAAAGGccG-AA AC-G;LUU GGGOAAU CUGATGGCCGAAA3GCCGAA AAGGG U GGGGCAA CUGAGCCr3AAGC7-C,,A~ AAAGWc,L AGGGGGU CGUAGCtrwXAAGGcGA

AAAAGG

GAGGGGG CAGCCGAAAGOCcGAA AAflAAG UCUGAAG CGUAGCCGAAAG~CCCCa A- ArGGU% GGUDCOG CAGGMGAGGCCGAA AC.AGG GM1GUC CUGDGWCGAAAGGCCGAA

AAGGAG

AC-AGGUU CUrADGCGCAAGGcc-A AGGU GCCAGAA CUU AGCCGAAAGGccGA~

AGGUUGAL

GAGCCAG CGAGAGCCGAAAGCCCGjAA

AGAGG-UU

UGAGCCA CUAUGGGCGAAAGGCC; AAGACGU UCUU U UGGGCCGAAGGCCGAA

AG-CWA

AGCCCCC CU GA~GGCCGAAAG-GCCGA ALUCU=u CGAC= C cGAGGCCGAAAGCCCIA

)GCCCCC

CCGALCCC CUGADGAGGCCAAAGG-rcAA

.AAGCCCC

GGGUUCC CGGACCGAAAGGCGA ACCCUaM AAGUUCU CGGAGCCCGMAGGcCG; -XCU=~ AAGUC CGAGGCCGAAAGG-Cc;

AAGCDUG

UOGC;U COAGGGCXcAMGCCCA AGtJUCUA GOUGCU COGAtXAGGCCGAAAGG--CCG-A

AAAGUUCU

GGUUUCG CMAGAGCCGAAAGCC

AC;UGGUG

AGGUUUC CUACA.CCGAAAGcC,-a

AAU

AUUCCUG C -rAU AGGCGccCGA,

AUCCCAG

CAUUCCU CtJGAfGAGGCCGA AGCG AATJCCCA GAAUUCEJ CLGUGGCCG k CC AGGGUU CAGUUG W-UAGCCGAAAC-CC At7UCVM~ CCAGUULJ CUGAGGCCCGMAAGGCCG

AAIUUCUU

AGUUCUG CUGUGAGGCCGAAAcCCG

AGGC

CCCAGU CUGAGAGCCGAXAGGccr-A A~uucLTG AAAGCUG CUGAU GCCGGCCC-r

AGCC

GGGAucA cUGmtJGAGGCG 'CGAA AGCtJGUA AGGGAUC CUAUAGGCCGAAGCCGA

AAGCJGU

u~uc~ CJGAA.A

AUCAAAG

GAX3UCCA. CUGAUGAGGCCGAAAG-GccA

A-CGUCAG

1067 1085 1086 1090 1091 11123 1124 1129 1135 1151 1152 1158 1159 1162 1164 1166 1174 1175 1176 1183 1184 1187 1208 1224 1228 1230 1232 1233 1234 1238 1239 1245 1251 1252 1254 1255 1256 1258 1259 1261 1262 1263 1265 1266 1267 1269 1270 1272 1273

GO=U(

AGAACC

CAGAAC

UGGC~

CCGC

CAALMC

OUGUGC

AAGGCC

GAAGGC

AGAGAG

GAGAGA

CUGGAG

AUCMG

ACEC

GUCCGG

AGUCUG

AAUCD

CUCAAG

UCrUCA;

GUGUCU(

CAYGG(

AMAC

AmJAAU ACAUAA3

AAACALB

CAAACAI

GCAAArC,

AAGUGCA

CAAGt7GC

UAAUCAC

AAUAAATJ

UAAMAA

AAUPIAU

AAAUAAU

UAAAUJAA

AALUAALT

AAAbAA AtUAAA

AALULAAU

tWAAUAA

AAAUA

AAADAA

MAMAAA

AAMUAA

AAALMhAA

ALAAA

AALUhAATJ 247 ACOGUGGCCGAAGCA

AUUCCAG

COAUAGCCAGCCA

AG-,-UCC

CGAGOCCGXAGGCCGA

AAGGCUTC

~CMGAGGC-,G AAC"

ACCAAAG

:A L"UAUAGGCCG AC-CCrGAA

AACCAA

C CA GGGCCGAACCGA

AGUCUG

uCOM~r-GG--CGAAAGGCCA AGW=uu ICUGAVGG-AAGGCC .3ccc AGGUGG rc CGAGG-G;, AUtUUa a VG GGL(-CGAA

AGGUCCA

CCU AAGAGGC GGCCGA

AAGGU'CC

CraG L CCG~%Gt--W

AGGCCUA

GCMAGACGGCCG-AAGCCC-X

AAGGCCU

ACOGAUI-ACC GCGAA

AGGAAGG

A TTUGrGCCGAAAG3C-CGAA

AGAGGAA

GCUCAGC-GAG XGC-GA

AGGG

ACUGAIMAGGCCGAAGCCGAA

ACA.U=U

CAUkGCCGAAGGCGAA

AAOWCU

CUGAVGWZCCG1AAAGCCCGAA

AAACAUC

3CDGMt=3GGCCGAAAGGCCGAA

AGUCUGG-

CUGU~kGCCAAAG,-GAAAAGUCUG COGATJGAGGCCGAAAGGCCGAA

AGGAAGU

COGAGAGGCGAAGGCGAAAGGGCTJG

COGALX~kGGCGAAAG;CCCGAA

AGCUGGC

CUGADGAGGCCGAAAGGCCG-AA

AGGMWG

LCGWXGCAGCGAA

AGAGGGA

COGAUCAG,-CGAAAGGCG AtJAGAGG

AAUAGAG

CUGGGCCGAAAGCCGAA AAAflAGA CLUGAUGAGGCCGAAAGGCCGMA

ACAAA

*aMAAGGCCGAAAGG-CGAA

AACAYJAA

CGUAGGCCGAAAGGCCGAA

AGUGCAA

COGAGGCAGGCCGAtA

ATUCACAA

CUGADGGCCGAAGccGMA AA~xcA CtUGGCGAAAGGCCGAA

AMWAXCA

COAGAGGCCGAJUAQGCCG-AA AAXrh 4

AUC

CUGAUGAGGCkUGCrAA

AAAUAAU

CCGUGrGC.AAAGGCCC-AA~ AtIAAAUA CtXGAtGAGGCCAAAGCCrA

AAMMAU

CUTGAGGCCGAAAGGCCGAA AAUAAk COGAGAGCCGAAMGGCCGAA

AAMAM

CUG-AGGCCGAAGCC

AAAUAA

COGAUGAGGCCGUGXGGCCA

AUAAXI

CLXAtkAGGCCGAAAGGCCGAA

AALMAAU

CUGAUGAGGCCGAAAG.CCCGAA

AAAUAA

CUar4=AG---CGAAAGGCCGAA

AUAAAUA

CUGAt3GAG---CGAAJGGCCGAA

AAUAAAU

CUGAUGAGGCCGAZGCC-A

AUAATJAA.

CUTGAOGAGGCCGAAGGCC-AA AAflAM 248 1274 AAAUAAA CUGAflGAC-GCCr=AAAGGCCCGAX AAAXJAU 1276 G7DAAAUA CUGAUGAGGCCGAAAGICGCCGAA AUAAAIJA 1277 u IAAU CUGAUAGGCCGAAAGGCCGAA AAUJAAAU 1278 C LMA A COGAUAGCCGAAAGGCCGAA AAAIIAA 1280 ACGLUAL GAGC~AGCG~ AUAAUA 1-281 C.-UGU CMUG~AGGCCGAJUGGCCGAA

AAAU

1282 UCAVU=

AAAXLAA

1294 AAATLAACGtAGAAGCAA

ACADUCA

1-296 CCAAAUA CGDGCA.GCGA

IJCAUU

1297 CCCAAAU CU~)A~CAAGCAA tCAU 1298 UCCCAAA CUGAXUGAGGC-CGAAAGG-CCAA AAAIkr_.

1300 uCcCCA C3AGccAc-cA AUAAAUA 1301 GCUCCC CUGXLAGAGCCGAGG%-C-cAA AuU 1315 CCCAGA CU-~PGx-CAGCA NCCG 1317 CCCA CGAUAGG-CcGAAAGCCGAA -NuA=C 1334 CAG-CUCC CUGAUAGG-CGAAAGGCcc-AA AcALDUOG 1345 C ;ASCC CUGAUAGCCAAAGGCCrGAA AGGCAGC: 1350 CI=LCU CUGAUAGG-CGAAAGG-cCGAA AGCCAAG 135.9 CACGGAA CJGAUGAGG-CC-AAAGCCcGAA ACA~xCUC 1360 UCACGGA CUGADIAGGCCGAAAG-GCCGAA

AACAXJGU

1361 UUCACGG CUGAt AGGCCGAAAGGCCGAA AAACAIJG 1362 UUUCACG CUGAVGAG.-GCC-AAAG,-CCGAA

AAAACAU

1386 AACAGCC CUGPJAGAGCCGAAACGCcGAA AUUOUU=C 1393 ACAM7GG CUGAUGAGGCC)GAAAGGCcG-AA ACAGCCU 1394 UACMG CUGAVGAGC,CGAAAGGCCGAA

AACGC

1401 AGGGGGC CtJGAJJGA=GCGAAAC-GCCGAA ACALUGGG 1414 AGGCAC CUCAUGAGGCCGAAAs-GCcGA AGC.CAG .1422 JCAAAAG CUGAUGAGGCCGAAAGGCCG-AA

AGGCACA

1423 AUCAAAA CUGAUGAGGCCGAAAC-GC CcG.A2 AAGGCAC *.:1425 UAAUCAA CUCG AGGCCGAAAGGCCGAA AGAAGGC *1426 AtUhAUCA CUGAUGAZC-CCvjAAAGcCG AAGAAGG 1427 CAThAVC CUGAUGAGGCCGAAAGG=-,CCGAA AAAC-AAfl 1431 AAAACAU CUGAUtAGCCCGAAAGGCcGAA4, AUA .1432 AAAAACA CUGAXr4AGGCCGAAAG-cGA AAUCAAA *1436 00 AAA c CGAUAGG--CGAA GCCG ACAXJAAU 1437 UUUAA CUAGGCGAA,-CA AAA 1438 AUUtUA CUGAGAGG-CCC-AA C-AA AAT3U 1439 U~O~CUGAV)AGGC-CGAAAGGCCGL

AAAACATJ

.:1440 A.fLMLVW CUGAtflACCCCGAAZ-CCGAA AAAAACA 1441 AALULUUU CLTGAtGAGGC-CGAAAGGCcWG- AAAAAAC 1446 CAGNAfAc CUAGGGCCGAAAGG-cCGAA AUUUUA 1448 AIUCAGAUJ CUGAUGAGGCCGAAGCCCGA AXTA=UU 1449 AAUCAGA CUGAUGAGGCCGAAAG cG AAIL.U= 1451 UM.AUCA CUGAtr=AGGccLGAACGCGcG ALTAmjA~U 1456 ACAACUU CUG UCAGC,3CGAA GCCCGA AjCAU 1457 C-AC;AC CUGA C-GGCCGAAACG&-AA

AAUCAGA

1461 UEJAGAC CUGALuGAGGCCcGAAAGGCCGA

ACUUAAU

1464 UUGjUU! CUc-A-AGccGAAGGCCGAA AjCAACUU 1466 CAUUGUU Cu GAGrcc-A.GG cG AGACAAC 249 1479 GOCACCA CUGAUGAC-CCCGAAAGGCCGAA ALTCAGC:A 1480 GGUCCC CUGAIUGAGGCCrAAAG~cCGAA, AAIC 1494 AAUGAGU CUGACAZG~CCGAAAGGCGAA

ACGU

1498 CAGCAAU CUCAUAGGCCGAAAGGCCAA

AUAC;

1501 CCUCAGC COGAUGAGCCCGAAAGGCcGA AUr:GU is=2 GGGAGCA CUuGAGC-CGAAAGGCCGA ACGCCE7C 1517 CCCUGGG CUGAUGAGGCCGAAAGGccGAA AGcAGA 1528 CAGACAC CUGALGA GcGA-c ACUCCCU 1533 GAUCMCA CU XUAG-CcGAAGGccA ACzACAAC 1537 GGCCGAU CUGA~AGrAGG c- ,AA ArBAC 1540 GLVC.G%-Cc CtrmAUGGGA AAG AA AXrjtCAG 1546 UAAUAG CUAtAGG-CGAAAGGcCAA

AGCCGA

1549 CAC A C G3ALGAGGCCGAAA cLGA AGUAGGC 1551 GCCAC= CUAGG-CGAG-C AIrhGUAG i552 CGCCACU CUGAtrNA -)GkGCCG AA akUAGL 1566 CAACCUU C GA~zWGGGCGAAGC ALXUUCc 1572 CCUAAGC CUGAUAGCCCGAA GCCGA AccuuaA 1576 CUU=c CUul-ACCAAGCA

AGCAACC

1577 UCUooCC CUAGGCrAGCCA

AAGCAAC

Fee.

be 4 $too e 250 Table 25: Mouse TNF-a EMTarget Sequences lit.

Posi±ti±on IR Target Sequence Ut.

Position HETarget Sequence 66 101 101 102 102 106 137 139 177 207 228 228 236 236 249 249 261 261 263 263 264 264 266 269 270 276 297 299 300 304 306 314 315 315 324 UgGAAAU i GGCAGGUt GGCAC~gUt GCAGGUU C gCAGgUU GOUCUgU UgUcCCTJ L gUCcCUU 1: gUCCCUlU v UcCCUuU C DUUCCU C GvCCaCAU C caCAuCU C GCAUGAU C AGGCaCU C GGGGCuU C GGGGCXU c CAGaaCU C CAG8-ACtJ c GGugCC a GjuGCCrJ a UCAGCC C UCAgCCU C AGCCUCU U AgCCrCU U GCCUCD C gCCUCUULT C CUCUUCU C UUCCCaU U UCUCaUc7 C UCCUGcU u CCACGCU C ACGCOCU U CGCUCUTU C CUUlCUgU c UcUGUcU a CtGaACU U UGaACUJU c uGaaCuu c gGGUGAU c LGcucCck ICUgUCC UgUcCCU ugUCCCU

CCUUUC

LCA~uCAc cACU AcU~gcc UCCcCC *CC~cAaA

CAGAACU

CAGaactJ

CAGG=G

cAGgcGg UgUCt~cA UGucUCa UUCVCaU MUCcau

CCUC=

CUCMuU UCaUUCC UcauUCc alUCtMG CCUGcUU

CUGCUUG

GUOGGCA

UCVUC

CUGUCUA

UGUCUAC

uAct3Gaa cUgAAcU cGGgGU GG9GUGA GGGgu~a GgUcC 324 347 364 366 366 369 376 390 396 401 404 406 406 407 409 409 409 432 444 501 5.60 560 564 567 569 572 572 572 579 580 580 582 582 584 585 608 615 61.5 618 GgGUGAU GAG~agU

UCCCUCEI

CAGuuCU AgACCCU ucaCAcU

CUCAGAU

AGAIUCAU

AUCMUC

AU~cAUcU T UCAUCUuc

AUCUUCUC

AuCuuCU atlcUJUcu c AGCCUGU A AcG3cGU A AcGCCCU C gG9UUGU a GGgUUGU A uGtkC=u U ACCEJUgU C CUugUCU A gUCUACU C GUCaCLJ c GUCUacO C CCCAGGU u CCAGguU c CCaGuU c AGGUUCtJ c AGGUuCU C GUuCLTCu U UuCUCUEJ C CCCGaCtJ a aCgUGcU c AcGUCCU C UIGCUCC

C

C GGuCCCC= U cCCAaaU C UCAUCAG c AUCAGUU arCAu AGUUCUa

SUG-GCCCA

AcaCUc.N

AGAUCAU

AUCUUCU

UUCUCaA I CUCaAAa I cUcaAAA UCaAAau aAAauuC *AaAALIUC *AAAauUc

GC~CCG

*GCAAACC

CUGG=c CCUUguC CCUUgUC gUCUACU

UACUCC

CUCCAG

CCAGGUII

CCAGguu CCAgGUu

CUCUUCA

UCtlUcAa UCuUcaa UUCaagg

UUCAAGG

CAAGGGa AAGGGaC CgUgCJC CCcCC

CUCACCC

ACCCACA

251

-I

630 638 643 645 647 663 669 669 672 674 681 681 682.

