AU744191B2 - Method and reagent for inhibiting the expression of disease related genes - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

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Name and Address of Applicant(s): Actual Inventor(s): Address for Service: Invention Title: Ribozyme Pharmaceuticals, Inc., of 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 Beigelman, Sean M Sullivan, David Sweedler, James D Thompson, Danuta Tracz, Nassim Usman, Francine E Wincott and Tod Woolf Spruson Ferguson, Patent Attorneys Level 35, St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method and Reagent for Inhibiting the Expression of Disease Related Genes The following statement is a full description of this invention, including the best method of performing it known to me/us:- [R:\LIBM] 0762.doc:SAK r .)11 1 i i: 1 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-ca, p210 bcr-abl, and respiratory 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 15 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 20 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.

2 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 nontargeted RNA. This cleavage mechanism is dependent upon factors additional to 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 15 catalytic activity and increased site specificity, ribozymes represent more potent and safe therapeutic molecules than antisense oligonucleotides.

Thus, in a broad aspect, this disclosure relates to ribozymes, of enzymatic RNA molecules, directed to cleave RNA species encoding ICAM-1, IL-5, relA, TNF-a, p210 b rabl or RSV proteins. In particular, applicant describes the selection and function 20 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-a, p210 b r b or RSV genes and that the catalytic activity of the S 25 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, TNF-a, p210 bcr abl or RSV encoding mRNAs may be readily designed.

According to a first embodiment of the invention there is provided an enzymatic nucleic acid molecule which cleaves respiratory syncytial virus (RSV) mRNA or RSV genomic RNA in a gene region selected from the group consisting of 1C, 1B, and N gene regions.

According to a second embodiment, there is provided a mammalian cell Scomprising an enzymatic nucleic acid of the first embodiment.

[R:\LIBZ]05575.doc:lam 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 S*

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S S tA\LIBZ]05575.doc:Iamf 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 15 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, S: 20 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- 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 Retroviruses 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 Biochemistr 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35 849, 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.

There is also disclosed a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target I CAM-1, IL-5, reLA, TNF-a, 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 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.

ynthesis 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, Virol, 66, 1432- 41; Weerasinghe, et al., 191, J Virol 65, 5531-4; Ojwang, et al., 1992, Proc. Natl Acad, Sc. USA 89 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver, et al., 1990 cience, 247, 1222- 1225). Those skilled in the art would realize that any ribozyme can be R4~ii expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from tihe primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT W094/02595, both hereby incorporated in their totality by reference herein; Ohkawa, et al., 1992, Nucleic Acids Symp.

Ser. 27, 15-6; Taira, 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 S. 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 1 5 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, p21obcr-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 Plipids, 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.

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 disclosure, ribozymes that cleave target 15 molecules and inhibit ICAM-1, IL-5, Rel A, TNF-oa, 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 ribozyme sequences are driven from a promoter for eukaryotic

RNA

polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol 1l). 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 EMBOJ. 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 O (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 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 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, Nucl. 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 0 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.

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 linkages. P=O refers to ribozyme without phosphorothioate linkages. P=S refers to ribozyme with phosphorothioate linkages. The sequence for site C Sis 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.

i O 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 suora).

The trans ribozyme domain extends from nucleotide 1 through 49. After 3'end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides 15 (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 o 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 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 O 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 11, II, 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 SRcal restriction enzyme. Transcripts from R templates contain 450 nonribozyme nucleotides at the 3' end.

20 Fig. 28 shows the effect of 3' flanking sequences on the transcleavage 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 1 i:i: 12 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.

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.

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

The A and B box are internal promoter regions necessary for pol III 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 O 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 promoter. 36) Expression of S35 constructs in MT2 cells. S35 (+ribozyme), S35 construct containing HHI ribozyme. S35 (-ribozyme), S35 construct 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 20 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°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 pooled cells 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. tRNAimet, refers to the O 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.

*CS. 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 S* 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.

20 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 i forms 5 bp on the 5' side and 8 bp on the 3' side of the cleavage site for a 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).

16 Figs. 63 and 64 show RNA cleavage activity of HH-I 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 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 esubstrate 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 basepaired 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).

i n:a~ l 'r;S 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 pL1 DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1IM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard Scleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCI2) by heating to 90°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 370C. Aliquots of 5 gI 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 S* "secondary structure of the hairpin ribozymeosubstrate complex. The 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 0 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 200C 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 I:1 Helix 4 is replaced by a nucleotide loop wherein q is 2 bases.

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 1 base. Hairpin 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.

1 I 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, 20, 3252) showing specific substitutions.

Figs. 81a-j shows the structures of various 2'-alkyl modified Snucleotides which exemplify those of this invention. R groups are alkyl 15 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.

Figure 84 is a diagrammatic representation of the synthesis of 2'-Cl methylene 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

I-

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 10 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 15 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 20 U7 position. U4/U7-ala, represents HHA containing 2'-NH-alanine modifications 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.

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.

Figure 107 shows a method for use of self-processing ribozymes to generate therapeutic ribozymes of unit length. This method is essentially 15 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.

SRibozymes of this invention block to some extent ICAM-1, IL-5, rel A, TNF-a, p210 bc r a bl 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-a ,p21bcr-abl, 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 lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

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 representing potential hammerhead or hairpin ribozyme cleavage sites are Ssynthesized. 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 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 15 high pressure liquid chromatography and are resuspended in water.

Example 1: ICAM-1 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 20 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 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).

10 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), S.:0 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.

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 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 O 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.

T h 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.

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 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 i 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 oe [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 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 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 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., 1991 Transplant. Proc. 23, 533-534) graft rejection in primates.

1- 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., 1993 Transplantation 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).

10 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 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).

SPsoriasis 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 Kawasaki disease Surface ICAM-1 expression correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989Lancet 2, 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. This disclosure features use of ribozymes to treat chronic asthma, e, by inhibiting the synthesis 20 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-aR 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 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 1 not conserved between the Human and the Mouse IL-5 sequences.) 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 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 I Pj -i i n ;1 1 lIY. S~ lrr ~y .I l 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., 1988 J. Ex. Med. 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. ExD. 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 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 O 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 10 observed between the methacholine responsiveness, a reduction in the 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 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 reactions associated with exposure to certain environmental antigens. One of the common clinical manifestations of atopy is eosinophilia *0 i (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 suera). 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).

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.

Thus, ribozymes 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 suDra) and can be used to optimize activity.

Example 3: NF-KB Ribozymes that cleave rel A mRNA represent a novel therapeutic 20 approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) 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 25 the transcriptional regulator, NF-KB. One subunit of NF-KB, the re/A 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-KB, 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 F, 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-iB2 (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 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-iB2/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 I' 1 1 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 (1.

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. ExD. 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).

The above studies suggest that NF-KB 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.

S"Activity of NF-B 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.

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.

*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 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-iB 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.

I v, 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-KB 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.

-Asthma.

Granulocyte macrophage colony stimulating factor (GM-CSF) is thought 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 25 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 that cleave re/ A mRNA and thereby NF-KB 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 -t 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-a mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.

Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated Sleukocytes, that is a potent mediator of inflammatory reactions. Injection of 10 TNF-a into experimental animals can simulate the symptoms of systemic and local inflammatory diseases such as septic shock or rheumatoid 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-8 (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. Hyg. 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 disclosed herein 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 15 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 20 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 25 ribozyme prior to testing in humans. The nucleotide base position is noted 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 5

'-GGCCGAAAGGCC-

can be altered (substitution, deletion, and/or insertion) to contain any i 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 one preferred arrangement, 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 to* 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 injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

S

9 In another preferred arrangement, a transcription unit expressing a ribozyme that cleaves TNF-o RNA is inserted into a retrovirus vector for sustained expression of ribozyme(s).

25 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 IB!r'-~ 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 localized inflammatory response are evaluated. In addition, we will 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 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 O 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 10 lipopolysaccaride (LPS) was added to each well to stimulate

TNF

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 -700C. Quantitation of TNF-a was done by a 15 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 0 from septic shock averages 50%, due primarily to multiple organ failure and disseminated vascular coagulation. Septic shock, with an incidence of 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-11 (IL-1B), 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 suora). 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-11, 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-13, 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/B 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-B, 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 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 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.

EXD. 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-a/1, 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.

lls 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 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 TGF-a IL-la, 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-1a, IL-113, 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 r I~ 1 i i O 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 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 20 endothelium and keratinocytes, and IFN-y expression by T-cells to maintain 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 1 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 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- levels, hypergammaglobulinemia, and lymphoma/leukemia (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 supra). 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.

II The role of TNF-a in HIV replication has been well established in Stissue 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 it 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.

15 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 20 chronically elevated TNF-a frequently observed in AIDS patients.

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.

l" !Bci!n! 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 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 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 Sura).

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 i 1 Il*Ui iil~:. :*l(i/li ii li4/ ljl$jj:i~ 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 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 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: p210bcr-abl Chronic myelogenous leukemia exhibits a characteristic disease 15 course, presenting initially as a chronic granulocytic hyperplasia, and Sinvariably 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 S2 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 (.g approximately 4 years). Consequently, CML patients are candidates for :i bone marrow transplantation (BMT) therapy. However, for those patients 25 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 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. ci. 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 (p210bcr-ab l in the evolution and maintenance of the leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p2 1 0 bcr-abl expression. These inhibitory molecules have been shown to inhibit the i vitro proliferation of leukemic cells in bone marrow from CML patients. Szczylik et al., 1991 Science 253, 562).

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 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 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 25 to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to extemal stimuli.

The disclosure 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.

This disclosure 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 :rl i iiiilili r i il i~ eradicate transformed cells from tissues such as bone marrow prior to reimplantation.

This disclosure 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 inhibition of translation of the fusion protein in treated cells.

This disclosure 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 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 Rossi et al., 1992 supra) is an in vitro transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments. This limits the utility of this approach. An alternative approach disclosed herein uses smaller ribozyme motifs and exogenous delivery. The simple structure of these -25 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.

Enzymatic RNA molecules disclosed herein 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 'aq replication. This is possible because the ribozymes are designed to disable those structures required for successful cellular proliferation.

Ribozymes disclosed herein block to some extent p210 b c ra b l 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 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 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 25 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 I r I; i r 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.

SRSV 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 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 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 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, 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.

Nat. 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 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 failure by 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 al., 10 1992 J. Vet. Med. Sci. 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in Ssubsequent natural infections with RSV (Connors et al., 1992 J. Virol. 66, '*7444).

The current treatment for RSV infection requiring hospitalization is the 15 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 20 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.

59 SWhile 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 al., 1987 supra). RSV proteins 1C, 1B and N are highly conserved among various subtypes at both the nucleotide and amino acid levels. Also, 1C, 1B and N are the most abundant of all RSV proteins.

S* 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.

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 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 G 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 S"" 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 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.

~I 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 1. 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 1 Spol 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. U S 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 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 20 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 10 with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules).

Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA associated with ICAM-1, 4 relA, TNF-a, p210, bcr-abl or RSV related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second 20 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 efficiently in terms of both yield and time, that correct exocyclic amino o. protecting groups be used, that the appropriate conditions for the removal .i of the exocyclic amino protecting groups and the alkylsilyl protecting groups on the 2'-hydroxyl are used, and that the correct work-up and 15 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 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 NH3/EtOH (ethanolic ammonia) for 20 h at 65 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 O 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-alkyltetrazole 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 analogue) amidite during the coupling step. (By delivered is meant that the 20 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 group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

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 cr! 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 x electron 15 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 20 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 °C for 10-15 m to remove the exocyclic amino protecting groups (vs 4-20 h 55-65 °C using NH 4 0H/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 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-

Q®.

Thus, there is also disclosed 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, there is disclosed an improved method for the 25 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 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 RNA molecule is produced in a synthetic chemical method and not by an 20 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 im, preferably 5 rm.

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.

O 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 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 eo 5'-end, and phosphoramidites at the 3'-end. The major difference used was the activating agent, 5-S-ethyl or -methyltetrazole 0.25 M 15 concentration for 5 min.

All small scale syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 pmol 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 lpL of 0.1 M 32.5 gmol) of 20 phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 UL of 0.25 M 100 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, 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 pmol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of O 0.25 M 1125 jmol) 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 accomplished by removal of the exocyclic amino protecting groups with either NH 4 0H/EtOH:3/1 (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845- 15 7854) or NH 3 /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 Deprotection of Exocyclic Amino 20 Protecting Groups Using Methylamine (MA) or NH4OH/Methylamine (AMA) SThe polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or (AMA) 55-65

O

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 °C 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 Alkylsilyl Protecting Groups Using Anhydrous TEA.HF To remove the alkylsilyl protecting groups, the ammonia-deprotected oligoribonucleotide was resuspended in 250 uL of 1.4 M anhydrous

HF

solution (1.5 mL N-methylpyrrolidine, 750 gIL TEA and 1.0 mL TEA*3HF) and heated to 65

O

C 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.

15 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.

e: *Example 10: HPLC Purification. Anion Exchange column 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 NaCIO 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 280% 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.

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 20 RPC column. A gradient from 20-55% B (20 mM NH 4

CO

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 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 gL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCl2. 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, deprotection of RNA synthesized on a large scale 100 pmol) becomes challenging since the volume of solid support used is quite large.

SIn an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant 15 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 OC 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.

20 The reaction is quenched with 16 mM TEAB solution.

Referring to Fig. 13, hammerhead ribozyme targeted to site B is synthesized using RNA phosphoramadite chemistry and deprotected using either 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 Fi. 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.

Examole 12a: Imroved protocol for the synthesis of phosohorothioate containing RNA and ribozymes using 5-S-Alkyltetrazoles as Activating Agent The two sulfurizing reagents that have been used to synthesize ribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu and Hirschbein, 1991 Tetrahedron Letter 31, 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).

15 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 o 20 and lyer, 1991 Tetrahedron 49, 6123). These conditions produced about 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 25 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 pmol 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 pmol) of phosphoramidite ls,. and a 40-fold excess of S--ethyl tetrazole (400 gL of 0.25 M 100 pmol) 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 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 snthesis of 2 '-N-htalimidonucleoside KM esis iof 2'-N-phtaIimido-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 supra. 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.

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 Sderivatives were formed with the major component being N-Pht-derivative 15. 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 CHCI3) and 57 g1 of TEA (0.1 eq.) was added to effect closure of the phthalimide ring. After 1 hour an additional 855 p. (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 mis). 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 eve* 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 15 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 20 There follows a method using the 2 '-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in Sparticular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable Sthroughout 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 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 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 20 Application 92110298.4.

This disclosure 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 25 tetrabutylammonium fluoride and SEM-CI. Those in the art, however, will recognize that other equivalent conditions can also be used.

In another aspect, the disclosure 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 Fiure 1, 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 3 i i- 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'protected moiety is converted into a phosphoramidite by standard o. procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.

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-((trimethylsilvl)ethoxvmethyl)-5'- 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 20-25oC) at which time (trimethylsilyl)ethoxymethyl chloride

(SEM-

CI) (487 LL, 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.

Examole 15: Synthesis of 2 '-O-((trimethylsillethox meth y) Uridine (4) inFiucleoside 2 was detritylated following standard methods, as shown in au Example 16: Synthesis of 2 '-O-((trimethylsilyl)ethoxvmethyl)-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 gL, 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

C

2 gave 10 20 mg of SEM deprotected nucleoside 6.

Example 18: Synthesis of 2 '-O-((trimethylsilyl)ethoxvmethyl)-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.

Example 19: Synthesis of 2 '-O-((trimethylsilvi)ethoxymethyl-5'-a Dimethoxvtrityl Uridine 3'-(2-Cvanoethyl N.N-diisoDropylDhosphoramidite) Nucleoside 3 was phosphitylated following standard methods, as shown in Figure 19.

0 20 Example 20: Synthesis of RNA Using 2'-O-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 p.mol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 iL of 0.1 M 32.5 pmol) of phosphoramidite and a 80-fold excess of tetrazole (400 gL of 0.5 M 200 lmol) 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- 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 uL, 30 pmol) was added to the solution and aliquots were removed at ten time points. The results indicate that after 30 min deprotection is complete, as shown in Figure 22.

III. 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, there is disclosed 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 .7 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 25 RNA transcript which is cleaved in situ or in vitro before or after transcript isolation.

Thus, this disclosure 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

CO-

LU

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 disclosed herein 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 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, or to produce a concatameric transcript for subsequent in vitro isolation of S...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 disclosure features a vector which includes a 25 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.

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 15 first ribozyme sequence is able to act, under appropriate conditions, to 4 cause cleavage at the cleavage sites to release the second ribozyme 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 disclosure 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 Sby 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 F 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, 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 15 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.

There is further disclosed 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, 25 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.

There is still further disclosed 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 I 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 supra; 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.

o. *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 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 .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 ii 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 S. 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 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 20 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.

S

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, I 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.

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 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 20 other embodiments can be readily generated using a variety of promoters, initiator sequences and stem-loop structure combinations generally known oin the art.

The trans-acting ribozyme used in this study is targeted to a site B (5'--CUGGAGUC'-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 i 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 su.ra. 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 su2ra) 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 supra). A tetra-loop at the end of helix 1 side of the cleavage site) serves to link 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 (Figure 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 supra) r 11' -I i :I 1 n i;, w 89 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 Fig. 26). The singlestrand portions of annealed oligonucleotides were converted to double- S. 10 strands using Sequenase® Biochemicals). Insert DNA was ligated into EcoR1/Hindlll-digested pucl8 and transformed into E. col strain .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 pM each NTP and 0.5 to 1 .ig 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 37 0 C. 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 ~I t lr I i;-j i;ill 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 (Hindill-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- S. processing and yield RNA products of expected sizes. Two nucleotides essential for hammerhead ribozyme activity (Ruffner et al., 1990 suDra) 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 (Fi. 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, 20 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.

91 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 .M CTP; 40 gpCi [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.l 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 (KaleidaGraph, Synergy Software,Reeding, PA) of the data to the equation: (Fraction Uncleaved Transcript) j (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 Hindll 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 *oe 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 suora). 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.

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 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 4l DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1IM) 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 MgCI2) by heating to 90°C for 2 min. and slow cooling to 37 0 C 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 b. 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 (Fiure 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. IUA 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 suDra) were grown in 6-well plates with 5x10 5 cells/well.

Cells were transfected with circular plasmids (5 jg/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 il/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 r "i I I I 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 g/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90 0 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 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'-CTCCAGTTTCGAGCTT-3'; HDV primer, AAGTAGCCCAGGTCGGACC-3'; HP primer, ACCAGGTAATATACCACAAC-3'.

As shown in Figure 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 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 (Fiure 29 "In Vitro +MgCI2" 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 29). 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 1 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 10 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 20 incubated for 1 hr in DEPC-treated water at 370 C prior to the standard primer extension analysis (Fiure 29, in vitro "-MgCl2" 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 (Eiure 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 intemallylabeled 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 oo \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 encoding such constructs, excellent for use in decoy, therapeutic editing 10 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 in a double-stranded structure in order to achieve enhanced

RNA

accumulation.

0* Thus, in one preferred embodiment the invention features a pol III 0@ 0 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 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 (Eia. 40) the 3' region is one nucleotide from the 3' terminus. In another example, the 3' region is 43 nt from 3' terminus. These examples are not meant to be limiting. Those in the art will recognize that other embodiments can be readily generated using o°elotechniques 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 Ill driven RNA molecule that is generally derived from any recognized tRNA gene. Those in the art will 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 othroughout 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 III 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 i' 4 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.

10 Biochem. 60, 631-652). By "enzymatic RNA" is meant an RNA molecule S.,with enzymatic activity (Cech, 1988 J.American. Med. Assoc. 260, 3030- '3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic 1: 5 portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target

RNA

through base-pairing, and once bound to the correct site, acts S: 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.

100 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 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.

In preferred embodiments, the 5' terminus of the chimeric tRNA includes a portion of the precursor molecule of the primary tRNA molecule, of which 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 molecule 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.

101 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 °lee 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, 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 Ill 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 Ill based genetic element derived from a human tRNAimet gene and termed A3-5 (Eig.3; 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"; Eig. 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 A3-5 chimera, the applicant made a series of modified A3-5 gene units l containing enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (Figa34). 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 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 10 expression observed. Thus, those in the art can now design equivalent 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 15 The use of a truncated human tRNAimet gene, termed A3-5 3; Adeniyi-Jones et al., 1984 supra), to drive expression of antisense RNAs, and subsequently decoy RNAs (Sullenger et al., 1990 supra) has recently been reported. Because tRNA genes utilize internal pol III promoters, the antisense and decoy RNA sequences were expressed as chimeras containing tRNAimet 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 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 (Figure 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 stably-transduced human MT2 (Harada et al., 1985 supra) and CEM (Nara Fischinger, 1988 supra) cell lines were established (Curr. Protocols Mol.

Bio. 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 20 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 .M 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 buffer containing 40 mM Tris.HCI, pH7.5, 20 mM MgCl2, 50 mM NaCI, mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 37°C 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/l 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 15 mixture was diluted with LB media and the cells were allowed to recover for 60 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.

20 Biology 1990, Wiley Sons, NY).

6 0The 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 (Fi 35.36).

In addition, these results demonstrated the relative differences in accumulation among the different constructs (Fiure 35.36). The pattern of 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 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 1 0 100-fold more abundant relative to the original A3-5/HHI vector transcripts (i35.3). This may be due to increased stability of the S35 transcript.

Example 28: Cleavage activity To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T 15 cells were incubated with a labeled substrate containing the HHI cleavage site (Eiure7). 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 20 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 (Fiure 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 (Fio.

-1 ii 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.

o 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 15 if ribozyme expression was stable over extended periods of time. This *o 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 10 generated using techniques known in the art, are within the scope of the lil 'present invention.

Example 32: Ribozyme expression in T cell lines Ribozyme expression in T cell lines stably-transduced with either a o retroviral-based or an Adeno-associated virus (AAV)-based ribozyme 15 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 20 derivative, G418 (0.7 mg/ml). Ribozyme expression in the stable cell lines was 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 .o 109 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 (Fig. 58) or long (622 nt) RNA (Fig. 59. 60 and 61).

Matched substrate RNAs were chemically synthesized using solid- 10 phase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res.

18, 5433-5441). Substrate RNA was 5' end-labeled using 32 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, 15 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.

Cleavage reaction was initiated by mixing the ribozyme with the substrate at 370C. Aliquots of 5 pl 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 Su.ra). 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- 32 p] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was 110 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 sural. 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 MgCl2. The RNAs were renatured by cooling to 37°C for 10-20 min.

Cleavage reaction was initiated by mixing the ribozyme and target RNA at 370C. Aliquots of 5 p.l 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 supra).

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 base-paired stem II region are catalytically active.

Example 35: Synthesis of cataltially active hairpin riboym RNA molecules were chemically synthesized having the nucleotide base sequence shown in Eig. 6 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 ribozyme-substrate 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 (fig,6 The target and the ribozyme sequences shown in Eig. 62 and 5 are 10 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 15 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 .0 provided at the 3' end of the ribozyme after helix 3 as shown in EiuruL 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 5' portion able to hydrogen bond to the 3' end of the hairpin or may have a 0 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 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 9 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 Fig._5, 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 disclosure also 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 can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

15 Also disclosed 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 °25 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 S..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, thedisclosurefeatures 5'-C-alkylnucleosides, that is a nucleotide base having at the S'-position on the sugar molecule an alkyl moiety. In a related aspect, the disclosure also features alkylnucleotides, and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the disclosure 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 are shown in Fi c ure 75, where each R 1 group is any alkyl. These examples are not limiting on the disclosure. 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 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 15 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 o substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, 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, 25 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.

In other aspects, also related to those discussed above, the disclosure 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 15 molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, thedisclosurefeatures triphosphates. These triphosphates can be used in standard protocols to orm useful oligonucleotides of this invention.

The 5'-C-alkyl derivatives described herein 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, thedisclosurefeatures 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 thisdisclosure is applicable to all oligonucleotides, applicant has found that the modified molecules disclosed herein are particulary useful for enzymatic RNA molecules. Thus, below is provided examples of such Z/ I^ 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 Figure 1, the preferred sequence of a hammerhead ribozyme in a to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. Herein, the use of 5'-C-alkyl substituted nucleotides 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 15 syntheses of the amidites.

Example 37: Synthesis of Hammerhead Ribozymes 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 io 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 25 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: Methyl-2,3-O-Isopropvlidine-6-Deox--allfranosde (4) A suspension of L-rhamnose (100 g, 0.55 mol), CuSO 4 (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 CO2 and evaporated to dryness. The residue was suspended in 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-Butvldiphenylsilyli-6 S. 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 CHC1 3 (200 mL), filtered and evaporated to dryness (below 40 °C using a high vacuum oil pump). The residue was dissolved in CH 2

CI

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 CH2C1 2 to yield 20.0 g of compound Example 40: Methyl-5-O-t-Butyldiphenvlsilyl-6-Deoxv-8-D-Allofuranoside 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 °C 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-Benzoyl-5-O-t-Butyldiphenylsilyl-6-Deoxy.- 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 h. 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 CH2C1 2 to yield 9.5 g of compound 7.

Example 42: 1-O-Acetyl-2.3-di-O-benzovl-5-O-t-ButyldiDhenylsilyl-6- Deoxy-D-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 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.

NaHCO3 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-Benzovl-5'-O-t-Butyldiphenylsilyl-6'.Deoxy-B-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 C1 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 Cl 2 Yield: 5.7 g Example 44: NA-Benzovl-l-(2'.3'-Di-O-Benzoyl-5-O-t-ButyldiDhenvlsilyi-.6' Deoxy-1-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 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), T 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 C12 yielded 1.8 g of compound 15 Example 45: /N -Benzovl-9-(2'.3'-di-O-Benzoyl-5'-O-t-Butyldiphenylsilyl-6'- Deoxy-1-D-Allofuranosyl)adenine (11).

NS-benzoyladenine (2.86 g, 11.86 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under S* 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 CF3SO 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: 6'-Deoxv-y-D-Allofuranosvyl)uanine (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 solution of of acetates 8 (3.4 g, 5.3 mmol) in dry CH 3 CN (100 mL) followed by CF3SO 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: Benzoyl-9-(2',3'-di-O-benzoyv-6'-Deoxv---D-AIIof rano syl)adenine 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 2. 15 Example 48: N~-Benzovl-9-(2'.3'-di-O-Benzovl-5'-O-Dimethoxvtritvl-6' Deoxy-B-D-Allofuranosyl)-adenine (19), Nucleoside 15 (0.55 g, 0.92 mmol) was dissolved in dry CH 2

CI

2 mL). AgNOs (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 20 stirred for 2h, diluted with CH 2 Cl 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-Dimethoxytrityl-6'-Deoxy.-D-Aiio furanosyl)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.

Example 50: A~-Benzovl-9-(-5'-O-Dimethoxvtrityl-2'-O-t-butyldimethvlsilyl- 6'-Deoxy-~-p-Allofuranosyl)adenine (27).

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

Pyridine (0.50 g, 8 mmol) and AgNO 3 (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.

NaHCO 3 (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 Example 51: A-Benzoyl-9-(-5'-O-Dimethoxvtrityl-2'-O-t-butyldimethylsilyl 6'-Deoxv--D--Allofuranosvl)adenine-3'-2-Cvanoethl N.N-diisopropylphosphoramidite) (31) 15 Standard phosphitylation of 27 according to Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield.

Example 52: Methyl-5-O-p-Nitrobenzovl-2.3-O-Isopropvlidine--deoxyv--iL- Tallofuranoside 20 Methylfuranoside 4 (3.1 g 14.2 mmol) was dissolved in dry dioxane (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 25 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.

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 described herein can be used to form Sstable 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 MgCl 2 as described above.

Applicant has substituted 5'-C-Me-L-talo nucleotides at positions A6, 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 Oliaonucleotides with 2'-Deoxv-2'-Alkvlnucleotide Also disclosed herein is the use of 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 in this application, 2 '-deoxy-2'-alkylnucleotide-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 o can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

Also disclosed 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 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 disclosure 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 disclosure 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 disclosure preferably includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.

0 0 'o Examples of various alkyl groups are shown in 000 Figure 81. 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 O is adjacent the 2'-position of the sugar molecule.

In other aspects, also related to those discussed above, the disclosure features oligonucleotides having one or more 2 '-deoxy-2'-alkylnucleotides 25 (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 havinq at its 2 '-position an alkyl group. In other related aspects, the disclosure features 2 '-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides.

The 2'-alkyl derivatives disclosed herein 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 disclosure 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 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.

15 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.

Referring 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. Herein, 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 25 possible, and were indeed synthesized, the basic structure composed of promarily 2'-0-Me nucleotides weth selected substitutions was chosen to maintain maximal catalytic activity (Yang et al. Biochemistry 1992, 31, 5005-5009 and Paolella et al., EMBO J. 1992, 11, 1913-1919) and ease of synthesis, but is not limiting to this disclosure.

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 O 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 ontainin 2-Deoxy- 2'-Alkylnucleotides Other 2'-Modified Nucleotides 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 S.V 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 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 10 assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCI 2 Reactions were 10 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 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.

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-(TetraisoDropyl-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-(Tetraisopropyl-disiloxane-1.3-diyl)-2'-C-Allyl -Uridine To a refluxing, under argon, solution of 3 disiloxane-1,3-diyl)-2'-O-phenoxythiocarbonyl-uridine, 7, (5 g, 8.03 mmol) and allyltributyltin (12.3 mL, 40.15 mmol) in dry toluene, benzoyl peroxide 15 (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-Allvl-Uridine (9) 20 A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry tetrahydrofuran (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.Ndiisopropylohosphoramidite) 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 OC) 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-(Tetraisopropyl-disiloxane-1.3-diyl)-2'-C-Alyl-N4- Acetyl-Cvtidine (11) 'o 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 oxychloride (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 0H 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-NV4-AcetvylCyidine This compound was obtained analogously to the uridine derivative 9 in 55% yield.