734 734 744 746 759 759 761 762 786 798 802 812 816 821 822 830 840 842 842 842 845 84.6 852 855 887 891 905 905 905 914 915 919 928 928 932 ACACCgU C AGCC#.-au ACACCgU C AgCCgaU agcCgAXJ u uGCtuauc alUUGcU a uCUcAiiA UuGCuaU C UCaUACC GCuaUCUT C aLMCCAG agAAaGU C AACCMC UCAACCt3 C CUCE;G UCAAccU c cUdUCtUG ACCUCCU C UCUGCCg CE3CCUCU C UGCCgLTC cDGCCgU C Aaga~cC CmGCgU C AACGAGCC- CU]GcCgU C aaGAgcC CCCUGGU A UGAGCCC CCCUGGU a ugaGCCc AGCL A a U.cC=G CCCDaU A cCUGGCGA GAgGAGU C UUCA~c GAGGaGTJ C UUC~C GGaGVCU U CCACG GaGUCUU C CAGCOJGG ACCaACU C AGCG CEJGAGgU C AAUCuGC- GgUCAAU C uGCCCaA CCCaAgU A cuUaGALC AgUAcuU a GACUJUUG uUaGACU U VGCgGAG UaGACUU U GCgGAGU GCgGAGU C cGGGCAG GGCAGGU C UACUUUG CAGGUCU A CUU~Ga CAGg=Uc a CUUugGA cagGUCu a CUUUgGA G0C~kCU U UGGagUC UCUPACUU U GGagUCA UUG-GagU C AUUGCuc GagU=f UJ GCuCUTGU AUCCaUU c ucuACC AuxucuCU a CCCaGC CCcCaCU C UgaCCC cCCCacU c UgACCC CcCCACrJ c uGAccC GAcCCcU U uacUCLTG ACCCCUU u acUCuGA CtJUUAcU c ugaCCcC GACCcCU u UaUugUC gAcCCE U UAUUguC CCUUtMU U guCuaCU 940 943 972 972 973 984 984 985 997 1.0i0 1017 '018 10=9 1!073 1096 106 1107 13 164 1203 1211 1214 1218 121 8 1218 1218 1219 1229 1.226 1226 1227 1228 1238 1262 1283 1283 1285 1.287 1287 1288 1.289 1293 1293 1294 ZMACccU UCL~aaCU tucUaaCtJ CUaACuU -lGgC~gAU ACGG-:gaU GGGGauU Uc~a.GAgU CugGu= AGAgCU GAgC-UL AAGgAct7 aLTGGGcU 1

UGGGCUU

GC-gCuUU

CCG-'AAU

CGAugu C gagtr~gU c UcUgUca c aaGAuCU c cAGGCCU U A GC-CCU =c CCOU'CCU a CcuACcU u CcuaCCEJ u CCUACCU u CCUacCU u CuaCCUU C C'.jAcCruU c CagACCLT U CiALccU U ag.ACCU u AGAccUu U GAccULTU C gACU~uU c CACCuu c CCC-ccu c CcCCCtJu c CCCCUCUT A CL-CUCAU u CCUCkUA u cUCj.UU u UCT"UU A UUtTAUaU U1 UUaUaU u1 UUAUaUjU U C CUCAa c AGarcc u AgAAAGg u AG-AaAgG A GAA-, gG U auG~c UJ allGgCUc a I-.CG~CUCa c C.AAcucu c AGAgCUU U UcAaCAA U cAaCA.AC c AaPCAACu a ucAUgCA J uccGAAU a1 ccGAAt~u cGaaUUC :ACtUGGaG

CAUUCCU

AgGUUTGC agaAQA

*AGGCCUU

Ct~acCUu

CCUUCAG

CaGACCu CAGACcu c'AgACCU CAkGAccU AGACcuu agACcUtJ UCCAgAC CC-AgACu CCAGACrJ C-AGACUc

CCAG

CUCAcaG uaUUUAU UAtUU)AU UtUAUaU UauAuUEJ TJaLaUU AUaUUUG LUaUUUoGC

UC-CA=U

JGcAcUu 3CACUUa 252 1300 1303 1304 1306 1307 1307 1308 1310 1310 1310 1311 1311 1311 1313 1313 1313 1314 1314 1315 1317 1318 1319 1326 1328 1329 1330 1332 1333 1337 1338 1346 1348 1349 1350 1352 1352 1353 1369 1398 1398 1412 1413 1414 1415 1415 1438 1451 1453

UUGCACU

C.cuUaU acUuATjU UuAt1=

UAUCUM

UatJUaIOU UauUuAU

AUUUAU

UUAUU

UULV=

UAUM=

U alluAtUUu u AuUuAIJU U UAU U AfltamU U AuuAULIU A UUPIDOC.

AUU!Th.U U AUUU U AUua;uu A UUCUUU A UUUaUU A UuUauUU u UauU~u 1462 1470 1472 1473 1474 1478 1479 1479 1484 1498 1511.

1514 M56 1529 1529 1530 1530 1563 1563 1568 1589 1592 1617 1623 1633 25 tUMIfU U AUU MUWLUU U AUUWMfl

AUMAUU

AUUMMl

AUM=U

UU

A UUfLMDM U UUU U AUUAUU A UCUUaUU U ~fUUOM accGuua u Gccuccu GccuccU C uuuUmcu CUCCUCU U TJUGCUUA UCCUCUU U UGCUCAU CCUCUUt3 U GctuuAuG UUUrJGCU U AUGtJUUa UUUGcEJU a UGuuuAa UUUGcULI A UGUUUaa uau=fGU u aaaAcAA AAAuauU U AUCUaAc AcCCAAU U GUCrUwA cAaUUGtT C UuAAuAA aUlUGUCU ui AAuAAcG CgcugAU u UGGiGAC cGCUGAU U UGGUGAC gCtJGAVU u gGUgacC GCUGAUU U craGAcc UgaAcCU c UrcuCCC ugaaCCU C UGCUCCC CUCUGCU C CCCAcGG UGaCUGU A ATuGcCC CtJGUAAU u GcCCUAC GAGAAAU A AAGaUcG UAAA~au c G-cuUaaa UUAaaaU a aaAAaCC AgGgaCU a gCCagGA AUUMUU U AUUUgCu UULIflUU A UUUgCuu mUmuLuU u UgCuuAU AUUM~IU U gCUUAUJG auUUGCU U AI1GAAUG uUUGCrUU A uGAAuGu UGAAbGU A UUaWUU AAU U DAUUGG AUGOAUU U AfUUGa WUGMU A UOUGGaA UAUUMU u UGGaAGG MUElUU U UGGaAGq AUUMMU U GGaA~gC GGG-GUgU C CUGGaGG gCUguCU U cAGACAg GCUGuCY U cagaCAG GACA1MU U UucuGU ACAUGUU U UCUGUGA CAUGUUU U cuGuGAA AUGUUU C uGUGAAA AUGUUUU c UgugAaA gaGCUGU c CCCAccU CUGGCCU c ucuaccu 9gCCUCU C UACCUUG 253 Table 26: Mouse TINF-a Hammerhead Ribozyme Sequences nt. Mouse BE R:ibozyme. Sequence Pos-4t-on UCCCGGC CUG UGAG-C-uAAGCcGA,:A

AGUCCC-J

66 UGGGAGC C~AG~ACGCtrAAAG~G.AA

AUUCCA

101 GGGACAG COAGGv-C-vuAG-Gk

ACCUGC-

102. GGMA=A CVGAUGAGGCCGAAAG,"CCAA

ACCUGCC-

102 AGGGACA CUG~AULGGCGAAAMGCCGAA

AACC=G

i06 UGAAAGG CUGAUlGCC,AAGGCCAA

ACAGAAC

210 U~vAGUGA CUGAtrAGG--CwutAC.CCGA;

ACGC,%CA

I GG CVGUAC-C AAACL-G AAG= 11 GUGAU CUGGCGNAAGCCGAA

AAGGGC

116 GGCAGU CUGAflGAGCLCAAAGGCCzAA AGUIG M- 2.37 C--ACGGA CrJGAGGC,-CGAAAGGCCGAA AU~GU=c 39 CtJGGAW CUAVAGGCCGAAAG CC AGU i-177 CGCGCG CUGAUGACGCCGA-AAG GCCGAA. AU c 207 UUUC-GGG COGA.rGGCCAAAGGCCGAA

AGUGCC!

228 AGUCUG CLAGGCAAGCA

AAGCCCC

228 AGUUCUG CUGAmrAGGCcGAAAGGCGA

AACCC-

236 CCGCCUG CUAiuGC-,AGCG;

AGUCUG

.:236 CCCI CtGUAG--AAC-CA AGUucr 249g UGAQICA MMG GCC,,A G,-r AGCC-C- 249 UWAGICA C~NUAC-CAA CA AGGCaC 261 AXrmAGAA C UGAr'GCCAAAGG-CcGAA

AGGUGA

261 CvA~A CU~JAG~XAAX:A

AGGG=

263 GA~rA CUA)wiGCAAcCA

AGA~G=

263 GAGA CU~GG.CGAA GGCCGAA AAAGGCt .264 GGAAUGA CU~rG----,AjGCA

AACAGGC~

266 GLGo *269 AAGAG COGALTGGCCGAAA&-CGcL)

ADG.AC

*.:270 CAAGCAG CtGAXUGAGGCCGAAGCCGAA

AWA,-GA

276 CUCC~CA CUGXfA~rGC,-GAJGCA

AGA

297 GACAAA CCAMt.-GAAGCA

AG=~

299 LMOIAG CUAG~~cAAGCA AGA~c~ 300 GUGACA COGAGAGGCCGAAAGCCQA

AAGAGCG

304 WtAGM CUAGGCAAGCA

ACGA

306 AGUCG CUCAU~w,=ccGAAGG,-CG AGA~nGA 314 CACCCCG CUuUAGccAAmCA

AGUUCAG

315 UCACCCC CUArAG-CCAAC-CA Ar~j-C 254 315 UCA.CCCC CUGAD~AGCCGAAAGGCCAA AA;UCA 324 GGGGACC CUAAG~AGcGAAUCACC 324 GGGGACC CUAGMCNAG= ADUCC 347 AUUUGGG CLMUGA-~trUAGGOOAA ACOCUC 364 COMMUA

AGGGAGG

366 AACUGAU CUA~AG-~rAAGCG AGAGMA 366 AACGAU

AGAGGA

369 tmAACU C Gr.NG AG 376 UGGGCC.

AGAACU

390 UGAGOGU COAGCSCCMIGO AGGCtJ 396 AUG1= UCAGGGOAAG=A ACGA 401 ACVa.GAU C A-CrAAGCA AflUC,- 404 UUGAGAA CUGAG GC.,AAAG CC=%A ADuAUC 406 UCUUGAG CUGAUAGGCCGAAAG~CaAA AGAMMD 406 VUUUGAG CUGUZAGCGAGGfG

AU

407 AUUUG;L~ ,G C' O- AAIA 409 GAUU Cu GAAGGCCAAAGGCccAA ArzA;L 409 GAAflUU CUGAUAGGC-CGAAAGCCcaA~ AGAAAU 409 GAP&U=U ~~~~AGcGA A.A U 432 C3GGGC

ACGGCU

444 GGUCUGC CUG?,XJGAGGCCGAAAGGccAA ACGA=G *.:501 UGCA CUmUwCC~AAGCA AGG *560 GACAAGG CU~G~GCvCAAAGcCGAA ACAACCc- 560 GACAAGG COGAU~X GAA cc AcAA~CC 564 AUAAC CUGwAG~XCGccCAAAGGccG-r.A AGZ~kA 567 GGGAGUA CCGAUGAGGCCCGAAAGGMWcA

AMA=GG

*569 CVtGG CUArXCCC; AG~,AA ACICAAC; :572 AACCUGG CrAGGCGAWCA

A~GC

S 72 AACCUGG CMUCflAZGCCGG- AGUAGAC 5712 AACCDGG CUG UAGGCCGA.AAGccc.). AGUACC 58 -nfl CUGAUCAGGCG~rAAAGGCCGAA A CG 580 UUGLGA CUCAUCAGGCCGvlA~kGcCG-AA. ACCU 80 CCUAA CUGMAGGCCAAAAGccGAA AACCLT 582 CCUUGAA CVCUA~lkGcGAAGcG

AGAACCU

*5a4 UCCCouo CUUAG-CAAGCA

AGGAAC

585 GU~CCUU CUGAGAGGC-AAccA AAAA 615 GGGUGIGG Uu UAGCGAAGcccA ACCA=~ .:615 GGGUGAG CUGAUGAGGCCG c c~ AGCACGU 618 UGr-GGU CUGAAGCCAAGGCQ AC~a= 630 AUCGGCU CUGAAG--CrAAGCC ACG 630 AUCGGCU CUGAflGAGGCCGAAA GCCGc ACGGUGU 638 GAUtrmCA CVUAAGG%-CCQa-ajGCCC ADCGGCU 643 UAUGAGA CUGAX3GAGGCCGAA~ GCCC-A AGCAAXJ 645 GGUACA CUGAflGAG.GCCGAAACCCGA

AUAGCA

647 CUCGUAU CUGAmxAGGccc-AA ,cGAA AGaM3GC 255 663 GGAGG-UU CUGAD~uGGCCGAAAG-GCAA ACUCUcr 669 CAGMAGAG CLAUAGC~rmA-

AC--C;

669 CAGAGAG CU~wAGCCGAAAGGCCOC,-A

UU

672 CGGCAGA CUCATr-uGGCCGAAAGGCCX;AA AGGk=G 681 GGCuCuU CUGArAGGcCcGActAA

-,CGC=

681 GGC-MUU COGUGA~G~CCGAAAGG;L

ACG

681 GGCUCOU c~xUAG-~'AGcGL

CG

734 GGCC CUAGGCGAAGCA AcCGAG 734 GGCCA CDGAUAGGCCGA G XCA=-G 744 CCAGU CU~rACCrAG-C.A -aGGC- 746 UCCCAGG C GAUGGCCGAAAGCCGMAAL-.- 759 GCtUGGAA Cl GAUGCCCGAAAGGCcGAA

ACCC,-

759 GCUGGA CUArAG~vAC--G

ACC,-C

761 CGCUGG L"GUAGC~~.C-C AGAC-,Cc 762 COO=~ CUGAUAGGC:CGARAGCG

ACC

786 CGCGCU LGAGfGCcrAr, -CGaA~ AGOUr.GU 798 GCAGnU CUGUGA GcCX;AA cr.A ACCUkG 802 UUGG=C CUGAUAG GCX:AA~ CA AUUGW-A 816 CAAAGUC COQAGAGG~AGcC M AACA= 821 CuccrG-A CUAGGGCXAGCCA

AGCA

822 ACUCCGC CUGUGAGCyAAr A; AAc= :830 CG CUGAI GCL

ACM=~

840 CAALACGUAGC~aUG-GAACc- 842 UCCAAAG CT AUAGGCCGA AGACCMt 842 UCCAAAG C GAUGAGGCCGAAAGCGAA

AGACCI

**845 GUCCA COGAX GAGGCCGAAAGGCGA

AGA~CE;

:845 UACUCCA COGAUGAGGCCG--A CCGAA AGkW 852 GAGCAAU COGAXGAGGCCGX;AGGCCGAA

ACUCC~A

855 ACAGAGC CUGAGAGGCCGAAG= A AUMAcT= :-887 GGGAACUG AMGGAAAGG GA AAUW.-t 891 GC-U= GGNGGCAAGCGk AG GA 905 GGGGUMA CVGAGAGCCAG GCCG AGO=,-- 905 GGGGUCA COUAGAGCC~k GCG AGG GGGCGUCA CMUGAxrGC=-UAGCCGla

AGOG=

91 *GG CUGAtX.AGGCcGAAAGGCCGAA

AW

*"915 UCAGAGrJ COGAflGAGCCGAAAGCC

AAGGG=

919 GGGUCA CUGAUGAGGCCC AG4=a AGUaAA 928 GACA:AUA CUQGraGCGXU CGAA AG==C 92 **AAJ CUGAUGAGGCCGAAAGcccGAA

AGWGC

932 A~kAC COGAflGAGGCCGAAAGCCGAA

AUU

940 CUCUGAC CUQAXJGAGGCCGAAAGGCYG17

AGAM

943 -GcU~CU CUGAUGAGGCCGAAAGGCCMAA AGU3 972 CCUUUCU CUAGGCCAAAGGCA

AGOA~

972 CCUUUCU CUGAWGCCG AGUrkam 973 CCCUUUC CUGAMAGGCCGA .GC;CGAA AAGUMC 984 GAIGCCAU CUGAUGACCC CGAA AUCC-t 256 984 GAGCCA~U CtAAGGGAACCGA Ancccc 985 LPZGCMACG AG~A~c~ AAUCCCC 997 AGAGUUG CG~.GcAA~ ACCUCGA 1010 AAGCUCU CUGAUAGGCCGAAAGGCCGAA AGccAG 1017 UUUG-CGUAG-CAAGCa AGCucUG 1018 GUGUU CUG~AUGGCCGAAAGCCGAA AaGCUCr 1019 AOUGU CUAUAGCCAAAGGCCGaA AAIGCU 1073 UGCAMA CVGGAGAGGCCGAAAGGCcGAA IGG-,_CCA 1096 CCCnUUU CU 8 GA~'G-,GAAAa c AGvcCU 1106 AfUCGM CUGAUAGGCUAAAGGCCGaA AC-CCM 1107 AAVUCGG GAmGGCCGAAGGCCGA GCCA U108 GAADU GAUAOCGAGGC; AAAcc- 1115 CtUCCAGU CUGAUGAGCCGAAAGOCGLa AAUlCC- 1133 AGGAAUfG CUGAGAGCCWL -au AckucM 1164 GCAACCU COGAUAGCCQ AGCCGAAC ACAC 1180 UADUC CUGAXUGAGGCCGAAAGOCCGAA AGACA= 120 AGGCC CUGAUGAGGCCGAAAGGCCGAA AcflCUU 1210 AAGGMG rCUG=LnAGGCCGAAAGGCC=A AGG=~J 1214 CUGMGCGACkiCGAG~=AGGAAC 1218AGG= CUAUGGGCCAAAGC- AGGaAGG- 1218 AGGU=CU vUAGCAA~cA A GuGG 1218 AGGOG COGAUGAGGCCGAAmGGAJra AGa ***:1218 AGGUCG CUGAUGAGGCCGAAAGGcCC=~ A~GG 1219 AAGGCU CUCAUAGGCCAAAGrCCGAA AAGaA i219 AAGC= CUGAUAGGCCGAAGGCGA AAGGC:;LG 1226 GUCUGGA CUC GAGGCCraAAAGGCCGAA AG,-,UG 1226 GUCtJGGA CUAGCGCGAGcp AGGU= 1227 )&GCG C ~vUAGtCGCCCAAGcCCAA AAGGUCU 1228 GAGUCUG COGUGGCCGAAACG-C AAAGurC 2238 CCUCAGG CUGAUGAGGCCrAAAG~CC~r AAGAGUC :1262 CUGGAG COGAUGAGGCCXMAAGGLCC" AAGGC3= .1283 AXLAAM CDGAUAGGCCGAAGGC AGG 2283 AfVLkMA CUGAUGAGGC'CGAA CCc=~ AGG 1.285 AUAA CUACGCXAGCA AAG= 1287 AA~AMMl CUGAUAGGCCGAAGGCCGAJA AUM~Gz 1287 AAMM CUAGGCGAA-XG AU--C 1288 CAAM CGrmCGCCGAAAG~C.CGM

AAUGAC;

289 GCAALCGUAGMkkcCA

AAA

*.1293 AAGUGCA CUGAGCCCAAAGGCCAp.) MAUaAA 1293 AAGUGCA CUGAVGAGGCCGAAA ac AUfAUa, 1294 UAAGUGC CUGAZraAGCCCCAAcC

AAAA

1300 AAAWAM CUG~AGrzGCC AAGGCA 1303 AALMAAU CUGAUGAGGCCGAAa,CCAZA

AIAAGLTG

2.304 MAMLA CUAGG~rAGCX;

AAMAGU

1306 AALXULA3A CUGAGAG-rAAGCA

AUAA

1307 AAGAU kGAUAc~ 1.307 AAALTAAU CUGAit .CAAG;CA AAcrnAUA 257 1308 UAA.AM LC~r~NIGCGA AGCCGAA AXAL~U= '310 AMAAAU CGAUaGAGCCGAAAGCCQA AMaU 1310 AAMLWt CUGA~vN CrAC--C,-a,

AUAAA

1310 AAm.UAD CUGA1GAGGCCr=GA-aGCCC U~~z 1311 AAAEAA CUGAUGAG-CrAAGGC-cG AAjMhAU 1- 11 1 AAAMQA CUrm GAGGCCAAAGG--cG2

,,,,MLW

131.1 AAAUAAA CMGAVGAGGCCr -tAGcMM AAflkaA 1'13 A~~C~~GcG~jca 21113 AMALI C~A mGCCGG CCGA AtMAtIAA 1313 AMAAXUL COG AGAGG-- AA~-Cr.,U ALMhA 1314 AAMAW C~wUAGCGAAr-rA

AARAX

1314 AAUAU CrIUu-GG-AAcC-; 1AAAj 1 115 MAMAA CUGAUAGCCujAAGG-_CA. AA;t.3AW 1317 AALUhAUA C~3UvtGCAAc-rA AUIz 1318 AAAI3AA CUGAUGG1CA~cG A 1 MLhIk 1319. UAAAUAA CD =AvGG,-CCAAAG CW.A -U,,XUAAA 1325 AAAMA CUGAUGAGGC-Cru., c,-CGAA AAAU= 1328 GCAAL CO~"GAGGrA GcC

,LVLAXU

1329 AGCAAAXj COGAUAGGCCAAAGGCCC.A

AAUAAW

1330 AACAAA COAGGCGAAG-G

AA~A

1332 AtAGCA CUAGG-CAAC-CA

AUAAALCA

1333 CAUAALGC CUGALMArGGCCGAA GC,.-GAA AAMAA~m 1337 CADUCAU CUCAUGAGGC-,AAGCGL AG-i 1338 ACDUC= CGWAGGXZCCAA

ACAAA

*1346 AAAM OA GGCGXr-6AAAUA 1348 CCAAA3 CDMVACAGG"-Mi AGG=A I

CA

*.1349 UCCAAAD CUGAOGAGCCGX GG AAM=u 1330 UUCCAAA CL-A3AUAG-GCcG jGGr-CGA AAArA 1352 CCUUCA CUGAUGAGGCC rAAG-rCGA AUAAAUA "2 CCUUCA CUAGG-'rXAG-a

AUAA~UM

!z253 GCCtJUCC CUAGG-CCAGCCA

AAAAU

*.:1369 CCUCCAG CUGAIGAGGCC GCC;-GAA

ACA.CCCC

.1398 COGUCUG COGAXGAGCCkJ CG= AMAOGC *1398 COGUCUG COGAUG-GCCGAAGCGAA

AGCGC

1412 CACAGAA CUGAUGAGGCCG

ACAUGUC

1413 UCACAGA CUGAUGAGGCCGAA GCCA AACAXJU i*i UUCC COGAtX2AGGC-CGAAAGGCCGAA

AACT

i42.3 UUUCACA CUGAGAGCGc; GGA AAU 1438 AGGU=G CUGAMflA=GA~GC

ACAG=~

1 451- AGGUakGA COAGGC-UG-CA

AGG=G

IA= CAGGMCOMADGAGGCCcGAAAGGccGAA AmAGGC 1433 AACAAGG aMUACCAAGCA

AGGA

1462 AZGAGGC CUArA---CAAGCA A~k= 1470 AGCAAAA COMAG-CGAAAGGCccL

AWAW

1472 UAAGA CUGAUGAGCCGXG-CA

AGGG

1473 ALMAGCA C GAUGA GC-AAAC,.-

AAGWA

1474 CAUAAGC CUGAUGGSCAG,-G AAcc AA hc 1478 UAACAU M-GAUrGGCGAAAGGCCGAA

AZCAA

258 14719 UCtaNACA CGtAcGAGcGAAGCAA 1479 UMLV=CUGUAG CGAGOCA AACAA 1484 UUGUUU CUGNOCAGCCCGAAGCCAA AAC>ALM 1498 GOUGAU CUGAWGAGGCCGAAAGGCCGAA Am~fu= 1511 UMLAmAC CGUAGGCCGAAGGCCGAA AflUGG 1314 UUnrD= CUGAUCMGGCCMAAAGGC-CGAA ACAAUD 1516 CGMUAGAAAU 1529 GUCACCA UAAGC~AGC~ AUCACG 1529 GCCAC. COG ~wGCCMAAAGCCGAA AtICAGCr 1530 GUCCC CUGAGCGAAAGGCMGA AAUCAGC 1530 GGCCACC CVGAUAkGGCCGAAAGCGAA AAUCAGC 1563 GGCGGCA CUGAMAGG-CGAAAGGCCGAA AGGOUCA.

1563 GGaGCA CU AV kGCCGAAGGCCGAA AGGUUCA i568 CCGWG CVGULrNC. CAA G-GA AGCAG 1589 C-GGCAAU CUAUAGCCrAGG c7A AmCAGC 1592 GaGGGC CDQGAGGCCGAAAGOCCGAA AuawAG 1617 CGAcuu U

AUUC

1623 UD~aLGC CUAUAGGCCMAAAGCCcGhA AUCOUCA 1633 GGUVuuU CUGAUGAGGCCMAAAGccaAA AflUUA

V,

.4 Ta'sble 27: llumnini'I'NF-.a Hlairpini Itihozyine Scqiteonccs nt.

Position 46 54 185 201 230 234 254 296 317 387 404 453 518 554 565 576 687 704 726 730 824 1042 1168 1178 1202 1220 1284 1340 1390 Hairpin Ribozyme Sequence AGCCGUCG AGAA GUAUCU ACCAGAGAAACACACG1UUGCGUAC-AUUTACCLUrGUA CAGGGUGG AGAA GUCCCU ACCAGA0ACAcUUGCuGAcAUACCLUrGUA GCACAAGA AGAA GAGGAA ACCAGAGAAACACACGtUC)uCCUACAUUACCJrGrUA CUICCACG ACMA GCAAGG ACC-AGAGAACACACG[JUGoUAc~JaACxTCaUrA GiJOCAGCA ACMA GAACAG ACCACAGAAACACACGUUUGrjAC~jlACCUGGUA CAAAGUGC AGAN GCCACA ACCAGAGAAAhCACJGJGuAcAUJACC1JGUA CCUCUGGG AGAA GAUCAC ACCAGAGAAACACACGJIGUGGUACAUUACCtLrJGUA GGCCAGAG ACMA GAUUAG ACCAGAGAAMCACAccuUUCUGGuACAutJACY21rGUA AGAACAUG AGAA GACUCC ACCAGAGAAACACtJIJC1GG!AJAUACCJUA GCCACUGG AGAA GCCCCU ACCAGAGAAACACCGuuJOUGuAcAuuACY21rGUA AUUGCCC AGMA GUUCAG ACCAGACAAACACACIGUGGGUACAUUACCJQGUA GCACCACC AGAA GCUUAU ACCAGAGAACACAGUUGCGUACAUJACCUGGUA GGUGGAGG ACMA GCCUUG3 ACAAAAAAGUGGUCUACG GGCGAUGC AGAA CAUGGU ACCAGAGAAACACACGLTJUGGTJAc1?AuACCUGA UIGGAGGA AGAA GCGAUC ACCACAGAAACACAC(;rJJUGGCUACM11JAc(Y1JGUA UGACCiJUG ACMA GGUAGG ACCAGAGAAACACACGUIJGuGCuAChACCUGGUA CCUUCUCC AGAA GGAAGA ACCAGAGAAACACCG1utCIu~uAUU1ACCUGGUA AGCGCUGA AGAA GUCACC ACCAGAGAACACACCu~juG~~uUGGUA~JJ~~QU GAUACUCG AGAA GAUCA ACCAGAGAAACACACGIJUGUG(JUAAUUACCJGCUA UCCACAUA ACMA GCCCA ACAAAAA~~uUGAAUC

M

GGGAUUGG AGAA GGGGAG ACCACAGAAACACAcGUUGJGGuACJAUCGUA GGAUCAA AGAA GUAGGC ACCAGAGAAACACACGUUtGUG~uCU!ACCUGGUA CUGCAAAC ACMA GGACAG ACCAGAGAAACACACUUJCVGUAJAACGGUA UCAAGCAA ACMA GGAAAC ACCAGAGAAcAccGJTJCGGuACAUJACCJGGUA AUGGGGAG ACMA GGGCUC ACCAGAGAACACACGU(GGUACAUUACCJGGUA AUAGAGGG ACMA GGCUCC ACCAGAGAAAanrrun uvy nirihn-.i,r" Substrate ACAUACU GAC CCACGGCU ACCCACG GCU CCACCCUC UUCCUCA 0CC UCUUCUICC CCUUCCU GAU COGOCAG CUCUUCIJ 0CC UCCUCCAC UCUCCU GCU GCACUUUIG GUGAUCG GCC CCCAGAGG CUMAUCA 0CC CUCUCGCC GCAGUCA GAU CAUCUU)CU AGGCGCA GCU CCAGUGC CUGAMCC 0CC GCCCMU AUAACCA OW CUCUGOC CAAGGCU GCC CCUCCACC ACCAUCA 0CC GCAUCGCC CAUCGCC GUIC UCCUACCA CCUACCA GAC CAAGGUCA UCUUCCA GCU GCAGAAGG GUCACC GAC UCACCGCU UCAAUCG CCC CGACUAUC UCGCCC GAC UAUCUCGA CUCCCCU 0CC CCAAUCCC GCCUACA GCU UIJCAuccc CUCUCCA GAU GUUUCCAG GUUIJCCA GAC UUCCUuGA GAGCCCA 0CC CUCCCCAU CCACCCA GCU CCCUCUAU AUUUACA GAU GAAUGUAU AGGAGCU CC uuCCCUCA AUACGCIJ GUU CCCAUIGIA

AUACAUUC

UCAGCCCM

UACAUGG

1GM

AGAM

AGCA

GUAAAU ACCAGAGAAMCACACGUJUGUGUACAUJ1ACYCJQGuA GCUCCU ACCAGAGAAACACACGU1JrLJCCUACAUUACC1J=UA GCCUAU ACCAGAGAAACACACGUrGUACAUTACCJCCUA *r r

S..