Example 62: '-O-Dimethoxtrityl-2-C-allyl- A-Acetyl-pytidine 3-(2- Cyanoethyl N. N-diisogroo~ylphosphoramidite)_(12) 2'-O-Dimethoxytrityl2'-C-allyl-N 4 -acetyI cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichloromethane under argon. NN-Diisopropylethyl.

amine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl NN-diisopropylchlorophosphorarnidite (0.38 mL, 1.7 mmol) 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 OC) and purified by flash chromatography on silica gel using chlo roform: ethanol 98:2 with 2% triethylamine mixture as eluent. Yield: 0.91 g white foam.

Example 63: 2 -Deoxy-2'- Methylene- U rid ine 2'-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 THE (20 mL) was treated with 1 M TBAF in THE (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

CI

2 Examgle 64: '-O-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. 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 eluant to yield 15 (0.43 g, 0.79 mmol, 22%).

Example 65: 5'-O-DMT-2'-Deoxv-2'-Methylene-Uridine 3 -_(2-Cyanoethyl N. N-diisoo~roo~ylohosphoramidite) 7J 1 2 Deoxy- 21 -methylene-5- O-d imethoxytrityl-..D- ribof uran osyl)uracil (0.43 g, 0.8 mmol) dissolved in dry CH 2 01 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 NN-diisopropylchlorophosphoramidite (0.25 mL, 1.12 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 vacua (40 00). The product (0.3 g, 0.4 mmol, 50%) was purified by flash column chromatography over silica gel using a 25-70% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.42 (0H 2 C1 2 MeOH 15:1) *Example 66: 2 .Deoxv- 21 Dif uo rom ethylen e3". 5 0-(Tetraisop ro 1yld isi loxane-1 .3-diyl)-Uridine *1 5 2 '-Keto-3',5'-O-(tetraisopropyldisiloxane-1,3-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 OC A warm 00) solution of sodium chiorodifluoroacetate 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 01 2 and chromatographed over silica gel. 2'- Dex-'dfurmtyee3, ttaspoydslxn-,3-diyl)uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.

Example 67: 2 -Deoxy- 2 '-Difluoromethylene-Uridine 2'-Deoxy-2'-methylene-3',5' O-(tetraisopropydisiloxane-1~,3-diyl)uridine (3.1 g, 5.9 mmol) dissolved in THF (20 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 silica gel column.

2 Deoxy-2'-d ifluoromethylen e-urid in e (1 .1 g, 4.0 mmol, 68%) was eluted with 20% MeOH in 0H 2 C1 2 Example 68: 5'ODL_-ex-'Dfur (16)leeUhbpL 2 '-Deoxy- 2 '-difluoromethylene-uridine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (1.42 g, 4.18 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 Cl 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'-difluoromethylene-uridine 16 (1.05 g, 1.8 mmol, Example 69: 5'-O-DMT- 2 '-Deoxy-2'-Difluoromethvlene-Uridine Cyanoethyl N.N-diisooropylphosDhoramidite) (118 10 1 -(2'-Deoxy-2'-difluoromethylene-5'- O-dimethoxytrityl-p-D-ribofuranosyl)-uracil (0.577 g, 1 mmol) dissolved in dry CH 2 C1 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 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 50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH 2 01 2 MeOH 15:1).

Example 70: 2 '-Deoxy- 2 '-Methylene-3'.5'-O-Tetraisoropyldisiloxane .3diyl)-4-N-Acetyl-Cytidine Triethylamine (4.8 mL, 34 mmol) was added to a solution of POCl 3 (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-(tetraisopropydisiloxane-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

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 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.

NaHCO 3 (5 mL). The mixture was concentrated in vacuo, dissolved in

CH

2

CI

2 (2 x 100.mL) and washed with 5% NaHC03 (1 x 100 mL). The 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'-c,.

(tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes.

Examole 71: 1 2 '-Deoxy-2'-Methylene-5'.O-Dimethox rity U--p-ribofuranosyll-4-N-Acetyl-Cytosine 21 2 '-Deoxy- 23 -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 1 0 residue was triturated with petroleum ether and chromatographed on silica .9::.gel column. 2 '-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'-methylene4N- .9..acetyl-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 *15 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 V. 9concentrated 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 999920 purified over a silica gel column using EtOAc:hexanes 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, Examlle 72: l-( 2 '-Deox-2'-Methylene5'ODimethoxtrityl-3DDribofuranosyl)-4-N-Acetvi-pytosine 3'-(2-Cyanoet hyl-N. N-diisoprooylphosphoramidite) (22) 1-2-ex-'mtyee5--iehxtiy---iouaoy)4N 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-d iisop ropylchlIorophospho ram id ite (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 OC). The product 22 (0.82 g, 1.04 mmol, 69%) was purified by fl .ash chromatography over silica gel using 50-70% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.36 (CH2CI 2 :MeOH 20:1).

Example 73: 23 -Deoxv- 2 disiloxane-1 .3-diyfl-4-N-Acetyl-Cytidine (24) Et 3 N (6.9 mL, 50 mmol) was added to a solution Of POCl 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'-diflIu oromethyle ne-3', 0- (tet rai sop ropyld is iIoxane-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.

NaHC0 3 The mixture was concentrated in vacuo, 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 and the residue chromatographed over silica gel. 2 Deoxy-2'-difl uorom ethyl ene.

31,51..O-(tetraisopropyldisiloxane-1 t 3 -diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol, 78%) was eluted with 20% EtOAc in hexanes.

Example 74: l-( 2 -Deoy-2'-Difluoromethylene-5'. -DimethoxytrityIlD ribofuranosl)-4-N-Acetyl..C~osine 2 Deoxy-2'-dif luoromethylene..3',5'-0teriooplslxa-1,3 diyl)-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 tri 'turated with petroleum ether and chromatographed on a silica gel column. 2 1 -Deoxy2'difluoromethyene4N.acetyl-cyidine (0.89 g, 2.8 mmol, 72%) was eluted with 10% MeOH in CH 2 01 2 2'-Deoxy-2'difluoromethylene-4-N..acetyl-cyidine (0.89 g, 2.8 mmol) was dissolved in pyridine (10 ml-) and a solution of DMT-Cl (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: 1 2 '-Deoxy- 2 '-Difluoromethylene-5'-O-Dimeth xzrityl-L.BDD..

ribof uran osyl)-4- N-Acetylcytosi ne -3'-(2-cyan oethyl- N. N -d i isooopylorhosphoramidite) (26) 1-2-ex-'dfurmtyee5--iehxtiy---iouao syl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in dry 0H 2 01 2 ml-) was placed in a round-bottomn flask under Ar. Diisopropylethylamine (0.5 mL, 2.9 mmol) was added, followed by the dropwise addition of 2cyanoethyl NN-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL).

After 10 mn the mixture was evaporated to a syrup in vacua (40 00). The 00: 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, containing 1% trethylamine, as eluant. Rf 0.48 (CH2CI 2 :MeOH /20:1).

Examr~le 76: 2 -Keto- 3 '.5'-O-(Tetraisopropyldisiloxane-1.3-diyl)-6-N-(4-t- Butylbenzoyl)-Adenosine (28) *Acetic anhydride (4.6 ml-) was added to a solution of 3 (tetra isopropyldisiloxane-1 3 -diyl)- 6 -N-(4tbutylbenzoyl)-adenosine (Brown Christodolou, Jones,S.; Modak,A.; Reese,C.; Sibanda,S.; Ubasawa A.

J. Chem .Soc. Perkin Trans. 1989, 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 vacua. 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 ',S'-O-(tetraisopropyldisiloxanel 3 -diyl)-6-N-(4-tbutylbenzoyl)-adenosine 28 (4.8 g, 7.2 mmol, 78%).

Example 77: 2' ex-'mtye -0 Ttaio qyl silxne .3diyl)-6-N-(4- t-Butylbenzoyil-Adenosine (29) Under a pressure of argon, sec-butyllithium 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 warm to -30 00 and a solution of 2'-keto- O-(tetraisopropyldisiloxane-l,-i.)6N-4tb1ybnzy adnsn 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 C1 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 NaHC03 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%).

Examnle 78: 2'-Deoxv-2'-Methviene-6-N-(4-t-Butvlbenzov)-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 .2 Example 79: 5'-O-DMT- 2 '-Deoxy-2'-Methylene-6N(4tButylbenzol) Adenosine (29) 2'-Deoxy-2'-methylee-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%).

ExamDle 80: DMT-2'-Deox2'-Methv ene-6-N-(4-t-Butbenzo)- Adenosine 3'-(2-Cvanoethyl N N-diisoprovpylhosphoramidite) (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, Ndiisopropylchlorophosphoramidite. 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 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 C1 2 MeOH 20:1) Example 81: 2 '-Deoxy-2'-Difluoromethylene-3'5'.O-(Tetraisopropyldisiloxane- 1.

3 -diyl)-6-N-(4-t-ButylbenzoyflAdenosine e e 2 '-Keto-3',5'-O-(tetraisopropyldisiloxane-1, 3 -diyl)-6-N-(4-t-butylbezy)aeoie28 g, 10 urmol) and triphenylphosphine (2.9 g, 11 e e, mmol were dissolved in diglyme (20 mL), and heated to a bath e e temperature of 160 OC. A warm (60 solution of sodium 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 vacuo. The residue was dissolved in 0H 2 C1 2 and chromatographed over silica gel. 2'- .20 4 -t-butylbenzoyl)-adenosine (4.1g, 6.4 mmol, 64%) eluted with hexanes in EtOAc.

~*Examole 82: 2 -Deoxy-2'-Difluoromethyene6N(4tLatylbeno) Adenosine 2 '-Deoxy-2'-difluoromethylene3',5' O-(tetraisopropyldisiloxane-1,3dil---4tbtlbnol-dnsn (4.1 g, 6.4 mmol) dissolved in THE ml-) was treated with 1 M TBAF in THF (10 ml-) for 20 m and concentrated in vacuc. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2 '-De oxy-2'-difl uo rom ethylen---4tbtlbnol-dnsn (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in CH 2 0 2 Example 83: 5-DT2-ex-'Dfuroehl e6 (jRW benzoyl)-Adenosinie 2'Doy2-ilooehln--N(--uybnol-dnsn (2.3 g, 4.9 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 vacua 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 vacua 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: '-O-DMT- 2 -Deoxy2'DIfluor methlen6(Buy benzoyl)-Adenosine 3'-(2-Cyanoethyl N. N-diso rooYIho-hoamidie .2 J2 6 -N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mnmol) dissolved in dry 0H 2 C1 2 (25 ml-) was placed in a round bottom flask under Ar.

D iiso propyl ethyl amine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N, N-diisopropylchlorophospho ram idite (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 vacua (40 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 MeOH/ 15:1).

Example 85: 2 Deox- 2 M ethi oxyca rbonylmethyl idine-KIS 5-(Tetra isopropyldisiloxane-1 .3-diyl)-Uridine (331 Methyl (tri phenylphosphoranyl idin e) acetate (5.4 16 mmol) was added to a solution of 2 -keto-3',5'-O.(tetraisopropyl disiloxane-i ,3-diyl)uridine 14 in 0H 2 C1 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% HCI. The organic layer was washed with H 2 0 (20 mL), 5% aq. NaHCO 3 (20 ML), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacua 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'- 1 ,3-diyl)uridine 33 (5.8 g, 10.8 mmol, 67.5%).

"I

W 138 Examile 86: 2 Deoxy- 2 Meth oxyca rbonylmnethyl idi ne-Uridine (34) Et 3 N*3 HF (3 mL) was added to a solution of 2 '-deoxy-2'-methoxy- ,3-diyl)-uridine 33 g, 9.3 mmol) dissolved in 0H 2

CI

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

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

CI

2 4:1.

Examlle 87: 5'ODT2-exL2-ehxgrbnlehldn-rdn 2 '-Deoxy- 2 1 -methoxycarbonylmethylidine-uridine 34 (1.2 g, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-Cl (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 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 an~d purified over a silica gel column using 2-5% MeOH in CH 2

CI

2 as an eluant to yield 5' -DM-'dex-'mehoycroymty di -urdi (2.03 g, 3.46 mmol, 86%).

Example 88: 5' ex-' etxyc bnlmtjldin-Urdin ***3'-(2-cyanoethyl-N. N-diisoprooylDho ,sphoramidite) (36) D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in dry 0H 2

CI

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 N, N-diisopropylchlorophosphoramidite (0.91 mL, 4.08 mmol).

The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mnL). After 10 m the mixture was evaporated to a syrup in vacuo (40 0

C).

5'ODT2-ex-'mtoycroy ehldn-rdn cyanoethyl-N,IV-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 (CH2CI 2 :MeOH 9.5:0.5).

139 Example 89: 2 '-Deoxy- 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'carboxymethylidine-3',5'-O-(tetraisopropyldisiloxane-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 disclosed herein 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.

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

Oligonucleotides with 3' and/or 5' Dihalophos honate 20 There is disclosed the synthesis and use of 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'- 25 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 disclosure 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-2,3-0isopropylidene-B-D-ribofuranose 5-deoxy-5-dihalomethylphosphonate 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'-dihalomethylphosphonates. 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 disclosure 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.

0 0

(R

3 0) 2

PCX

2 R2

(R

3 0) 2

PC

(R30)2PCX

B

R R 1

CX

2

R

1

CX

2

R,

20

(R

3 0) 2 P O (R 3 0) 2 P O 0 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 disclosure in particular features nucleic acid molecules having such modified nucleotides and enzymatic activity. In a related aspect the disclosure features a method for synthesis of such nucleoside 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 10 triphosphates 1, where the bridging oxygen atoms are replaced by a difluoromethylene group, have been employed as substrates in enzymatic processes (Blackburn et al., 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, 20 9125-9128), but can still form stable complexes with complementary sequences. Heinemann et al. (Nucleic Acids Res. 1991, 19, 427-433) S.found that a single 3 '-methylenephosphonate linkage had a minor influence on the conformation of a DNA octamer double helix.

NH

2 0 o o0 N I It II N O-P-X-P-O-P-O IN J O' O N 0 0- 0 N OH OH 0

N

(HO)

2

OPCF

2

NNH

2 2 (ETO),POCF2Li One common synthetic approach to a,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,a-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 V143 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).

Example 90: Synthesis of Nucleoside difluoromethylphosphonates Referring to Fig. 87, we synthesized a suitable glycosylating agent from the known D-ribose a,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-P-D-ribofuranose a,adifluoromethylphosphonate was synthesized from the according to the procedure of Martin et al. (Tetrahedron Lett. 1992, 33, 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 20-25C), 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 (Vorbr0ggen, 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 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

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 recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H 3

PO

4 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 J 2 3 4.9, P3H2), 5.67 (dd, J 3 2 4.9, J 3 4 6.6, 3H3), 5.63 (dd, J3,2 6.7, J 3 4 3.6, aH3), 5.57 (dd, J 2 ,1 4.5, J 2 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 31p 7.82 (t, JP,F 105.2), 7.67 JP,F 106.5). 6a: 1 H 8 9.11 1H, NH), 8.01 11H, 20 Bz, H6), 5.94 J1', 2 4.1, 1H, 5.83 (dd, J5, 6 8.1, 1H, H5), 5.79 (dd,

J

2 1 4.1, J 2 6.5, 1H, 5.71 (dd, J3', 2 6.5, J 3 4 6.4, 1H, 4.79 (dd, J 4 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: 3 p (vs 0SS) (D20) 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 I I -I 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 Triphosphate The triphosphate derivatives of the above nucleotides can be formed as shown in Fig. 9, 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, pp. 211-218.

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

The nucleoside dihalophosphonates described herein are S.o. 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.

20 These compounds may also be of use as monomers as antiviral and/or antitumor drugs.

Oligonucleotides with Amido or Peptido Modification This disclosure replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and S* 25 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: 3 W 146,

^O

0

N

H RN Ra R 1

R

3 0- I *0I 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, 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, Le, 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 San 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.

.idr~i~ i~iUi 147 Oligonucleotides of this disclosure 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 disclosure features an oligonucleotide containing the modified base shown in Formula I, above.

In other aspects, the oligonucleotide may include a 3' or 5' nucleotide 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 phosphate moiety may be modified to include other substitutions (see 15 Sproat, supra).

SExample 93: General procedure for the preparation of 2'-aminoacy2' :deoxy-2'-aminonucleoside conjugates.

Referring to Fi.2, to the solution of 2 '-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or amino acid (1 mmol) in methanol 20 [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1ethoxycarbonyl-2-ethoxy-1, 2 -dihydroquinbline (EEDQ) [or 1isobutyloxycarbonyl-2-isobutyloxy-l,2-dihydroquinoline (IIDQ)] (2 mmol) is Sadded and the reaction mixture is stirred at room temperature or up to SC from 3-48 hours. Solvents are removed under reduced pressure and i 25 the residual syrup is chromatographed on the column of silica-gel using 1- 10 methanol 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 1H 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, 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 3 2 p] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace 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 o* min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37 0 C for 10 min in a buffer containing 50 mM Tris-HCI and 10 mM MgCI2. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 370C. Aliquots of 5 pl 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 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.

L Preparation of aminoacyl-derivatized solid support A) Synthesis of O-Dimethoxvtritvl (O-DMT) amino acids 15 Referring to Fiq. 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 20-25oC) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was partitioned between 5% aq.

20 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 column 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 Fiq. 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 using standard solid-phase synthesis protocols described above.

II. Referring to Fig 99, 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 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 80, 1990, describes targeted gene modification. It reviews the use of 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 Soligonucleotide. In this methodology a gene or gene segment is S: essentially replaced by the oligonucleotide used.

This disclosure 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 1 0 RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying 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).

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 .el: mutations are introduced this may cause cancer, mutate other genes, or ol. 20 cause genetic changes in the germ-line (in patients of reproductive age).

However, if the base changing activity is a specific methylation that may modulate gene expression it would not necessarily lead to germ-line transmission. See Lewin, Genes,1983 John Wilely Sons, Inc. NY pp S" 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 USA 90:8673-7. Analogously, in one preferred embodiment of this disclosure a complementary oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by 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 (see Figure 102), by appropriate positioning of an enzyme (or ribozyme) 15 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 20 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

Sbase-paired intron-exon structure determines position and efficiency Cell 75:1361-1370. The machinery involved in RNA editing can be co-opted by S. 25 a suitable oligonucleotide in order to promote chemical modification.

The changes in the base created by the methods of this disclosure 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, this disclosure 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 disclosure creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base.

This includes correcting a sequence instead of inactivating a gene but can also include inactivating a deleterious gene.

Thus, in one aspect, this disclosure 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 enzymatically alters the targeted 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.

°r 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 ''i i 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 disclosure 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, this disclosure features correction of a mutation, rather than inactivation of a target by causing a mutation.

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 25 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 disclosure features only the addition of a few bases (1 3).

Thus, in another aspect, this disclosure 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 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.

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 properly regulated by its natural transcriptional promoter. Lastly, reversion 20 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 necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.

*Endogenous Mammalian RNA Editing System 25 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 rtNAs (four) have been reported to undergo editing in i mammals (Bass, supra). The predominant mode of RNA editing in mammalian system is base modification (C U and A The mechanism of RNA editing in the mammalian system is postulated to be that C-4U 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) Qell 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 conversion of Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.

In the present disclosure, ine endogenous deaminase activity or other such activities can be utilized to achieve targeted base modification.

i 15 The following are examples of the disclosure 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 disclosure and are not limiting to the disclosure. Those in the art will recognize that equivalent methods can be readily devised.

20 Example 97: Exploiting cellular dsRNA dependent Adenine to Inosine .converter: An endogenous activity in most mammalian cells and Xenopus S* oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. Weintraub, H. (1988). An unwinding activity that 25 covalently modifies it double-stranded RNA substrate. Cell, 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 l's and read as G, converting an A to I cannot create a stop codon. The A to I transitions i r 'i li r~ i E: ~bl!-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 noncomplementary 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 S. 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.

S. 20 The following detail specifics of the methodology:

RNA

oligonucleotides were synthesized on a 394 (ABI) synthesizer using *o 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) I,T 1 158 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.

S: supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 250C. 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, 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.

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, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226- 230.) e 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)).

i 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 be utilized. 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 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 25 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 A IInTrasversion Transversion |DNA 3RNA3 T(U) Transversion IDNA5/RNA 7 Transversion C Transversion

RNA

2

/DNA

6 I. Transversion G

DNA

6

/RNA

6 Transversion Transversion

I"

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 S' 0 2 -methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron 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, S521:770-778 (1978)).

7. Amination of uracil to cytosine. Bass supra. fig. 6c.

In Vitro Selection Strategy Referring to Fiure 105, there is provided a schematic describing an 15 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.

4 162 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) 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 (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 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 position within the random region) or the degeneracy can be partial (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635- 641). In this disclosure 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 include certain cofactors like ATP or GTP or an S-adenosyl-methionine (if methylation is desired) in order to make the selection more stringent).

25 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 0: 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 7 acids with the best base modifying activity is cloned in to a plasmid vector 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.

VIII. Administration of Nucleic Acids Applicant has determined that double-stranded nucleic acid lacking a transcription termination signal can be used for continuous expression of 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 portion of the R-loop can be used to target the whole R-loop complex to a S• desirable intracellular or cellular site, and aid in cellular uptake of the S• 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 disclosure 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 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 10 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., 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 92110298.4 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.

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 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 %vith 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, this disclosure 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.

In another related aspect, this disclosure 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.

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 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 intramolecular and intermolecular cleaving enzymatic nucleic acids to allow release of therapeutic enzymatic nucleic acid in vivo.

In a further related aspect, this disclosure 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 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 general is described by DeWet, 1987 Methods in Enzymol. 145, 235; Neuwald et al., 1977 J. Virol. 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 S 20 of an R-loop between the promoter and the reporter gene increased the 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 25 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 00 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, supra).

One of the salient features of this disclosure 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 process will continue until a termination signal is reached, or the plasmid is degraded.

This disclosure 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 15 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.

Ligand Targeting Another salient feature of this disclosure is that the RNA used to 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 25 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 figure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 J_,iol. Chem_. 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.

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 S. 20 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 .1 0 1169 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 position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 Science 257, 635-641). In this invention, the 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 20 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 population of nucleic acids by using a variety of methods (Joyce, 1992 "sulora). 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.

I

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 (M1 RNA) Size: -290 to 400 nucleotides.

RNA portion of a nrbonucleoprotein 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.

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

14 known members of this class. Found in a number of plant Spathogens (virusoids) that use RNA as the infectious agent (Figures 1 *0 a0 and 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 e 172 Table 2 Human IOAM HH Target sequence nt. Position U1 23 26 31 34 48 54 58 64 96 102 108 11.5 11.9 120 146 152 i58 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 Target Sequences CCCCAGU C GA C-CUG CUGAG7CU C CJC'GC-U AGCE7CC C MCL-Ut CtCUGC-U A CUCAGAG UGCUALU= C AGAGUG UCAGAGU U GCAACCU GCAACCU C AGC=tCG TJCAGCCU C GCtADGG CCUCGCU A DGGCUCC UJAUGGCU C CCAGCAG CCGCACTJ C CUGGUJCC UCCUGGU C CUGCUCG t7CCUGCU C GGGGCUC CGGGGCtJ C UGUUC CUCUGU U CCCAGGA CtJCEGUU C CCAGGAC CAGACAU C UGUGUCC UCTJGTGU C CCCCUCA UCCCCCT C AAAAGUC CAAAAGU C AUCCUGC AAGUCAU C CUGCCCC GGAGGCU C CGUGCUG AGCACCtJ C CtJGOGAC CCCAAGU U GUU;GGGC AAG=Ut U GGGCAUJA UGGGCAU A GAGACCC ACCCCGtJ U GCCtLA GUUGCCU A AAAAGGA AAC-GAGU U GCUCCtJG AGUUGCU C CUGCCUG AAG-GUGU A UGAACEJG AGAAGAU A GCCAACC AUJGUGCU A UUCAAAC GUGCUAU U CAAACUG UGCE]AUUt C AAACUGC GGGCAGU C AACAGCtJ AACAGCU A AAACCUU AAAACCU U CCrJCAC AAACCUU C CUCACCG CCUUCCU C ACCGUGU nt. Position 386 394 420 425 427 450 451 456 495 510 564 592 607 608 60.9 62.1 65 6 637 668 67 7 684 692 693 696 709 720 723 735.

738 765 769 770 785 786 792 794 807 833 846 851 ACCIGU=t

CUGGACU

CACC=

AGAACCrJ GIAACCUrJ UtT.CCCt CCAACCtJ LJGCLr7CU CE7G-AGCGU

GAGAGWU

AGCCAXI

GCCAAUt7 CCAArjUUU AAUUrjtj GAGCLTGt7

AGCUG-U

AACACCUc

GCCCCCJ;

ACCAGCrJ C CAGACCu 1L AGACCtUu i CCUUT~JG C AGCGACU C CACAAtJ U AACUUGU C CCCGG~TJ C C-GGUCCU A CCGUGGrj C GCrJCUGEJ U GUCUGUU C C-GGCUTGU u GGCUGUU C UCCCAGtJ C CCAGUCEJ C CCCAGG-U C CAGAGG'J U CC-ACACtJ C GUCACCU

A

A GjGACrJ C CAGAACG C CCUMU U GGCAGCC- U ACCUAC A CCtJACC :L CGCUGCC C ACCGUGG C CGUGGG C AC =-CA C A6CCAUGG U UCUCGU7G U CtJCGUGC C UCGJCc,

:GUGCCGC

3 UGAGAAC

JGAG-AACA

GGCCCcC CCAGCrJC CAGACCtJ

UGUTCCUG

GUCCUGC

*CUGCCAG

CCCCAG-A

GUCAGCC

AG-CCCCC

CrCAAGG-

GAC-GUGG

UGUUCCC

CCCUGGA

CCUGGAC

CCCAGUC

CCAGUCU

UCG-GAGG

C-G-AGGCC

CACCCG

GAACCCC

ACCUAUG

UC-C-CA-A.C

Target Sequences 173 .00* .000 .:Ooo: 863 866 867 869 881 885 933 936 978 980 96 987 988 1005 1006 1023 1025 1066 1092 1093 1.125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 1334 1 344 1351 1353 1366 1367 1368 1380 1388 1398 1402 AACGACJ C CU C GC-;LJCCU U Ct3CGGCC ACtJCCLT~t C UCGGCCA TJCCtIUCT C GGCCA-AG AAGGCCU C AGUCAGU CCtJCAGU C AGtJGEGA GUGCAGtJ A AD)ACUGG r-~JA A CUGGGGA UGACCAU- C MCArCu ACCAUCt) A C-aZCtjU UACAGCt) U UCCGG,-G ACAGCUU U CCGGCGC CAGCUUJU c CGGC-GCC ACG;GAU U CUGACGA CGUGAUU C UGACGAA CAGAGGEJ C UCAGAAG G-AGGUCJ c .AGAAGGG CCACCCtJ A GAGCCAA AUGGGGGU U CCAGCCC UGC-G-=U C CAGCCCA CCCAGCU C CUGCUGA CGCAGCU U CUCCUGC GCAGCtIJU C UCCUGCtJ AGCEJU C CLICCuCL tJCCUC-U C DGCAACC C-CCGCUu AUrAcAC_ CCAGC--U A t7ACACAA, AGCUrJAr- A CACAAGA C-C-GAGCtJ U CGtUrCC GG-AG=J C GuGuccu UUCGUGU C CUGCATUG GIUCCUGrJ A DGGCCCC G-AGG-AtJ u GuccGGG GCGAUtJGE c CGGcAA, AGAAAAtJ U CCCACA G-AAAAIJU C CCAGCAG GCAGACU C cAAuUG CCAGGCU U GGGGGAA AACCCAU U GCCCGAG CCGAGCU C AAGtWC CAAGTJGU C UAAGA AGEGUCU A AA.GGADG UGCA-BCU U UJCCCACUj GGCACtU U ccC-AcuG G-CACTJUt C CCACUGC UGCCCAU C GGGGAAU C-C-G-GAAU c AGU3GACEJ UGACUGU C ACEJCGAG UGUCACU C GAGAIJCU 1408 1410 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 1518 1530 1533 1551 1559 1563 i565 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 UCGAC-AU C UJUGAGGG GAGAUCY U GAGGGCA GGCACCU A CCUCtJGU CCt3ACCtJ C UGUCGGG CCUCLUGU C GGGCCAG GRAGCACTJ C AAGG-GGA GGG;fLG-U C ACCCGCG AUGtJGCU C UCcCCC~C TGrGCUcu c CCCCC,-, CCCCCGU A UGAGALUu AUGAAU U GUCAXJCA AGATJtyGU C AUCAUCA: UrUtCAU C AUCACUG UCAUCAt) C ACUGUGG CUGUGG.U A GCAGC:CG CCGCAGU C AUAAUGG CAGTJCAU A AUGGGCAD CAGC=t C AGCACGU AGCACG;U A CCUCrUAU CGUACCtJ C UAtUXACC UACCUCu A3 uAAccrC CCUCtrhij A ACCGCCA GGAAGAT- C AAGAAAU AAGAAAU A CAGACUA ACAflALrJ A CAACAGG CACGCCEJ c c~t3AAc UGAACCtJ A UCCCGGG AACCUAU C CCGGGAC AGGGCCu c UUCCUCG GGCC-UCEJ U CCUCGCC GCC3CU C CUCGGCC UCUUCCtJ C GGCCT-.7C UCGGCCtJ u CCCAATJ CGGCCEJU C CCAUALrj UUCCCAU A UEJGGuGG CCCATJAU U GGUGGC:A AAGACAU A UGCCAUG UGCAGCU A CJACCUAC UACACCU A CCGGCCC AGGGCAU U GUCCtJCA GCAUUGU C CUCAGUC UUGUCCU C AGEJCAGA CCUCAGU C AGAUACA GUCrAGAt) A CAACAGC AC-AGcAU U UGGGGCC CAGCA-TU U GGGGCCA CCAUGGU A CCUGCAC CACACCU A AAACACu AAACAkCU A GGCCACG 174 ::O.o oo..