.5e S S

S

S

*55 S

S.

555 1452 1475 1513 1541 ACAACUUA AGAA GAUAAU ACCAGAGAAACALCACGU(GUGGUACAU1ACCUGGUA GtJCACCAA AGAA GChUUG ACCAGAGAAACACACGUTUGUGGUAC1WUACCUIGGUA CCCUGCGG AGAA GAGGCC ACCAGAGAAACACACGtAJGUGGUACAUACCTIGGUA GAAUAGUA AGAA GAUUAC ACCAGAGAACACACGUTGJGGUACUUACCUGJGUA ALJUAUCU GhU UAAGLIUGU CAAUGCU G1AU UUG;GUGAC GGCCUCU GCU CCCCAGGG GUAAUCG GCC UACUAUuc S S S

S

0 0@S

S

*5* 5 0

S

S.

*5* 5*5 S.

S

S

00*

S

*5 S S* S SSS S 0

S

S SeS Table 28: Mouse TNF-a Hairpin Ribozyme Sequences nt.

Position Hairpin Ribozyme Sequence 103 256 272 301 325 370 383 3917 467 546 549 598 603 631 634 675 691 764 803 895 906- 920 953 1175 1L220) 1230 1256 1274 GfLAAN3 AGAA GAAcatU AAAACCCLGLna- U3AGAAG AA GAGACA CWGACCOLIGJGAAUCUG CUXXCACA AGAA GGAAUGAI GGAAAA)U3XXCAXCLX GUCa AGAA GAAGAGACGGACCC3LLXMAUCUG CC JUG AGAA GALU2AC ArGGACCCUOXXrAUOLG GOCAUAG3 AGAA GAXflAG rW-AAAwuamxn m GQaAGG AGAA GOWMA3

GAACC

AGAAGAUW AAA GALIU ACG~bCCOLXJGAALOU OC~=AA

~MACAAAAAAGLX~LJAUCUG

AACCCAL72 AGM~ OCALUArGGACCOUUUGAAUC~ UACAACtC AGAA GUAGU3 PLSAA Gctvw W-WA~xmammm~ AOAGLA AGAA GO CAWNCC~XUajpWG AGCAAANC AGA GA= GUAGAA AGAA GCU3AC QLuafAC AGM GGAG GCUU= )V3AA GOMU CCLUCUtC NGAA GGAAA AGJACtXU3 AGAA GAUMJA AGAGUOM-AGAA

GOGUAG

GLAANYG3 AGAA GAGUGG AUAAAM AGAA GBaM AGGACACA AGAM ::MOC CAUtMU3AGAA

GAGOM

CUOGAANG AAA GAogj )Afl3AAGA )I3AA GGWAAA CA~~m~ UG3A AGAA QXOCAU Substrate AGGUX'U GU OCL1J)AC CUXtU GCU UGUOOC2G cGnAtU0 GUC OAAAGG LI)ALCA GLU QUJGOJ3X U30Xr2A GAC CCLIEACAC ACACUtA GNU CALXU1CU AGACAGU 0GNXfl GGUGlX& GTC GAfLX~nu GXCAGC GAU OO3MA CAAGOCU GC OCACUAC QDXO= GAC UNAxGUO ACOGrA GO GAIIXXU GUCAGQX GAU UL~P UCXtCA GCU GOAGAAGG UAAUCU OCC CAGUAC CCCLXU GAC CCUULIAC UUCXU GAC 0CtUIAU LCA GAC WAXXr3L3 -UXXCA GAC UCUttCC VOAMGC ULXXDJZc Y3G 0 CC COCCIXIJ 262 to 0. 111 qn .n .n 0) LA L7* I 263 Table 29: Human bcr/abl HIH Target Sequence SeqLuene =f No.

HETaxget Sequence b3-a2 'Rincti GkA-AA= CU C~AGJ3=~ AAkA =i AAOAGMj tUAA=AG~ =3 AAA= 264 Table 30: Humawn bcr-abl E Bibozyme Sequences Sequence Em i1bozyme Sequence 26 GGCUUUCC COGAUGAGGCCGAAAGGCC,A

AUUGAGGJC;

27 AZUGGCCG-CG CUGAVGAGGCrcGAAAGC-j AGGGjU=C 28 UACOGGCCG-IY CUGAGAGG=c X=,GccA AACCGCUUO=r :.29 GAGCUUU CUGAUGAGGCC-G AAGGccrcAA AACUCWGCUrjA *31 UACOG-GCCGCU CUGAU GC-CAAAG-,-ccU AAGWcUUUUG 265 Table 31: RSV (1B) E Target Sequence nt Position EH Ta~get Sequience GGAAU A AAUCAAU nt.

Posi±tio n AAtD.AAU

AWUCAAU

AUCAA1U

CAAUGAU

GAUDAU

AGACCGU

UGDCACU

C AADCALG U CAGCCA C AGCOAAC A AM~CACC A CACCAJCX C ACAiGA.CA U GUCACU C ACDUAG a a a..

a a a 110 13 .18 122 134 137 148 149 150 i52 154 157 161 165 176 188 208 209 210 214 215 221 226 239 241 242 251 261 265 267 274 AGACCAU A AMAUcAX ~C&AW A ACAUCAC ARACU C ACMLZC CACACU A A~CCAG GAGAAU C AUAACAC AC7AflAU A ACAOCA CAAAAU U MUM=~h ACAAA13U U AMUcrj CAAADUU A MU= AAfUUW A LULCUUGA VUM A CUUGAM AMV9= U GAM.AAI ACUUGAU A AAUCX3G GiAXMAAU C AUfGAAYUG AAXGCA A c7JGAGAA GAAAACUT U cGA).

GCAM Uj m~LUC CCACAVU U AcAuJFC CACMUU A CAVDUCCEU UUAM= U CCOGGUC UMAAUU C COGUCA UCUGGU C AACLU GUAACU A UGAAAUG UAAACG A U~CAA AAACL.U U ACACAAA, AACUAUU A CACAAAG ACAAAGU A GGAGC AAGCACU A AAUAMA ACULW A UAAAAA UAAAIWI A AAAAAXJA AAAAAWU A tACtMA 276 283 295 303 304 305 309 317 319 320 323 327 337 338 340 341 350 356 357 363 372 375 380 383 385 391 396 398 402 406 410 411.

412 421 423 424 432 434 446 448 454 IMTarget Sequence AAAAflAU A CUG-AALTA ACL'AAU A CAACACA ACAAAAU A UGGCACUtJGGCICU U UC--t-hmU GG~CA= U CC.rIA GCACUUU C CCCAtJGC tU XCCCU A UGCCrAAII LrrCCAAU A UUCIt7C CCAAUhU U CA3C.AAXJ CA),MJI C AUCAAIC aUCAU C AADCAUG CAUC2AU C AflGA=G G-AUGGGU U CU~kAA AUGawU C UtmhGAAU GGGUCU U AGAAUGC GGUCCU A GAAXJGCA AAUGCAUW U GGC.-XVj UL; G=C U AAGCCTJA UGC-CAI3U A ACCEIC UAAGCCUM A CAA-AGCr.l AAAGCAU A CUCCCAIJ GCAtUCu C CCAUADU CUJCcCAU A AU~IJACA CCAkA A UACAAGEJ AflNAMX A CAAGUAU UACAAGU A UGATUCC GUk~U= C UCAAUCC AUGAUCEJ C AAUCCAU UCUCOAU C CAXUAA)J AAI3CCAU A AAVUUCA CAUAAAU U UCAACAC AUAAAUU U CAACACA UAAAJUU C AACACAA ACACAAU A UUCACAC ACAAXMU U CACACA6A CAA~UA C ACACAAU AcACAAU C tUhAAACA ACAAUCU A AAACAAC AACAACU C UAUGCAU CAACUCEJ A UGCAlUAA tMt5GCA A ACtkUAC 266 458 CAXMhAC A akcoCA 460 UAAC A ;LCOCCA 463 CMM1McU CA~M= 467 ACUCCWJ A GUC= 470 CCAMW c cAOcfl 489 cGAAuw U AM;M~ 490 GUAIJAU A UMaMUa 492 AAAOD A GMDUU 495 U MW A AUUA 267 Table 32: RSV-(i.B) HIH Ribozymie Sequence Ut. E R:Lbozyme Sequence Position AflUGADU COAGGCM-AGC'k AfltUCc 14 CUGAAUU CLAGG-CGAGCA

AIJUUAU

18 UW-GCEr COGAflGAGGCCGAAAGcCA AftUUU 19 GUUG=CU GfGA C GAAGGCQ AAIUG=n 54 GGUGM.U CMUGA AGCCGXXUZCCGQ

)AUG

37 UGGMG CUGAUGAGGCC--UGCCA t..

77 UGUCUGU CUGAGGG AGC AU~fCW 94 AAGGAC CUGAXGAGGCCGAAAGGCGA

ACGGUCU

97 CUCAAGU CUGAUGAGGCCGAAAGGCCGAA

ACAACGG

110 )Aucua COAGGCGAAGC AU1G==C 113 GG&UGU COGAAGOCCGkAGA rrrAUW=~ 137 UUUWGUCUGADMAGGCCGAAAGCcGAA r= 148 GUMU37L C GACAAGGCCGAAc

AUCMC

148 AGM CUGWXGAGGCCGA-AG GCrGA AnUrJU= AG D CUM AGCCGAAAGGCCGA ADAUtj 152 CTJAVGAGGCCGA. AUkAAMf 154 U7WfCAAG CtMAXGAGC4CGAA GG-CGAA

T

tAMA 157 AIU~kC CUGAUAGCGAACGAA, AGtMhlUf UGAGG CUGGCA

AUU.AMT

176 UOC=cC CUGALIGGccGAA GCUGAzA

AU-

*.188 UUU=Cc CWAGr-GAAGCA

G

GAMAGUA CUMUMVXGCCGA QacA

IXIUG=

209GGV=CVGAUflGGCCAAAAGa=A

AAG=

210 AGGN.UG

CUWAGC~~~CAAAXU=

214 GACCGG CUGAX AGnCCCAGCMM

AUGMCQA

215 UGACCAG CU~-GCGAGCrA AAUGtJAA 221 CAM~U CUGWMWGCCXAGG CCGA ACCAGGA 226 CAUUUCA CL1GAIADCCAA

AUUGAC

239 U=ckGiA CUGAMW4CMXV4X=M A UtAr~ 241 UUUGUG-U CUMGatGGCCGAGGCmr3A AMQrUrUr 242 COUU=U CUXfGAMWGCCQ

AAM=U~

251 UGCUUjCC COAGGCCAGCrA ArCUUEI 261 uraAnu COGUGAGG~CCGtAAGrY2 A AYJGr 265 UUUUUUA CMAGGGCcCGA AGCG

A~UA

267 UUU UMM- AflfU=M 274 UUCAGM CUGAIX GCCAAG~L

AVUUUJUU

276 tWUCAMG CUGAtJAGCCGAAG,- AUtThIrjUr 268 283 UGG CUGA AMGGCCGAAAGCcQM

AUUCAGU

295 AGUGCCA CGUAOCAACCM ~o 303 AWkCGGGA CUAGGCCAGCM -icuG= 304 CMEMM GAU aG-- AGCC- AAUcC 305 G7UOGG CUCAXXAGGCc AA.AGCX

AAA=

309 ADUGGCCA CUACOCCAAGCG

AGGGAA

317 MAM UAGGCCMAAG3CAA

AVGG

319 AtmUw-a CUGAUGAGG-CCGAAAGmcC3mAaw= 320 GAUUG3AD CUGALGAGOCCGAA AAflAUUG 323 CUGAWl CUGAUGAGGCCCG-,,., AAGcCGAA 327 CCMUCAU CUZGA cOGA AGCG AUDG=~ 337 UUCMLAG CCUGA GCCGAGCV-

AC=LUC

338 AflUCMA CrJGAJAUGCX-AGGM AACCc= 340 G=flUc CUAGGCMLGCCA

AGAM

341 UGfUC CUAGGCGAAGC

AAGACC

350 UAUCC COAGGCGAA~rA AUGCAUry ~356 UAGGQCUGAGGCGAAOC

AUGCCAA

363 r u CUUGAVAGCAWCA AGM= 372 AUGGA CUAMMCW==

AU

375 ADMU -uMM~cuG=uAam :383 ACVUGI COAMGCMAGCA AnrCu 385 AMCauG -o~moaAG=A AM 391 GAGflMA COAM- tAGGCA -U 396 GGAUUGA CL1GAXU GCCMLAG AU VA **398 AUGGAVU CU~GAUkC<X&AGCC

AGA=

402 AUM CVUGAAGGCCG AAGCCGAA

AVUMG&~

406 UGAAADIU CUGAtGAGGCCMMAGGCOGAflAUGG .410 LTAUU CGGUGAGCCM6%=C

AUM

9421 UUGCUGADAGGUAGA

AAUUM

412 OUUU CUGAt7GAGGccGJ AA.AfU 421 XGGCUAGC.

UGU

9423 uo~ua co-mucPA -CA AMDU= AV--G CUAGG-GAX -CA A. M 432 UOUACGUGOCGAGGCA

U

434 GUUGUUU CAM*Mua-

AA=

446 iUCCAM CL1GAfGAGGCCCG 3CCcAA AGUUU 448 uUC UAMGMUG=

AGAGOUG

454 GU CuATAG c aUcc A

M

UGGAU COAMMCGUGC.i

GM

460 UAUGGA CMUAGGA~A

AUAGVMA

463 GACLG CUGAUGGGGAAGGcCGcr trGrM,,

M

467 UCOG=A CVUGAAG~ccMWMCGAA

AUGU

470 CCAflU= CtXGAXGAG=MUJAGGCGA

ACMW

489 UL~caw CWAMGCGAGCA

AUDUUC

490 AUU~caA CUGATX.AGGccr )rYGCCGA AAnuutC 492 AAAVUAC COG1WGAGGCCrGJU GCG AMA=ur 495 Uu~u CUGAGAGCCC-AkG cA AcaAuA 269 Table 33: RSV (10) HEtarget Sequence Ut.

Posi±ti±on Target Sequence nt.ic

S

i6 17 21 31 32 36 37 38.

42 46 50 51 67 68 71 76 81 87 88 92 93 100 101 104 105 120 125 128 129 135 143 145 151 155 156 159 163 164 tAAGAAU

AGAULTW

UACCAuGLT A AGAAMUU

UALAG

U GALAU A AGMCCIA A CCACDUA *.fl ACCA= U AA~nnrM.