0 0 0 0 1856 1861 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 21.76 2183 2185 2186 2187 CACGCAU. C UJGAUCUG AUCUrGAU C uat-GUC GAUCUGU A GUCALC;tY CUGT.AGU C ACAUGAC CAUGACU A AGCCAAG CAAGACU C AAGACAU ACAUGAU U GAUGGAU UGGAUGU U AAAUCE GGALUG=X A AAGUCUA UUAAAWt C UAGCCUG AAAGUCU A C-CCGAU GAGACAU A GC--CAXC AGGACAt3 A CAACUGG GGGAAAU A CUGAAAC UGAAACU U GCt3GCcu CUGC=-t A UUCGGEJA tJGCCUALU U GGGUAIJ AkUUGGGU A UGCEIGAG ACAGACU u AcAGAAG CAGACUE) A CAGAAGA, UGGCCCU C CAtkGAC CCUCCAU A GACALUGU CAUGUGE) A GCAUC.AA GUAGCAtJ C AAAACA CCACACU U ccUGAcG CACACUE) C C,-CACGG GCCAGCU U GGGcACEJ Ct.GCEJ C UACEJGAC GCUGUCU A CuGA;CCC CAACCCU U GUGAUJA UGAUGAU A uGfLurJ GAUALGU A UUEUTUC UAUGUAU U UALuUCAU AUGtJAUU U Aluc AtjU UGUJAUEIU A TUCAUUU UALEJTU U CAuUcUUG AUUUATUE-J C AUUUGUU tJAUUrCAU U UGUUALUU AUEJCAUU U GuuAtuU CAUUUGU U Auututi.C AUUUTJU A UUt7UACc UUGUUAU U urJACCAG UGUUAUEJU tJACCAGC GUEJAEJEJ u ACCACt) UUtAtU= A CCAGCEJA ACCAGCU A UUUUUG CACUATU U t3AUUGAG AGCUAUEJU AuuGAGU GCEJAUUU A UEJGAGUG 2189 2196 2198 2199 2200 2201 2205 2210 2220 2224 2226 22-3 22 42 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 UJAUUUAU U GAG-,G-,C LMAGUGU C UUUM.UG AGUGTCE U uUAUGUA GUGUCU U U;UAGh UGUCELtJ U AUGUAGG GUCDUUU A UGtkG,-C EUQAMUE A C-GCaAAA GUAGCTJ A AUGAAC UGAACAU A GGCCUCj CAUAGGU C UCUGGCC UAGC- C c GGCCjC CUC-GCC= C ACGGAGC CGAC.II-- C CCAGUjCC UCCCAGU C CAUGUCAL UCCAUGE) C ACALUrJC GUCACAu u CA;AGGUC UJCACAUt) C AAGGCA UCAAGG.U C ACCAGGU ACCAGGE) A CAGUUG GUACAGU U GUACAGG CAGUUGU A CXGGEJLG tIACAGGU U GUACACEJ AGIGUUGU A CACUGCk AAA.AGAU C AAAEIGGG UGGGACU U- CUCAflUG GGC-ACLtU C UCADUGG GACUUCU c ALUEJGGC UrJCtCAU U GGCCAAC CCUGCCU U UCCCCAG CE3GCCtUU U CCCCAGA UGCCE=J c CCCAGAA GAGUt U UUuCEhU AGTJGAUU U UUCUAhUC GUGAUU U UCUAUCG UGAUUU U CUAUCGG GAUUUUU C UAUCGGC UUUU.UCU A UCGGCAc UUJUCUAU C GGCACAA AAGCACU A TJAUGGAc GCACUAEJ A UGGCACUG GACUGGU A AUGGUUC UJAAUGGU U CACAGGEJ AAUGG7UU C ACAGGUU CACAGGE) U CAGAGAU ACAGGEJEJ C AGAGAUU CAGAGAt) U ACCCAGU AGAGAUU A CCCAG;UG GAGGCCtJ U AUUCCUC ALGGCCUE A UUCCUCc 175 2451 2452 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2 545 2568 2579 2585 2588 25921 2593 2596 2601.

2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2691 2700 2704 2711 271.2 2721 2724 2744

GCCUUAU

ccritJUU uAUUjCCU ccUcccT ctucccut

GA.CACC

ACA.CCU

CCrUrU CUUUGUt

GCCACCU

CCCACAU

CAUACAU

AUIACAUU

UACA=3r

CCAGUI

CAGu~uU TJGACA=t

CAGCGG-UC

GUCAUGUC

AGGGAAU rccucccrj

CUCCCUU

CCUt7ccc

CCCCCCA

CCCCCAA

UGUUAGC

GUr.AGCC

AGCCACC

GC--ACC

CCCACCC

CATJLLCU

t7CtGCC:A

CUGCCAG

UG%-CAGU

CACAADG

ACAAUGA

AGCGGUC

At7GUCUG

UGGACAU

CCAAGCt7 A UGCCUUJG

UAUGCCY

GCCULIGU

UUGUCCt7 GtTCCt7CE I CE7CUUGU GtJCC7GU I UCCUGUU I

UTJDGCAUI

TUGCAUUEt UGCAUM c GGGAGCt3 t UJTGCACJ n.

GCACUAu L t7GCAGCU c Ct3CCAGU U tJCCAGut TU CCAGUUU c CAIGUGAU c UCAGGGU c CCAAGGu A AAGGt7AU U GAGGACU c ACUCCCEJ c CCCAGCU U CCAGCUU U GAAGGG~u c GGGtJCAU C UGUGUGU A cUGUCCUt CriGUUUG 3 tJGCA.UJU 3 GCAX3UUC I t7CACUGG I CACUGGG

ACUJGGGA

TGC.ACUU

LUUGCAGC

I GCAGCrJC UCGtC

CCUGCA

CUGCAGU

AGGGEJCC

CtIGCAAG

UUGGAGG

GGAGGAC

CCUCCCA

CCAGCEU

UGGAAGG

GGAAGGG

AUCCGCG

CG-GTJG

UGUGEJAG

2750 2759 2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 291.5 292.6 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 UAUGUIt .ACAAG,-t

AAGL-CUT.

GCUCUGt GtJGCAAJ

UCALUGGU

CATGG.UU

(JGCAGU

GCAGUCU

UUGACCJ

UGAC-UU

GACCUUU

UtIGGGCU

AAGMIAU

UGAUCCtJ

CCCACCTJ

UCAGCCJ

CCUGAGU-

GGACCAU

AUkGGCU

GGCAALAU

GCAAAUU

AUUtJGAU UUUGAUU1 UtUUM- UGAUUtU I

GAUUUUU

AUUUUUU

UUUU(= z UEUUUtUU L

UUUUUUUL

UULUI= t:J UUUUUUtJ UUUUUUtY u UUUUUUU u UUUUUUU u UUUUUUU u Tuuuuuuu c ACGGGG-U C GGGGUCty C GCAACAU

U

CCAGACTJ U CAGACUU c ACUUTCCr U UUUGUG~U u TUCGUGUU A UGULTAGU

U

IA GACAAGC 7C UCGCUCU C GC-UCUGTJ C TJGCAC- IC ACC-CGG IC AUGGU,7c U CACUGCA C ACUGCAG C UrJGAcCU u CGACcuuu U UGGGCUC U GC-GCUCA C AAGUGAU C CUCCCAC C CCACCtUC C AGC=C C CUGAGUA A G--trG A GGCtICAC C ACAACA~C U UGAUUUU UI GAUEUrJ u uuuuuuu .3 uuuuUU .3 UUUr,'Uuu '3 MIXTE=U 3 UtJEULuuut

UUMULTU

UUUUCAG

UUCAGA

UCAGAGA

CAGAGAC

AG-AGACG

UCGCAAC

GCAACAU

GCCCAGA

CCUEJUGU

CUUrjUGUG

UGUGUUA

GUGUUAG

AGUUAAU

GUUAAUA

AAU~AAAG

176 2966 GUtLhZU A AtUPAAGC 2969 AGUEM.AD A AAGCrjUU 2975 tUhAAGCU U UCLMAAC 2976 AAAGC-UU U CUCAACU 2977 AA-GtJUU C tTCAACUJG 2979 GCEJUUCU C AACUGCC 177 Table 3 Mouse IOAM HH Target Sequence nt. Position Target Sequence nt. Position Target Sequence CCCugGU CaGuGgtJ UGgUuCtJ CUjtJGCU UCtcaU gCAcACtJ agACCU tUggGCCU CaUgcC-U cAcccCU cucugcu UgCcaGU acC-GULiG

CLJCUGCTJ

LUG-tUcCU CUCcaca AC-gGT~cG GuAgCCtJ -A GCCEgG GugAUGG UaG,-UCC

CCAGA

CTJGGcCC CUGCUgG *foe O*9V 1 0664 96 102 108 115 119 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 785 786 792 794 807 833 846 851 UGGuuCU G-gaaUTJC; CUCt3GCU CAGuCgIJ UL"UGTGT7J UCCUguU CAgAAGU AAGcCuU GGUGGgt7 gcCACuU CagAAGU

I

AAGtJUGUI UGuIGCut3i AaCCCaUc ccUGCCU; AgGGuuU AGggGCtj

C

AAGcUGTJ u AGgAGAU A ct3GUGCtJ u GUcCaAU Uj aGCt~gU.U u GuGCAGLT C GGcCUGU u GcC7GUU u UggagGU c CugGgCU u CuCgGaU a CAaAGcU c C-CcugGU C Gag;LCCU c C t7CtT-CCU C aCCAG4,- C CU9Gcc C cGc-UtCC C agCCaCu AAAAacC gUuUUGC C CUGCCCC CCGTJGCaG CCUCUGgC J GUUUGC J UuGCucc 2 GAGAaCu -uCCMAA LAggAaGA UCUaCLTG CUGCCUa UGAgLt3G UigagAAC

CA~CACUG

9AgCt3Ga GUCcGCtJ uCCuGcC CCuGcCEJ UCGCGAaG GGAGaCu UACC7GG GAcaCCC ACCguUG UacCAgc 367 374 375 3718 386 394 420 425 427 450 451 456 495 510 564 592 607 608 609 612 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 1353 1366 1367 1380 1388 1398 1402 AAuGcu U CAACCcg gAA9Ccu U CCUgcCC- AACcuu C CTgcc CuacCay C ALCCGEJGU AC--LJGU A uUCGUUU C=GGACT U UCGC-U, CACaCuU C CCCcCg CaCCCCtY C ccaGC.= Ca9CtCU c aGCAGug AGgACCU c ACCCT~gc GAAaCcU u uccuuuG UUIACCCU c aGCcaCu CUAcCaU C ACCGUGu UGCt3GCU C CGUGGGG CUcAZGGU a uccAuc GAaAGAU C ACaugGG AGCCAAU U UCUCaUG GCCAAT-U U CtCaUGC CCAAUUUE c UCatGCC- AAUUUCU C allGCCGC aAGCU-Vu U UGACu;rg AGCUG7Gtj U GAGcugA cgagCcu a GGccaCC: GaCCUCU A CCAGCcu UUCAGC-U c CgGuCCT CgGc-;-uU U cGaucu AGgaCcU c acCCUGC CCUgUuU C CUGCCuc gGCGgCU C CaCCuCA uACAACU U UUCAGCu AACUUuu C AGCuCCg aCCaGaU C CU9GAGa u-GC-gCcu c GuGaUGG CaGUcG;U C CGcUuCC GGcCUGU u uCCUGcc UuUGcU C CCtGGAa AGUCggu c g.AaGgUG UaaCAgu c UaCaACU aG"A6CCU C CCCACcu GuACUgU a CCACUCU UG-CCCAtJ C GGGGugg GGaC-Acu C AGUGgCEJ tGgCLTGU C ACagaAc UGT~gcurj u GAG-AaCU 178 863 866 867 869 881 885 933 936 978 980 986 987 988 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 1.334 1344 1351 1793 1797 1802 1812 1813 1825 1837 1845 AgCcACU u CcUCt~gG GAagCCU U CcuGcCC Ant3CgUU u cCGGagA UCUT~cCtJ C augCAAG AuGGCutJ C AacCcGrJ CCt~ugGU a gagGUGA cUauAaU c AtUcoGG uAat~cAU u CUGGuGc tUaACagt7 C UkC-AaCtJ ACagUTtJ A CAaCUUU UACAaCU U UuCaC-u ACAaCUTJ U uCaGCuC CAaCUUJ u CaG--ucc ACcaGAU c CUGgaGA uGaGAgU C UGggGAA ugG-AGGU C UCgGAAG GAGGUCU C gGAAGGG CCACUCU c aAkauAAL AcuGGat) c ucAGgCc UGGacct3 u CAGCCaA CCCAaCtJ C utucuUrA CGaAGCU. U CtUuuuGC GaAGCUU C UjuuUGCU AG-CUUCtI UuUGCUCU UCCt~utj u aaaAACC cuCuGCU c ctjcCAcA 9CuGC-Ur u UgaACAg AcutUu u cAc.AGu GGuAcatJ a CGUT~gC GaAGCUU C uuugCt UtYCGUuU C CgGagaG GUgC3GU A UGGuCCU GAaGGgU c GtlgCaaG uGAgaGU C uGGGgAA AGgAgAtJ a CugAGc GAggggT c ucAGCAG GCAGACtJ C ugAaaJG gaAGGCU c aGGaGgA JAACCCAU c uCCuaAa auGAGCtj C gAGaGUg ugAaUGU a UAAguuA UgGTJCCU C gGcugGA CacCAGU C AcALQAa acCAGAt) c CuggAGa ACuGgATJ c UcaGGCC CAGCAUEJ U acccuCA CCAcGctJ A CCUcugC CAUgCCti u uAgCuc cgAgcCt A GGCCACc 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 GAGgtj c GgaaGgg ccCACJ A CUuuGU aCt~gCCu u gGUaGaG uCUCt~aU u GCCCUG GAaggcu C AgraGGA GGa~uGU C ACCaGga AgUtJGu U u gCuCC cUGutJCU u CCUCauG Cu~guGcU u UGAGAac AUGAaAU c algguiCc gGActyaU a AUJCAuuc UUat~gutJ u AUaACcG cuAcCAt) c ACCGUGu ucaUG-,tJ c CCAGgCG CuauAaU C AUucUGG ugGUCADJ u gUGGGCc CAuG.CCLT u AGCAgcU AGCACCt3 C CCcaccU CULTAugU uU TAtLACC TMUgUuU A t7AAtCGC UgUUMM A ACCGCCA GaAAGALU C AgGAUAU AgGAUAty A CAaguuA ACAaguU A CAgaAGG CcCaCCU c CCtUGAgC gaAACCU u UCCUUUG AACCE~uU C CuuuGAa AGGaCCU C agCCUgG aGCCacEJ u ccucuGg GCCaCUU c CUCuGgC aCUtCCU C ucgCtugu cCGGaCtJ U uCgAUcU CGGaCUrJ u CgAUcUU UgCCCAU c ggGGUGG C9gAUAU a cc7GGag gAGAccu c tUaCCAgc gGCgGcu c CACC~ca gAagCCU u CCuGCCC gaGaCAyU GtJCCcCA GCAUtIGE u CtJCuaau UUagagty U UUACCAG TUagagoty U UACCAGC agagUUrJ U ACCAGCU gagttUtjU A CCAGCEJA ACCAGCU A ULTUAUUG CAGCUAU U UAUUGAG AGCTJAUU U AUUTGAG;U GCUAUUU A UUGAGUa 179 a a 1856 1861 1865 1868 1877 1901 191 1922 1923 1928 1930 1964 1983 1.996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2 115 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2 166 2167 2170 2171 2417 2418 2425 2426 2433 2434 2448 2449 CggaCuU AcaUGAU cAcu=GJ.

CaccAGtJ i CAUGcCTJ I uAAaACU AuAt~agU i UGaAVGU UGAUJGct Ut-AgAGUJi AgAGUuU t AGG-AAU aGGAgATJ UGgAgCU a GCt~auuU

A

LUGCCcAU c ggUGGuU c gCuGgCU a CuGACctJ c LTcuCCtJ C CuaCCAtJ c CAcuUGt A GUAGCct3 C CaACuCEI U CACA-kLt C GCC.AGCtJ c CaGCt~aU u CAAuCtJu u u cGAUCUu a UccALGta h~ GcCuCAg ACADaAa a1 AGCagcu :AAGggAc L GAUcagLI LuAAGUua AgGUat~c iUuaCCaG iaCCaGctJ L GuCCCca LCAAgLUa 6CLrGAgcC *GCgGaCc *UU~aG3A GGGgugG

IUCLGAG

gCAGAgG CuGgAGg CAcAucCacCg7Gt GCcUCAg ANgAgCuia Ccccl-'cG GGaggau U~uUGAg Ct7GCICI CutJGAug 2189 2196 21-98 2199 2200 2201 2205 221.0 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 2691 2700 2704 2711 2712 2721 2724 2744 UAtJTJU U C-AGUacC CaAcUctJ u CUE~gAUG gcaGcCtj c Lt~hGju GccuCau a tUgUuEThu Uct~uccU c AUGcAaG aagUTttjEJ A: UG;UCGGC UUAUGj c GGCcugA GgAGaCtJ c AgtGgcu 0cugcA~U u GLUCtJCJ LCCArGU a UCcauCC UgGaUC~J C aG-GCCgC CL-,GaC-,rJ c CUGGAGg UC-G;GC-U a gCgGaCC UauCcaU C CA:uccCA UCCAauU C ACACUgA allCACAU u CACGG-Ug UCACAUt7 C AcGGtJgc ggAAUGu C ACCAGGa ACCAGaLI c CuGgaGa GaAggGU C-GrgCAaG aAGCUGU u ugaGCUG UJAUAaGU U allggcCUj CaGUgGLI u CUCUGCU gAAAGAU C AcAUGGG tJGaGACU c CUgCCt3G GaaACcU u UCCULUG GACcCC a ccaGCCu UUucgAU c UuCCAgC CCcagCU c UCagCAG CUGCuUEJ U gaaCAGA aaCCUUrJ C CuuuGAA agGU;GgtJ U CUUCUga gGUGgUU c UUCt~gag agGgUtU c UCUTACUG UGCtJUUU c UC.AUaaG aAgUUUU a UgUCGGC alUUCU A UuGcCCC atlcCagu a GaCACAA AAaCACU A UgUGGAC aagCtlgU u UGagCtJG uACUGU c AgGaUgC AAUGtJcU c cCGAGCcC GAaGcCU u CCUgCCc gacCucrj a CCAGccu CCCAGCU c UcagcaG gagGucU c G-AAGGG GAAGGG-U C gtgCaaG.

GGuaCAU a CGuGUGc gGtTGgGU c cGUGcAG uugAUGU A UU!aIJUa gAUGUAU

AUGUAUU

UJGUAUUU

UAUUUAU

At~gUAUU acUUCAU AkUguAUrJ uALUUUaU AgUTJGUU gAAUGGU AcUGGaU CAugGGtJ .kuuaaUEJ uAG-AGuU AG-AGuT.TU G-AaGCCU 1 AaGC=UU MUaAU AUr~aAUU UUaAUUTJ aAWUUag AtJUaaULJ cucjUpj aUUCAaUUJ AaUUrJg tUgcuccc CAuAcGU UCAGGcc gAGgGuU AGAGuTU uaCCAGC aCCAGcu CCUgcc CUgcCC 180 e* 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 CCtlguut C gAagCCU u CCaCaCt TU CaCaCUU C GAgACCtJ c UCACmgu U CCaaUG;U c CUUUUUU c agCACCU C CC-CACcU A UAt~cCAU c ut~gAg3 U TJAgAgUU u CUUUEJGT U CA~caUtJ u tIGAugCU C CAGCaGU C Gt~gcUGU a guGaAgU c cCUgcc CCgCCCc UaccAGC GugAnCC

AGCCACC

aCCAguc CCCACCu CuUUUgU caUcCCA uUaCCAG UaCCAGC

CCCAAUG

ACccUcA AGguaUC cgcuguG UGGuCcU UGuCaAA GCCr~gUt U CCUgCC auAAGUU A U~gCct7G cugGCaU U GUuUCU 2750 2759 2761 2765 2769 2797 2803 2804 2 8-'3 2815 2821 2822 2823 2829 2837 2840 28B47 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 CCggaCU AgGacCtJ TUUUG-tJ agJCUGU allGaAAU

UJCALUGGTJ

ggUG~gU CcCGcu aCAGUCtJ ctJGAC-tJ gGAgccu UgCCJuucUGGacu AgGUGgU UGAgaCUC CCaAugU C gCAGCL cU gCcaAGU GGACCuU c UUcc-Ctj a cGgAcuU U UUAAUU a ACEUCAU u CUUCAJEJ C U=J~3U a UJGUatUj a GAagctj-U c AUcuucu u tUgUaUUU a UgUaUUU a tuugUtctJ c UtUUCUU a UgcUUUtj c alUUaUUy a UaUE~cgu U aUlUcg~tj U UTJcgJLuU c UtjctcaU a ugGaGGU C GaGGUCu

C

u GAguAcC u UCGaUCEJ C aCcCUGc c uGccgcu C AaaCAGG C AUGGUC c CcagGCg C cEGCA-:G C CUGAc a cAaCtUU CCUGGagg :CGC-ac~Ju GcmCCCA 3. uAat~cAU I CUu~uga CugCCtug AGCCaCuUauGUu LaCUGuC-A aGCcaAg cCAuCAC cGAUcEU GAgUUrjtJ UcUaUU UtjUaTjUg TJUaaUUU UtjmugcrJ Utgcuctj UT~aaUUUj TJUaaUUU tUaaT~gtC cL~ggucA UcaUaAG aTj-UuAGA tUcCgGAG C~gCAGAg A'ZgGuCG UCGgAAg GgAAggg GCaJTGU u~ tUgGtuCU C cuCucU u CtUUUUGU u acCgUE'GU a UCCaGctJ a CtUcGgAU a caGCAgU c gGaAI~gU C aGG-AcCU c UUuCgaU c GCACacU U UuCAGCU C ggCCuGU U cCCAGcY c CCuGUUU C uAcU'GgU C gaAGGGU C' tCucuaau :ugcUCCU rGcuCUGc CccaaUG uCgDuu CCAUccC UacCUGG CgCUGuG ACcaGGA aCcCUgc UUcCAGC

GLAGCCU

CgGwccu UCCUGCc uCaGCAG CUGCcuc AGGaUgC gUGCAAG CUAAUGU c UCCGAGG GagACAIJ U CCAcgCU a CAGcagu C AgUgaCtJ c uUtJCCUU U UcUGUGU c atJGUaUUj u UuUgAaU c

GUCCCCA

CCUCUGc C9CUGUG UGUIGUcA GaaUcAa AGccAcU aUlUAA~ju

AAMAAG

181 2966 GcUgGcU A gcAgAGg 2969 AaUcAAU A AAG6UCT 2975 UAgAGUU U UacCAgC 2976 gAgGgrJU U CUCuACU 2977 AAGCUgtJ U tgAgC3G 2979 uCaU7C C uAuit3cc T able 4 Human LOAM HH Ribozyme Sequences *t S a. S S nt. Position 31 23 26 31 34 48 54 64 96 102 108 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 CAC--C CUGAUGAGGCCGAAAC-,-CCAA AC~roCGG AGCAGP.G CU rGAGCC,A--,CCA

AGCUCAG

AMMAGCA CUAGGC,-; C:A AGGAGC-U CUCEJG CtJ AUGAGCC-AAAC-GCCCGAA,

AGCAGAG

C.AACt7Ct CUCALMAGGC_-CAAAC,-CCGAA

AGJAC,

AGGULT7GC C G;L G G C ,GAAGGCC G AcG A =CUC-A CGAGG-tJ L-JGAUGAGGC--c- ALG CC).AUCC CCAUAGC CU7GAUGAGGCCAAAGGCCAA

AGCUGA

GGAGCCA CUGAGC- AACG:=AA

AGCGAG

CM7CUGG CUGAXIGAGGCCC AGCcU GGACCAG CrMAUGAGGCCGAAGC,-I

AGUGCG

CC-AGCAG CUGAtMAGGCCGA GGCGA

ACCAGGA

GAGCCCC CU-GAUGAGGCCGAAAGGCCGA AGcAGG GGGAACA CtJGAUGAGGCCGAAAGGCCXA

ACCCCG

UCCYGGG CUC- AGCCG-AAG-.CCA

ACAGAGC

GtTCCUGG CCAUGAGGCCAGCA

AACAGG

GGACACA CLJGAU;GAGGCC-,.AG MA AUGUCUG UC-AGGGGC-- CUGAfGAGGCCZ'AGC-CGAA

ACACAG

GACUUUU CUGMAW.AGCCGA -GCCGAA

AGGGGCA

GCAGGAU CL"GAUGAGGCCGAAAGGCC;L

ACUUGU

GGGGCAG CUGAUGAGG;CCGA G cGAA. AE7GACUU CAGCACG COGAUGAGGCCGAG~ C~rAA

AGCCUCC

GUCACAG CUGAUGAGGCCGAA GGCCGAA AGGUGCrj GCCCAAC Ct3GAUGAGGCCGAAGCCGAA ACtJUGGG UAtJGCCC CUGAUGAGG-CC-AA GCCCGA.

ACAACUU

GGGUCt3C C UC-QAGGCCG-AA G-CCGAA AUGCA UUUAGIGC CU QAGGCCAGCCA;

ACGGGGT

UCcuutjt CtJGAUGACGGCCCGAAAC7-rGCC-AA

AGGCAAC

CAGGAGC CUGAUGAGGCCG-AAAGGCCA

ACUCCU

CAC-SCAG CUGAUAGGCCCAAG,-~, AGCAACrJ CAGETJJCA CUr-AUAGGCCGAAGGCCA

ACACCUU

GGUJUGGC CUGAUGAGGCCGAA-GCCCAA

AUCUUCTJ

GUUtUGAA CUGAUC-XGCCGAAAGGCCA

ACGCACAU

CAGtUUUG CUGAUGAGGCCAGCG

AUAGCAC

GCAGUTJU CUGAUi~GG GAGGCA

AAMGA

AGCUGEJE CUGAUGAGGCCGAAGCGAA

ACUGCCC

AAGGUtJU CtJGAL-AkAAGGCCGAA~cG AGCEJGUrJ GGUGAGG Ct3GAUJGAGGCCC-LAGGCGAA AGG-UtUj CGGUGAG CUGAUGAGGCCAAGCA

AAGGUEU

ACACGGU CUGAUGAGGCC-AAAGGCCAA

AGGAAGG

AGUCCAG CUCAUGAGGCCAAACCA

ACACGGU

CGUEJCtJG CUCAUGAGr.CCGAAAGGCCGAA

AGUCCAG

AAGAGGG CGGAGGCCGAAGGCCCGAA

AGOGGUG

CUGCCAA CUGALMAGGCCr AAGGcCCC.A

AGGGGAG

Ribozymne Sequence

A,

C

427 450 451 456 495 510 564 592 607 608 609 6i1 656 637 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 867 869 881 885 933 936 978 980 986 987 988 183 GGCUGCC CUGAUGAGGCCGAAAGGCCGAA AGAGGGC; GCAGGGU CUGAUGAGGCCGAAAGGCCGL AGGUUCUj CGUAGGG CtUGAUGAGGCC,-AAAGGCCG;A

AA).-,WC

GGCAGCG CUGAUGAGCCGAAAGGCCGAA ACGA CCACGGTJ COGAUGAGGCCGAAAGGCCGA

AGGUUGG

CCCCACG CUG AGGCCGAAAGGCCGAA AGCAGCk UGGUCGU CUGAUGAGGCCGAAAGGCCGAA ACCUCAG CCAUGGU Ct3GAUGACGGCGAAAGCrG;LA

AUCEIC~C

CACGAGA Ct3GAUGAGCCGAAAGCCGAA

AUGCCU

GCACGAG CMUG GAAGk" CGAA AAUUGGC C-GCACGA CCGAflGAGGCCAAAGCcA AAALUrGG GCCGCAC CUGAUGaAGGCCGAAAGGcCGAA AGAAALuu GUUCtJCA CUGAUGAGGCCGAAAGcCGA ACAGCrIC UGUUCUC CUGAt aAGGccGAAAGGcCG;LA AArkCU GGGGGCC CUM~r=AGGCCGAAGGCC- AGGUGUrJ GAGCUGG CUGAUGAGGCCGAAAGGCCGAA

AGGGGGC

AGGUCEG ClrJG GAGGCCGAA).GGcCCAA AG,-UGGj CAGGACA CUGAtraAGCCGAAAGGCCGAA

AGCUCUG

GCAGGAC CUCVJGAGGccGAAAGGCCA

AAGGUCTJ

CUGGCAG CAGCCGAAAGGCCGAA

ACAAG

UJGUGGGG CUG ;LGAGGCCGAAAGGCCGAA AcuCGCJ GGCUGAC COAUGAGCCGA)AAGGCCGAA

AGUUGUG

GGGGGCU CUGAUGAGGccGAAAGGCCA

ACAAGUU

CCUCUAG COGAUAGGCCGAAAGGCCGA

ACCCGGG

CCAccuc CUGAUGAGGCCGA G~CCGAA AGACC GGGAACA CGAcGAGGC-CGAA GCCGAA ACCACGG UCCAGGG CUG.,XUGAGCCGA.GGCCGA ACACrzC GUCCAGG CUGAUGAGGCCGAAGCCAA

AACAGAC

GACUGGG arUGAGCGCCGAAAGr.CCAA

ACAGCCC

AGACUGG CUGA GccGAAAGGCCGAA

AACAGCC

CCTJCCGA CUGAUrGGCGAAAGGCCGAA

ACUGGA

GGCCtJCC CUGAUG-AGGCCGAAGGCGA AGACtJGG CCAGGUG CUG;tJGAGGCCGAAAGGCCG.AA

ACCUGGG

GGGGUUC CUGAUGAGGCCGAAAGGCCGA ACCtJCUG CAUAGGU CGUGAGCCGAAGCCA

ACUGUGG

GUUGCCA CUGAUGAGGCCGAAAGGCCGA AGGtJGAC CGAGAAG CUGAUGAGGCCGAAAGGCCGAA AGtJCGrUU GGCCGAG CUGAUGiAGGCCGAAAGGCCGAA AGGAGCc UGGCCG-A CUGAUG.AGGCCGAAAGGCCA AAGGAGu CUUGGCC CuGAUGAGGCCGAAAGGCCGAA

AGAAGGA

ACtJGACTJ CUGAUGAGGCCGAAAGGCCA AGGCCtjE UCACACU CUGAUGAGGCCGkA GCGAA ACUGAGG CCAGUAU CUGAUGAGGCCGAAAGGCCGAA AcuGCAC UCCCCAG CUGAUGAGGCCGAA£GGCGAA AUtUhCJG AGCUGL1A CUGAUGAGGCCGAGGC

AUGGTJGA

AAAGCUG CUGAUGAGGCCGAAAGGCCA

AGAUGGU

CGCCGGA CUGAUGAGGCCGGCGL

AGCUGTJA

GCGCCGG CUG-AUGAGGCCGAAAGGCCG6

AAGCUGU

GC-GCCG CUG-AUGAGGCCGAAAGGCCGAA AAAGCtJG 184 1005 1006 1023 1025 1066 1092 1093 1125 1.163 1164 116 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 1503 1506 UCGt7CA CUGA GAA' C"'CGAcc AJCAkCGU UUCGtJCA CUGAGAGGCCGAAAGGCC;L

AUCACG

CtJUCUGA CUGAGAGCCQA A AC=CUG CCCUUCU CUGAUGAGGCCGZAAGGCCAA

AG-ACCJC

ULtC-GCUC CUGAMCAZGCCGAACCGA GC -Crj GGGCtJGG CUGAOAG GAAAGGCGAA a.CC-CIu t2GGGCTG CW.At GCG CG AACCCC; UCAGCAG CUGAM GA-GGAJ AC-Ctj, GCAGA CUGAMG GCMA CGAA AC-Jr.CG AGCAGGA CUGAM GGCGA AC,-JCc AGAGCAG CUG GGGCCGAGGCC.A

AGAAGCJ

GGUUGC CUGAUGAGGCCGA AGG,-CCcGAA

AGCAGGA

t3GUGtU.U CUGAtJGAGGCCGA CCGAA

AGCUGGC

U UGGGA CUAUAGCCGAAAGGCAA

AAC-=

uCOG CUGAtArGCC CZAA AUAAGCtJ GGACAICG CUar-AGGCC AGCAA AGCUCc AGGACAC CUGM;AGCCQ lGG AAGCrUCC CAUACAG CUGA-GGCAGGCcGA

ACACGAA

GGGGCCA cuGfGAua~Gcc G Gr~cGAA

ACAGC

CCCGGAC CUGWAvGGCCGAAGGCC;L ATCCCtUC UUUCCCG CUJGA=GGCCAAC ACAAtICC UGCUTGGG CUGAUGAZZCCGAA GCCGALA AUUUUrcu CWGCUG CUG;LUGAGGCCGAAAGCCGAA AAUUUc CACAUUG CUGAtJ'LGGAG GCCC~ C;AA AGUCUGC UJUCCCCC CUGAUGAGGCCGAAAGGCCGAA

AGCCUGG

CtICGGGC U uUAGCAA~,C-L AtC-GGWu GACACUU CGAOGAGCCGCG

AGCUCCG

UCCUUtA aUGAUGAGGCCGAAGGCCUAA ACAC=tG CAT3CCUU CUGAtGAGGCCGAAACGCCGA AGACA=t AGUGGGA CUAGGrCAAr-G; AGtUGCC 'MI-GG CUA6UGGGCXCGAGGCCA

AAGUGC

GCAGUGG3 CUGAr-GGCCGAAGCGAA AAAflUGC AUUCCCC CuGAUGAGGCU CGAAA -C

AL'GGC.