CUV.AAU U M~CUCC mu~aiU U AACUCCC MLIDU= A ACUCCCU UOM.ACU C CCOU= ACDCCCU U GGOtAM- CCUUGGrj U AGAGA13G CUUGG1UU A GAGAUGG CAGCAU U CAUAfl AGCAAU C AULX3At3 AAtUCAU UJ GATh= AUUGAGU A UGATM TUAU=~ A AAAG=U UAAAAGUJ U AGAVUAC AAAAGUU A GAUDACA GUAkAU U ACLAAUt UUaAfU A CAAAfU ACAAAAU U VGuDuGA CAAAM U GCUUGAC AAUUWU U UGAAD AUUUGUU U GAvVMi AflGAAGU A GCAfUGU GUAG=~ U GOUAAA GCAUtGI U AAJAUM CAUU= A AAAAA UAAAAU A ACU ACAUGCU A UA.COAI AUGC---U A CUGAMA UACtJGAz A AAUUAAU GAUAkAAU U AAXJACU AULAUM A AX~hCAUU AADUAAU A CAMMJAA AAUACAU u UakCUIAA AUACAxJ uU AAcuAAC 1 65 169 175 1.76 181 2.92 196 201 206 216 221 222 231 2-2 234 235 241 247 249 250 256 259 262 265 267 270 273 278 283 284 285 300 303 316 317 319 321 338 339 346 Target Sequence tmhCx=U A ACUAACG UUUAC A AL-CCUU UAAC~C,- U UG=G-A AACGC,.-a U GG-CUWaG UUUGCu-- A AGCAtj CAGaGAU A CAUW-CA GAUACAU A CAAUCAA AtU.GAU C AAALJUGA AUCAAAU U GAAXJGGC AUGG=~ U GUGUU= AUUGG U UGUG=A UUGMGUU U GUGCAt1G UGCAUG-t U AUUACAA GCA=GU A UtUC.AAG ALUUUAU U AC-NAGuM M2UCAMj A CAUAG UACAAGU A GmrGAMLU UAG~-AU AL VUUGC-C GGUA= U UGCCCUA MGAMMU U G-.CCM-A UUGCCCU A AfLALIMA CCCtUhAAU A IAXAU UAAMW A AUAUUG UAAU A UU~amGU AuAkkmu u GakGUAA AflAfUcu A GUAAAAU UUGLUhjEJ A AA~AUCCAL GMAAMU C CAAflUUC AUCCAAU U TJCACAAC UCCAAUU U CACAAcA CCAAI C ACAACA UGCCAGU A CUACAAA CAGCACU A CAAAAIJG VCGAGCU U AtmIAUAU GGA-GGUU A LAMuGG AZGUU=~ A t3AUGGGA GU AMA A UG.GGAAA AUGGAAU U AACACALU UGGAAUU A ACACAtU AACAC;LU U GCUCUCAL 270 350 UGU C UCAACCU 352 OuG=cU C AACCaA 358 UCAACCuT A AUGC 364 UAD C UACCAGA 366 AUGC A CCAGAJ 369 GUCaAC A CAGACA 379 UACAAU U GUAAAW 387 GUAAU U AAADUC= 388 UAAA33U A AP&UUCC 392 AUMAAU U CUCOCAA, 393 UL.JPM C UCAAAA 395 AAAflMU C CAAAAA 405 AAAMACEI A AGUAU 412 AAGUGAU U CAACAAU 413 AU C AACAAUG 427 GACCAAW U AXJATGAA **428 ACCATIU A MWGJ 430 C 4 AflU A UGAAWCA 4236 WU)GAAl C AAU1 1

D

440 AAUO. U UCUAA 441 ACAA1U A UCUAAU 443 C7AACM C UAA=t~ 449 UCUGAAU U ACUWGA 450 CUAADU A CUUGGAU a.*453 AAvaACU U GGD .458 CUGGU U UATCUU *459 UUoGnu U GAW 463 AUO~tMW C UUAUCC 465 UUflCU U AAUCCA 466 UGD0Uu A AflCCAU 469 UCUMLU C CALMAAU 473 AAXKX2M A AUflM 477 CAEAAAU U AUAXC *.:478 AUAAAUU A WAM 480 AAAUMU A AUCAM 483 UMLAfl U AARDM 484 UWA= A AAUCA 487 AADMAW A UCAACA 489 UMLMW C AAC~kGC 494 AflCAACU A GCAAWCX 501 AGCAAAU C AAflGUM 507 UCA= C ACUAAA 511 tUUCCU A ACCA 519 ACACCAU U AGUMW 520 CACCAVU A GUCMAUA 523 CAUUAGU U AAA 524 AUMLGU A AUAMAA 271 Table 34: RSV (10) HE Bibozyme Sequence Ut.

Position 16 17 21 31.

32 36 37 38 42 46 51 67 68 71 76 81 87 88 92 93 100 101 i04 105 1.20 225 1.28 129 1.35 143 145 i=1 1536 i59 2.63 2.64 1 65 H R;ibazyzu. Sequence AAAflUCT CUAU GCMLAC,-GAA

AUUUGCC-

CU UCA CUXAGCCGAAG~a-,

ADMCUU

ACOUAUC CO-IGGG AAcr.A

AAU=

UGGUACtI CUGAUGAGGCCGAA CC-AA MAIOAALT UAAGUr.- CMAXM.CC iA1tUMr~ aAA=U CCAM ,C.AGCCG

AUG

UMAAUU GCAWGCCGALCc-.

AGUG

GGAUI CWMGGccGAXA~oCc-=

AU~A

GGGAG G AU OCGAAGGCCGk

AAULA

AGGCQ GUGA cGC;-=aAGCCG

AAAXUM

CCAU=u CUAUMIGCCGUGI--

AM=CAG

CCAG CG GCCGXUGG-cA

AMC

ACUCAU VGUXCGcGGAC=A

AALWCU

CAMCuc CGUGGCGcAC,-CG AUM~fUM UUCAUCA CGUAGGCCGAA-CGAA

ACAAU

tUhACDUU CCGUGAG--CAGCG AUA1A GMA=UCM AGCCMUG;-C-

ACUUUUA

UGMWAtJCM~AUAG--CGXUGCAA AACUUrUu AUUUt2G CUGAU GGCC rAAG-CGAA AUClkAC AAUUUU CUGAtGAGG,-CGXGGCXA AAUCrA UCAAA CUGAGG-GA AGGC; fYCr CAUUJGUC COAGGGAAAGOCCM

AACLUV

UUCt.UAC CUMAXGAGG. rA-CCGA AflCu)= ~UUUA UGA vGG-CcGAAAGCGCM

AUCAC

VfUUAU CUG UGCC-,-GA~r-CC

AACAAU

AGCAzuM CUGAGAGCCQUxG A AfUUOUUA AUCAGGm, CUA~GCGXGCA

AG

UUAUCAG CUAGCCGAACCGA

AUAGCAU

AAzu UGAGGCCGAALCA AflCGUA AUU CAUGGCCGAAGCCGA AtUXC AAt2GU= CrAUAGCCAA-CcGAA AAUt7Mu UUAAAUG CC3GGtAGG CCCAA Am3u U~kGOLA CUk GGcGU A)CM AUGUAUU GU~kGuW LM AAGCZAAGCCA AA~7L CGUJUAGU CUG-XUGAA-z-Cc AGGCCCA ;LAAUrGu a. a 272 169 AAAG= U -GAGOCAA CA Aja3.AM 176 CULVOCC CUAGGCr.A~,-G

AAGC=U

181 ACUG= CUG r'G~rXajc-CA

A~CCAAA

192 UUUA CUGUGAGCC AAAcGCCCA 'dTCU= 196 UUALDG CVGAUXwAGGxCCC CC AGUAC 201 UCAPADOU CUAAC-aIAGCA

AUGD

206 GCCAUUC COCADGAGCCCGAA GC ATIOUGAU 216 CAAACAC Cr GAUCAGGCCCGAo cGA Aflr,,MU 221 AMCA, CUArGC-AA~CA ACMC~Xjk 222 CN3GA C'AUGZAGGCCAGcA C U AAC.ICAA 231 Uuuk CUCAGAAG-GaAAAGCW-U ACAUWc.

234 TU.CUC CVUGAGOAAGGCCGA A AUAAC U 235 CLMCUOG CUGAUGAGGCCCAAAG=CGA

AAM

241 AUAUCAC CuAGCCGA GrAA AcrouA 27GGCAAAC AGG~-NAGCA AUCALtJh 249 UhGGGCA CVGUGA~GC<cGAAGC .A ADUXjCAC 5..250 UMGG=COGUAGCUGcr

A--

256 UUD VNGGXjAA=r.AAMA 259 ALUWI Ct G~ACaCCMVG=CGM AUlGG 262 AOCAWAU CUAGGCCUZCa

AD~UMZ

265 AC~kCAA CUMGAXWAGtcc AD~U74.

267 UtC~CA COGAUGAGCCGAAGCC AMMArU AflUUAC oUAMGccGaGcrc AcAA)u *278 GAAAflD CCGAUGAGG--CGXXAGGCCG-AA

AUUUMAC

283 GEUGUGA CODGA GAGGCCCAAAGCCC AfUGUM 284 UGUGG CUGAXMIGGCCGAAAlGCGA AAflUGA 300 UU)G7AO COGGAGAGGCCGAA rcGA ActuGGCA 303 CAUUUUG COUGCCCAGCGA A Grkrj 317 CAua CUGALXGACG cc

ACCUCC

319 u CUM~AGGCCGAA=GA

AMU.

321. UUMccA COGAUGACCAAGG C~CGA AUAMLAAC 338 -~uu CUMAXGAGGccc Q AUUCCA~U 339 AAUG= CLJGAXGAGGCCGU GrAA AUC 346 UGAGC CUGAGAGCCCG AGGCCrAA AX~UGUUj 350 AGGUWGA CMLJGAGGCCG AGGCCCUA

AGCAADM

352 UG=U CMUAGCGAGCA

)GAGCAA

358 AGACAU CUGAUWAGCC

AGM=

364 UC AGM COGAXUGACGAA A rCrmA ACCfltA 366 CAUCUkG Ct7AUGAG~-AAccGA .A AMXCU 369 UGUCAUC CMUGAGAGCCAAGG'CGAA AG~t4AC 379 AUUUCAC COGAflGAGGCCGAAAGCCCAA AX3UG=c 387 AGAADUU CUGADGAGGC~CGcc OCC% AU CA 388 GAGAAflU CUAMGC

AAUC

392 UUGGAG CUGAUGAGGCCGAAGCC

AUULTAAU

273

S

393 395 405 412 43 427 428 430 436 440 441 443 449 450 453 458 459 463 465 466 469 473 477 478 480 483 484 487 489 494 501 507 531 51-9 520 523 524 UuuuGA CO-UAGGAAPGG

AWUUMA

uuauuo CUAM4GCA~cC;

CAU

AAUCCU CUArGCCXVAAC-A~

AGUUU=L

A~uutaUUGADMWG&cA~r AflC= CAUUOUU CUAGCG LAAA~CA

AAU~C=

u MW UG rAG~NAaVGCA A~yG= ADUCAM- -UAKLG~AA~cA MAU= UGAUacM CUAXAGCAAGCA AfLAflU GAvAD, UGXMGCAAGCw,

U=

U GAU CUArA CGASC-CA AUUGAIUu AVOCAGA CU%~aGCCAAGCA AAtVLX,= MLAU~cA CUAGG-CXAGOA AMA=fU UCCAZGU CUUW-CGAACCGA AfUCA AVCCAAG CUAAC^-MAGCA

AAU~CAG

CAAAUCC CUAU GCCGAAACA

AGMUAA

AAGAUCA CUAGGGUGCCGAA

ACAA

M~AGAV UCAUXG-r

ACA

GG&UUAA CMVV*

AUCAAA

AUUM=CM)MZG

AMG

MWAAUU CAAGGCCGAAAGCCG~t AUGGAflU Ukmw A UMVGCCGAAAcG AfUUM UtMAM CWAGGCCGAA)C- AAUUtL tUAUlUAU CUGADAGGCCGAAAGxM;

AM=

UGAMU ccGACkAGCA

AULTA

UDGA3U COGGAa--AGGCC G AADUAMl GAGOUGAL CUAOAGCAG=AA~ AMMAfU GcaGu UG GGCAAGGCAA A~ujA GAVOUGC CUAUVCGAAG

AGUMAJ

UGACAUUG JGGCCGAACM; AUCU~CrC UGOUAGU CUA~GCCMUGCCX;

ACAUA

AUGGUGU CMrAGCCGC-A

AGUGC

~AAU=COWGCGA== AccGM UVkC CGOCGAAAGC=

AACUA&

C

274 Table 35: RSV HETarget Sequence Ut.

Position 9 21 23 24 32 37 66 70 .73 82 89 108 31i1 '113; 117 120 123 126 127 146 150 154 155 166 167 169 170 173 186 189 192 196 197 205 206 209 213 HH Target Sequesnce GGCAAAU A CAAGAU GAuMG-t C UCAGCA UGCU= IU A~C-APAAG GGCOCU A GCAAAGU GCA.AAGU C AAGOU Mm GUCAMG U CNIAUGU GAAUGNU A CACUCAA AUACACU C AACAAAG CAAAGAU C AACUUCU AUCAACM U CWU= UCAACU C UfC CUUCG C .ADCC COGUC;L C CAGCAAA AGCAAAIJ A CACC

CAACGGA

AGM'-AU A GaUXUGA AGAU A VUGAWLC AU U GAACOC UADUGAU A CDCCLAA UGAflCE C CUAAM MkCCU A AUUGA "~CAM U AUGAlU -!CMAAfU A UGAXG 217 218 220 229 231 235 236 254 260 263 277 279 284 299 305 315 318 326 327 346 347 355 356 361 370 371 383 384 389 395 401 406 408 415 418 431 449 453 460 472 474 ont.

Po±t ion HE Target Sequence GGUAUGU U AIIAUG-CG GMMMGU A mumGc-,A ArUUMI A MG=~r GC.-A=G C MkGL GAtUG= A CGUMCG UCUAGG U AGGAAZA CUAG=U A G-GAAGAG ACAC=A A AAAAVAC UX3AAAAU A C"CGL AAAtMAC C AG~AA GGGUA UCAGMh GGGAIU C AfLGUA .AIUGU= A AAAGCAA AMGAG A GAUGUAA UAGCncu A ACAACAC AACACAU C G-CAA; ACAUCCU C AAGACArJ AAGACAU U AZLGAA AGACAXUU A AfLrGGAAA AUCAAAU U UCGAA=U tG-AAUU U CGAMGGU GAAGUGtJ U AACA=t~ AAGU=U A ACAUMG UUCAU U GGMAGC GCAAGCU U AAc=AACLT CAADCUU A ACAACrUG CUGAAAU U CAXA UGAA7,fl C AAAUCAA UUCAAAU C AACUUG UCAACAU U GAGALTAG UUGAGA=- A GAAUCUL AUAGAAU C tPIGAAAA AAAMJ A GAAAAUC AGAAAAU C CUACAAA AAAUCCU A CAAAAAA AAAIMCU A AAAGAAA GAGAGGU A GCtUCA G-GUAGC-U C CAGAAtIA CCWAAU A CAGGCAU CAUGACU C UCCUGAU UGACUCU C CUGAUUGTJ S. AACA= C CAUCAAU A

U

AMAV= A GG=k=~ U G"AUGOUj

A

AAM~kGU

AGDM=

AUGtGC

UMUCA

AUM=. U AAUCACA UEOAUU A AflCACAG tUAUtwA

AGAUGCU

UAAUA

UCAUCAU

ACUGGGU

CUIG=~

AGGMU-AU

C ACAGAAG A AflCA C AUAAAUUJ A AAUCAC UJ CACUGG C ACOGGGU U AAU7AGG A At~hGGMA A GGtJWGtJ A UGUjA 275 .480 491 494 496 497 501 503 511 '512 515 518 522 526 527 544 549 551 552 563 564 ucc~aw u GUGGGNU GGAfU A AtiflMU tGA1JALW A UMiUa, AAU U AGlA MAMWUl A UGUAAG AUUAMU A URGCAG UAGM A GCMG=.

GCAGCXU U AGCADS CGCA~UU A GUALA CAMO A AUACaA MhUAA A ACCMAAU AAUAAM A AAPCGC ACUAAM U AGCAGA CAXU A GCAGCAG GACAGAU C UGGUUr B B A. V>

A

B

B

*BB.

A

B

AUCUGrU C cCGaCU u t~UCU= A CGUU= A UACAGCi

AGCCG

GGAGAG

GAGAGM~ A ADUA= 1IGCUAAU A AlGU AURA=U C CUMAL AGCU A AAAAALIG GAAAGU U CAG AAAGU A CAAA=C AAAG=U U ACU.CC 696 698 706 708 709 711 726 731 740 741 742 743 751 754 755 756 766 787 788 800 802 803 831 815 816 822 824 825 829 830 840 866 869 875 876 87 883 895 913 914 916 .921 923 925 943 946 947 949 950 UUUUGGU A UAGCA2-A u ;G A GCACAAU GCACAAU C UUCACC ACAA=C U caCAG C7AAUCUU C MCAcA AUCUUCJ A CCAA2AG UGGO.AU A GL~UrA Gt~aLU-U U GC.AGGG AAGGGAU U UUrJGCAG AGGGA.UU U UUG=AGG, GGADU U tUGCAGGA GGAnrUt U GCAGGAU GCAGOAu U GUUUJG GGAfUGU U UAflGAArJ GAUwrGrJ U At3GAAG MfUGUU A UGAAUGC- AAUGCCU A UGGUGrCA GUGALTGU U ACGG UGAZU= A CG-GUGG cGGG C uaWCAA GGAGUCU UJ A~CCAAAA GAGUCU A GAAAD GCAAAAU C AGUtMhAA AAUlGU~ u AAAAAUjA AUCAGUtI A AxzAAMU MAAAAIJ A UtMlUX AAAAIAU U AUUUUAG AAAXZ;U2 A UGUUAG AIuakJG U AGGACAU UXaVUU A GGACAtG ACAXGCU A GUGUGCA AACAAGU U GUrJGAGG AAGUUGU U GAGGUE) UGAMGU U UMGAU UGAGrGUU U AflGAATUA GGJuA UGAAMLU MU3JGAAU A UGCCCAA CAAAAAU UJ GGCGUGG GCAG=A U cUACC;LU CAGGAUU C tiACCAUA GGAflUC A CCAMUJ; CUaCCAU A UMI3UGAA ACcAUAU A UUGXACA CAIUAU U GAACAAC AAAGCAU C AUtM~XUA GCAI3CAIJ U ALUWJC CAUCAUu A ULTAUC~U UCAUUJAU U Ar-CUUJEG C;LUUAUU A UCUUUUA AAGGC=r

GCUMBCU

AGGACAU

AACAGCU

ACAGCOU

A CaThCCCA A CCCAAGG A GCCAACA u caAuGAA 643 652 653 663 670 671 672 674 680 681 682 683 686 AGCDMU A UGAAM GAA= U tflAAAAA AAGMU U GAAAAAC AAAACA.U C ~CCCCUU CCCACU U WAUGAU CCACUOU A EMAGU ACUUMU A UAGATU t AGAUU U GAUU U AUGUUU U uuuuoaU U

GAUMMO

UUGUC

GUUCAU

UUUU C AUUOWGG UGUUCAU U UUGJJ GUUCAIJU U UGGUA UUCA=U U GGU3A=A 276 952 ucMDAW c UOUGAMC 954 AUCI.UCU U GACUCA 955 UVCAUIU UJ GaCCA 960 UGACU C .AADUCUC 964 ACOCAAU TJU CCA 965 CUCAALTU U CCCC 966 UCADOU C ~CU 969 AfUUU C ACOUCC 973 CCUCACU U CUCCAG 974 CUACU C UCAGUG 976 CACU= C C*AG M 983 CCA= A GCMDMG 986 GUGU A UC 988 GaG U AGGCAAU 989 MDU; A GGCA 1007 CUGCCU A GGCWAIA 1013 TAGGCW A AflGGGAG 1024 GGAGAGUT A CAAGGU .032 CAGR=G A CAC~AG 1044 C AAAUL 1050 UCAA3D C UAOG 052 AAA A UAGU *1054 GflCCfl A UGA 1072 AAG A UGCOA 085 AACACLT C AA~k 1103 GUUA U AACA 1104 UGUGAU A AUACA _U08 AUAC A AUCI 11 A ACArU A CCAACCU 1118 Gt3GTACU A GACUTJGA 1.123 CUAGACU U GACAGCA.

AAGAACU A GAGGCD..

2146 AGAGGCU A U~CA; -1148 AGGML C AAADDC 1155s CAAACAU C AGCUAA 1160 AXUCAGCr U AAWCCAA 1161 UCAGCULT A AUCCAAA 1164 GC"UUALU C CAAAAGA 1173 AAAGAU A AUA 1181 AUGAU A GACUU 11.87 tUhAGCUM U UGGC 1.188 AGAGCUU U G~AGEA 11.93 UUOGGU U AAUAAAA 1194 UUGAUU A AUAAAAA 277 Table 36: RSV E Bibozyme Sequence lit H Ribozyme Sequzence Position 9 ATJUUUUG CUAGGCGAG-CA

AUUUGCC

21 0 ~CUAA, CUGAGAGGCCG aAGG.cA AGCCAUC 23 COUUGCE7 CUGAGGCCGAAAG=C,-aAA AA-CcA 24 ACM=UGC GUAGC-LAG-.A

AAGAGCC

32 UCAACUU CUGAUGAGGCCGAAAGGc--.A

X-CUJG,

37 ATJCAMUC CU-Ok-CGAGC.A

ACUUGAC

UUGAGUG CO AGCCGAAGcc. AUDAVUc so 5 CUUUGUU (ACCGAAAGG-'CAA

AGUGUU

AGAAGDU CUGAUAGGCCGAAAGGCC-.AA AflCUUUG *66 GAUGAC? CUAGGCC~G=A

AAU=

GCU~GA CtGAAGcG LGc~jA A~kAG 73 UUOG=1A CMAVMGGCTY CGA ArGACAG 82 0 GAUGGU GAUAGCAAGCa AJUtM.Cu 89 UCCGUUG Cr3AU GcGUM G- CGAA~ AUGGt3Gr 108 UCAU3AC CUGAGCCGA GC.. AUCuE *.1GMM CUGAUGAGGCCGAAAGGCCGAA XMCcIJ 1 GAGMAUC COUGk~~GGAAAGG-CQA AflCn 117 uakGGAG CUGA GAGGC"CGA AC-CGAA

ADCAA

1.20 UAAUMG CUGAMfAGGCC A~GAkX,,-. A=tUCA 3.23 U~dD7'AU CU~aGCXAGCA

AGGAGUA

*126 ACAUCAD CUCGAUGAGGCCGAAcGA A~jkGG 127 CACA<DCA. CU Vr-G----XCG -C.A AAUU(AGG 146 ACUMUJ Cr3GACAG cc

AMOGUU

150 CAT3AACrJ CUGAGAGGCCGAG~c; ADuGAIJG 155 GCCACAJ CUGAUAGGCCGA AGGCGAA

AACUAU

166 GAUtUAAU CMGAFZCCGAA~J3CGA A=CCc 167 UGAUUA~A COGAUGGGGAAAG~G.

AACAUGC

169 UGUU CUGMAGGAAGCCC-

AMAA

170 CUUk CMUAr-GAGC.A AAWkA 173 CMUGU~ CUGAUGAGOCCGAA QGC=AA AUMWA 186 UtUGAU CUGADAG~C;-Cv'GccGL A~ uc 189 AAUUAU CMUAGCaAGCGA

AMMGCA

192 GUGAAjUU CGCUXWMNAGCA A IUGAU 196 CCCAGUG CUGAfGAGGCCQ c- AuucAUG 1.97 ACCCAGU CUG-AtAGGCCX"GCGA

AAUUUAU

205 ACCU70IU CUGAUGAG AGC~GAY2

)ACCAG

206 uACCUAT CUGAGGCCG kG-CrAA AXArA 209 ACAM=C COGAflGAGGCC rG-C,-AA AUUAACC 213 UATUAACA CtJGAIUAfG~cCGXAAGGCCGAA ACCUAUu 278 217 CGAM CUGMtflAGGCCAAAGGCCGAA

ACAM=C

218 VUL ,MCA CUr3GA~GGCCv'GGCCAAC 220 CAtUCMA COMIUAGOGAGM

AWAA

229 ACC CUVMGCMAXG-AA 231 ACC =DAMM A

W

235 0030~r CTJGAUnM AGCGA AC UA 236 rlruucCOG

AGCAGCXAAC

254 nUUU CCGAtGAGCLraa.GCC

CMAC

260 C UGAG CUQGAccaAAAG c uuu=~ 2-63 CACC CUGU)MM GAAA c.A AGu~iuu 277 UAAA

WGCAAC=AACCC~C

279 u ACAU COD~gJG'GAOCC r caAAUCnc 284 DUWCUOU COAGGICGG=A

ACG

299 gUarCA CrCAUxACG~COMIG CCCAA ACUCCAU 305 Goa CVGAUGAGGCCGAAX=A

ACL=

315 UUCUMZC CUxGAEGAGGCCG A ai AoUGUM 318 AUGO CUGAUGAGGCCGkAAGG~CCGA 326 UUCCA1U UMr3CkAGUAGCU 327 UucfLu CUMGA~GGCXAAAGMA

A&U

346 AOILCGUWCGRM=

UM

347 A.CDCO

CUW-G-MQGCCAWUUCA

S355 CAAflGU CMUW<.AGCA

ACACUUC

:356 CCAAG CUkMMCAAGCA AAACULj 361 GCUOGCC CVGAXXMA4GcAGC

AUMUA

370 AGUGU CUGWMGCLAGCGx Acc cuUGC *371 CAUG ;MGCAA~aAAAGC=U 383 UGVNM= UGADAGCAAG-G A U )OrJG 384 UO1tflU CWUZC-AGCA

AAUCC,

389 CXW=U CMGAGCXAAct Cr AUUA 401 c~a=C UGAXMGCAAG AUM 406 UUU a~wvC~AGaA AUUC~lhU 408 GUUMXCUMnWG~CCMGMQCcGAAAGU 415 **UUA CUM-X PAAG aAAUUUUU 418 Ut.UUU= UL Mar-AGGCU jW 431 UUOUCMMMMAGCA

GAU

449 CLMGMCU -CVLM ACCUCuC 453 uu= lGcaAa=. A~caA=c 460 AG=CUGAMALGcGCAAGCM

D

472 AU3kGGA UMMOCkAGGA;L

AUCAU

474 CAAFCG COGAMGGMAGG CCAA AGAUC 491 AULMUCMrAG.%AGCC

UUCC

494 UACAMA. CWxAx7AC JIf.A.VC7 AMUMc 496 akk~ aruAGCGAV<= fLVm~ 497 CMCLX~MGCGAAGCA AA k~ 501 GCtJGCUA COUGAOCCCAAGCC

ACUXLU

503 AUGC CMAU3GAG-

ACU

51i UAUUACU CGUGAGGA~AAGGCG

AUGCUGC

279 512 Uafl~c CUG~AGGCCGAAAGGCCZ AAUG=u 515 MLua CUGAX 3kGGCCGAAAGGCCCQN ACWAlG SIB ADuaw UGAMG--lGGCA

AUM~CM

522 GCMAW U cDAGAGGCCGAAGC ;L AUU 526 UG=Cwc CUAG.GCGAGCG AnUr~kG 527 CuGCUGC CDGAGGCAAAG GA AAUU= 544

AAGACACGUAGCAAGCGAACU

549 GCUGMA -COCAG~AGG-iAGC

ACCAA

551 CG=~ CUGAGGGCCGAAACaM

AGCA

552 ACGGCUG COGAUAGCCG AAGCC~i

AAGA=C

563 C~UCcU COAGGCGAAGC

AUCA=Q

564 GCUCUCC COUGAGGAAAG C.A AAUCACCG 573 ACAUW CLGAGGCGAAGCGA

AGCC

576 ~~AGAMU CUGAXAGGCCG~aGGCCmQA

AULVZCU

581 Uuuk CUGAUGAGGCC--XAAGGCCGAA ACAIUrM.

584 c~nuuuu CUGAX GAGCG .Ar

AGM=CA

:603 ccuuOUa CUAGGCGAAG=A ACGUUtC *604 GCUU CUGAUGAGGCC AAGXA AAMUUu 613J GG VW- GCUAGr

AGCCULT

614 UGG UAGGCMA

AAM=~

617 CCUUGM CUIAGCCWGOCGcy AGaAAGC *629 UGuuGGc COGAflGAGCc

AUUC

.:640 UUAM UAMGCGAGCGA

AGCMUU

641 CUUA LUTT CCt- AA== 643 CACUUCA CtXAUGAGOGAAG A AGAC ***652 uuuuuc;La~mC<MA.CG

CCU

**653 GUUUUUC CUtAUGXAG-CGAAGCC

AAC

663 AAGUGGG CW'UA AC AUGoUvu 670 AX3ctah CUGAEIG GCGGC C GAc AGGG ***671 cACU CUGZUGAGG,C UGCGA AAGUGG 672 ACAUCUA CUANkGCAA,,CA

AAGG

674 AAACXUC CO4MGCGAAGCA

AMA

680 GXaLUA CW-DUG GCCGXAGC

ACAU=

*.:681 UW.AMCUGUAGAGAAGCO AAOlU= *682 AAACA CMUAGGaCCrkAAG A AAACAflC 683 AAPGAAC CUDAG:MAGCM

AAAACAIJT

686 CAAAAflG CUGAtRMAMCCckA=C Gr3-rA2A 687 CCAAAAD CUGUMTGTGXGG-CAA rCA 690 AMCCAA COUGAGAGcCC AG3C AfGAAC; 691 m.11jor~c~)~~

AADGAAC

692 Cmmic CUkGGCCAAGG

AAAGAA

696 UGUGC~Mk CMUMV=GGG c

ACMAA

698 AUGrC CGAlGAGGCcrkArGCM

AUACCAA

706 GGGAA CUGWJClGACccrJ J AflUGUGC 708 CUGGG CUGWtUAGkccc AGAU1UGr 709 UCUGGUA CUGWG Gc

AAGA

711 CCUCUUG CUAGCGAAAGGCCGAA AG AGA6 726 UCACU CWAG'MAG=.A

ACGC

731 CCU CUGAGAGGAGC=A,

ACUCUAC

280 740 CUGCAA CUCAUAGC-CCAAAGGCCMA. AflCCCrUu 741 CCUGCAA CUGUM-GGCCu-lGCGA AAUCcu 742 ="MMGC CUGArAGGCCGAA-cr.