AGUCACtI CUGAtJAGGCCGAAGCGAA

AUUCCC

CUCGAGU CUGAt GGCCGAAGCGAA

ACAGUCA

AGAEJCEC CUGAUGAGGCCGAA GGCG6 AGUGACa CCCt3CAA CUG.AtJAGGCCAGGCCA AUCtJCGA UGCCCtJc CUGAUGAGGCCGAAGGCCGAA, A-AUCt3C ACAGAGG CUGAUGAGGCCG_-GAAC

AGGUGCC

CCCC-ACA CUGAUGAGc CGAAAGGCCGAA AGGUAWc CUGGCCC CUGAUGGCC AGGCCGA

ACALGAGW

UCCCCUU CUGAt3GAGGCCGAAGGCCGAA*AGUGCUc CCGGGE CUGAUG )CAA ACCtICC WWGGA CUGAUGAGGCCGAAGCAA AG CACAU CCGGGGG CUG-AUGAGGCCGMAGC-,,CGAA

AGACCAC

AAEJCECA CUGAUGAG-CC-3-GGCCA

ACCGGGG

UGAUGAC CUGAUGCAAGGCCGACA

AUCUCAU

UGAUGAU CUGAUGAGCCGAAGGCCGA ACAAUCrJ CAGUGAU CUGAUA-GAkXCCGAC.

AMJGA

185 1509 CCACAGUJ CUGAlrmACGCCZAAAG,-cCAA AUGArGA I518 CGGCUGC CUAGGCGAAGCA

ACCACAG

1530 CCAULIhU CUAGCCC-AAGCA A~r 1533 UGCCCAU CUGAUGAGGccGAAAGGcCGAA

AUGACUG

i551 ACGGCU CUGAGAGCCGAGCCG

AGGCCUG

155 9 A~GAGG CUGAGAGGCCGAA GCG ACGG- 1563 GGUCAUA CUAGCCGAGCGA

AGG

1565 GCGUET CUArGCGAA~C-A

AGAC-J

I57 TLGC-CGG-7-U CL7,UGAGGAAAG-,GCC

AUAGG

1584 AIJUUCUU CUGAUGAGGCCGA A-CcGA

AUCUUCCC

1592 uAGU=UG cuGAM-AGGccG~cccA Ariuju i399 CCtGUUG C CAUGAGGCC--AACG- AGUCt-i 1651 GUUCAGG CUGALrAG- GAAG c CG- GU 1661 CCCGGGA CUGAUGAGCC-AGCGA

-ZLGGICA

1663 GUCCCGG CUGAUGAGGCCGAAAGCGA AtUhGGU 1678 CGAGGAA CUGAt7GAGGCCGAAAGCCGAA AW.C=cr 1680 GCCGAGG COGAIXGAGGCCGAAAGGCCAA

AGAGGCCC

168 GGO. ~CCGAG CUAGGCGAAGCA

AAGC

*.1684 GAAGGCC vG; 1690 AUGGCUGAUGAC-GAGCCG A AGGCCGA ***1691 AAMDGG CUG AGCCGAAAGGC.

AAGGCCG

1696 CCACCAA CcGAGAGGCCGAAGCGA

AIGGA

1698 UGCCA C GGGCAAGCCA

AUAUGGG

1737 CAIJGGCA CUAUAGL-r CC-AA ALTGUCUr_ *1750 Gt~kGUG CUGAMAGGCCGAAfGCCG

ACQC

1756 GGGCCGC;GAUuCCGAAGCA

AGGUGUTA

***1787 UGAGGAC CUGAI.X CCAGGC CGAA AJGCCCrJ 1790 GACUGAG cuGALGAGGCCGA GccGAA AcAAIJGC 1793 UCUGACJ CGAU GC GA c- AGGACAA 1797 UGUIjCU CUGAUGAGGCCGAGCCGAA

ACUGAGG

1802 GCtJGUjUG CUGAGAGGCCGAA).QGCCGAA AUCUGAc 1812 GGCCCCA CTUGAUGAGGCCGAA GCGAA AUjGCUGEJ 1813 UGGCCCC CUGAUAGGCCGAAGGCCAA AA3GCt7G :.1825 GUGCAGG CUGAUGAGGCCGAAGCGAA

ACCAUG

1837 AGUGUUU CUGAUGAGQCCGAAGCGAA AGGC3GUG *1845 CGUGGCC CUGA GAGGCCGAA GCGA AGU.rIrUrj 1856 CAGAUCA CUGAIGAGGCCGAAAGCCGA

ADGCGUG

1861 GACLTACA CUGAUGAGCcGA cc AUCAGAU 1865 AUGUGAC cuGA1UGAGGCGAA GrccGAA

ACAGAUC

1868 GUCAUGtJ Ct3GA~kGGCCGU-GCG. ACUM2AG 1877 cuuGGCU CUGAGGccGAA .ccA AGucAuG 1901 AUGUCUU CUGAGArG~CCGAAAGGCCGA AGt3CUtJG 1912 AUCCAUC CUAGGCGAG;CA

AUCAUGU

1922 AGACtJUU CUGAUGAGGCCGAA CcGCCA

ACAUCCA

1923 UAGACUU CUJGAUGAGGCCMAAGCCG

AACAUCC

1928 CAGGCUA CUGAUGG-AAGGC CG A ACUUU~AA 1930 AUCAGGC CUGAfGAGGCCGAA~.CCC-A

AGACUUUT

1964 GGGGGC CUGAGAGGCCGAAACA

AUGUCUC

1983 CCAGUtG CUAGAGGGCcG

AUGUCCU

186 1996 GUUUCAG ClJA= -C-CGAAGGCC~k

AULUCCC,

2005 .AGGCAGC CUGALr-4G=GC-AA G--C-AA AGUUUc-z 2013

TU

1 CCCAA CLTJGACCG---XAG ,a CCC 2015 CAtzkccc CUGAUACAACG-CA

AAGGCA

2020 CuCZAGCA CUGA~rGACr-GA -CGAA.

ACCCAAU

2039 COUCUGU COUG~AGGCC--AA GCCG;AA

AGUCTJGT

2040 CCUCUG CUGAUGAGGCC -j AAGtJCr.G 2057 GUC3AXG CUGAflGAG----AcG G-C- AGGC 2061 ACAtJGUC CUGAUG~~ccGA

AUG

2071 UtJGAUGC CUGAUGAC-GCC,--A GCCGAA

ACACAUG

2076 GUGUUMtJ CUG"L(;L-A2G,--CC GGA CC-AA AUG-CTJA'C 2097 Crz-CAGG Ct3GAVGAGGCAG CGAA AGUGtx-G 2098 C-G3CAG CCAGGCAAGCCA

AGUG

2115 AGt.GCCC CUCGA C-CcGAAGG.CCr,;, AGCtJGGC 2128 Gt3CAGUA CUuGAAc GcCGC,-rAA

ACAGCAG

*2130 GGCGUCAG Ct3GA.UGAGCCGAG,--A

AC

2145 tMhJCAC CUGAMAGGCCAAGGCGAAx AGGCtJEG :2152 AAAkCA CUAGClC CA AUCATJrA 2156 GAAULUA CUGAUCGAZGCC -AAGCGAA

A(CAMUC

215 CUAGG,-GAA-GC A~kCAUA 2159 AAIJGAAU CUGAUGAGGCC-A AGCCCCAA tMCAU *2160 AAAXUGA.A CUGAUGA-GCC GXAC=CGA;L AAAMVc', 2162 ACAAAXIG CUGAGAGC,-CGA CCCG AUAAAUA 2163 AACAAAU C GAGCCr.,z GAA

AALUAAAU

9.2166 AAXUAACA CUG-AUGAG-C .CGA UG-CGAA

AUGAAU.A

2167 AAAAC CUC-ALM;LCCCGA 'G-CCGAA

AAUGAAJJ

2170 GUAAAAU CUiGAflGAGGCCGAA C-GAA ACAAALUG 2171 GGUAAA CMAUGAGGcGAC--CGA.A

AACAAAY

2173 CUGGUAA CUC-AUG-AG,^-C-,AC~CC-AA

AIIAAC.AA

217 9~a CLT;LGAt~ACCGAG-CA AA~kACA .*.2175 AGCULG%-U CUGALUGAGCCAA CGA AlA 2176 LMGCUGG CUC-AUrIGCCc-I C;CG AAAAUAA 2183 CAAUAAA CUGAt GAGGccGaAxax,-C AG~CEGut .2185 CUCAAUA CUGAUGAGGC CAAG-CGAA AEUhGCtJG 2186 ACUCAAU CUGAIr-GC-C- AGC

AAGCU

2187 CACUCAA CUG~r.ifl GA AAG CGAA AAAUAhC- 2189 GA-CACUC CUGAL'GAGGCCG AG-CGA AUAAAt7A 2196 C.AUAAAA CUGAtA CG ACACUC 2198 UALCALMA CUGAL7GAGGCCAAACGW

AGACACTJ

2199 Ct3ACAUA CtJGGArur-=XAGGCC A AAGACAc 2200 CCtJACAU CUGAGA,- GAACC

AAAGACA

2201 GCCLVACA CJGAUGAGGCCGAAAG-4-

A.AGA

2205UUUAGCC CUGAUGA GC GGCCGAA

ACAUAAA

2210 GJUCAtJU CtJGAUGA r.CGAAA-CAA

AGCCUAC

2220 AGAGACC CUGALIGAGSGCC-,. AUGUrUCA 2224 C-GCCAGA C'TUGAGCACGCCGAA

ACCTJAUG

2226 CGGCCA cuGTJ-GA CCGAA AGA~a CLU 2233 C-CCCGU CtGAL'AGGCCCG AGGCCC AGGCCAG 2242C-ACGG CUGA UGAGGCaGA.A A A AG=tCCG 187 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 233 9 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 2504 2508 2509 TUGACAti(

UGAAUGI

GACCUUC

UGACCUE

ACCUGGt

ACAACUG

CCDGCC

CAACCUG

AGTJGUAC

UGCAGUG

CCCALWr

CAAUGAG

CC.AALGA

GGCCAAU

GUUGGCC

CUGGGG

UCUGGG

UA~CGA

ALGAA

GAUGA

CGAUAG

GCCGAA

GGCCA

GUGCC

t3GUCC

AGCCA

GAArCCA

GACCU

ACCUGU

UCCUG

AUCJCUj

AACUCU

ACUGGG

CAGGA

GAGGAAC

AGGAGG

AGGGAG

AGGGAG

C

GGGGG

C

UGGGGG

GUGGGCA

;CUGtGCGA AACGCCGAA

ACUGGG

3 CGGC-GCCGAAAG CGAA

ACAUGGA

CUGAUAGG-CGAAAGGCCGAA

AUGUGAC

7CUJGAUGAGGCCGAAAGGCCGAA

AALUGUGA

CUGAUGAGGCCGAGCGAA ACCirA CUGAUGAGGCCGAAAGGCCGAA

ACCUGGU

*CUGAUGAGGCCGAAAGGCCGAA AC3GtMC COCAGAGGAAGGCCGAA

ACAACUG

*CLUG DAGGC-CGAAAGGCC;L

ACCUGUA

CUGAWAGC<4-GAAG-CCGAA

ACAACU

CtGAUGAGGCCAGGCCG-AA AUCU~Ut) CUGAEGAGGCCGAAAGGCCGAA

AGEJCCCA

CUGAUGAGGCCGAAAGGCCGAA

AAGUCCC

CUG-AW.AGG-CCGAAAGGCCGAA

AGAAGUC

CUGAUAGr-CCGAGCCGAA AtJGAGAA CrJGAUGAGGCCGAA.AGGCCGAA

AGGCAGG

CW-GUGAGGCCGAGCCGAA

AAGGCAG

CUGAUGACGGCCGAAAGGCCAA

AAAGGCA

CtJGUAGGCCGGCCGAA AUCACUyC COGAUGAGGCCGAAGGCCGAA

AAUCACU

CUGATJGAGCCAAGGCCGAA

AAAUCAC

CUGAUGAGGCCGAAAGCCGAA

AAAAUCA

CUGAUGAGGCCGAAGGCCGAA

AAAAAUC

CUGAUGAGGCCGAAAGGCCGAA

AGAAAAA

CUGAtLrAGG--CGAGCGC

AUAGAAA

CJGAtGGAAGCCAA

AGUGCUU

cLUGA~GGCAAG~cAA AtTAGUGC C~UGArGiCCty-A GCCGAA,

ACCAGUC

CJGAUGAGGCCGGCCA

ACCAXJUA

CUGAIJGAGGCCGAAAGGCCA

AACCAUU

EJAIGGUGGACCA ACCt3GUG UGVAUGAGGCCCGAAAGCGCCGAA

AACCUGU

EJGAUGAGCCGAGrCCGAA

AUCUCUG

UAUGAGCCGAAAGGCCGA

AAUCEJCU

"UGAUGAGGCCGAAAGGCCGAA

AGGCCUC

%J]GAtJGAGGCCGXAAGGCCGAA

AAGGCCTJ

-UGAUGAGGCCGGCCG AtAAGGC

E

7 GAUGrAGGCCGAAAGCGCCGAA

AAUAAGG

GAUIGACGAGCCGAA

AGGAAUA

UGAUGrGCAGGCCGAc

AGGGAGG

uG-AUAGGccGAGccr.A

AAGGGAG

UGAUGAGCCGAAGGCCG;L AGGtJGUC

U

7 GAUGAGGCCGAAAGGCCGAA

AAGGUGTJ

L2GAUGAGGCCAGCGA

ACAAAGG

IGAUGAGCCGAAAGGCCA

AACAAAG

JrGAUAGGCCGAApGCCGA

AGGUGGC

JGAUGAGGCCGAAAGGC-CGAA

AUGUGGG

IGAUGAGGCCGCCGAA

AUGUAUG

YG;L3rAGGCCGAAAGGCCGAA

AAUGUAU

GGCLULAC C GGUGGCU

C

AGGUGGC

GGG,-UGGG C AGAAAUG

C

UGGCAGA

C

CUGGCAG

C

188 a.

a a a 2510 2520 2521 2533 2540 2545 2568 2579 2585 2588 259i.

2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2 691 2700 2704 2711 2712 2721 2724 2744 2750 2759 2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 ACtJGGCA CUG;LAuGGCCGA AL-~CCAA

AAAUGUA

CAUUG CUA-ZCGAAACCCG-A

ACACUGG

UCAUUJtT CUG UGAGCCGAAAGGCCrA

AAAU

GACCGCU CUAW-GCCAAAGX-GAc

AGUUC-A

CAGACAU CVfGAAG CGAAc AC-CCU AUGUCCA, CUGAVJGAGGCCGAAGCCA

ACAUG.AC

UUGGGICA CUGADAGGCGAGCCAA

AUMCCU

CAAGGCA CUALGGCCGA Gc-A AGCUG AGAGGAC CGCCGCAA,

AGGCATM

ACAAGAG CMAGGCGAAC-GL

ACAACGC

AGGACAA CUGAUGAGCCaGA--AA

AGSACA

AC-AGGAC CUG;LUGAGGCCGAA ,CG-AA -AuAGAC CAAACAG CUAGG-CGAGC-A

ACAAA

AAAUCCA CUGAM AGGCCGAA CAA ACAGGAc GAAA=G CuUGAGGClAXCC AA AACAGGA CCAZUJGA CtIGAUGAGGCCGAAGGCCGAA

ATJGCAAA~

CCCAGUG CtJGAXGAGGCCGAAGCGA

AWCAA

UCCCAGEJ CUGATJGAGGCCGAA CCL

AAWJGCA

AUAGUGC CUGAXAGGC=AWXCCAA

AGCUCCC

GCUGCAA CUGAU GCCGAAAGGcC-A AGuccAA GAGCUGC CUACAGCCGAAAG--GCA

AMAGUC

GAAACUG CUG GAG-CGAAAGGC-CGAA

AGCCGCA

UGCAGGA C~-AGAGCCGAAC,-CA ACt3GGA CUGCAGG CUG UAGCG-

AACUGGA

ACUGCAG GArNCCXAr.-GA

AUG

GGACCCt7 CW-AUAGGCCGA AGGCCGAA AUCAXrJ CUUCAG CrGGGCCCAAGC~rA

ACCCUGA

CCUCCAA CL'GAGAGCA G~ccGAA ACCMuW GUCCUCC Ct1GAI2GGCC A~,-CA

AU;CC

UGGGAGG CUGAUGAGGCCGAAAGGCCGAA

AGCC

AAGCUGG CUAGGCGAAGCA

AGGGAGU

CCUUCCAL CMUfGAGGCCAAGGc

AGCUGGG

CCCEJucCc CUAUCAGGCCGAAGCCGL

AAGCOGG

CGCGGAXJ CUG A-,GGCGAA GCGAA,

ACCCUUC

ACACGCG CUaMMAGGCC GGCGA

AUGA=C

C~CACA CUGAXGAGGCCGAAGGCGAA

ACACAA

GCUUTGUC. CUGAWAGGCCGA GGCCGAA

ACACAUJA

AGAGCGA CLGUAGCAAGCA AGCUUGu ACAGAGC CLTJAUCAGGCCGAAGGCGAA AGAGCUrJ GGUGACA CUGAAUCGAAGCCGAA

AGCGAG.A

CCUGGGU CTGUAGCGAZ

ACAGAGC

GAACCAU CtJGAUGAGGCCGAA GCCGAA AUtr-AC UGCAGUG CVGUCGGCCCAA

ACAG

CUG-AGU CJU GGCCGA. GAAc

AACCAUG

AGGTJCAA CEUGAUGAGGCCCG AGGAA ACrJGCAG AAAGGUC CUGAUGAGCCG .GCCGAA

AC-ACUGC

AGCCCAA CUGAUrGGCCGAACGAAZA GAGCCCA Ct3GAUGAGW=AGCCA

AAGC

UGAGCCC CUGAUGAGGCCGAAAGGCCGAA

AAAGGUC

189 0 1 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 AUCACUU COGAUCGCCGAAAGGC= GUGGGAG CUGAUGAGGCCGAAAGGCCG

GAGGC

GGAGGC

tTL=c

UCCCAG

GM7AGC

GUGUUG

AAAAUC

AAAAAtJ

AAAAAA.

AAAAAAL

AAAAAX

AAAAAX

AAAAA~i

AAAAAPU

AAAAAAU

AAAAAAJ

GAAAAA;

UGAAAA;

Ct7GAALJ UCrJGAAA

CUCUGAA

UCUCUGA

GUCUCUG

CGUCUCU

GUM=CG

AUGUJUGC

UCUGGGC

ACAAAGG

CACAAAG

tUhACACA

CLU.ACAC

AUUAACTJ

MUL17AC

CUUUAUU

GCUUUAU

AAAGCUrJ

GUUGAGA

AGUUGAG

CAGUUGA

GGCAGUU

G CGAGCCAACCGA

CUGGGGCC--AZG-CA

'CUGAtrAGGCCGAAAGGCCGAA SCUGAUGAGGCCGCAAkAG-CCG-AA

UCUGAUGAGGCCGAAAGCCAA

ACUGAUGAGGC-CGAAAC,-CCGAA

CCUGAUGAGGCCGAAAG,-C-CGAA

ACUGAGAr.GCAGCC,-,A ACtGAUGCGGCCGGAAAGCCC--AA

ACUGUGAGCAGGCCGAA

SCUGAUGAGGCCGAAAGGCCGAA

CUGAUGAGGCCGkkAGGCCGAA~

COGAUGAGGCCGAAAGCCGAA

CUGAUGAGGCCGAAAGGCCGAA

CUGATJGAGGCCGAAAGr.CCGA

CUGAUGC--CGAGGC;

LCUGAUAGGCCGAAAGGCCG-AA;

CUGAUGAGGCCGAAAGCCAA

CUGAUGAGGCCGAAAGGCCGAA

CUGAUGAGGCCGX.AAGCCGAA

CUGAUGAGGCCGAAAGGCCG;,AA

CCGAUGAGGCCGAAAGGCCGAAA

CUGAGAGCCGAArCCGA

A

CUGAUGAGGCCGAAACGGCCGJ;

A

CUGALrAGGCCGAAAGGCCGAA CUGAUGAGGCCGAAAGG~ccGAA

A

CUGAUr.GGCCGAAAGGCCGA CUGAUGAGGCCGAAAGGCCGAA

A

CUGAUGAGGCCGAAGGCCC,

A

CUG.AUGAGGCCGAAAGGCCGAA

A

CUGAtIGAGGCCGAAAC,-CCGAA

A

CUGAUGAGGAGCCGAC

CU-AUGAGGCCGAAAGGCCGLA

CUGAUGAGGCGCC-,-A

CUG-AUGAGGCCGAAAGGCCGAA

A

CUGAUGAGGCCGAAAGCCG

AA

CUGAUGAG-GCCGAAAGGCCGAA

A

CUJGAUG-AGGCCGAAAGGCCGAA AaA

UL

AGCCCCAA

AUCACtU

AGGAUCA

AGGTUGGG

AGGCCUGA

ACUCAC-G

AUGGOCC

AGCCaAu AkumtGcc AAUtJCC-C

AUCAAAU

AAUCA;A

AAAUCAA

A.AAAUCA

~AAAAZC

kAAAAAU kA.AAAAA

LA-AAAAA

LAAAAAA

LAAAAA

L.AAAAA

AAAAA

AAAAAA

AAAAAA

AAAAAA

AAAAAAL

CCCCGtJ

GAC

U;GUUGC

,UCUGG

kMGCUG ;GAA~rJ k.GGAAG

:ACAAA

LCACAA

kACA

LCEUAC

Tt7AACE

'CUUUA

GCDuEJ AGCtU

AAAGC

190 Table Mouse ICAM nt. Position 23 26 31 34 48 54 58 64 96 102 108 115 119 120 146 152 158 165 1.68 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 394 420 425 HH Ribozyme Sequence Ribozyme Sequence CAACGGY CUGAUc~zzG-cCGAA GCCCAA

ACCAGGG

AC-AGAG CUGGGCC,--AA- -CAA~c kCCACjC, AW-Ar--A C"-Mt G=GCWAAG:--CGAA

AGAACCA

UGUGGAG C~xUA--CCA~cA

AGCAGAG

CGAccc CUGAI;GACCCCG -a-CCrCA

AUGIGAA

AccacAUAGGC GCcGa-GGCCM

AGUGUGC

CCAGG=t CUAUcGAAG~kGCCGA ACZUcc CCAUCAC CG~ ,GCCGAAAGGCC

ACG~CCC;

GGAGCUAL CUAUAGCCG

AGGCAUG

CUGCUGG CUGAGCGGCC

AG,

GGCCAG CUAGGCCAA-CG

AWCAGAM

CCACAG cuGAuGAocCc or~cc-i Aarzr-A GGGCCCAG CUGAXJGAGGCCGXZCt:

WM

AGGCAGCB, CUGAflGAGGCCcXAG=A

AWACCA

UCCUGGU CtJGAI3GA GCG ACAtUCC GGGCCAG CUAGAGCAAGGCoCGA

AGCAGAG

GGCGz--. C'JGAflGAC---GXUC--

ACGAMM,

AGUGGC CUGAGAGGCCGAAAC-ZCCA

ACACAGA

GUUUUJ CMUGIAGGCCAAAGCCGAA

AACAGGA

GCAAAAC CUGAUGAGGCCGAX CCA ACUUCuG GGGGCAG CU 3UAGCCGAAAGGCCCA

AAGGCUU

(;CAM CO GAUGAGGCCGAAAGGCCA

ACCCACC

GCCAGAG GCCGAAAGCCG

AAGUGGC

GCAAAAC CVGAXJGAGCCGAAAGCCAA ACUUCtJG GG-AGcAA curGAtGAGGccGAAG c.A AcAAcuu AGUtJCUC CUG-AUGAGGCCGGC C AGUCACA UUUGA CUGUGAGGCGAGGCCGAA

AUGGGUTJ

QCCUUCCU Ct3GAUGAGGCCGAAAGCCG

AGGCAGO

CAGUAGA aUGAGAGCCGUGCCGA

AAACCCU

tIh.GCAG CUG-AUGAGGCCGAAGCrA

AGCCCCU

CAGCTJCA CUG-AUGAGGCCGAAGCCG ACAGCUUr GGCEJCAG ctJGAtUGAGGCc AAC,,C

AUCUCCU

GUUCtJCA CGAUGAGCCGAAGCCGA

AGCACAG

CAGUGTJG cuGAuGA~ccGAAGccr-A AuuGGAc tJCAGCUC CtJGAUGAGGCCGMAGlCGAA

AACAGCU

AGCGGAC CtJGAUGAGGCCGAAG CGA ACJGCAC CGGGRJUG CUGALUGCGAAGGCCGAA~C

AGCCAUTJ

GGGCAGG CUGAUGAGGC-CGAA GGCCGA.A

AGGCUEJC

GGGGCAG CUGAUGAGGCCGAAAGCCA

AAGGCUTJ

ACACGGty CUGAUGAGGCCGAAGCUGAA

AUGGUAG

AAACGAA CUGAUGAGGCCGAAGCGAA

ACACGCTJ

AGAtTCGA; CUGALUCGrGGCCGAA GGCCGA

AGUCCGG

CGGG CUjGAUCACGCCAACGCCG.A

AAGUGUG;

Ct7G%-C--G CUGA GAGGCCGA-AGCCCGA

AGGGGTJG

427 CACUGCU CUGAL-GGGCCGAAGGCCGA

;CAGC-LG

450 GCAGGGU C-UGAIUGAG,-CGAA GCGA AflGEUCCU 451 CAAAGA CUGA7GAGCCGAAGGCCGA

AC,-=L-C

456 AGUGGCU CUG~u-AGc-c AG_Cr.G

AGUA

495 ACA.CGGU CUGAL GAGGCCGA GGC-GAA AUGGLEAG 510 CCCCACCG CUACGCGAAGC^ ArGr~k 564 GAGGA CUArA-CGAGCGL

ACC'J,G

592 CCCAJGt CUGAUGAGCGAAC-GCC-G A7,C',=JC 607 CAUGAGA CUGAUGAC-ZCGAAGA A T,7,rZUGCJ 608 GCAUGAG CUGAUGAG,-,--aaGGCCGA

.AJLTLIC

609 GGCAUGA CUG UGAGCCGAAGC~C,,A

AAAUL=

611 G-CGGCAU CUGAUGAGC-CGAACCGA AGAAAUrJ 63 6 CAGCUCA CtJGAUGAGCCAAGCG6 ACAflCUr 657 UCAGCUC CUGA~r=GGCCGAAGCCA

AACAGCU

S..8 c-n UGCC C~AU AGGCCGCCA

AGGCUCG

677 AGGCUGG CUGAUGAGGCCG AGGCCGAA

AGAGGUC

684 AG-ACCG; CUGAC AGGCCG-GA kGCtJGjA 692 AAC-AtCG CtJGAUGAGCCCGAAAG,^CcGAA

AAGUCCC;