AAAZCC_-

743 At1CCC C-v-DZCGAAO~A

AAC

751 C?.MAAC CUwGAGGCCGAAj3ccmj, AtX.Cc 754 AUUA3 CDGAUGAGGCrGGC

ACAL

755 CMUDCU CUAGZCGAGCC-

AA

756 GCAUUCA CUGAVGAGGCGrAAAoCCXDA

AAACAAU

766 UCACCA, CUGAUAGCCGA ACCA AJGGCAU 788 CCCAcc CU~vGAGGccGA~Accc;A. AACMrcA Bo0 UOGCaA CUGAUGAGGCCGAAAGGCCcMA. A~Cccy 802 UUUU CUGAXU~uIGCCGAAGGCCMA.

AGACUCC

803 AUUUUGC COGAUGAGGCC~waAaGGcCGA AAGACurC 81 LIDuuuu CUGAC~ajGCCGAAAaccGC

ACGU

:816 AMVUMM CUGAdrAGGCCGAAGccaGA

AACUG~AM

824 1TA MU 825 CCCACA -U~X.GCAA~cM ~a 829 AUGCCU COUGAGCCGAA oCCX ACt *830 CAU= CUGAUCGAGGLCCAGA CC AC B, 866 CC.AC CGUAG~=jA~~.

ACU~U

869 AAACMC CUGAGAGGC :AAACcr

ACAACU

*.:875 AfUUCX CUA~'GCGAGCC%

ACCUA

876 MUCU CUGAXrzAGGCCOAAA .CG; AACCUCA 877 ATIDUh CUGAXrAGCCAA GCGAAUACCrjC 883 u 3GGCA CtrGAtrAGCCrNGGA A AfUcUCA *895 ACCACCC CUAAGC~G-= i wcUuuG 913 AUGGEIhG CUGAtMAGCC

AUCCUGC

914 CUIfG CUGA rAGGCCAGcrA A AIICCMj 916 M= CU3GAr.AGG AAGCcr.. AGgaADCc.

*921 UM.AD -U-GGCMAGC= UG *923 L&AUCMA CUAGGGCAAGCG

AUU

925 -JGU UAMXrVAGCA

UM

943 MAMLW CUGAGGCMUAG C=A .AU U 946 AGAUAAU CUGAUGG.GAAo GGCCAA -jrGAUGC 947 AAAA CGXUGMcG 949 CAAAGAXU CUGxxMMGAAGG =GAGL AULA 950 UCAAGA CUVMGCAAG=

AMU

952 AG~r-AAA COAGGCMAAGCA AMUAtm~ 954 UGAGUCA. COGAUGAGGCCMGG=A

GU

955 u GAGUC CUAGZCGAAOCA

AAGAMA,

960 GGAAA33U CUA~ZCGAGcrA AGucA;A 964 GUGAGGA CVGAUGAGGcCCGA

AUUGAGU

965 AGUGAGG COGAUGAGGCCGAAAGCGA AAflUMG 966 AAGUGAG Clr-VJtGAGGCCGA tAGGA AAAfUUGA 969 GAGAAGU CUGLUGAGCCGGCCG

AGGAAAU

281 973 ACOGGAG CUGAUACGCCAAAGMGCCAA -iGCAWG 974 CACUGGA COGAUGAGGCcG GCGAAGrA -UUCCj 976 ECACJG CU'JGGCrAGC1_-CG

AGA

983 CaAXIC CUAGGCGUCCG

ACAC=

986 UGCCLIA LVAUAGCCAl'GGCGA Acakc 988 AUUGCCU CUGfAL=CG=,AG AaacE.c 989 CAflUGCC CUGAUGA~rGC GCGA Aak= 1007 umuAGCC CUGAUflAGGCCGXUGQCCAA

AGC-=

1013 CUCCCAJJ CUGAUGAGGCCGAA GC-CGAA A~c~ 1024 ACCUCUG CUG~AGG-CC-AA G--CGAA ACUCUCC- 1032 CucGrG m CUAGGC G.G-AA AcCUCU 1044 AGAU= CGGGG C,

ADLCC

1050 UCAUM CUGAGAWCGcA~C-GA

ACCUUMA

1052 CAflCA~ C GUGAvGCCr=AAGG,-'.

AGO

1054 UGCAIUCA CUGAUAGGCCGAAA,-CGAA ALZAiM **1072 UUCAGCA CUGAUGAGGCCXXGt-AA ALUGC-rJU *1085 UUUCUUU CUGrflGAGGCCGk GCGA AGUMj 1104 UGkG CUG~AUGcc -CGA AA~c *1108 CUUA CUGA4CC=AAGC

AAUA

VI8UCAAGOC CUGAA--CGAA-CrXri

AGU

123UGCUGUJC CUG flGAGGCCG AGC-,W

AWUA

'1119 UGCCCC COGAflGAGGCCG~GCGA A GrCUJ 116**UG CUGAUGAGGCCGAAAGGCcGAA

AGCLM

**.1148 GAIUGUUU CUA.G--rUGCCA AaaTj 1160 UWrGAUU c rALGAW-CGAAGcc--A AcG-,GAU *1161 UUt3GGAU CUGAGAGCCCCAAGCCGXZ

AC,-UGA

*1154 UCrUUU CM~A-G~aAGr AU Gc 1173 ACAJCA CVAAG=%C-CA

AUC~UC

U0181 AAAGCUC CVGAUGAGGCCGAAGCCGA ACAflCAU 1187 U~kCUC CGAUGAGGCcGAAAGGccGAA

%-LM

1188 UMCCCA AGCXc-CAAAGCC= 1193 UUUt~flU CUGAMGGCCGXMCGA

ACUCAAA

1194 MUUUU CW-MAGGCCGAXIcGGCC; AACtCA

S..

0 6

S

0 0 S S S S *@0 5 00 S 00 0 505 5S S 050 0000055 0 S 0 50 50 0 50 0 0; 0~ 000 S 0~ 0 0 055 0 5 555 Table 87: RSV (111) HIP Ribozyrne/Substrate Sequence Position HP Ribozyme Sequience Substrate CUGUGAUC AGAA G;UCUUU ACAAAhAAGUGGUCUACGU CMAGUGAC AGAA GUCUCA ACAAAAAAGU~GUCUACGu CAGGCUCC AGAA GGACUA ACCAAGAAACAC. T" WI ?irhAff Avn"-" 77 AAAGACU GAU GAUCACAG UGAGACC GUI)

GUCACUUG

UAUC AU GGAGCCUGw Table 38: RSV TIP RiLbozyne/Stibstrnte Sequence nt. ha~irpin Ribozyme sequence Substrate Pos it ion 476 AUCCCACA AGAA GGAGAG ACAAAAAAGW~)CUAa U CJuCU GAU UGUGGGAU 540 AAGACCAG AGAA GUCCCC ACAAAACCC GUGAAU JCIGA GGGCA GAU CUGuCUU 554 C13AAUCAC AGMA GUAAGA ACAAAAAAIJGGUCUAa U UCUUACh GCC GuGAUUAG 636 UUCAUAGA AGAA GUUGGC ACCAGAGAAACACACUUGUGGUAhUAAUtIGQA GCCAACh GCU UCUAuGAA 990 CCUAGGCC AGAA GCATJUG ACCGAGAAACACCX3!JrGJ~r-IAUtACUGGA CAAUGCtj GCU GGCCUAGG 1156 UUGGAUUA AGAA GAMGU ACCAGAGAAACACACMM=ACAUUGTAMJIJAMUA AACAUCA GCU UAAUCCAA 284 Table 39: Large-Scale Synthesis Sequence A9T

A

9

T

(GGU)

3

GGT

(GG U)3GGT CgT CgT

U

9

T

UgT A (36-mer) A (36-mer) A (36-mer) A (36-mer) A (36-mer) Activator [Added/Final] (min) T (0.50/0.33] S [0.25/0.17] T [0.50/0.33] S (0.25/0.17] T (0.50/0.33] S (0.25/0.17] T (0.50/0.33] S (0.25/0.17] T (0.50/0.33] S (0.25/0.17] S (0.50/0.24] S (0.50/0.18] S [0.50/0. 18] Amnidite (Added/Final] (min) [0.1/0.02] 1/0.02] [0.1/0.02] (0.1/0.02] [0.1/0.02] 1/0.02] (0.1/0.02] [0.1/0.02] [0.1/0.02] [0.1/0.02] (0.1/0.03] [0.1/0.05] [0.1/0.05] 15 m 15 M 15m 15 m 15 M 15 m 15 m 15 M 15/15m 15/15 m 15/15 m 15/15 m 10/5 m 89 78 81 97 21 38 Time* Full Length Product *Where two coupling times are indicated the first refers to RNA coupling and the second to 2'-O-methyl coupling. S 5-S-Ethyltetrazole,

T

tetrazole activator. A is 5' -ucu ccA UCU GAU GAG GCC GAA AGG CCG AAA Auc ccu where lowerecase represents 2 '-O-methylnucleotides.

285 Table 40: Base Deprotection Sequence iBu(GGU) 4 iPrP(GGU) 4 cqu Deprotection Reagent

NH

4 OH/EtQH

MA

AMA

MA

AMA

NH

4 OH/EtOH

MA

AMA

MA

AMA

NH

4 OH/EtOH

MA

AMA

MA

AMA

NH4OHIEtOH

MA

Time (min) 16 h 10Mn 10 M 10 M 10in 4 h loin loin l1in l10M 4 h 10Mn l1in 10Mn 10Mn 55 65 65 55 55 65 65 65 55 55 65 65 65 55 55 T C Full Length Product 62.5 62.7 74.8 75.0 77.2 44.8 65.9 59.8 61.3 60.1 75.2 79.1 77.1 79.8 75.5 22.7 28.9 A (36-mer) 4 h 10Mn 286 Table 41: 2'-O-AlkylsilyI Deprotection 0 S 0 Sequence A9T

(GGU)

4

C

10

U

10 B (36-mer) A (36-mer) Deprotection Reagent

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF Time (min) 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 20 65 20 65 20 65 20 65 84.5 81.0 60.9 67.8 86.2 86.1 84.8 84.5 25.2 30.6 TaOC Full Length Product

TBAF

1.4 M HF

TBAF

1.4 M HF 24 h 1.5 h 24 h~ 20 65

GAAAGG

29.7 30.4 COG AAA AUC CCU B is UCU OCA UCU GAU GAG GCC Table 42 NMJLData for UC Dimers containin~g.

Phosphorothioate Linkage Synthesis at Type Delivery Eq., Wait ASE 3524 ribo 2 x3 s 10.4 2 xl100s 95.9 3625 ribo 2 x 3s 10.4 2 x75 s 92.6 3530 ribo 2 x 3s 10.4 2 x75 s 92.1 3526 ribo I x 5 s 08.6 1 x 3003s 100.0 t 3578 ribo 1 x 5 s 08.6 1 x 250 s 100.0 3529 ribo I x 5s 08.6 1ix 150s 73.7 Table 43: NMR Data for l 5 -7ner RNA containing Phosphorothioate Linkages syndiegis 3581 3663 T ype ribo ribo Delivery I x 5s 2 x4 s Eq.

08.6 13.8 Wait 1 x 250 s 2 x 300 s

ASE(%

99.6 100.0 3582 3668 3682 2 '-O-Me 1 x 5 s 2 '-O-Me 2x4s 08.6 13.8 08.6 I x250 s 2 x 300 s 1 x 300 s 99.7 99.8 99.8 2'-O-Me Ilx 5s 289 Table 44. Kinetics of Self-Processiig.In Vitro 0. -k represents the unimnolecuiar rate constant for ribozyme self-cleavage determined from a non-linear, least-squares fit (K-aleidaGraph, Synergy Software, Reeding, P.A) to the equation: (Fraction Uncleaved Tanscript) (-e'kt The equation describes the extent of zibozyme processing in the presense of ongoing transcription (Long Uhienbeck, 1994 Proc. Nat]. Acad,. Sci. TUSA 91, 6977) as a function of time and the unimnoleculax rate constant for cleavage Wk. Each value of k represents the average range) of values determined from two experimnents.

290 Table Entry Modification tI 1 2 (in) t 1 /2 (in) =tSA Activity Stability (0A) (ts) x1 1 U4& U7=U 1 0.1 1 2 U4 &U7 =2'-O.Me-U 4 260 650 3 U4 2'=0H 2 -U 6.5 120 180 4 U7 2'=0CH 2 -U 8 280 350

U

4 &U7 =2#CH 2 -U 9.5 10130 6 U4 =2'=CF 2 U 5320 640 7 U7 2=~U4220 550 8

U

4 &U7 =2*CF2-U 20 320 160 9 U4 =2'-F-U43280 U7 8 400 500 11 U4 &U7 4 300 750 12 U4 2'-C-AIly-U 3 >500 >1700 *13 UP 2'.TC-AIlyl..u 3 220 730 14

U

4 &U7 2-C.jJIYl.U 3 120 400 :15 U4 2'-araF-U 5 >500 >1000 16 U7 2'-araF.U 4 350 875 17

U

4 L7 =2'.arF.U 15 500 330 18 U4 =2'-NH 2 -U 10 500 500 19 U7 2'-NH 2 -U .5 500 1000 U4 U7 =2'-NH 2 -U 2 300 1500 21 U4 =dU 6 100 170 22 U4 &ULPdU 4 240 600

Claims (110)

1. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, IL- mRNA, rel A mRNA, TNF-a mRNA sites shown in Table 23, 27, or 28, CML associated mRNA selected from those identified as SEQ. ID NOS 1-25, or RSV mRNA or RSV genomic RNA in a region selected from the group consisting of 1C, 1B and N.

2. The enzymatic nucleic acid molecule of claim 1, the binding arms of which contain sequences complementary to any one of the sequences defined in any of those in Tables 2, 3, 6-9, 11, 13, y 23, 27, 28, 31,33, 34, 36, and 37.

3. The enzymatic nucleic acid molecule of claim 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 15 RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA or RNaseP RNA motif.

5. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 12 and 100 bases complementary to said mRNA or genomic RNA.

6. The enzymatic nucleic acid molecule of claim 5 comprising between 14 and 24 bases complementary to said mRNA or genomic RNA.

7. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 5 and 23 bases complementary to said mRNA or genomic RNA.

8. The enzymatic nucleic acid molecule of claim 7 comprising between and 18 bases complementary to said mRNA or genomic RNA.