693 GCAGGCt3 CUAGGCGAAGCA

AGGEJCCU

696 GAGGcAG cuGAUGAGGCCGAAAGGCCr.A AA6AcAGc 709 MGArGUG MM-~AUCAG^--CGAAAGGC:GA6A

AGCCGCC

9720 AGCt3GAA L-GU-G-CGA~CG6 AGtUUA *723 CGGAGCU C'6jGAUGAGGCCGAA GGCGAJA AA;AGtJU *735 UTCUCCAG M-GAUGACSCCGAALGCCGAA6 At7CUG.G,_ 0 V 38 CCAtCAC CUGAUGAGGCCGu AGGCCGAA AG-GCCCAL So: 765 GGAAGCG.CUGAUG-AGGCCAAAGGCCGAA

ACACIG

GGCAGGA CTJGAUGAGGCCGAAGGCA

ACAGGCC

egos 770 uuccAGG CuGAUG-Ac-ccGArccc-t

AGAA

785 C-GCAGGA Ct3GAUGAGGCCGAAGGCCGAA

ACAGGCC

786 AGGCAC-G CUGAUGAGGCCGAAAGGCCGA

ALCAGC

:.*792 CUtICCGA CUGAUGAGGCCGAAAGCCGAA

ACCUCCA

*:e:794 AGUCC CUG-AUGAGGCCGAAA-GCCGA6A

AGC:CCAG

807 CCAC-GUA CUG-AUACGCAAG%-CCCA

AUCCGAG

833 GGG-UCtJC CUGAUGCGCAGGCCGAA AGCtJEJEG 846 CAACGGU CUGAUGAGGCCGACA

ACCAGGG

851 GCUGGTJA CUGAUGAGGCCGAAGGCCGAA

AGGC;UCC

863 CCAGAGG CUGAUGAGGCCGAAGGCCGA

AGUGGCU

866 GGGCAGG CtJGAUGAGGCCAAGC -A AGGCUUC 867 UCtICCC-G CUGAUGAGGCCGAAGGCCGAA

AACGAAU

869 CEUUGCATU CUGAT-TCAGGCGAAAGGCCGAA

AGGAAGA

881 ACGGGUUj CUGAUGAGCCGAGCCC.A

AAGCCAEJ

885 tJCA6CCUC CUGAUGAC-CCGAAAGGC

ACCALAGG

933 CCAGAAU CUGAUGAGGCCGAAAGGCCGAA

AUUAUAG

936 GCACCAG -UGAUGAGGCCGGCG

AUGAUUA

978 AGUUtGUA CUGAUJGAGGCGAAGGCCGAA

ACUGUTA

980 AAAGUUG CUGAUGACGGGCAA

AGACUGU

986 AGCUGAA CUr-AUCAC-GCCGAAGCCCGA.

AGGUA

987 GAGGC,-UG-A CUGAUGA CCGC-AGGCG.

A.AGTJGU

88a GGA CUGAUG.AG CCCG;AGGCCA

AAAGUUC

1005 UCUCCAG CUGAUGAGGCCGAAAGCCGAA

AUCTJGTJ

1006 UUCCCCA CUGAUGaAGGCCGAAGGCCA

ACUCUCA

1023 CUUCCG1L CUGAUGAGGCCGAAAGGC-CGAA

ACCEJCCA

1025 CCCtUUCC CtJGAUGAGGCCG cGGA AGACCUC 1066 UUAUUUU CUGAUGAGGCGAAGGCCGAA

AGAGUGG

1092 GGCCUGA CUGAL'GAGGCCGAAGCCGA AUCCAGrJ 1093 UUGGCUG CUGAUGAGGCCGAAAGGCCGAA AGG-UCCA7 112)5 UC-AGAA CUGAL-AGGCCAAAGCCA

AGUUGGG

1163 -CAAAAG CUGAUGAGGCCGAAAGGCCGAA

AGCUUCG

1164 AGCAAAA CUGAUG-AGGCCGAMGGCCGAA

AAGCUUC

1166 AGAGCAA CUGAUGAGGCCGAAAfGCCAA

AGAAGCU

'0001172 GGUU U tGAUGAGGCCGAAAGGCCAA

AACAGGA

1200 UGOGGAG CUGAtG-AGGCCGAAAGGCCGAA

AGCAGG

1201 CUGUUCA CrUGAUGAGGCCGAAAGGCCAA

AAGCAGC

1203 ACUGGUG CUGAUGAGGCCGAAAGGCCGAA

AAAAAGU

227 GCAC-ACG CUGAUGAGGCCGAAG~CCGA.A AuGUjACC .1228 AGCAAAA CE7GAUGAGGCGAAGGCCGA

AAGCUUC

1233 CUCUCCG CUGAUGAGGCCGAAAGGCCGAA

AAACGAA

1238 AGGACCA Ct3GAUGAGGCCGAAAGGCCGAA

ACAGCAC

1264 CUUGCAC CUGAUGA GGCCGAAGGCCGAA

ACCCUUC

.1267 UtJCCCCA CUGAflGAGGCCGAAAGGCCCGAA ACUCTJcA .*1294 GGCUCAG Ct3GAUGAGGCCGAAGCCA AEJCLCCtJ 1295 CUGCUGA CUGAUGAGGCCGA AGGCCGAA ACCCCtJC 1306 CAflUCA CUGAUGAGGCCGAAA.GGCCGAA ACtJCUGC *1321 UCCtJCCU CUGAUGAGGCCGAAAGGCCGAA Acccuujc 1334 tJUUAGGA CtJGAUGAGGCCGAA GGCCGAA AUGGGUU *1344 CACUJCUC CUGAUGAGGCCGAAGCCGAA

AGCUCAUJ

1351 UAACUtJA Ct3GAVGAGGCCGAAAGGCCA AcAuuCA 1353 CACCUEJC CUGAUGAGGCCGAAAGGCCA

ACCCACTJ

.:1366 AGtJUGUA CUGAUGAGGCCGAAGGccGAA AcuGuTJA 1367 AGGUGGG CtJGAUGAGGCCGAAAGGCUA AGGTJGCrJ 0-1368 AGAGUGG Ct3GAUGAGGCCGAAAGGCGA

ACAGUAC

1380 CCACCCC CJGAUGAGGCCGAAGGccGAA AuGGGCA 1388 AGCCACU CUGAUGAGGCCGAAAGGCCGAA AGUCtJCC 1398 GUUCUGtJ CUGAUGAGGCCGAAGUCGAA

ACAGCCA

1402 AGTJUCTJC CUGAUGAGGCCGAAAG-cGAA

AAGCACA

1408 CCUCCCC CUGAUGAGGCCGAAGCCA

AUCEC

1410 CCCUCC CUGAUGAGGCCGAAGGCCGAA. AGACCtJC 1421 ACAAAAG CtJGAUGAGGCCGAA GGCCGAA AGGGGG 1425 CUCUACC CtJGAUGAGGCCGAAAGCCGA

AGCAGU

1429 CAGGGGC CGAUGAGGCCGAAAGGCCA

AUAGAGA

1444 UCCtJCCU CUGAUGAGGCCGAGCGA AGCCUUjC 1455 UCCtJGGU CUGAUGAGGCAACU

ACAUEJCC

1482 GGGAGCA CUGAUGAGGCCGA G~CcGAA AACAACrJ 1484 CAUGAGG CITGAUGAGGCCGAAAGGcrAA

AGAACAG

1493 GUUCUCA CUGAUGAGGCCGAAAGGCCGAA

AGCACAG

1500 GGACCAU CtJGAUGAGGCCGAA GGCCGAA AtUtJCAU 1503 G--'UGJCAU CUGAUGAGGCCGA-,A-CGA

AUAGUCC

1506 CGGUUAU CrAUGAGGCCGAAAGGCC,A

AACAUA

C. 193 1509 ACACGGEJ C UA~jGGCCGAAAGGCCGAA AUGGUTA 1518 CGC-CUGG CUGAAUGAGGCCGA AGCCAA ACraUjGA 1530 CCAGAAU CUG GAGGCCGAAaGCscGL

_AUUAUAG

1533 GGCCCAC CUGGGc-C~AAGcCGAAL ArX= 1551 AGCUGCU CU -AUAGGCGAAAGCCGAA

AGGCPAUG

i559 AGGUGGG CUCAU-ACGCC-A.AAG-CGAA a.G,-UGCU 1563 GG%-UUAUA C-,-LGGC,%AA=-.A

ACIJTAA

1565 GCGGUU tJGX;AC--Cr AC-cA 1567 UGGCGGJ CUC-AUrGGC-AAGGCCG;.

A;LCA

.1584 ALMhXCCU CU'UGAGGCCCLA CC.CG AUCUUUC 1592 UAACUUG CUGAUGAGGCcGAA ,CC AUAUCCU 1599 CCUUCUG M7.GAUGAGGCCGAAAGGCCGAA

AACUUCGU

1651 GCUCAGG CUGAUGGCC AGCCGA AGUG 1661 CAAAGGA CUGAXGAGCCGAAAGGCCC-A AGGUrtjjC 1663 Ut3CAAAG CUGAUGAGCCGAA -GCCGAA AAAGG.-, *1678 CCGW =CAGGC GAU AGGCCG A A AGGUCCU 1680 CCAGAGG CUuGAGGCCGAAAGGCCAA

AGUGGCU

1681 GCCAGAG CUGAUGAGGCCC-AAAc r: AATGGG 1684 ACAGCCA CUGAflGAGGCGAAAGCCA

AGGAAGU

*1690 AGAUCGA Ct)GAUGGGCCCG AG,-CGAA AGrJCCG 1691 AAGAUCG CUGAlUGAGGCCGAAAGG-CCGAA

AAGV=CG

:1696 CC-ACCC CLGAUCGGCC~j-GGCC-A

AUGGGCA

1698 CUCCAGG CLMAGAGCGAAGGCCG

AUAU=~

00 1737 GCUGGUA CUGAIUGAGGcCGGG-_)CCGA kAUCUC o..1750 UGAGGCUG CUGALMAGGCCGXAGf-CGAA

AGCCGCC

o.1756 GGGCAGG CUGAIr.AGG-CGAAAGGCCGAA AGGCUUC 1787 UGGGGALC CUGAUGAGGCCGAAAGCCCGAA AUGUCtJC 1790 AUIUhGAG CUGAUGAGGccGAAGGcCGA

ACAAUGC

*1793 tTCCAGCC CUGAUG.AGGCCGAAAGGCCGA

AGGACCA

01797 UUUAUGU CtJGAtJGAGGCCGA AGCCGAA ACUGGt3G 0: :1802 t7CUCCAG Ct3GAUGAGGCCGAAGGCCA

AUCUGGIJ

1812 GGCCU7GA CUGAUGAGGCCGAAAGcCCGA AtJCCGU 1813 UGAGGGU CUGAt GAGGCCGAAGGCGAA

AAUGCTJG

%1825 GCAGAGG CtJGAUGAGGCCGAA GCC-AA JAGCGUGG 1837 GGAGCUA CUG-AtGAGGCCGAAA-GCCA

AGGCAJG

1845 GGUIGGC CUGAUGAGGCCGA AGGCCGAA AGGCTJCG 1856 AAGAUCG CUGAUGAGGCCGAA GCCGAA AAGUCCG 1861 UACUGGA CUGAUGAGC-CCG ACCCCGAA

AUCAUGU

1865 JUGAGGC CUGAUGAGGCCGAAAGGCCGAA

ACAAGUG

1868 UUUAUGU CUGAUGAGGCCGAAc-GCCC-A

ACUGGTJG

1877 AGCUGCEJ CUGAUGAGGCCGAAG-CCGAA

AGGCAUG

1901 GUCCCUU CUGAUGAGGCCGAAAGGCCA

AGTJUUUA

1912 ACtJGAUC CUGAUGAC-GCCGAAAGGCCW.A

ACUJAU

1922 UAACUtJA CUGAUGAGGCCGA GGCCGAA ACATJucA 1923 GAUACCU CUGAIGAGGCCGAGCcGA A~~c 1928 CUGGUA CUJGAUGAGC-CGAAC4CGAA ACtJTA 1930 AGCUGGU CUGALGAGGCCCGA GC-G-A AA~ 1964 MGGGAC CUb~'CGCCCAACGCC-M ktCtC 1983 UAACUUG CUAUAC-GCCGAAAZ-CC-GCCk AUAtUcCU 194 1996 GGCUCAG CUGAUGGCCAGC, A~jUCCT 2005 GitjUCCGC CUGAtGAGGCCGAAG.-CCG

AGCJCCA

2013 tUhCUCAA CUGAL'GAGGCC-.AAGCCGAA

AAAA

2015 CCACCCC CUGAUGAGGCZAA;LGCC" AUiGGGc- 2020 cr-r-AGAA CUGAMGAGC::AAG -CAA uACCA 2039 CCTJCrGC CUAGGC:AAGCA

AGCCAC-C

2040 CCUCCAG Ct3GAtGAGCG -Gc-ca AGG-uCA--G 2057 GGAUGUG CU;IGGC=AA-.----

AGAC

2061 ACACG~TJ Ct3GAUGAGGCCZ GA CCAA AUGGMJG 2071 CtJGAGGC CUGAUGAGGCCGA GC-A ACAAGtjG 2076 LVLGCUCU CUGUGAGCC-kA CGAA AGGc uAc *2097 CAUCAAG CUGAt7QAGGCCG.A AGGC-C,- AGA.GUtG *2098 CGGGGGG CUGAGCAGGCC CGAA AAGUGJ 2115 AUCCtJCC CUGArGGCGAAAGG c,-A AGEIGGC 2128 CYCAAUA CUGAUGAGGCCGAAGGCcGA AUjAGEJG **2130 GAGGCAG CJG;LUGAGGCCC-AA~CGAA

AAACAGC;

2145 CAUCAAG =UAUGAC-GCCG- GGcG;

AGAGUUG

2156 UAA~kAACUGAUGAGGCCGAAAGGCCG.AA C u A 215 ALUAUA CGUAGGX

GC,.AAAJA

2159 tAhAUAAU CUGAUGAGGCCC AAGCGAA

ACACAA

*2158 AUAAUAA CUGAUGA GCAAGC-A AAUArt7C 2159 CAAUU CUGAUGAGGCCGA~cGA

AAAAU

2160 AAU)AUA CUAGGCGAGcz AAUAcA *2166 AAtUhGAG CUGAUC-XGWC-AAGGCGAA

AUGAAGU

2167 AAUUAAU CUGALUGAGGC-AGGCCGAA

AAUACAU

2170 CUAAAUtJ CUGAUGA-CC- caAA AtJAAAUA 2171 GGGAGCA CUGAUGAGGCc-AA 'c,-,GcAA AAcAAcu 2173 CUGGUAA CUGAUGAG~cCGAA cc-- ACcuA 2174 GCUGGUA CUGAUGAGGCCGAGCUA

AACUCUA

2175 AGCUGGU CUGALUGAr.GCCGAAAGCCC-A

AAACTJCT

2176 UAGCUGG CUGAUGAG CCGAAAGGC

AAAACUC

2183 CAAUZAAA CUGAUGAGGAAC -GCCCGAA AGCUGCrJ 2185 CUCAAUA CUGAUGAGGCCCGAA CCA AUAGCtJG 2186 ACUCAAU COGAUGAGGCCGA AGCGAA AALUAGCtJ 2187 tUhCtCAA CtJGAUGAGGCCGAAAGGCCU

AAAUC-C

2189 GGUACUC CUGk=GGUAG CGAA AUAAAUA 2196 CAUCAAG CUGAUGAGGCCGAAGGCGA

AGAGUUG

2198 AACAUAA CUGAUGAGGCCArGACCAA

AGGCEGC

2199 AUAAACA CUGAUG-AGGCCGAA GGCCG-AA AAGkGGC 2200 CUUG-CAU CUGAUG-AGGCCG AAGCGAA AGGAAG A 2201 GCCGAC-A Ct3GAUCAGGCCC-G%-.CCGAA

AAAACUJE

2205 UCAGGCC CUG-AUGAGGC CGA.CA

ACAUAAA

2210 AGCCACU CUGAUGGCAGGCCGAA~AGUCtJCC 2220 AGAGAAC CUGAUGA GCCGAACGAA

AUGCCAG

2224 GGAUGGA ClJGAUG-AGGCCCGMAGGCCG.AA

ACCUGAG

2226 GCGGCCTJ CUGAUGAGGc-AA GCG-;

AGAUCC_;

2233 CCUCCAG CUGAUGAGC -r7-.C-CC:G- AGc-tJCAG, 2242 GGUJCCGC CUGAuc.A.CCCc

;ACUCC:A

195 2248 UCGG.UG CUGA~XGrGCC-.2 GAA AtIGGATUA 2254 UCAGUGU CUAGGCCAACCA

AAUUGGA

2259 CACCGUG CUAGGCGAAGCA

AUGGAU

2260 GCACCG;U CLUcrGAGGCCG GCGA AAUGGA 2266 UCC--UGt3~GAVGAGGCCG AAGC.A

ACAUUCC

2274 UCtJCCA CUGuNUGAGGC-CGAAAGGCCGA

AUCUGGU

2279 CUUGCAkC CUGAUGIr-AAGG---%

ACCCUTJC

2282 CAGCt3CA CUAr.c%,cAAGC~ ACAGCrUU 2288 AGGCCAU CUGAUGAGGCCGAAGC~r- ACrUUAj 2291 %GCAZ7AG CUC -Ar-GCGcckAGCCG

ACCACIJG

2321 CCCAflG CUfGAGCCGAAflGCGAA AUCUt3UC 2338 CAGGCAG CUkGAGGGAGGCCGGAA Axr=CA 2339 CAAAG CUGAUGAGGCCGAAGCCA AGGL=Cc 2341 AGGCUGG CUGAUGAG~ccGAAG CGL AGAGGTJC *2344 GCtJGGAA COGAGAGCCGa~~kGCC AE7CGAAA *2358 CUGCW-A CUAGGCGAAGCA

AGCGG

2359 t3CGUC CrJGAUGAGGCCGAA -CGA AACA 2360 UUCAAAG CUGAI7GAGGCCGAAGCGA AAAGGUrJ 2376 UCAGAAG CUGAfGAGCCAGCC;

ACCACCU

2377 CtJCAGAA CUGAUGAGGCCGAAGCCA

AACCACC

2378 CAGLVZGA CUGAUGAGGCCGAAAGGCCGL

AAACCCU

2379 CUtUhflA CUGAUGAGGCCGAAGGCCG

AAAAGCA

2380 GC-CGACA C GUGAGGCCGA CC,-;L AAAACU 2382 GGGGCA CUGAUGGCGAGC ZA AGAGAA *2384 UUGUGUJC CUGAUGGCGAAAGcGCG.,

A~CUGC;A

2399 GUCCACA CtJGAUGAr-GCCAA -C-A AGUG=~ 2401 CAGCtJCA CUGAUGAGGCCGAA GCGA

ACAGCUU

2411 GCAUCCU CU~GAGG-CAGCCG LA ACCjAGU .2417 ACGUAtJG CUr-AUGAGGCCGAA GCGAA~ ACCAUUC 2418 GGCCUGA CUGAUGAGGCCGAACCGAA AtJCCACTJ 2425 AACCCUC CUG~AUGAGGCGCCGA

ACCU

2426 AAACUCU q~-UAG.CAAGCA

AAUUA

2433 GCUGGtA CUGAUGAGGCCGAA GGCGAA

AACUCUA

2~434 AGCtJGGU CtJGAGAGrGCCGAA GGCCAA

AAACUCU

2448 GGGCAGG CUAGGCGAAGcA

AGGCUUC

2449 GGGGCAG CtGAtGAGCCGAAAGCCGA

AAGU

2451 AGGCAGG CUGATJGAGGCCGAAGCCA

AACAGGC

2452 GAGGCAG CUGAUGAflGCCGAA~cA AcG 2455 GGGCAGG CUGAEJGAflGCCGAAGCGA

AGGCUTUC

2459 GGGGGGG CUGAUGAGGCCGAAGGCCA AGUrJGG 2460 CGGGGGG CUG3UGAUcCAnAAccG)AA AAGUGtTG 2479 GCUGGUA Ct3GA AGGCC G c CCA AGGUCtJC 2480 GGAUCAC CUGAUGAGCcCGAAGGCCGAA

ACGGUGA

2483 GGtJGGCU Ct3GAUGAGGCCGAAGcA AcuG 2484 GACUGGU Ct3GAUGAGCCCAAAG,-CCG

~AAA.AAA

2492 AGGUGGG CUGAUGAGGCCGAAGCA

AGJUG

2504 ACAAAAG CUCAUGACGCCC--GCCC-A

AGGUGGC-

2508 UGGGAtJG "UCAiGGCa~ -c G;cck A.u~cA~ 2509 CtYGUr-A CU AC-GC--S .C-GCCCA ACUCUA 196 2510 GCUGGUAM CDG-At AGGCCGAAAG,,CCGAA

AACUCUA,

2520 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA, AcAAAAG 2521 UGAGGU C~rGAUGAGGCCC-AAAG~CCGAA

AALTGCL-G

2533 GAUACCU CUGulUGAGGCCCGAAAG-,-CGAA A~CaZCA, 2540 CALCAGC%- CUGAUC-GCCGAAAGG-_CGAAJ ACLTCU 2545 AGGACCA CUC-AUGGG-CCAAAc-GCCGA

ACACAC

2568 UUUC-Ar-A CUG-AU-AGCCGAAC-,GAA

ACUUCAC

2579 CAGGCCA CTAGG,-GAcCA AACUU 2585 AGAGAAC CUC-ALGAC-GCCGAAAGGCCGA ATJGCrC.G 2588 AUt3GAG c AGAGCCGAAAGGCCrA

AAAUGC

2591 AGGAGCA CLGAU-AGCCGA A~C.CGA AGAACCA.

2593 GCAC-AC-C CtUGAGGCCGAAGcCGA

AGAAG

*2596 CAUUGGG CUGALMAGGCCCGAAAGGCCGAA

ACAAAAG

V 2601 AAACGA6A CtJGAUlGGCCGAAAGGCCGAA AA C-,U *2602 GGGAUC-G CUGAT3AGGcCGAAAGGCCA; AfCUCGA *2607 CCAGGtJA CUGA.UGAGGCCGAAAGGCCA

AUCCGAG

2608 CACAGCG CUC-AUGAGGCCGAAAGG CCGAA. ACUGC-UG 2609 UCCtJGGtJ CUGAUGAGGCCGAAGCCA

ACAUUCC

2620 GCALGG-.rJ cUAUGAGGCCGA '-CGAA AGGUCCU *2626 GCUGGAA CUG At7GCGAAA-r-CA x~.A 2628 AG=~JC CUG-AtGAG~CCGAAGCcGA

AGGG

*2635 AGGACCG CUGA6T A~C.-CGAAAGGCCGAA AGCUGAA 2640 GGCAGGA CUGAUGAGGCCGA AGCCGA ACAGGCC **2641 CTJGCUC-A CUGAUGAGGCCGA GGCGAA AGCUGGG 2642 GAGCG C-UGAGAGCGAA-C-C-GAAG

AAACAGG

2653 GCAUCCJ CUAGGCGAG-CA

ACCAGUA

2659 CUUGCAC CUGAUGAGGCCG AGCGCCC-NA ACCCUUC 2689 CCUCGGA CUGAUGACGGCCGA AGCCCGAA ACAUU'AG 2691 GGCCE7CG CUGAUGAC-GCCG ACCGAAL A-AC-ATjU 2700 GGGCAC-G CUGA6UGAGGCCGAA .C-CGAA AGGCUUC *2704 AGGCUGG CUGAUGALGGCCGAAAGCCGAA AGAGGUc 2711 CUGCUCGA CUG-AUGAGGCCGAAAGG,-CGAA

AGCUGGG

2712 CCCUCC CUGAUGAGGCCGAAAGGCCGA AGACCUc 2721 CUUGCAC CUGA6UGAGC-CCGAGGCGAA ACCCUtJC 2724 GCACACG CUGAUiGAGG;CCGAAAGGCCGAA

AUGUACC

2744 Ct3GCACG CUGAUGAGGCCGAGCGA

ACCCACC

2750 GGUACtJC CtYGAUGAGGCCGAAA-GCCGAA

AUAU

2759 AG-AUCGA CUr-AUGAGGCCGAAGGCGAA

AGUCCGG

2761 GCAGC-GU CtJGA6UGAGGCCCGAAAGGCCGAA

AGGUCCU

2765 AGCGGCA CUGAUGAWXAGGCCGA A AGCAAAA 2769 CCUGUUUJ CUr-AUGAGGCCGAA GCCGAA ACAGACU 2797 GGACCAU CUGAUGAGGCCCGAAAGCCA

AUUEJCAU

2803 CGCCYGG C 3GAUGAGGCCGAA GCGCA ACCAUGA 2804 CUGCACG CUGAUGAGGCCGAA GGCGAA ACCCACC 2813 GGGTUCAG cuGAuGAGGCcGAA GGcrAA AccGGAG 2815 AAAGUTJG CUC-AUAGG--CCGAC-GC AZACUrGUJ 2821 CCUCCAG CUACGCCAG -CCCGA AGGUCAG 2822 AGtUCC GU Cr-!C-;-CCC Ar;CGCCG AGC-C=CC 2823 MC-CC- C GA;GWCMCGC CG 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 AUC-AUUA CUUGAGGCCGAAAGGCCA AGtICCAG UCAGAAG CUG!4UGAGCCG AGC ACCACCrJ CAGGCAG CUGAUGAGGccGAAAGCA rAcrJA GGUGGCU CUGAUGAGrCCGAAAGGCCGA

ACAUGG

AACAUAA CUGAUGAGGccGAAAGGCC-,A AC-GCtJGr UCACAGtJ Ct3GAUGAGGCCGAA GCCGAA

ACUUC;

CUUGGCU CrJGAUGAGCCGAAACCCAA

AAG,-UCC

GUGAUGG CUAUGAGCCGAAG -CGAA AGCGGAA AAGAUCG CUGAUGAGGCCGAAGCCzJ

AAGUTCCG

AAAACUC CUGAUGAGGCCGAGCGA

AAAUUAA

AAUGAG CUTGAtGAGGccGAACA;Lc AtJGAAGU CAAUAGA Ct3GAt3GAGGCGAAACCCGA AAtJGAAG UAAtJAAA CUGAXUGAGGCCGAA GCCCA ACAtJCAA AAAUUAA CUAUGCCGAVCCA AAAMVhCA AGCAAAA CUC-AUGAGG--CGACCA AAGCUtJC AGAGCAA CUGAUGAGGCCGAAGG-AA

AGAAGCU

AAAUEUA CUAUAGCCGAAAGG,-CGA

AAAUACA

AAATJUAA CUAUAGCCGAAGGCCA AAA7jACA GICAMM3 CUGAUGAGGCCGAAGCCA

AGAACAA

tiGCA CUGAUGA .G.CAGGC CG A AGAGAAA CUTJAUGA CUGAU G~AGGCGACc

AAAAGCA

tJCtAAAU CUAJArCCAAGCA

AAUAAAU

CE7CCGGA CUGAUGAGGCCGAAGCCA

ACGAAUJA

UCUCCGG CUAGGCGAAGCA

AACGAAU

Ct3CUCCG CtJGAtGAGGCCGAA GGCGA AAACGAA CGACCCU CUGAUGAGGCCGAAGGCGAA

AUGAGAA

~CCGA CUAGGCGAAGCA

ACCUCCA

CCCtUUCC CtJGAUGAGGCCGAAAGGCCGAA AGACcCc UGGGGAC CUGAUGAGGCCGAAGGCCr-AA AUGYCt3C GCAGAGG CUGAUGAGGCCGAAAGCCGA

AGCGUGG

CACAGCG CUGAt7GAGGCCGAAGGCCGAA

ACUGCUG

UGACACA CUGAUGAGGCCGAA GGcGAA AGucACU ULTGAUtUC CUGAUGAGGCCGAA GGCCGAA

AAGGAAA

AGEJGGCU CUGAUGAGGCCGAAGGCCGAA

ACACAA

AA.UUAAU CUGAtJGAGGCCG AGCCA

AAUACAU

CEUUUtAUU CUGAUGAGCCGAAGGCCGAA

AUUCAAA

.CCUCtJGC CGAUr-AGGCCG AGGCCGAA

AGCCAGC

AAAAC!3U CtJGAUGAGGCCGAAGGCCGAA AJGAUtJ GCTJGGUA CUGAUGAGGCCGAAG CGAA AACUCUA AGUAGAG C 3GAUGAGGCCGAA GGCGAA

AACCCUC

CAGCUCA CGA3GAGGCCGAAAGA

ACAGCUU

GGCAAUA C GAUGAGGCCGAAGGC

AGAAUGA

S

Trable 6 H-uman ICAM Hairpin Ribozyme/Suibstrate Sequences nt.Hari ioyeSqec PositionHarnRboyeSqnc 86 343 635 6 5 782 920 1301 1373 1521 1594 2008 2034 2125 2132 2276 2810 GGGCCGGG AGAA GGAGUGCG

AGAA

CCCAUCAG

AGAA

GCCCUUGG AGAA UGIJUCUCA

AGAA

AGACUGGG

AGAA

CUOCACAC AGAA ACAUUGGA

AGAA

CCCCGAIJG

AGAA

AUGACUGC

AGAA

CUGUUGUA

AGAA

ACCCAArJA AGAA UUCUGUAA AGAA GGIJCAGUA AGAA GGGUUGGG AGAA ACCUIGUAC AGAA

GCUG

GCG;C

GCAG

GCUC

GCCC

GCCG

GCUG

GUGG

GCUA

GUAU

GCAA

GUG

GCAG

GUAG

ACCAGAGAAACACACGUUGUGGUACAUJ!ACCUGGUA

ACCAGAGAAMCACACG[J[JIJQGI AC/WIJACCIJGGtJA ACCAGAGAAAcAcAGUUGUGUACAUUACCUGGUA ACCAGAGAACACACGUUGUGUACALUhACaJGUA ACCGrAGAAACAC-ACG1JUGUGUAr-AUUACCUGGUA ACCAGAGACACACGUUGIJG~uACAUUACCUGGUA

ACC-AGAGAAACACACGGUGUACUACCUGGUA

ACCAGAGAAACACACGUUGTJGUUAC~cuG~uA

ACCAGAGAAACACACGUUGUGUACAUACCUGGUA

ACCAGAGAAACACACGUTGUGUACAUUACCGGUA

ACCAGAGAMCACACGUJuGACAUU!ACCGGUA ACCAAGAArACACLGGJJAACCUGUA

ACCAGAGAAACACACGUUTGUGUACAUUACC.JGGUA

ACCAGAGAACACACGGUGUAJuAccuG~uA ACCAGAGAAcACACUUGG~uACUUACCUGA ACCAGAGAACACACtG1J1GUGGUACAUUIACCUGUA

ACCAGAGAAACACACGUGUUACAUUACUGUA

Substrate CAGCA GCC CCCGGCCC GCGCIJ GCC CGCACUCC AAACU GCC CUGAUGGG CUGCG GCC CCAAGGGC GAGCU GUU UGAGAACA GGGCU GUll CCCAGLJCU CGGCU GAC GLJGUGCAc3 CAGCA GAC UCCAAuGu CCACU GCC CAUCGGGG IJAGCA GCC GCAGUCAU AUACA GAC UACAACAG UUGCU GCC UAUUGGGLJ CCACA GAC UUACAGAA CUGCU GUC UACUGACC CUACTJ GAC CCCAACCC GUACA GUll GUACAGGCU CUGCA GUC UUIGACCiUU AAGGUCAA~ AGAA GCAG 9 Table 7 Mouse ICAM Hairpin R ibozymne/S ubst rate Sequences nt.Hari ioyeSqec PositionHariRioyeSqnc Substrate 76 164 252 284 318 447 804 847 913 946 1234 1275 1325 1350 1534 1851 1880

GGGAUCAC

UGAGGMAG

UCAGCUCA

GCACAGCG

AAGCGGAC

AGAGCUGG

UCUCCUGG-

UCUACCAA

AGGAUCIJG

AAGUUGUA

CCCAAGCA

AUIJUCAGA

UGCCUUCC

CCCCGAUG

ACAUAAGA

GUCCACCG

AGAAUGAA.

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GUGA

GUUC

GCUUr

GCUG

GCAC

GCGG

GCAU

GUGG

GCUA

GUUA

GUCU

GCUG

GCAG

GCAG

GCCA

GUAG

GCGU

ACCAGAGAAACACACG1JUGUGGUACUUACCJGGUA

ACCAGAGCACGUUGUGUACAUACCUGGUA

ACCAGAGAACAACGUUGGGUACAUACCUGGUA

ACCAAGAAACACAGUUGUACAACC~GGUA

ACCAGAGAAAACGUUGUGUACAUACCJGGUA

ACCAGAGAAICAACGUGUGGUACAUACCJGGUA

ACCAGAGAAACCACGUUGGAJ1JACCJGGUA

ACCAGAGAAACACGJJGUGUAUACCGGA

ACCAGAGAAAACACGUUGUGGUACAUACCUGGUA

ACCAGAGAAACACACGUJGGGUACAUUACCJ)G.UA

ACCAGAGAAACACACGUUGGGUTAAUUACC?1GGUA

ACCAGAGAAACAACULGGUAUACCGUIA

ACCAGAGAAMCACACGUUGUGGTJACUUACCGGUA

UCACC

GAACU

AAGCU

CAGCA

GUGCA

CCGCG

AUGC

CCACIJ

UAGCG

UAACA

AGACG

CAGCA

CUGCA

CUGCU

UGGCA

CLJACA

ACGCU

GUU

GUI)

GUI]

GUC

GUC

GAC

GAC

GCC

GAC

GUC

GAC

GAC

GAC

GCC

GCC

GCC

GAC

GUGAUCCC

CUUCCUCA

UGACuGA CGCuGuOC

GUCCGCUU

CCAGCUCU

CCAGGAGA

UUGGUAGA

CAGAUCCU

UACAACUU

UGCUUGGG

UCUGAAAU

GGAAGGCA

CAUCGGcG

UCUUAUGI]

CGGUGGAC

UUCAUUCLJ

Table 8 Rat ICAM Hairpin Ribozyme/S ubst rate Sequences n t.Hari ioyeSqec PositionHariRboyeSqnc Substrate AAAGUGCA AGAA 59 84 295 329 433 626 806 849 915 1182 1307 1357 1382 1858 1887 2012 2303 2539

GGAGCAGA

GGGAUCAC

GCACAGUG

AAGCCGAG

IJUCCACCA

CAUUCUUG

UCUCCAGG

UCCACUGA

AGGGUCUjG

ACCUCCMA

AUGUAAGA

UGCUUUCC

UCCCGAUA

GCCCACCA

AGAAGGAA

GAGUUGGG

AGACUCCA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCAG

GCALJ

GCGA

GCUG

GCGU

GCGC

GUGA

GCAU

GUGG

GCCA

GCAG

GCUG

GCAG

GCGG

GUAG

GCCU

GLUGU

GUG

GCUU

ACCAGAGAAACACACGUUGUcYGUACAUUACCUGA

IACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGUAhTJACCUGGUA ACCAGAGACACACGUGUAJuACCUGGUA ACCAGAGAACACACGUGU~uACAUUACCUGGUA

ACCAGAGAAACACGUUGGU~UACCU.JGUA

ACAA -CCCUGGUcu~CGU ACC-AGAGAAACAcAcGuuTJGu1JAuQJAccuGuA ACCAGAGAACACGUUGUJ~AuAccUGUA

ACCAGAGAAACACACGUGEJAAACCUGGUA

ACCAGAGAAAcCACGUIJGTJIAJ1ACCUGGUA ACCAGAGAAACACACGU

JJG~AUACCUGGUA

ACCAGAGAACAACGUGGGUACAJACCUGGUA

ACCAGAGAACACACGIUGUGUAAUACC1JGGJA ACCAGAGAA1ACACACGUUTGUQ1AAUUACC.JQJUA ACCAGAGAACACAcGUUGUJAAJACCUGGUA

ACCAGAGAACACACGUGGAUACCUGUA

ACCAGAGAAACACACGUUGGGUACAUUTACUGGUA

ACCAGAGAAACACACGUUGUGUAAUUTAQJuG~A

CUGCU

AUGCU

UCGCC

CAGCA

ACGCA

GCGCU

UCACLI

AUGCU

CCACU

UGGCG

CUGCG'

CAGCA

CUGCA4

CCGCUI

CUACA

AGGCU

ACACrJ

CCACA

AAGCU

GC(

GCC

GUL

GAC

GUC

GCC

GJUU

GAC

GCC

GAC

GCC

GAC

GCC

GCC

3CC 3PC Juc 3cc ;ui

UGCACUIUU

UCUGCUCC

IGUGAUCCC

CACUG;UGC

CUCGGCUUI

UGGUGGAA

CAAGAAUG

CCUGGAGA

UCAGUGGA

CAGACCCU

UUGGAGGU

UCUUACAU

GGAAAGCA

IJAUCGGGA

UGGUGGGC

UIJCCUUCLJ

CCCAACUC

UGGAGUCU

GUGGGAGG

-CCJCCCAC AV," 201 Table 9: Rat IOATM HH Ribozyme Target Sequence p.

nt. EN Tar~get sequence Position U1 GAIUCCAAU U CkAMCUGA 23 GCUGACCU C CUUCUCUA 26 GAACCG=~ C UUCCUCU 31 CCUCOGCUT C CUGGUCCU 34 CUGAAGCU C AGAMhXMC CtJCAAGGU A CAAGCCcc 48 GAGAACCU C GCtMGG 54 CCCCGCCU C CCUGAGcC 58 CCGGCCU U MGCUCCC 64 CAAUGGCU U CACCCGU 96 CCUCUGCU C CUGGUCCU 102 CUCCMGU C CUGGUCGC 108 GGACUGCU U GGGGAACU UCCUACCEU U UGUUCCCAL 119 GACACOCU C CCCAACUC 120 GMXUGGLn C CCCGGGC i46 CCAGACCU U GGMACUCC i52 ACCCGGCU C CAcCCWA 158 AfUUCU3 C ACGAGUCA 165 UGACG A CUUCCCCc 168 GDGcco7U C CUGC=CcG 185 GGGUGGAU C CGUGCAGG 209 CAGCCCCU A ADCDGACC 227 GAXCCAAGU A ACUGUGAA 230 CAAGCOGU U GUGGGAGG 237 CUGAAGCU C GACACCCC 248 GGCCCC A CCUEMGG 253 CACUGCCU C AGUG= 263 GAGCCAAU U UCtUCAG 267 GAAGCCUU C CUGCCUCG 293 GAAGCUCU U CAAGCJGA 319 CGGAGGAMU C ACAAcGA 335 ACUGUGCU U UGAGAACJ 337 UGUGCChU A UGGUCC 338 AAGCUCLuU C AAGCUGAG 359 CACGCAGU C CUCGGCLrj 367 CAAUGGCJ U CAACCCGU 374 UUACCCCU C ACCCACCJ 375 AGAAGCCU U CCUGccuc 378 ACCCA.CCU C ACAGatjU, 386 CGCUGJGU U tXIGGAGCrj p nt.

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 GCACCcCU

UCCC,,GU

AAGAACCU

GGU-MCUU

CUCGGC-IuU

GCC-ACCAU

GOUGrCU

GGGAGMW

-ACCAAU

AGCCAAUU

CCAAUUUC

CAAUUTU C UCACUGU C GAACtJGCU

C

CCCCCU C AGGCAGCJ

C

CCAGACCU U CGGACUUU

C

GCCUGUUU

C

CA~CInUL A CUACAACU U CAACUUtU C CUCCUGGU c UCCtJGCU C ACUG3GCU

U

UJCUUGU-GU U CUIUGUGrJU

C

AGGCCUGU

U

GGCCUGU Uj CUCCM3GU C UCCt3GC=U C1 GCLTCAGAU A CCJGGG.GU u CJGACAGU

U;

3CUCACCU U :AAUGG;CU U C :CAUGC=t

CC

U CUGACA C CCACGr U C-GCCACC U AAAAJACCA C AUCtri'- C Lt-c-A C ACOGUGMA C CGtJGGGA U CCXACCAC 7 LUCL'AUGC

UCAUWGCJU

AUGCA

CAAICAAUG

UUCCUCD

CC-AGCGCA

CGA.CO

GGAACUCC

GAUCLUC

CCGCCUCU

CCCCUCAC

LMUCAGCUC

AGCE3cCCCA C-lG-UCGC

GC-GGUGGA

UGAGAACU

CCCt3GGAA CL-taGGAAG UCCUGCCtY

CC"UGCCUC

CUGGUCGC

IC'GAAGCUC

j;ACCUGGA

"CAACUA

LUUUAXUG

);AGCAGCU

:AACCCGU

7t7CUGCACA 13Target Sequence 202 .*see: 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 i092 1093 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 "334 1344 1351 1353 1366 1367 1368 1380 .3 88 1398 1402 1408 1410 GACCA=C C CCCUz= CUCUUCCLT C UUGCGAAG AAUGGCUU C AACC=~G GACCAAGU A ACUG~rAA TUGGlU C GOUCCCAG GCAGAGA.U U UUGUGOCA UUGAGAADT C MWCAACUU GACAAlU A CAAZUOU CtkCAACU U UUCAGCU UCAAC-.U U UCAGCUCC ACAACUULT U CAGCUCC UUCMM~U C GUGGCGOC G3GGGAGU A UCACCAG CCGGAGGU C UCAGAAGG GGAGGUCU C ALGAAG=G CCrkCcu uJ GUCCC AGAGGGMu C UCCCO AGGGAAU C CAGCCCCU CCCCAACt3 C UUGUA ACGACGCU U CUUUWU CA~CG=r C DUUUGCuJC ACGCUUC U VuGCUCUG, CUUUGCU c uGcGGr, AUCCAADU c AcAcrJGAA UUGGCU C UCCAAG G-GGCOUCU C CACAGGUC UtJGGAACU C CAnGtMCU C-GGGCUU C GUGAU=G CUCCOGG3 c CVGGUCCc UGUGC~kU A UGGuccri GGAAAGAU C AUAC'G= GUCACUGU U CXAAG CAGAGAUU UJ UGUGTUCAG AGAGGGG;U C UCAGCAGA AGCAGACrJ C ua~CAuc AACAGAGUT C UGGGGAAA GMAUUCG U CCCAGAGC UCGGUGC C. ACGMUC UCAGGcc A AGAGGAcUy tMGCAGC C AACAA=G AGGUAC U CCCCCAGG GG~tkCUU C CCCCAGGC G-AtGGWGU C CCGCUGcc CUGCCUAU cC GAX73r UGGAGACcJ A ACUGGAUG, CUGGCUGrJ C ACAGGACA, CUGUGCLJU U GAGAACrJG tUtCGUGAU C GUGGCGUC' CGAACUAU c GAGuGGAc 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 1518 1530 1533 15951 1559 1563 1565 1567 1584 1592 1599 165i 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802.

181-2 1813 1825 1837 1845 1856 1861 GGGUACUU C CCCCAGC ACCCACC C CUCUGGCrJ AU&CUUGrJ A GCCUCAG AGAAGC-U C AGGAGGAG GGGAGUt C ACCAGGGA AGGGa~CU u cccccAGG ACUGC-UC u CCucu=G CCGGGUr U GGACA CGUUGAAAU U ALTJGGUCAA GAAAAUGT U CCAACCAC UGGGCU A AUUGUTUGG GCCACCAU C AC'JGUGUjA GUCCUGGrJ C GCCGUU-,r ACCUGGGU C AL~AtU=t CUGAUCty U GCGGGC=r GGGC=C C UrJCCnA UGGG~aA= C CCUGuuua, UCCccrJ U ucurCCCA.

U~kCA=c A UUACGCC ACACCEkU U ACCGCCAG; AGGAAGAU C AGGAUA CAGC-A= A CAAkGUUjAC tUhAAGUU A CAGAAG CCC~Ccr C CCUGC-C CUGMCACtJU U GCCCUGGU GAACAGAW C AAUGGAA GAGAACCU C GG;CCUGGG GGGCDOCU C CACAGGUC GG.CCUG=r U CC~r,C COGCUCG A GACCUCLTC CCCCACCU A CAakcAUr CCGGACU U CGAJUC CUCCUGGU C CUGGUCGC UCAGCAU~ A CCUGGAGA GAUCACAU U CACGGUG GUCCAUUM A CACCrJAJ CCUTCUGCU C CUGGU.CCU GAGAACCrJ C GGCCJGGG GACACUGrJ C CCCAACTJC ATJGGUCrj C ACCGGAC UCCCOGUU U AAAAACCA GCUCAGAU A UACCuGA AACAGAGrJ C UGGGGAAA GCGGGCU C GUGAUCGU GCCACCAJJ C ACUGUGUA ACCCACCrJ C ACAGGGUA, AGAGGACrJ C GGAGGGC CCCCUAAU C UGACCUGC CAUGUGCU A UJAUGGUCC 203 *see 0000 *0 1865 1868 1877 1901 1912 1922 1923 1928 1930 1964 1983 '!996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 215 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173- 2174 2175 2176 2183 21.85 2186 2187 2189 2196 MUfCOCG A GACACM.AG UCACCGAGU C AMUAAAU AcAGTa.Ct U CC-CCCAGG CUAAAACE C AAGGOUCAL GAACAGAUT C AAIJGGACA AUGUAAGU U ADE7GCCLUL CGMGACG-tJ C ACCDVUAG A UC-,UGA GGA=C A ACUGGAMG

G=GLCAGC

C-ACC',-'J C GC-CCCGGG MGGAAGCUT C tUCAACU- AXVGUAAGU U AUUGC=tA CC--=CU A LCsGGAU CCGCCMU c GGG'AUG -r3 MUCGw%= A CCC~JC CGGAGGAU C ACAAA=G C'Cr=XCCU C CUGGAGG CCGGUCCJ C CAAUGGCU GCGrUC=A U w~cCACCE A~kCU~UG A GC-CUCAWG L'GUAGCCUJ C AWGCC~MA CCAAC=Ct U GUMtAlU CZUGA.CU C CUGGAGCGU UXCCWACU A GCGUCCL-G AZGGU A CC;t7GAajc GCCtJGUMr C CUGCCUCEj CCAACUC U GUUCGA=~ UUGAGAAU C tUhCAACD tJGACAGCU A UDMUUA UGAtJGU U Uft~AUt~a G-ALMM=AU U AfLMA=U AUGU=U A UOAVnUM~ ACAVUCC A CCUUGU TZLUMIA= A AUUCAGAG MAUGMUt U UAUAI -AtUGtfU= U AMIAAXU GUAUUtUL U AAUUCAA CAGua~rDU U AUUGAGUA UGUGC~kU A UGC,-CUC UJCUCtahUU A CCCCEJGCu AUtJECUUU C ACGAGUCA GAAAALTWG U CCAACCAC tJGACAGUU A UZUtMUA ACAGUUAU U T-VUUG-AGtJ CAGtUWU U AMjUAGUA AGUMUUU A UUGAGkC UM~UULMU UJ GAGUACCC CUGACAGU UJ AUUUJAUrJG 21-98 23.99 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 2431 2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 GAAfLMUCU C CG-AGC,,.C AC-ACU=tI A CAUGCCA GG.Mk=U C CCCCAGC.C GGCU-UCu C CAXCAGG,-C UUUUGOGU C AG,-rACrG VGGAGACE) A ACUGGAUG GAGAACCEJ C GGC-,aC- ACAUACAIJ Ui C=t-CC C.UGGACCE) C AGGCCACA UCALLGCUU C ACAGAACJ ACCAGCU C UC;UA~U CECCEJ'GGE C CtGGUC,-f AUCCAAUU C ACACUGAA GAUCA=c~ U CACGM-GC AUCACAXUt C ACGGU~C,-j AXJCAGGAU A tahCAAGUU GAGCAGGrJ U AACAUGr3A GGAAAC.AU C AUACGG=t ACAGtUAW TJ U aLGAGU GCCCtGGU C CDCcAu= CAGGAU A CAAGUEC GCAAAGAU C AIUhCG,-:tJ TU7GCGCUU C TjCC;=AG GGCMuU C CCCCAGGC GGCCE)GrJ C GGUG--Vc;, CLUCCGU A GACMCUE CCCUGCCU C CE7CC=ILc CCAUCCAU C CCACAGA CUUGUU C CCUGGAAG GAACUGCU C UuCCU=ur GACUUCC-U Uj CUCEktJA CMGAUU C tJUUCAC-A CU~Crj-Vc C -UtrCG UGAtUE)CU U UCACGAGEJ AUUUCEJIJU C ACGAWjC.L CAUCCC-GO A GACAc;AAG UAAAIJAC! A UGUCGACGC UGUGCUALU A UGGCUC CAAfUU= C AUGCrUC;L AX3CAGGArj A UACAAGLJTU UCAUG=tT C ACAGAACU UOAUUAAU U CAGAGEJE) CCUGG-GGU U G-GAGACEJA UCAGAGUtj C UGACAGUEJ CGGAGGAU C ACAAACGA, UGAACGU ;LcuUCCCCC GAAGCCEJU C CUGCCUCG GGCCUGUU U CCGC GCMM'tUU C CUGCCUCtJ 204 a a.

a a. a.

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 ACADUCCUT A CCtUrUUGU CCCUGC-CU C CUCCCACA CCMACU U GUUCCCAA Ur~hCACCU A Uta.CcGC GUCGCCGU U GUGA=CC ACCOUt7U U CCCAAUGU CCUU3GOEJ C CCAAtUC G-ACCACC C CCCACcUa; ACCtMU A CAUUCCEC, ACAkC Ur CCUACUU cAc~fl c ct~ccuuu GECCA=O A CA~CCL-U ACCUUVW U CCCAAL=G CCUUUGUUT C CCAAXUTC ACAGCALUU U ACccaxCA uacG-CU C AGGtUW=C AGGCAGcU C CGGACUU CAGAGAUU T UGUGUC CCUGCACU U UGCCCUG= CUGCUCGU A GACCCUC TJGCCUCCU C CCACAGCC cUCUUmc C JOCGAAG tUCUCMvu A CCCCWCU CUCCUGGU C CUG.GUCGC UGtJGCLW A UGGUCcUC GUCCtUGG C GCCGUUG GUGSGAGET A UCXACCG CUUrkGCU C CCGUGGGA t7GGAGACU A ACUGGAIJG UCAGAGUU C UGACArU CUCUC=G A GUGCW.CU MkCACU U UCAGCUCC UCACAGAty C CAAUUC= CCAGGU A UCCAUCCA CCCCACCU A CAUXLC= GCCUGE r C CUGCCUCrJ CCACAGG!J C AGCGUGCrJ AGAAGG-GU C CUGCAAGC ACUAGG=t C CtrAAGCLT UCAGCCU A AGAGGACU AGGtkCu U CCCCCAG GXACCAC C CCC~aCC CCC!U.CCU AGGaaGG CCMCCUU A GGAAGGUG GGAAAGAU C AUACGG=t AAGAUCALT A CGGGUUJLG GGGUGGAU C CGUO=G GUCCCUGu U UAAAAACC GACGAACU A UCGAGGG 2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 29 1.1 2912 2913 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 CGGA=t~

C;UEUUGC

UUCUCM

CGUGAAA

CUICAtJGC

UCAUGCD

GCUcCCA CGGA=u

CCUGACC

UACAAcU

CAACUO=

LUCGGU~a

CACAGQ=

GCACCCcL UtACCCC

UUCGAUCU

M.ukCujuG GGGCCtUGU UGGAGL7CU

AGGCAGCU

GGCUGAcU

GAACUGCU

GGCWA~CU

GtUAUGU CuGCUCruU

UGAUGMAU

GAACUGCU

ACUUCCU

uUiCCUUCU AUG~UUMy

UGUGUAULT

GUALUUAU1

UAUULATU

CUCUCCU

CUUCCUC t

AUUUCUU

UUUUGUGUC

GAUGGUJGU

C

TGGAGUCU

C

CAGtTACUU c ACCAUGCU

U-

CCGGACTJU

U

UGCtJUCcu

C

CUUUCCUU u UUUVGtJGEI c UGtJGUAUTJ

C

CUU~jGAAU

C

UGGAAGCU

C

GAAUCAAU

A

7C GAUJCUuCC uC UGCCGCUr DU ACCCCUGC ~U AUr,-UCA UU CACAGAC UC ACAGAACU UC CLGACCCu UC GATIJLICC C CLMGAGWy :U lucaGc-Ucc 3C AG,-t7CCCA.

7C AGGUJAUC A CUUiCCCC- C CCAGCC C ACCCACCTJ U CCGACEIA U CCCUGGA C GGCCCUCA C CCAGCACC C CGGACUUrJ U CCLUCUCU C uuccucuty U CCUriuctJE A UUtUALMAA C CUCLTGC-G U UAUUAATJU C ULTCCUCU C UCIAULTh.C C MkUUACCC A~ UtUhAuUCA C UCCCAG :7 AAt3UCA k AUt3CAAG

UUGCGAAG

YGCGAAGAC

ACGAGUCA

AGCCAICUG

CCGCEJGCC

CCAGCACC

CCCCAGGC

CCUCUGAC

CGAUCEJUC

TUGACAUGG

GAAUTCAAU

AGCCACEJG

GUUCCCAG

AAUAAAGU

UtTCAAGCU AAGUUUrUA 205 2975 UGGAAGCU C UUCAAGCUT 2976 ANGU *C CUCACCUG 2977 G-AAGCUCU u cAAGccaA C 0 00 *0 7" 206 Table 10: Rat 10AMI EH Ribozyine Sequences mt. Rat HE Ribozyme sequence Pos- tion 21 UCAGUMTJG CUGAUAGGCcC,=U-MCCGAA AUCGGC.nA 23 UAhNAGAAG C~mUA= -vu~c-r-A

AAG-OCA

26 AAGAGGAA CUCNAr3AG-GCGC.G-,GAA.,

A~C,.G:

31 AGGACCAG CUGAIUGAGGCCG GGAA AC-CACG 34 GtAUAUCU Ct3GAXGAC-GCCGAAAGCCG-AA

AG---UCAG

GGG-GCUUG CUGAUGAGGCCGAAAGGCCGA

ACCUULGAG

48 C CCAG.GCC CUGADUGAGGCCG GACGCrC-

AGGUUCLIC

54 G-GCtCAGG CUGAXGG GAGGCcG ~GCWa 58 GGGAGCGA CU43ACCvA~-=,

AG~CAC

64 ACGGGUUG CUGADGAGGCCG AAGCt'rA

AGC

.96 AGGACCAG CUGAXUGAGCG-CC .AGGCCGAA ACaz 102 GCGACCAG CDGAXJGAGGCCAAGCCGAA

ACCAGGAG

*108 AGUTCCCC CUGuXTIGAGGCCGAAACGA-,mA AGCAUj US11 UGGGAACA CUGAflGAG7CCCGAA GGC-A ACGUAGGA *19 GAGUUGGG CUGGXUGAGGCCGAAGCC

ACG-,=

*120 GGCCCGGG CUC-AUAGGCC GAGGAWA

AUCACAAC

146 GGAGUU=CC UAGCGACGCA

AGGUCUGG

*152 UUGW--UG CUGAMACG-CcGAAGGCC;A

AGCGGGJ

i 58 UMA~CU CUGAUGAGGCCGXCG A X MAGAU 165 GGGGGAAG CUAGGCC-AGCCA ACuGU-jUCA 168 CGAGGCAG CUAGGCCXWG-G;

AAG-

185 CCUGCACG CLTGAUAGC fCCACCC 209 G-GUCAGAU CUGAUGAGGC c AVCCGCC 227 UUCACAGU C7AXGCQ t J ACUUGGUC 230 CCUCCCAC CGXI"

A~AGCUUG

:237 GGGGUGUC CMUGAGGCCGA XGCGALA

AGCUCAG

248 UCCUAAGG CtJGAUGAGGCCGAAGCCGAA

AGGG-GGCC

253 C~JcaCU CtJGAUGAGGCC -GAA

AGCAGU

263 GCAUGAGA CUGAXJGAGGCCG AGCGAA

AUUGGCEJC

267 CGAGGCAG =GXGCGAGCCA

AAGCUUC

293 UCAGCUUG CUGAIJGAGGCCG a=CGAA,

AC-AGCUTC

319 UCGUUUGU CUGAflGAGCC AUCCUiC 335 AGUUCtJCA CUGAMJAGGCCGAAGGCGAA

AGCACAGU

337 GAGGA.CCA CUGAIUGAGGCCMAA GCGA AlAZcCA 338 CUCAGCUU CUGAUGAGGCCGAAAGCC;L

AAGAGCUTJ

359 AAGCCGAG CUGAXUGAGCCWAA CA AJCGTGG 367 ACGGGUUG CUGAtGAGGCCXAAG CCAR AGCCAJTJ 374 AGGUGGG-I CUGAUGAGGCCGAAAGGCCGAAk AGGGGtUhA 375 GAGGCAGG CUGAUJGAGGCCGAA AGGC.CUr.~ 378 UrACCCUGU CUGA UAGcCCCAAGGCCCA

ACUC

386 AGCUCCAA CUGAUGAGGCCGAWC-L

ACCA--

207 1 9 394 420 425 427 450 451 456 495 51 0 564 592 607 608 609 611 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 867 869 881 885 933 936 978 980 CUOOCaG CMuAGWGCCGAAAGMCCGAA

AGCXCCAC

UGCGCUGG CUfGAGCCGAAAGGA

AGGGGUGC

GGUGGCAG CGDAGCCGAAA~oCGAA

AGCCGAGC;

UGGUOUU CUtrACCGAAAGCCGA

AACAGGGA

CGCAGG;LU CUACAGCCrAAAGGcCCA

AGGUUCUU

GCCUGGW G UA GAMGGAcL .aAGa;CC UCGG; UGGZGGCCcGAAGGCrA

AAGCCGAG

UACACAGU CulrGCCCAAAGCCCC AlGGUGC UUCCCACG CAGGCCGMAGGCCGA

AGCAGCAC

GUGGUMG CUvdG~fG,-.C~ CGCrC Ak~kUUrJ UCCCUGGU cUGA~wGCCGrAAGccGAA

AUACUC-C

GCALMAGA CUGACCGAA 'CCCriA ArULGGr AGCAUGAG CGUGGCCGAAAGCCGA

AAUUGGCU

AAGCAtJGA CUAU CGCCGAAAGGCCA AAAXUGrC UGAAGCAU CWAGAGGCCGAAGCGA AGAAAUu CAUUCUUG CGUAGCCCAAAGGCC=-

ACAGUGAC

ACADUCUU CGUAGCCGAAAGCCGA AACkGUGAL AAGAGGAA C-UAGGCAGCGAA AC==u~ UGCGCDGG CGUAGCCGAGccA

AGGGGOG

AAAGUC CGAWw CC-AGC

ACDGCCU

GGAGUUCC CO I c AUAGCGAGCCG

AGGUCU=

GGAAGAUC CUMWkGCqGXUG=

AAAMXCQ

AGAG-GCAG CUAGGCCAAGOA

AAACAGGC

GUAGGGG CAUWCCc AACOcccXM= GAC CWGAGCOCGAAA C~.A AGUUGUAG UGGGAGCU

AAXAGUU

GCGCCAG CGVAGCCGACa

ACCAGGAG

UCCACCCC CUWAGCCGAAGCCA

AGGCAM

AGUUCUCA. CMMGCCGcAWcc AGCAcAGU UUCC&GGG CUVAGCCGAAAGCCCa AcACAAGA CUUCCAGG CGM GCCGAGCCC.A

AACALA

AGGCAGGA CW AGGCACC

ACAGGCCU

GAMO AUAGG CCOAAGCCGAm

AACA==C

GCGACCAG COAGCCGAGC~CGA

ACCAGGAG

G-AGCUUCk aAGGCccAAGCCGA

ACAMA

UCCAGGUka CAUGAGCCGAAGcCA

A~~.G

UAGDCU=CC UGGCCGXAGCCGAA

ACCCCA

CAAUkAAU CWUGCCGGCCA

ACUGUCAG

AGCUGCA CMU GCCGAACCCL

AGGUGAGC

ACGGGUM AU G GAA=GcAA AGCCAnJUG UUCAGAG COA GGCvA.CCGAAc

AAGCA=G

UAG-GUGGG.CGUGGCCGAAGC~rA

AGGUGGUJC

CUUJCGCAA =MCAGGAAAGGCMc. AW~iG CACGGT= C~UGAGGCCGcACCCA AAGCCAnU UUCACAGU CUGAnGAGcGAAGCC ACUU.GUc CUGGGAAC COAGGCCGAAGCA