9. An enzymatic nucleic acid molecule consisting essentially of a sequence selected from the group of those shown in Tables 4-8, 12, 14-16,

19-22, 24, 26-28, 30, 32, 34 and 36-38. 10. A mammalian cell including an enzymatic nucleic acid molecule of claims 1 or 2. 292 11. The cell of claim 10, wherein said cell is a human cell. 12. 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. 13. A mammalian cell including an expression vector of claim 12. 14. The cell of claim 13, wherein said cell is a human cell. 15. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, relA, TNF-a, or RSV by administering 10 to a patient an enzymatic nucleic acid molecule of claim 1 or 2. 16. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, relA, TNF-cr, or RSV by administering to a patient an expression vector of claim 12. 17. The method of claims 15 or 16, wherein said patient is a human. 18. The method of claim 17 wherein said condition is selected from the .group consisting of atherosclerosis, myocardial infraction, stroke, restenosis, heart diseases, cancer, rheumatoid arthritis, asthma, reperfusion injury, inflammatory or autoimmune disorders, transplant rejection, myocardial ischemia, stroke, psoriasis, 20 Kawasaki disease, HIV and AIDS, and septic shock. 19. A nucleoside selected from the group consisting of alkylnucleoside, 2 '-deoxy-2'-alkylnucleoside, nucleoside nucleoside methylphosphonate, nucleoside 3'-deoxy-3'-dihalo- methylphosphonate, and 5',3'-dideoxy-5',3'-bis(dihalo)- methylphosphonate. A nucleotide selected from the group consisting of alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, methylnucleotide, 5'-deoxy-5'-difluoro-methyinucleotide, 3'-deoxy- 3 '-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)- methylphosphonate. 293

21. A nucleotide triphosphate comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, methylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy-3'-dihalo- methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate. S 22. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in a talo configuration.

23. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in an allo configuration.

24. An oligonucleotide comprising a nucleotide selected from the group consisting 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and dideoxy-5' ,3'-bis(dihalo)-methylphosphonate.

25. An oligonucleotide comprising a moiety having the formula I: wherein B is a nucleotide base or hydrogen; R1, R2 and R3 independently is 15 selected from the group consisting of hydrogen, an alkyl group containing between 2 and carbon atoms inclusive, an amine, an amino acid, and a peptide containing between 2 and 5 amino acids inclusive; and the zigzag lines are independently hydrogen or a bond.

26. An oligonucleotide comprising a 3'-amido or peptido group.

27. An oligonucleotide comprising a 5'-amido or peptido group. 20 28. The oligonucleotide of claim 24, 25, 26, or 27 having enzymatic activity.

29. Method for producing an enzymatic nucleic acid molecule having activity to cleave an RNA or single-stranded DNA molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'-position. [n:\libc]00132:MER 294 Method for conversion of a protected allo sugar to a protected talo sugar, comprising the step of contacting said protected allo sugar with triphenyl phosphine, diethylazodicarboxylate, p-nitrobenzoic acid under inversion causing conditions to provide said protected talo sugar.

31. Method for the synthesis of a nucleoside 5' or a 3'-dihalo- methylphosphonate comprising the step of condensing a difluoromethylphosphonate-containing sugar with a pyrimidine or purine under conditions suitable for forming a nucleoside or 3'- 10 difluoromethylphosphonate.

32. The oligonucleotide of claim 3, wherein the normal hammerhead U4 and/or U7 positions are substituted with 2'-NH-amino acid.

33. A method for the synthesis of RNA comprising the step of providing at a delivered 0.1-1.0 M concentration for the 15 activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

34. A method for the synthesis of RNA comprising the step of providing at 0.15-0.35 M effective, or final, concentration for the activation of a RNA amidite during a coupling step for less than 20 or equal to 10 minutes. A method for the deprotection of RNA comprising the step of providing alkylamine (MA) or NH40H/alkylamine (AMA) at between 70°C for 5 to 15 minutes to remove any exocyclic amino protecting groups from protected RNA; wherein said alkyl is selected from the group consisting of methyl, ethyl, propyl and butyl.

36. A method for the deprotection of RNA alkylsilyl protecting groups comprising, contacting said groups with anhydrous triethylamine.hydrogen fluoride (aHF-TEA) trimethylamine or disopropylethylamine at between 60 OC-70 °C for 0.25-24 h.

37. A method for the purification of an RNA molecule by passing said enzymatic RNA molecule over an HPLC column, wherein said HPCC column is an anion exchange chromatography column. 295

38. Method for one pot deprotection of RNA comprising, contacting a protected base with anhydrous methyl amine at between 60 °C for at least 5 min, cooling the resulting mixture and contacting said mixture with TEA-3HF reagents under conditions which remove a protecting group of the 2'-hydroxyl position.

39. Method for synthesizing RNA containing a phosphorothioate linkage comprising the step of contacting 6-10 equivalents of 3H-1,2- benzodithiole-3-one 1,1-dioxide (Beaucage reagent) with the growing RNA chain for 5 seconds with a reaction time of at least 10 300 seconds. Method of synthesizing RNA containing a phosphorothioate linkage comprising the step of achieving coupling with 5-S-ethyltetrazole or prior to sulfurization.

41. Method of claims 38, 39 or 40 wherein said RNA is enzymatically 15 active.

42. Method for synthesizing 2 '-deoxy-2'-amino-nucleoside phosphoramidite, comprising the step of protecting the 2'-amino group with a N-phtaloyl group.

43. The method of claim 42 wherein the said nucleoside lacks a base.

44. Method for synthesis of RNA comprising the step of: protecting the 2'-position of a nucleotide during said synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group. Method for covalently linking a SEM group to the 2'-position of a nucleotide, comprising the step of: contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions.

46. The method of claim 45, wherein said conditions comprise dibutyltin oxide and tetrabutylammonium fluoride and SEM-CI.

47. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, comprising the step of: contacting said molecule or oligonucleotide with boron trifluoride etherate (BF 3 'OEt 2 under SEM removing conditions. 296

48. The method of claim 57 wherein said (BF 3 .OEt 2 is provided in acetonitrile.

49. One or more vectors comprising a first nucleic acid sequence encoding a first ribozyme having intramolecular or intermolecular cleaving activity, said first ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif; and a second nucleic acid sequence encoding a second ribozyme 10 having intermolecular cleaving activity, said Second ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif and said second nucleic acid being flanked by other nucleic acid sequences encoding RNA which is cleaved by said first ribozyme to release said second ribozyme from RNA encoded by said vector; wherein said first and second nucleic acid sequences may be on the same or separate nucleic acid molecules, and said vector encodes mRNA or comprises RNA which lacks secondary structure which reduces release of said second ribozyme by more than Cell comprising the vector of claim 49.

51. A transcribed non-naturally occurring RNA molecule, comprising a desired therapeutic RNA portion, wherein said molecule comprises an intramolecular stem formed by base-pairing interactions between a 3' region and 5' complementary nucleotides in said RNA, wherein said stem comprises at least 8 base pairs.

52. The RNA molecule of claim 51, wherein said molecule is transcribed by a RNA polymerase III based promoter system.

53. The RNA molecule of claim 51, wherein said molecule is transcribed by a type 2 pol III promoter system.

54. The RNA molecule of claim 51, wherein said molecule is a chimeric tRNA. 297 The RNA molecule of claim 53, said RNA having A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases.

56. The RNA molecule of claim 53, wherein said desired RNA molecule is at the 3' end of said B box.

57. The RNA molecule of claim 53, wherein said desired RNA molecule is in between the said A and the B box.

58. The RNA molecule of claim 53, wherein said desired RNA molecule includes said B box.

59. The RNA molecule of claim 51, wherein said desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA. "i

60. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 12 bases of said 3' region. 15 61. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 15 bases of said 3' region. o 62. DNA vector encoding the RNA molecule of claim 51

63. The vector of claim 62, wherein said vector is derived from an AAV or adeno virus.

64. RNA vector encoding the RNA molecule of claim 51. The vector of claim 64, wherein said vector is derived from an alpha virus or retro virus.

66. The vector of claim 62 wherein the portions of the vector encoding said RNA function as a RNA pol III promoter.

67. Cell comprising the vector of claim 62.

68. Cell comprising the vector of claim 53.

69. Cell comprising the RNA of claim 51. 298 Method to provide a desired RNA molecule in a cell, comprising introducing said molecule into said cell a RNA comprising a desired RNA molecule, having a 5' terminus able to base pair with at least 8 bases of a 3' region of said RNA molecule.

71. The method of claim 70, wherein said introducing comprises providing a vector encoding said RNA molecule.

72. Hammerhead ribozyme having 2 or 3 base pairs in stem II with an interconnecting loop of 4 or more bases between said base pairs.

73. Hairpin ribozyme lacking a substrate moiety, comprising at least six 10 bases in helix 2 and able to base-pair with a separate substrate q RNA, wherein the said ribozyme comprises one or more bases 3' of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

74. The ribozyme of claim 73, wherein said ribozyme comprises six bases in helix 2.

75. The ribozyme of claim 73, having the structure of Fig. 3, wherein each N and N' is independently any base and each dash may represent a hydrogen bond, r is 1-20, q is 2-20, o is 0 20, n is 1 4, and m is 1

76. Method for increasing the activity of a hairpin ribozyme by providing one or more bases 3' of helix 3 able to base-pair with a substrate RNA to form a helix

77. Trans-cleaving Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

78. Trans-ligating Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

79. The ribozyme of claim 73.having the structure of Fig. 73. The ribozyme of claim 73 having the structure of Fig. 74.

81. A cell including the ribozyme of any of claims 73-80. 299

82. An expression vector comprising nucleic acid encoding the ribozyme of any of claims 73-80, in a manner which allows expression of that ribozyme within a cell.

83. A cell including an expression vector of claim 82.

84. Method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule, comprising the steps of: i contacting said nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid able to form a duplex or 10 triplex molecule with said nucleic acid molecule, wherein formation of said duplex or triplex molecule directly, or after nucleic acid S 0 repair in vivo, causes at least one base in said nucleic acid molecule to be chemically modified to functionally alter the nucleotide base sequence of said nucleic acid sequence.

85. The method of claim 84, wherein said oligonucleotide is of a length sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine in an RNA molecule.

86. The method of claim 84, wherein said oligonucleotide comprises an 0 enzymatic nucleic acid molecule which is active to chemically modify a base.

87. The method claim 84, wherein said nucleic acid molecule is DNA or RNA.

88. The method of claim 84, wherein said oligonucleotide comprises a chemical mutagen.

89. The method of claim 88, wherein said mutagen is nitrous acid. The method of claim 84 wherein said oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methylation of cytosine to

91. The method of claim 84, wherein an endogenous mammalian editing system is co-opted to cause said chemical modification. 300

92. Method for introduction of enzymatic nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of 10 RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under 0.0 ~conditions in which said enzymatic nucleic acid molecule is produced in said cell or tissue.

93. Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic ~acid molecule so that it is able to form an R-loop base-paired S. 20 structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under conditions in which said desired acid molecule is produced in said cell or tissue. 94 Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired 301 structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and wherein said second nucleic acid further comprises a localization factor; and contacting said complex with said cell or tissue under conditions in which said desired nucleic acid molecule is produced in said cell or tissue. 10 95. Complex of a first nucleic acid molecule encoding an enzymatic ;nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

96. Complex of a first nucleic acid molecule encoding a desired nucleic .oo acid associated with a second nucleic acid molecule having S" 20 sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

97. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said 302 first nucleic acid under said conditions, and wherein said second nucleic acid further comprises a localization factor.

98. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, mRNA, rel A mRNA, TNF-ao mRNA sites,

99. Method for producing an enzymatic nucleic acid molecule, substantially as hereinbefore described with reference to any one of the Examples.

100. Method for conversion of a protected allo sugar to a protected talo sugar, substantially as hereinbefore described with reference to any one of the Examples.

101. Method for the synthesis of a nucleoside 5' or a 3'-dihalo-methylphosphonate, substantially as hereinbefore described with reference to any one of the Examples.

102. A method for the synthesis of RNA, substantially as hereinbefore described with reference to any one of the Examples.

103. A method for the deprotection of RNA, substantially as hereinbefore described with reference to any one of the Examples. 15 104. A method for the purification of an RNA molecule, substantially as hereinbefore described with reference to any one of the Examples.

105. Method for synthesizing 2'-deoxy-2'-amino-nucleoside phosphoramidite, substantially as hereinbefore described with reference to any one of the Examples.

106. Method for covalently linking a SEM group to the 2'-position of a nucleotide, substantially as hereinbefore described with reference to any one of the Examples.

107. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, substantially as hereinbefore described with reference to any one of the Examples.

108. A transcribed non-naturally occurring RNA molecule, substantially as hereinbefore described with reference to any one of the Examples.

109. Method to provide a desired RNA molecule in a cell, substantially as hereinbefore described with reference to any one of the Examples.

110. Hairpin ribozyme lacking a substrate moiety, substantially as hereinbefore described with reference to any one of the Examples.

111. Method for increasing theactivity of a hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples.

112. Trans-cleaving Hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples.

113. Trans-ligating Hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples. [n:\libc]00132:MER

114. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule, substantially as hereinbefore described with reference to any one of the Examples.

115. An enzymatic nucleic acid molecule having at least one binding arm, which cleaves IL-5 mRNA.

116. The enzymatic nucleic acid molecule of claim 115, wherein said binding arm(s) comprises a sequence complementary to any one the sequences in Tables 11 and S io 13.

117. The enzymatic nucleic acid molecule of claim 115 or claim 116, wherein said V nucleic acid molecule is in a hammerhead motif.

118. The enzymatic nucleic acid molecule of claim 115 or claim 116, wherein said RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA, S. s15 or RNase P RNA motif.

119. The enzymatic nucleic acid molecule of claim 115 or claim 116, comprising between 12 and 100 bases complementary to said mRNA.

120. The enzymatic nucleic acid molecule of claim 119, comprising between 14 and 24 bases complementary to said mRNA. 20 121. The enzymatic nucleic acid molecule of claim 115 or claim 116, comprising between 5 and 24 bases complementary to said mRNA.

122. The enzymatic nucleic acid molecule of claim 121, comprising between and 18 bases complementary to said mRNA.

123. The enzymatic nucleic acid molecule consisting essentially of a sequence selected from the group sequences in Tables 12, 14, 15, and 16.

124. A mammalian cell comprising an enzymatic nucleic acid molecule of any one of claims 115-123.

125. The cell of claim 124, wherein said cell is a human cell.

126. An expression vector including nucleic acid encoding a least one enzymatic nucleic acid molecule of any one of claims 115-123 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

127. A mammalian cell including an expression vector of claim 126.

128. The cell of claim 127, wherein said cell is a human cell. [l:\DayLib\Ll BC]43638.doc:ais

129. A method for treatment of a pathological condition related to the level of by administering to a patient an enzymatic nucleic acid molecule of any one of claims 115-123.

130. A method for the treatment of a pathological condition related to the level of IL-5 by administering to a patient an expression vector of claim 126.

131. The method of claim 129 or claim 130, wherein said patient is a human.

132. An enzymatic nucleic acid molecule according to any one of claims 115-123 for use in the treatment of a pathological condition related to the level of IL-5 in a patient.

133. An expression vector according to claim 126 for use in the treatment of a pathological condition related to the level of IL-5 in a patient. S:i• 134. An enzymatic nucleic acid or an expression vector for use according to claim or claim 133, wherein said patient is a human. 1 135. Use of an enzymatic nucleic acid molecule according to any one of claims 115-123 for the manufacture of a medicament for the treatment of a pathological i5 condition related to the level of IL-5 in a patient.

136. Use of an expression vector according to claim 126 for the manufacture of a medicament for the treatment of a pathological condition related to the level of IL-5 in a patient.

137. Use according to claim 135 or claim 136, wherein said patient is a human.

138. An enzymatic nucleic acid molecule according to any one of claims 115-123 when used in the treatment of a pathological condition related to the level of IL-5 in a patient.

139. An expression vector according to claim 126 when used in the treatment of a pathological condition related to the level of IL-5 in a patient.

140. An enzymatic nucleic acid or an expression vector when used according to claim 138 or 139 wherein said patient is a human. Dated 25 March, 2002 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [I:\Dayib\L BC143638.doc:ais