AAJA

UGACACAA CUA AGCCcAAGCCGAA AfCG AAGUUJA CUGAUGAGcGC AC

AUUCUMA,

AAA.AGUU CC,;uAGGCCrcaGCCGA AGcLJ.UCUC 208 4 986 987 988 1005 1006 1023 1025 i066 1092 1093 1125 1163 1164 U166 1172 1200 1201 1203 1227 1228 1233 1238 2.264 1267 1294 2.295 -1306 1321 2.334 1344 1351 1353 1366 1367 2.368 1380 1388 1398 1402 1408 1410 2.421 1425 1429 1444 1455: 1482 1484 1493

GAGCUG

GGAGCO

GGGAGC

G-ACGCC

CCU=G

ccuUco

CCCC~U

UUCGGGS

LUCUGCC

AGCGGC7 AtLCxaAO-

AGCAAA;

GAGCAAJ

CAGAGCO

AG-GCCGC

UUCAGG

CCacuGG

GACC=

AGCACAUj

ACCAUCA

GCGACCAU

GAGC-C

ACCCGW

CAUUCUUJ(

CEtGACAC Uctrxc=G

GICLUGU

umTcccc;

CUCDGUC

GGACct AGCCUc=

CCAOTUGM~

CCUGGGGG

GCCTJGGGG

GGCAGCGG

ACCAflCC

CAUCCAGU

UGUCCOGY

CAGULTCUC

GACGCCAC

GtJCACUC GCCE7GGGG

AGCCAGAG

CCt7GA-GGC

MCUCCU

UCCCtGGUu

CCUGGGGG

GCAAGAGG

tJAGUCUCC COA UG GC--AAG--CGc COG UGGGCCG-CA ar-CAXJGAGGCCA

GGA.

uCUIGCCAGCG;

GCDAGAGGCG-CCA

LCGAUGAGCCGAAAGG.CCG-AA

LCUGAVGAGGC

-CGAA~~

ACOGAUGAGGCCGAX

G.-C.A

ACUGAMAG AA G.cAA u LTGAV(Gf-CCGAACA GCUGAUGAGAA

GCCGAA

G UGAG~CCGG-CA CCUGAVGAGG=UAZ flCGAA

SCUGAUGAGGCCGAAAGGCCGAA

ACUGAXJGAG

A

SCUGAUGAGGCCGQAGG-,CC-A

A

SCUGAMGr-&AGC

CCAAA

SCUGAflGAGGCCGAA

CGAAA

SCtJGatGAGGCCAAAGGCCGAA

CUGAUG=GCCGAXAAGGCCAA

CtJGAtJGAGOCCGAAAGGCGAA

A

CUGAtIGAGGCCGAAAGG--CG.A

TCUGAUGGCCAGCGA

CTJGAtJGAGGCCGAAAGGCCGAA

A

CUGAVGAGCCGMGGCGAAA

CUGAUGAGGCCGAAAGGCCGAA

A

COGA

3 GAGCCGAAAGG*CCGAA

A

CUGAUGAGGCCGAAAGGC-CGMAA

CUGAMJAGGCCGAAAGGCCGAA

A

CVM'UGAGGCCGAAAGGCCGAA

A

CUQAAUGAGGCCGAAAGGCCGAA

A

CUGAUGAGGCCGAAAGGC,-CGAA

A

COGAUGAGGCCGAAAGGCCGAA

A

CUGAUGAGC-(CGAAGGCCGAA

A

CUGAUGAGGCCGAAAG-GCCGAA

CUGAUGAGGCCGAAAGGCCG;A

G

CUGAUGAGG-CCAAAGGCCGAA

AG

CUGAUGAGGCCGAAAGGCCGA

A

CUGAU;GAGGCCGAAACGCCGA

ACC

AGOUG=A

AAGUUGUA

AAAGUG

AtJCACGAA

ACUCCCAC

ACUCI-c

AAGG

AcCCCC=

AGUUGGG

ACGUCGU

AAGCGUCG

AC-AAGCGU

kGCAAAAG

MVUUGGAU

LUGCCCAA

UNAkGCCC

LUUCCAA

LAGCCCGC

CCAGGAG

MGCACA

CAGUGAC

CCCCt3CE

-CUGCU

XAAUAC

;CAZ=G

;GCCUGAL

C"UGCUTA

ACCAUC

MGG=A

VCUCCA

7=1CAG

CACGAA

AGUUCG

~GACCC

LkAGUAU

:C=CU

LGCAGU

Y-r.

209 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 1793 1797 1802 1812 1813 1825 1837 1845 1856 1861 1865 1868 1877 1901 1912 1922 1923 1928 UUGACAU CUGAUGAGGCCGAAAGCC-AA

AUUCAM

GUGGtJUGG CUGAUGAGGCCGAG-CC-. ACAUUrUc CCAACAA~U CUGAX~GAGG--cGAAAC;CGAA,

AUGACCCA,

tUhCACAGU CCOGGG6CGAAGC;AAC.-G ACAACGGC CUGWUGAGCCQ

ACCAMA~C

ACAALW UGAGGCCAAGCr

ACCCAC,-U

AACCCGC COCAUAAG -c

AUGAZCA

UCGAGCAL CUGAUG GCC -AAGCGA ACGcC.AC UAAACAGG CUGAGAGGCCGAA ,,-CGAA AcUUCCcA UGGGAACA CCAGGCC--A~,CA Ga~.

GGCGGDAA CUA ~GCCAAGCA AGGGut CUGGCGGU CUAGCC-.,aGCA

ALMAG=G

MUCCU COGU GGCCGAAAGGCCA AlCUrcU= GtUhACDU CUGAXUGAr-C-CGAA GCG

AOAUCCUG

GCCUUCU CUG GGCGcAGCCC.

-AACUUU

GGCUCAGG CUA G ccGcc

AGGXGG

ACCAGGGC LGUAGC cMGC-CCGA

AAGA=

UrZCCA=l COGAUGCCAGGCCrAA AUCUGOUc CCCAGGCC cuGAZJGAGGccrGA c; AGGUU=U GACCUGUG CUGAUGAGGCCGZa-mCCCGL

AGAAC-CCC

GAGGCAGG CUGA.GAGGCCGAAGGCCAA

AACAWGCC

GAGAGGUC CMMGCCGAAGCGL

ACGAGCAG

AAUGtkUG CWAGGCAA-,CA

AGGTJGGGG

GAAGAUCC UGG cGCCAAACCG

AAGUCCGG

GCGACCAG L"G~!C*CGAGC

ACCAGGA

UaCtCA Ct3GNAGAGCCX" CCA AMUCUGA GCACCGU CUGAUGAGGCGAjGGCCG

AUGUGAIIC

AAUkAGGUG CJGAUG GGCCGACCCA

AAAUGGC

AGGACCAG C~AGGCGAGCrA

AGCAGAGG

CCCAGGCC CWV=CAAGCA AGGUUCtTC GAGUGGO GAUAWGAACC

ACAGUGTJC

GOCCAGGU ~CUGUAGCAAGCCA

AGGACCU

UGGUUUUU CUAGGCCAAGW

AACAGG

UJCCAGGUA CVAGGCCCX-.CcrG

AUTCGC

UUUCCCCA CUArAC-CAGC~CGA

ACUCUCUU

ACGAUCAC CUGAUGAGGCCCAGGL

AACC

UACACAGtJ CUGAI GAGGCCGAAG,-CGAA

AUGGGC

UaCCCUGU arUAGCAACL

AGGUG=~

GCCCCUC CUGAtJGAGGCCGAAGGCQ

AGCCUL-J

GCAGGUCA CGGAGCCGAGCCG

AUW=

GGACCUA CMWCGAGGCACCA

AGCAW

CLTUGUGUC C"UGAflGAGGCCGAAGCCA

ACCGGATJA

AXU~UGjx CUArGCrmkGC ACUCTLGUrA CCUGGGGG CUCAI~GAGGCCGAACCCA

AGUACUGU

UGUACCUEJ CtJGAUGAGGCCGAAAGGCCGAA

AGOUUG

UGUCCAUE) CUGAUGAGG-CCGAAAGGCCGAA

AUCUGEJUC

LGGoCAAU CUGAUGAGGCC GGCGAA

ACUUACMU

COA=G CUGT(AflGAC,;CGL(;A aL

AGCGEJT=

UCCAGGEJA CUGAGAr.GCCGAAGCAA

AUJCUGAGC

210 a 1930 1964 1983 1996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2115 2128 2:30 2145 2152 2156 2158 2159 2160o.

2162 21.63 2166 2167 2170 2171 2173 2174 2175 2176 2183 2185 22.86 2187 2189 2196 2198 2199 2200 2201 2205 2210 2220 2224 CAUCCAGU CulGCCGAAAGCGAA

AGUCO=~

GCE'GACAC COGAGCGAAGGCCAGcA AAAflUC= CCCAGGCC CUGPJJGAC-GCCGAAAGGCcGAA

ADG~U

AGCDUGAA CG zCCCGAAAGGCcGAA

AGCUUCCA,

URCGCAATJGU GAGGCCGAAAGCCGA ACUUacM CAUCCCGA CUAGGCCGAAAGGCCGAk

AGCGCACCG

ACCAUCCC CUAUAGCCGAAAGGCCGAA ADGGcA= GLVLCAGGG CrUGGGCCGAAAGGCGAA

ACUCAAU

UCCUUUGU CUAGGCCGAAAGacCGAA

AUCCUCCG

ACCtJCCAG CUACAGCCAAC~A

AGGUCAGC;

AC-CALUG CCG rAGCCAAAGGCCA AGGACCAG UAGGOGUA CGUAGCCGAAAGccGAA

AUCGACG

CCLTGAGGC COGAUGAZGGCCGAAAGCCAA

ACAAGUA

ULVGGCU CUGAGAGCCGAAfGCCCA

AGGLCILC

AC=AC CU i 1 AGAGrU ACCUCCAG CGUAGGCAAGC~ra

AGGUCGC

CAGGACCC CA GCCGA.GOCCGAA AGUCGGA CNAMUMG CUC%~rGGCCGAAAG~C4:CA

ACGC

AGAGGCAG CA GCCGAAAGGCCGA;

AAACA=G

ACAUCAAC CUA GAGCCGAAAGGCCGA

AGAGU=

AAGUU CrUAGCCGAAAGGCCGA

AJUCUCAA

UCAAA CA GCCGAAAGcCGA AAtUGU AAUAA CUA GCvIGaAGCCr. AtpCwLCp GAAKZAU CUGAAGCAGGcCGAAc AAUACAjC TGAAUMIA CUuGGG--rAAGtA -jAACL AACAAAGG C GIGAGGC CAAcCCCAA AGAA~rU CUCELGAAUT CUAUA CCGxazcAGC AAZ~aADA AAUCAAlL~ CUArjGCCGAAAG~cCGA AjOCAUc GAAUMLZU VGUGGCCGAAAGGCCGA

AAUACAG

UCtJGAAUU CUA;AGGCCGAAAGCz AUAAfAC UACUAY OAU GCCGAGG AAtMACU CICuCGAUG~GCCCCAAAGCCGAA AflACCAA AGCAGGGG COGAUGAGGCCGAAGr MA AAU.A UC-ACUCG;U GcAA c AAAGA GUJGGUUGG LCCG ACAnuuu UCAAEThAA, CUG IUGAGGCCCAA -CA AACU;UCA ACUCAWUA CUAUGCCGAAAGrC-Cc AUACtU UkCUCAAUT CGUAGCCGAAAGGCCGA AAfl~UG GLMCUCAA CUG GGCC GAA AAU GGGUACUC CUGAGAGGCCGAAGCCA

AUAA=A

CAAUAAAU CGUAGCCGAAGCCGA

ACUGUCAG

UGACCUCG CUGAfGAGGCCGAXGCCGA;. AGAC=flC CUGvGCAUG CUA GCCGAGCCA

AAGAGUCU

GCCUGGGG CUA~kCGACCCA

AAGUACCC

GACCtJGUG CUGAUGAr-CGGCCGA A AGAAGXC CAGUGGCU UAGAGCaAGCGAACAA CXDCCAGU CUGAUGAGCCGAAGC

AGCC;

CCCAGGCC CUGAX3GAGGCCGAAGCCA ACGuuCUC AAGGUAGG CUCAGAGCCGAAAGCCr-A

AUGUAUGU

211 S. 9 a.

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 VU7GCCL CGV =CAAAGGCC-AA A~a AGUU3CMU CUA~GCGAA--CA

AAZUC

ACtACUGA CUAGG;CAAGCA

AC-UGU;

GCGACCAG CJGAAGGCGAAZCCCA ACCIGGAcG UCAGGU COG flGAGGCCGAAGCCA

AATI

GCACCGUG CUGAtUGAGCCG -CGAA

AGUMAC

AGCCACCGt7 CUGAUMGrGCGAACC,-la AALMUGAu AACUUGM, LUGAUGAGGCCAAGCGr,.A AMC

U

UACAUGOU CMUAG-GAAGCA ACMtGcC ACCCG.U CUGAUGAGGCGcAZ: A AfLC=UMc ACt7CAAtE, CUGAMlAGGCCGA GC. A;crJArJ CAUL3GGAG COAUA GCCAAG-CcA ACCaG GM~ACUU CWAGGGCM"GCCA -cU~j ACCGU CUGAUGAG-GXU CGkA~ AVCZUuuC- CCCGUGGA COGAXGAGCAA G,-r-AA

AAGCCCAA

GCCUGGGG CGtAGcr AAGUaccC.

UGAGCACC CDGAM3AMCCg

ACGC-

GAGAGGUC ~COMGGCCUCCGM

ACGAGMGC

UGGGGAG CUC-AMAGGccC A

AGGCAGGG

UUCUGUGG CUGUAGCMG

C

CrA7CAGG CGUACCGa c AAAAA AAGAGGAA ~CUGUAGCGACCGA AGCArjc UAaW~AG Cr3GMnAGGCCM aCGC=AA

AGC-AAG'UC

UCGUGAAA CUMVUGAGGCCGA

AAAUCAGC

CGCAAGAG CUG DGAGGCC AG A AGAC ACUCGOGA CGUAGCCGXGCCA

AG-AUC

UC-ACtCGt3 CUGAUGAGGCCG .AGCGA; AAGAAUr CUUGUGUC CtJGAfGA~rCGAG CCGAC ACC-GGAjjA CGUCCACA CUAGGLGUGMk A~UtjM GAGGACCA CUGA GCA.CCG;L

AXMCC

UGAAGCAU

CMUTGAGWGXUGCCCAGAW

AA=UUA~ CUACACCGACCA AUCCUrAr- AGUUCUGU COAAGCAAGCGL

AAG-CAC

GAACUCUG CUGADA GCCGAAA OCCA AXn3UA; tUhGUCUCC CJGAL7GAGGCCGAa CGaA.

ACCCCAGG

AACUGUCA CMAGAGCCGMa GCr

AACUCUGA

UCGUUOGU CUG-AUGAGCCCAAGCCG

AUCCUCCG

GGGGGAAG CUAG GCCAGGA

ACCCUUCA

CGAGGCAG CUGWXGAGGCCGA AAGGCD

ACUC

GAGGCAGG CUGAflGAGGCCGAA GCGL AACAGGCc AGAGGCAG CO MGXGGCCGAA

AAG

AACAAAGG AGGAAUGUy UGUGGAG CVGMTJAGGCCG XGCCGAA AGGC a UUGGGAAC CUGAGAGCCGAGCC A.AZ,-GrZ GGCGGtTAA GUA~ GC G=CGc

AC,-UGU

GGGAUCAC CUGAGCGCCG~aM-GGCcA. Ac-GCG;LC ACAUUGGG CUGAUGAGCCGCCGA;

ACXU

GACAtIUGG CUGAUGAGGCCG GC,4=

AAG

UAGGG CUGAUGGCC AGGCCGA AC-UOCO7c 212 5* 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 2761 2765 2769 2797 2803 2804 2813 -2815

VGGAAIIG

AAGGaAGG AAA-GUGr.

AAtMG=U

ACAUUGGG

GACAUUGG

T

JGAGGGGU

GGAMVCU

AAAGUCCG

CtrzACACA

CCAGGGCA

GAGAGGOC

G-GCUG=

CUUCGCAA

AGCAGGGG

G-CACCAG

GAGGk

ACAACGGC

CCUGGUGA

UCCACGG

CAUCCAGU

AACUGUA

AGCAGCAC

GGAGCUGA

GtGAAUM UGGAtJGGA AATJGM=fl

AGAGGCAG

AGCACCCU

GCOUGCAG

AGCU=

AGCCUCU

CCUGGGGG

UAGGUGGG

ACCUUCCU I CACCUrUCC

ACCCGUAU

CAAACCCG

CCUGCACG

G-GUUtC

CCCUCGA

GGPAAGAUC

AGGCCGCA

GCAGGGGUC

UUGACCAUC

GUUCUGtJGC

AGUUCUGUC

AGGGUCAGC

GGAAGAUJCC

COGAUGAGGCcGAAAGG-CcGAA

AUGU

COGAUGAGGCCGAAAGGcG.A AflGfl= CDGAUaGACGCCGAAAGC<CCGAA

AAG~

COGAUGAGGCCGCAAGGCCGAA AAAflGGAC CW-NOGAGG~cGAAAGGCCGAA

ACAAAGGU

CUGUGAGCCAAA~ccAAAACAAG CCJMAUAGGCCGAAAGG-CCGAA

AAVGCUGU

CUGAUGAGGCCGAAAGGCCrAA

AGCACCGA

CUAGGGCCGAAAGGCG

ACUCC

COGAUGAGGCCGAAGGCCGAA AAI3CUCUG CUGAUGAGGCCGAAAGGCCCGAA

AGGCW

COGAUGAGGCCGAAAGGCCGAA

ACGAGCA

CUGAUGAGGCCGAAAGGCCGA.A

AGGACGGCA

COGAUGAGGCCGAAAGGCCGAA

AGGAAA

COGAUGAGGCCCAAAGGcCGALA

AAAGG

COAUGAGGCCMUAGGCCGAA A~CrAA COAGGccG ccc.A Ac~cAcA CMUGG

ACTCCAC

CCGAUGAGGCCGAAAGGCCGA

AGCMLA

COUAGAGGCCGAAAGGCCCGAA

AGUCUCCA

COGUGCCGAAGGCCGA&

AACUG

C-NAG~CCGAXAGG~rAA

ACCUGAG

CVGAUGAGGCCGkUZG=cGAA

AAGU~UM

CUGAUGAGGCCGAAAGGCCAA AflCUGMA CUGAUGAGGCCG~aAGGCCGAAL

ACCUGAGCC

MMAUGAGGCCGAAAGGCCGAM

AGGOGGG

CGAUGAGGCCGAAAGGCCGAA

AAACAGGC

CGAMVCCcAAG A ACCaGQ "GAUGAGCCGAAGOCCCAA ACCCUUCu "MUAGCCGAAACGCGAA ACCrM= -DGAGAGCCGAAAGGCCCA;L

AGGCCUA

-TMGAW4=~cGAAG=CcAA

AGMCCU

UVGAUGAGGQGCCGA= AGMGuGLc -VWXGAG~cIcGAAGcc

AGM=

TGAUGAGGCCWAAWX.CcGAA

AAGGUG

.VUMACJW GGCCA,

AUCUUUCC

-GUGAAGGCCG~aAGGCCGAA

AMAWCUU

-WAUGAGGCCGM"CGAk

.ADCCACCC

-UGAUGAGGccGAAAGGCCGA"

ACAGGGC

UGMAAGGCCAGGCCGAA

AGUCGUC

UGAUGAGGCCGA~aAGCCGAA

AAAGUCCG

OAUGGCCGhkAGGCCGAA

AGCAAA

GUGAGGCCGAAAGGCCGAA

AUAGAGAA

UGAUGACGoCCGAAAGGCCGAA AULt7CACG MGAUGAGGCCGAAAGGCCGAA

AGAUGA

MGAUGACZGCCGAAGGC

AAGCAUGA

MAflGGkAGGCGA

AUGGAGC

IGAUGAWXCCGAC

AAACGUCC-G

n NO 213 0

C

2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 291.8 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 2975 2976 2977 ACCUCCAG CUGAVGAGGccGrAAGCCLGAA

AG,-,C,-GG

GGAGCUGA CVGAVGAGG CcGAAAWG-ccGAA AAGUUGUA UGGGAG'%CCU CUGAUG--AGGCCCGA Ac AAAAGUUG GGAUACCUGAG CGAAAGCC-L

AGCACCGA

GGGGGAAG CGA CCCAAAGGcCG;

ACCCU=~

WGCGCUGG CUG~.CGflGGGC.... C r AGGtjGGGU CUAGG,-CGzcCC

AGCGGUAA

CLAGT2CGG LCj~ALGCG-CGAAAGccGCA

AGADCGCAA

UUCCAGG UUGG GCCGAACGCC A~~aAGA3 TJGAGCACC CGGGGjtrA 1 jCCr. ja.CCC AAAGUCCC;CUAG -CNAGAGCCG

AZCUGCCU

AGAGAAG CUGCG--Za-AC7C AGrJC,,GC, AAGAGGAA CU~G'GAGGCGZA AcC-A A~CAGUtC AGAGAAGG CUvJGG-cGAAr.-Crc;

XGLTC;.CC

UUAAkA GA Gc-ccAAAGGcc

AA=XUCAAC

CGAGG CUGAflGAGGCCCGA ~Ccc~A AACAG=tI GtAAMA CrUtTAGCCGAAAGGCCr.A ,t~kCAAG AACAGAA CUuJAGCCGAAAG~CCCC

ACCGAM

GAfLUA CUATA7C~CGACGLCG AXGAAlj CGGGAk CGLT G.CC-AAAGGCCGA

AAACCA

UUGAUU; Ct3GAIGA~GCGGCGAA AtAA CtjCAAU CUGAUGAGGCCGAG-,CA

AA~UAC,

CUCGAA CU GCCCGAAG~C AaLUAC Gt'jCUF3CGC CGUAGCCGAAAGGCCGA

AGAGGAAG

tJGACCGU CUG UAGGCCr.NACGAA

AAAGAAAD

CAGUGG=CUC% rCcrACCA

ACACAAAA

GGCAGCGG CIGccGAAAcc Ac-c~p G-GUGCCGG CUGAGAGCCGAAAt AGACU=c GCCUGGGG CUGATJAGGCCGAAAGGCC .GAA AAGUMCU GUCAGAGG CUG GAGCGACMCc

AGCAGU=

GAAGIAUM CG aG=GAG,3C

AAGUCG

CCAUGUCA CUGAUGAGGCCGAG Ca AGGAAGCA AUUGAUUC CtGOGA GC

AAGGAAG

CAGUGGCU CUGA A--AAGGCCG CA -AACAAAA CUGGAAC CUAUAGCCGAAGGCCG

AAUMAMA

ACUUWLUU CUGAGAGGCCGAAAGG

AUUCAAMG

AG%-tUUGAA CUAGGCCGAAAGGCc ACuUCC UAAAAACUU CUAUAGCCGAAGCCA AflUGAfU AGCUUGAA CUGAGAGCCGAAAGCCA

AGCUJUCCA.

CAGGUGAG aCUGAGCCGAAMGGCCAA

ACCAUATJ

t7CAGCUUG COGAXGAGGCCGAG-CUAA AGaGCUUC CC C

C

CC

'A

214 Table 11: Humann IL-6 HIH Target Sequence nt.

Position a.

8 9 i2 13 36 37 38 56 57 63 64 69 74 78 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 192 200 201 206 207 212 216 222 EX Target Sequence AUGCACU U UCUU=C UGCACETJU U CUCC GCACUUU C UOUGCCA ACDUUCU U UGCCAAA CUUUCU U GCCAAAG AGAACGU U UCAGAGC GAACGUU U CAGAGCC AACGUU C AGAGC; GGAUGCU U CUGCAU GAYJGCUU C VGCAUU UCUGODL U MG=~U CDGCAUU U GAGOUUG UUUGAGU U UGCUAGC UUGAGt3U U GCaAGCU GUUUGCU A GCOUUG G-t-MZ= C UOGGAGC Mra=C U GGAG GCUGCCU A CGUGUL UACU A UGCCAUC AUGCCALU C CCCACAG CAGAAAU U CCCACAA AGAAAUU C C~CACAAG AGUCAU U GGUGAA GAGA=C U GGCACUIG CACUGCU U UCC= ACUGCUU U CaACUCA CUGCUUU C tUICUC.X GCUUU A CUCAUCG UUCtJACU C AUCGAAC M~CUCAU C GAACUCU UCGAACU C MaCU LMCJGAU A GCCAAXJG UGAGACU C UGAGGAIJ UGAGGAU U CCUGUUC GAGGAUU C CUGUUCC TUCCGU U CCUGUIAC UCCUGUU C CUGUACA Ut3CCUGU A CAMAAA UGUACAU A AAAAXJCA MAAAAU C ACCAACUT mt.

Position 245 247 248 249 257 273 291 305 307 308 316 319 322 323 326 334 338 380 388 389 392 397 409 410 411 413 419 437 440 447 454 462 463 466 479 480 481 497 498 499

AAGAAAU

GAAAUCU

AAAUCrU

AAUCUUU

AGGGAAU

GAGAGU

AGGGGGU

AAAGACU

AGAcUaU GAcaAuu AAAAACU 1 AACUUGUi UUGUCCU I

UGUCCUU~

CCUUAAUJ

AAGAAAU.

AAUACAU

GGAGAGU;

AACCAAU Z

ACCAATUC

AAUUCCU CUAGACtJ A CAAGAGU 1: AAGAGLJU TJ AGAGUtJU C AGUUUCrJ U UUGGUGU A AGUGGAU A GGAVAAU A AGAAAGU U TJGAGACU A AACCGG u ACUGGMr. U GGUUG U CAAAGAU UJ AAAGAt7U U 'aAGAUUU U AGGACAU U GGACAUJU U GACAtUt U EM Taxget Sequence C UtUCAGG U UCAGGA U CAGGQ-AA C AGGGAAU A GGCACAC C AAACUGtJ A CUGUGGA A UUCAAAA U CAAAAAC C AAAAACU UT GUCCUQA :CUt3AThA J AAUAAAG k ATIAAAGA k. AAGAAAU k CAUUGAC I G-ACGG-CC

~AACCAATJ

ccaAGAc

COAGACU

GACE3hCC CCUGCA6A UCUUaGGU CtUGGUG

UUGGIIUG

GGUGUAA

AUGAACA

AUAGAAA

C-AAGU

GACRACUA

AACUGG-U

UGUUGCA

GUUGCAG

GCAGCCA

UUGGAGG

UGGAGGA

GGAGZGAG

UUIACUGC

UACUGCA

ACUGCAG

215 538 539 542 543 544 545 549 551 554 555 556 560 561 573 577 579 580 581 588 597 598 611 6i6 617 6i9 620 625 627 629 630 631 636 638 644 647 653 655 656 657 658 661 672 676 678 S81 682 AC&flDU A CUGCAWU AAAGAW C AG~C.c= CAGG=~ U AAUUUU AGGCCLUU A AflOUC CCULEhAU U DUCUM~ CUtUA=l U UCAAMW UUAAU u cxjm ~axuuuu C AAMA UOUCAAU A MLflOaA UCAMO A AUUL~AC AUAU U MLACDUC MUMLU U AACU= ~AU= A ACCUCAG UMLhACU UJ CAGG UMLACUU C )AGG= GGAAA= A AAMU=U AGMAAU A UUUCAGG UiAAMW U UCAGGMk AAAmkuu u cAG= AAT~flU C AGGCMM CAGGCAU A CUGACAC UMCACU U UC-CCAGA GACACO u GccAGAA AAAGAU A AAAU=cc.

AtIAAU U CUMULAA UUAAUU C UauAAA AAAUUC U AAflAU AAUUCL A AXflA uauhuwA* A uiKAucA AAAAMIJ A UUUCAM AAMZAU U UCAGAM, AMfU U CAGAA UflUU C AGaMMM UUCAGAUT A UCUMMA CAGAMM c AGAAucA.

UCAGAAU C AflUGAAG GAAUCAU U GAACU UtJGAAGU A UUUUCCU GAAGtUAU uucCC AAGMW~tU TU uccucC; AGMVfU= U CCMG GLMUfUUU C CUCCAM UUUUCCU C CAGGCAA G=AAAAU U GAmiC AAUUGAU A MkCM=U UUMMU A CLUUU AUUCE Ur UUUUCULT M~U= U UUUCDUJA 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 MkcmOU u UCUtMhn ACVUU u UCVUcXn CCUUUUU C UutM~tA uuuuucu U AfULkAc TUUUU A tJVUAACU UUCUUAU U thUUa uc~uxU

AACUA

CUMxUrj A ACDUAAC UOt~kACU U AACAx=u UMA=~ A AC-ALWUC u~acA U CUGWA.

MLIOCU C UGUAAA AUUCUGU A MAAJUC AAAAIJGU C UGUAAC UGUCUGU U AACULCA GUCWWO A ACtUAu GUUAACU U AA~MkGLA UMAAC A AaGUA= ACUMW~ A GnU3UM AMkG A UUUAUGA AT7L hx U U7WGo;LAM MkGUaUU U AUGAAAUT AGtU= A UGAAALTG AAAUGGU U AAGAAUU AWtJGGUU A AGAAflU UaAGAAU U UGGLVAA AAGAAUU U GGUAAA AUUGGU A AAUAGUj GGUA U AGUArUU GUAA A GMfUMA AAUrM= A UUkuAUU Uuak~tU U UAUUMA MGAU U AnUrM.au AGMfU= A UUUATM3 AUUUU UL AAfGUU UUUAUDU A AUGU=A UMA=U U AUGUUGU tUAUGU= A UGOUUG GUMVDG U GUGUUCUJ GUUGUGU U caXAAh UUGUGUU C UAUAA GuGukUCU A AULajaA.C UUCUAA A AAACAAM CAAAAU A GACAACU 216 Table 12: Human IL-5 HIH Ribozyme Sequences a.

a.

a a a a a.

a Ut.

Position 8 9 i2 13 36 37 38 56 57 63 64 69 70 74 78 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 192 200 201 206 207 212 216 222 245 GCAAAGA CUGAUAGG,C-AAAGG,-C---A

AGUGCL

GGCAAAG CUAV AGCG,-AAA,-CaA

A(;GC;

UGGCAAA CU; ;AGC-AAG-CC:,,AA j-;C UUGGCA CGUAGCCwAAAGGCCG-

AGAAGU

C;UUUGG CGAMAGGCCGAAA

AAGA

GCU3CuGA CUAGGC-AAGC-A ALCG-Uu GGCDCUG CUAGG-C,~G-CA AACSrJ~c MuG-cu C UGGCGAAACC--A AAACGUcy AAflGCAG CMUGtGAGGCC--A AM~CM-M AGC.XXCc AAAflGCA. CU~GAGCAGGC C -A AA~c.Oc AAAC~rCA CGUAGCCGAGC(i AVlC- CAAACtJC C~3VAWCAA~

AALUGCAG

GCUkGCA ~CUGUA-CC-XCC-A ACtUCAAA AGCL~r7C CUGAUGAGGCCGA3M CGM Ax CCAA, CAAGAGC COU GC-L G GC AGCAC GCUCCAA CGUAC-CGG-CcA

AGCAC

CAGCUCC COAGGCCC-GCCC-A

AGAGCUA

AUAC1ACG CUG AG=-C A CCA AGGCAGC GAkUGGCA, CU;LMA CGkA-CC--A

ACACGQA

CUGUG CUGAUGA=GCk ZCGC

AM~GCAU

UUGGG CUGAtr-AG--CCGAAG-.C-c-, AUJUCtjG C.UGu G CUG UAWCCCcAUGr

AAUOUCU

TUUCACC CUAAGCCGAAAGGCC

AUGCC

CAGUGCC CUGAGAGr-CG rGccc- A==Xrc GAGUAGA 'UAr: GAAc.GCc,.A

AGCA=U

UGAGTJAG CUGAWZCGCAAC-,-,

AAGCG

AUGAGUA CUGATDCa"GAAAGC

AAGA

CGAUGAG CUGAGAGCCGGG-CC-L

AGAAG

GUE3CGAU CUrkUGAGGCCCL CGaA

AGUAGAA

AGAGUUC C GAUGGCA AAGC-A

AUGGTJ

AUCAGCA CUGAUGGGCC' AGCG-

AGTUJCGA

CA=UGGC CDGAI3GAGGCCGA GCC.AA

AUCAGCA

AUCCUCA CGAIGCGAGGcCGA AG ICA GAACAGG CUAGGGCCGAGG

AUCCUCA

GGAACAG CUGAMAGCCGA &CcG

AAIUCCUC

GMACAGG CUGAflGAGGC-CQGAGCQAA

ACAGGAA

UGUACAG CUJGAUGAGG-CC-kAGCA

CGA

UUUE]AUG CUGAtGAGGCCGAAAGGCCGA

ACAGGA

UGAUUUU CU;UA CGAGCA;A

AUGUACA

AGLUGGU CUG GGGCCGZACCGA

AUULTC=

CCUGAAA CUGAUGAGGC-CAAAGC,,CGA~ AUUrUCuu H Ribozyme Sequzence 217 247 UCCCUGA CUAG~cGAG-L.p A~GruC 248 UUCCCUG CO GAGGCGCCG A f AQ G 249 AflUCCL CtLA AGGQ CCG AAGAU 257 GtLU'rG GGCC AUUCCCtJ 273 AM= U IAGCCG GC ACUCC 291 UCCACAG CUamG GcG r c ACCCCCU 305 UOUGAA COUGAGCCGAAAGGCA A GUCJJ 307 GUUUUUJG CLUGAUGA-CGAAAGGrCCGrA At3AGUCUr 308 AG~UUrjr CLGUA-C-GAGCG AAUkGUC 316 ULAGAC COGAUAGGCCGAG CC-A AGUUU 319 tAUflAG COAUAGGCCGAA GCGAA ACAAGUU 322 Cutn COAGGCGAAGCA

AGGACAA

323 rjuuCD.UGAG -CAAGCA

AAGGAC

326 AUUUCU CUG UAGGCCGA 'GGCGAA AIJMTA 334 GUCAAMl CUGaAGAGCCGAA GCGA AUUUCL~r 338 GGCCGUC COGAUGAGGCCCAAGCC AUGtUTU 380 ADlUGGU CUGAMAGcGCCA AG ACU=Cu *388 GUCCAGG COGAUGAGGCCG .AGCCGAA

AUG=U

389 AGOCUG COGAZ3GACZCCWAAGCCGAA

AAUGG

392 GGCGUC CUGAtUaAGGCCGAAA~GCA

AGA~J

397 U GCAGG CUGAUMZCGGAGGCC cGAA

AGUCA

ACCAAG CUG UGAGGCCGAAA CCGAA ACUCUU *410 CACCAA~ CUGAtUGAIGGC AAAG ~rCrUC 413 TurkcAcc CUfLVA*GCC

AGAAACU

419 U UCAX3 CUUAGAZCAGGC C AAAACCC.AA C.437 UUUICUAU CtJGAfLrGCGAAAGGCGA

AUCCACU

CC.*440 AACUUC CLUGAGGCCGAA rG-AA AUtMhICC 447 LMGCUC COGAflGCXUG GCCJ A ACUUtJCU 454 ACCAGUU CtXaxJGAGCCGAAAGOCCGA

AGUCUCA

46 C C AeM COMAUGAGGCCGA--IG-cCCG-AA

CAU

463 CUGCAAC COAGAGCGAGCCGAA

AACCAGU

466 UGGCUGC CUGAUGAGGC~CGcc GCG

ACAAC

479 CCUCCAA CUGAUGAGGC GCCGAA AUCUUU :480 UccUCCA CUGAIGAGGCCGAAGCCGAA

AAUCUUU

#*d:481 Lucc~xc CUTGAt1GAGCC CCGAA AAUCUU *497 GCAG3AA CUGAUGAGGCCGAAG.CGAA

AUGTCCTJ

498 UGCAGtU, CUGAUGAGCGGGCCGAA A ALU=~c 499 CUGCAGU CUGAUGAGr-CCGAA~CCGAA

AAAUGUC

500 ACUGCAG CUG-flGAGGCGGC~ rA A A)AUr 531 AAGGCCU CUGAUflAGGCCGAAGCCr.A ACUCUaU 538 GAAAAUJU CUUGWGAGGCCGAAAGGrCGA

AGGCCUJG

539 UGAAAAIJ CUGAUGAGGCCAACGCGAA

AAGGCMT

542 tLhLUUGAA CUGALIGAGGCCG 'GCGrAA AflUAAGG 543 ALUh.UUGA CVAGG-CAAGCA

AATUA

544 LUMUUG CUGAUGAGGCc

AAAUUAA

545 ULEh.UU CUGA G c- c AAAAUUA 549 UAA.AUUA CUAGGCGAG;CA

AUUGAA

551 GUtAAU CUGAUGAGr-Cc CGCCAA

AUAUUGJA

ovyvfOw ria y~vvayy anowmn MODWYvvmflW flVV=Y~EX~

DVYM

~1nnvvw vnvv VV~fl~ y ~V

WVU

OrDawmy m flmDD monm~y vmo flonlYvm vvmoy vtOwfm3vv no vm~v vnvfl flyl YnmfDw VVDO Y~nfl flaalODy narmm floolZmy n DDvDvy Ononvfvy

OC.M

0=0=1 OvonmDl £DvflD MOM=j mamvv =--WYv xnmDmyW W v 869 £.69 £69 Z69 L69 689 889 989 '89 T£89 E89 Z89 8L9 9L9 ZL9 T99 899 LS9 9 £9 I £9 019 8E9 L9 TE9 OE9 6Z9 LZ9 S99 119 86S 889 T8S 61.5 19S 09S 9ss v.s 0 *000 0000 0 0000 0 9 0 0 00 0 0 0000 0*0 *000 0 00 0 00 0 0 0000 0000 00 00 0 *000

STE

219 703 UU3ACAG CMGUGGAC-c~ GC CGA AUGUM~A 704 UUOUACA ccOGAGGCCGAA. ,craA AA~urum 708 GACAIJOU CUGAUGAGGCGAAGGCCA

ACAGAAU

715 GO MACA CUGAtGGGA AAGc:GA ArC-n= 7119 UEMAGVU CUANI-

ACAGACA

720 AULMAGU CUGAGAGG-CGc GCCA

AACAAC

724 MCMnu Ct AWGAGGCCGAA GCGA AGUUAAC 725 ALMCLIrU CUGAGAGGC-CAAGVCL

AAUA

728 UAAEC COUGAGGC-A-NAGGCCGA AI~A(rJ 731 UC~A CCGAX GAGGCCGAAGCGA

ACMUM~

733 UU~CAM C-,AGAG-GGGCCG-.c

AUACU.AU

734 AUU CLUAGGC-ArC,-c-A AAkam 735 CALMUMA CUAUG=AGcACGCCG;L

AAAC

745 AAUUCUU CUGAUGAGGCCG AGCM

ACCAUUU

746 AAA==C CUGAUAGGCCGAAA=rA A CCJnU 752 UriACCA CtGUACGA AC--c-c GCcAA

AUUCUUA

*fee 753 ADuumccCOAMOC AAM *757 Ac M=n COM JGAGGCCGAAGC(;AA

ACCAAAU

**761 AAADkCU CUAMZCGAGCA

AUUM=

762 UAAAUAC CUAGG-CAA-CrA AAIMuAC 765 AAAflAAA ar AIXAGGCCGJ rArGC i ACLMAUTJ 9767 tJMAAXM CCAGCC AflACA 768 ADJCAAAU COGAUGAGGCCGAAAG~rA AA k 769 CAuaAA CUGAW-AGGCCGAAAG ?JG; AAA= 771 AAAUM CCGAUAGGCGAAAGCA

AL

*.772 Maz= UGAG CGAAGCA

AAMAAU

773 AMW-AU CUGAUGAGGCCGAAGCA

AM

9.9778 ACAACAIJ COXtAjGGCAGGC GA ACALVL 779 CACAACA COGAXJGAGGC~CGAAAGCC-,.A

AAA

*783 AGA.ACAC COGAUMGGGCCAAGM AAMAuC *.:788 UMXUMG COWAGCAAGCA

ACACAAC

UUr~KMM CCGAXXGACCt

AACAC

794 UUUGOUU COGAVMAQCt

AGAM

805 AGUUGC CGADGAGGCCGAAAGGCCG.AA

AUUUUG

220 Table 13: Mouse IL-5 E Ribozyme Target Sequence nt.

Position E Target Sequaence Ut.

Pooltion HE Target Sequence cCCU C CtUcCD-U U GAAgacU U GaAgAcrJ u AAgacLlt C UcaGaGU c GGAfU U CUUG..i GCUgAA

CAAG

CAGA7uC cAwGA AGAg"C AUGCA6CU CGcU UGCAcUU

GAULT

GGUGu 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

GANUGCUU

gAUGcUrJ CUGCAcU U9ACuU GCUgUGU UgGC-gAU gGAgAU

GAGACCU

GAgACcU guccWCT CCGAgCU UGAGGcU 1 GAGGcUJU i gAC-GCuU UUCCtUG' TCCIGj UGUCcU I

LUCUCAU

UacuAtj Uaa.AaaU UAAAU c UAAAAAU c uaUGCAU

V

gAC-AAAY C gAgAaAU c gagAAA6U c gA.AaAt3 c gaAAucU U GAAAtJCU U AAAUCU U AAUCUUU C C uggGCCAL U CCCAU9A Z CCAugAG J GaCACaG JGaCAcAg :AcCGAgC UUtAC 3 CCUGUC

CUGUCCC

CLTGuCC CCUacLC CCt~khC cuCAUAA aAAaUCa AAAALUCA6 aCcAGCU ACCAgCU acCAgCu GGaGAAA

UUUCAGG

UUucAGG tUUCAGG UUUCAGg UCAGgGg UCAGGGg CAGGGgc AGGGgcU 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 AGGGgCr.

UagACAtJ GaAG-AatJ GaAGAau GAAgaAU uGGGGGtJ AAkugCty AAAugCLI AUGCuAU AugCU U6C=uu AACcUW~ CLCGUCaU UGUCaUU CaUM-AtU

AAGAAAU

AAUACAU

AAUaCaU AggCAgU1 9gCAgUU C'3SGAU

CAAGAGU

AAGAGUU

AGULCCtT t UcaCAAU L cAcAAUU L AcAAUU

A

AcAAUUU a AA6ATUgU c GCUGuUt7 c UuUCCAU U UUauAU u ULUuaUU ui uaU;LJE= a LTAULAuMjE a UUAUG;U C UJUAuG~u C AA.ACGT U AAgUGuU u A GaC-;uAC 8. C-,C-aAgA C AAACUGU A;AaLugU a.AAct~gu~ A CtUGUGGA A WJCcAA a uUCaa u CCaAaAc U CcAAAAC C cAAAACc C aruammJ U AAUAAAG -A AUAVAA A AAG-AAU A CAUUA U G-ACCGCC U G-ACcgCC U CCUgG;Lu CguU~ kCCUG,-cA 3CCUUGG-U G-UG~gA UAAgUEIA AA9UUaA ALgUt~aAa aG*LJAAa AAcAgAU CaUuTh.U UauaUtUU alggUCcu AugUecri UgUCCuG, UgUCcUg cUGt~aGtJ cUGUagU uaaCCUU aACcUUU 221 620 793 816 818 825 825 839 840 863 864 864 913 917 957 960 960 962 975 987 990 1000 1027 1034 1037 1039 1 039 1041 1051 1148 1213 1213 1214 1215 1234 1236 1275 1276 1280 1298 1310 1310 1310 1350 1358 1370 1375 1377 1383 1405 UMLACcU u uUuGMU caAC-gCU u UGUGcALT Ct3GagUU a UACUCcc GAgut~I3 a cT3CCcuC ACt7cCcU c CccCUCA aCUccCU c CcCcUCa AuCcucU U cGUUt-CA uCcucUU c GUtUGCku QzLAgtLU U cCAGCu AAgUAUU c CAGGCug AAGUAflU c caggCug gAaCtJCU U G-%-ucCaG UcC~uggU c CAG~uGG t3UagcAU c CUUUcUc GCAucc-U u UcUcCuA GcaUcCtl u uCDCcUa AUCuuU c Ur-cUaGC gcccCUU u AgA~1AgA aGarJGAU A cuuAAUG UAuAC7 u AAugacU UGACuCtJ c UugCLIGA CgggGCU U cCUgCUC UCCrJGCU C CUaUcuA UgcUCcU A UcUAACu cUccuAU c MACUUC cUCc~kU c UAACUc CcMUcU A ACUr~cAa UcAAuU UJ AAuAcr-C uGACUU u cUuaUGU GCUgGaU u UUGGAaa gcUGGAtT u uUgGAAA cugGAUJU U UGGAaaA ugGAUUU U GGAaaAG gGGACAU c UccuUGc GACAX~cU c CUUGCAG ugGGCC U AcULcUC gGGCCD A cUUcUCc CtULIhcUU c UCcgUgU UgAACUU a AGAa~cA gcAAAGU a aAuACcA GCAAAgU a aALAcca GcaAAgU a AAUCA AAAGCAU A AAAUggU AAAUGGU U ggGAugU UgUuaUU C AC-gL~kfc UUCAGgU A UCAGggU CAGgM~U C AGggUCA tJCAGggU C AcC-gAG cccCAgU U MACUCA 1407 1407 1410 1434 1434 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 CC~gUUU AL CUcCAGg CCAgUGJU a CUC-CAGG gUUUaCU C CAGGaA AtgCUuu U alluUaAUy allgcuU U AUuxAu allgcuU u iiAUrLAAU TUgCUUoU a UuUTAU ugcUuu a uOuLaaLUr UuULIAUU U- AA~urcug uuua;Luur U AAUucUg UUTJAGUU A At~ucUgU UJUUaAuU c UGuaAGa AUUCUGU A AgAUGUu UgUrcaE a tUtThIUUA 'gUUcAU A uUAXUUA UucATkU u AUUUJAu~g UcAU A UUtUtJGA AMW UJ MifGAug AuQgAVU c aGAgU ATJUcaGU A AgUrUAaU aGuAAGu u AAuAmmU aGUakgu U Aa~UU GUA'kgtXY A allAUUUA agtUtkAU a UUuUuUA AgO~a7 A tUtJU~a UELAUaY u uAuuACA UUAAuAU u t3ATJUaCA UUAaUAU U tTAUUacA UAAUaUUu AUhcAc MUAUU U AUuAcAc TJAatM= U AUt~acAc AA~U= a uuaCAcg AALUUU a UuAcAcg AaUM=~ A UuAC~cG AaU~flUU A UJ3acAcG AUUtUTJ a CAcgU cACGUaU A UaauAUu cAcg=~ a UAAuaUU AUAUJAaty a UiUcuaaU ATJAuAU U CUaAu.AA allaaE~aU U CUAAXMh UAAAUU C UaAuAAa ML~AUU c UaauAAA t~~UA~ut7 c UaaUAAA UaaUaUU c UAAUAAA allaTiUCU A AUrAAAgC UUMULAU- A AAgCAgA

S

Table)I 14: Human 1L-5 Hairpin Ri)ozyrnec Sequences nt.

Posit ion 86 151 172 203 Hairpin Ribozyme Sequence UACAOS1A AAA GUC GAGUAGAA AGAA G~-c UOGlWr NAM GAGU) LUWAO AAA GGA~uC

ACGAACQU~A~MM

ACCAk-AUnaWXflXG3 Substrate GACUIU 0U GAUAWCrA GAULXU U JJ cMCtA 9%* 0 *0* 0

S

*00 .0 0 0 0* 0* 0 0 ~0 Table 15: Mouse 11,5 Hairpin Ribozyine Sequences nit.

Position E3 147 150 154 168 199 274 381 454 499 5d8 701 710 870 919 1030 1170 1205 1402 1421 Hairpin Ribozyme Sequencq AGMGA )A3AA GAACAC CrAGACAC AGMA GNJ M GAGCOGAo AGAA GfLXWA C 3 G jm rm GGUAGOwx AGAA GaUG CAUrh Th GCOOMn AAA GACAGC A A j~~ UXUGM AGAA G1~AU CC 3 GA wm UGAG~kG AGM GGAGC CCCOCACG AGAA GUAU3rA ACG PL3AA GCM CACAM3 PLAA GCUAG UAAAUGGA PAGAA GCAUAU GCAS3 AGAA GAAAUrJU lN A i~ GAAGAGnp. PIAA O3GO3?G PLGIJ2AAA Pd3AA GCCUXG QXXGUC AGAA Q3ACCh UAGAUAGG PA3AA GGAG AUGO2ACA )A3AA GAflU CC 3AjA CAAMLOC PA3AA GQXTCA CLUAGM AGNAA

QOOGA

AAGCAUAC PAGAA GAJJW ACAAAAAAULXXgCXCLa Substrate GUM=U GAC UCrWU ACCLA OCU GLUU EACIA CL GUCUu CACGCU GU osa~Acp GCGC GCU CACGAC G~rLvC GUJ GACAA32A GCUOU U OUaPLW U2AACU GUC OaXnggX OGAGOCA QAJQ~oawuu CQAGU GCU CCALfljG GZ2AAO GAll GCAAAAC ALMw3Jc GUU UCCUUL AUJXXU GAllam LXUC GCC LUXCU OCAGOCU GAO MAACflJ LUGUOCA GAll G3ACCA WAAWA GAO LU~_N LXMtO2 GUl UACUz -AAACA GAll GQIACL 224

CD)

a S.

U,

V

V

0~

V

C12

V

N

0 .99 -l

V

U,

0 ~0

I-'

V

(U

I ifC)

C

I

A

C

C

fill 11111

CA

C

2 ii H1

'N!

HE

0 05.

zr 225 Table 17 Mouse re/A nt. Position HH Target sequence HH Target Sequence nt. Position HH Target Sequence 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

AAUGGCE

aGCU-Ct CcUCcat GAuCUGr.

AUCDU=

UUCCCCU

CUCAUCU

UCAUCUEJ

CAGGCLILJ

GGgCCut3

TJGGAGAU

AGACU

AtJGcGaU UGCGaUrJ

UUCCGCU

GGGCGCU

GCAQuAU

CACAGAU

CCACCAU

UCAAGAU

;'ALtGGCU UUCGaAIJ CGaAUCtJ

CCCUGGU

GGCCCu uCCaCCEJ CCGGCCU C AuGAaCUZ AGaUcaU c C-AUGGCU a AUGGucu c GCaCU A CUGACE C GCaGuAU

C

CCgCAGLI a CAuAGcU u AuAGCruU c UG-GGgAU C GCUCCU u CUCCULU U CtCCUUU C 3a caCaGgA 3a cGUgGC u GcGgACa U LCCCCUC U CCCCUjCA, C AUCUUuc C urUCCC..I U UICCCUCA u CCcUCAG C UGGgCCu A UGUGGAG C AU~cGAaC C GAaCAGC U CCGCU.u C CGCthuA A uAAaUG C aGCGGGC u CCuG-GCG A CCACCAA C AAGAUCA, C AA~rGC A C;LCAGaA C UCCCUG C CC-UGGU

CACCAACG

CUCCuga

ACCGGC

ACCaCAL GUgGWgA GaAcAGc CtkGAG UccGgaG

UGAGGCU

UGC-CCaG CAuAGcU UCCAuAg

CCAGAAC

CAGAACC

CAGUGUG

TJUCUCAA

UCUC;AG

UCAAGC

467 469 473 481 50i 502 B08 509 512 514 534 556 56i 562 585 598 613 61 6 617 620 623 628 630 631 638 661.

667 687 700 713 717 718 721.

751 759 761 762 763 792 1167 11168 1169 1 182 1183

CCAGGCI

AaGCcAJ TUU9AG( AC-CGaAI

AACCC=

UUCAC-rL UC-XcGULT M:CCUr-

GG-GACEU

UGCGcCU

CZ;CUGCEU

UCUGCLU

aAgCC,-Au GGCCCct3

CCCCUGU

CLMUCCU

UcCCUU

CCULCC?

UjCCUgcU AUCCgAEJ CCgAUU CgAIuuuu 1 U-GgCcAt7 CCGAGCu

UCAAGAXI

CGgAACU C GCUwGCCUC AUG-AGAU

C

GAGAUcU UJ AC-AUcUt C UUCUCCEJ C AaGACAtJ

U

GAGG-UG A GGUGUAU

U

GUGIA~UU lU t7GUALT~Ur

C

CGAGGCU C GAUGAGrJ

U

ATUGAGLrJ U UGAGULTU u AUGcUGtj

U

U-GCUGUEJ a -C CUgUUCg Ju AGCCAGC 3C AGaucAg 7C CA;GACC U uCAcGUJC u CACGUUC U C-CUAmA C CUAUkAA A UNCGAgGA A GAgGALGC A uGACuUG C L-G-UUjCc U CCAGGTJG C CAGUrGA U AGCCAGc C CLUCCUGa C CfcuCaC c uCaCAUC C Ct7CAgCC- C A4CCaug U CC.!LCc u UUUGAuA J UGAUAA U G-AUAAcC .1 GUGuuCC

AAGAUCU

LUGCcA LMGgAGC

GGUGGGG

UUCuUgC C.UgCUG Ut7gCUGY CauUGcG

GAGGUGU

UUUTCACG

UCACGGG

CACGGGA

ACGGGAC

CUUUU=CU

UUCCCCC

UCCCCCA

CCcCCAY aCCaUCa CCaUCaG UGCCU U GUGUCC 226 834 835 845 849 872 883 885 905 906 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 1125 1127 1131 1132 1133 1137 1140 1153 1158 1680 1681 1683 1686 1690 AtMURN3 U CCGGA~u UDG= c CcGGACUC GACLICCU C CgUACGC CCDCMgU A cCCGAC CCAGGCUT C CUG~uCG UuCGaGU C UCCPAflG CGaGUCU C CADGCAG GC-GGCC U CuGAuCG ClGoGCU C UGAnCGC GcGAGCU C AGGG AtJGGCg UJ CCAGC UGGAgUU C CAG~kCu UUCAW A CutGCC-A GCCucAU cC Aj=AuGA AGAuGAD C GCACCG Cagt~acU u gCCaG~c ACCGGA U GaaGAGA GAqACcUT AGGACcU.

GAGACCU 1

AGACCUU

AAGuAU

GAAGAGU

GAGtU

AGUCCU

GuCCuuC CCGG-C C UaCA=C t GgCQuAUt UGUGCCUa aaGCCU C CGaAaCrJ C CUCAaCU Uj UCAacuu c CUUCUGU C CAGCCCU A GCCAUAU a CAU'CCCU c AcaCCUU c UCCaUcU c UUEVhCuU u cCagCAT C GCACCAU c AUCAACtI u GAAGACU U AAGACUU C GACU C UtJCUCCU C CCUCCAU

U

u1 ckaAagu h~ UGAGAMc U CAAGAGu CUUUCAa 7 UCAauGG 7 CAauGGA AauGGAC CAaCCG L GAUCCAa T cm'G= LCt~aAa CCGaAGu AaCUtU

CCCAAGC

caCCOc 9cuahc agCacCA ccagCA CagCuUC AgCgCgc CCUcAGC

AACUUG

UGAUGAG

cuccucc ucctJCCA CUCCmuU

CAUU=C

GCGGACA

1184 1187 1188 1198 1209 1215 1229 1237 1-250 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 GGCCCCU C CrjcCUGa GUcCLuu c CUcaGCc UJUaCCaU C aGGGCAG, GC-gAGUU u AGuCuGa CAGCCCEJ a caCCUlUc cuGGCCU U aGCaCCG, GGuCCCU u CCUCAGc CCCAgcEJ C CUGCCCC CcAGcCU c CAGgCuC CCCaGCU C CuGCCcc CCAUGGU c CCuuCcu gUGGgcUi C AGCUgcG ALugAGuJ u ucc-cCa.

CUCCt3GU u CgAGtTCu CCCCAGEJU C7~~aC CAGUuCU A aCCCCgG G~UCCU C CCCAGuC CUUrJUCU C AaGCUra ACGCUGU C gCaGCc- CUGCAMG U UGAUGcU UGCAGUU U GAUGCUJG GGGGCCU= U- GCQUMGC CCUUGCLY U GGCAACA GgaGUGU Uj CACAGAC gaGU=U C ACAGACC CUGGCAU c uGUgGAC CuUCgGU a GggAACE GACAACY C aGAGUUU UCaGAWG U UCAGCAG CaGAGUU U CAGCAGC aGAGUUU C AGCAGCEJ 9GuGCAU c CCtJGUGu AUGGAGU A CCCUGAa UGAaGCU A t3AACUCG AaGCUAU A ACUCGC UAUAAC C GCC:UgGu CUJCUCCU A GaGAggG CCCAGCU C CUGCCC UCCUGCEJ u C99UAGG CGGGGCU u CCCAAUG cUGaCCU c ugccCA cCUgUCU U cCAGGUG UCUgCrJU c CAGGuGA CUCgcUU u cGGAGgU 227 1704 AT3GGAC T CUJ~jGCu 1705 M=GCD C UCjGCUC 1707 GACt=CE C uccCCut 01721 UUUGAGEU C AGAUCAG '1726 GUCAGAU c AGC-U=C 17"O1 ALTCAGU C CUAGU 1734 ACCUA AGGuGcU 1754 CaGugCU c CCaAGAG .00 228 Table 18 Human re/ A nt. Position HH Target Sequences HH Target Sequence nt. Position HH Target Sequence 19 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

AAUGGCU

GGCU=G

GAACUGU

AACUGUU

tICAUCUU

CAGGCC

GGCCCCU

TJGGAGAU

AGAXAU

UGCDU

GGGCGCLT

GCAGCM

CA MGAU CCACCAV C

UCAAGAUC

AAUGGCUI

UGCGCAUC

CCAU=C

CCCUGGU C GGACCU C cccOcc

C

CCGCCU C ACGAGCU TJ AGCUUGU A GAUGGCU T AUGGCU c GGCUUCU A CUGAGCu c GCUGC-AU c CCACAGU U CACAGtIU U ACAGOULY c UGGGAAU C GGCUCCU U GCtUCCtUr U Ct3CCUTJ U t7CCUrUrj C UGGCCAU u AUUGUGU U C GUCDGUA C UGCAGUG A GUGCACG Uj CCCCCUC C CCCCUCA C AUCUUCC C UUCCCGG U CCCGGC2A C CCGMcA C UGGCCCC A UGUC4AG C AUtMAGC U GAGCAGC U CCGCAC k CAAGUGC

CGCGGGC

:CCAGGCG

kCCACCAA

AAGAUCA

AAflGGCU

LCACAGGA

CCUGGUC

ACCAAGG

CUCACCG

ACCCCCA

GOAGGAA

GGAAAGG

*CELhDGAG

UAUGAGG

UGAGGCU

UGCCCGG

CALCAGUU

UCCAGAA

CCAGAAC

CAGAACC

CAGUGUG

UUCGCAA

UCGCAAG

CGCAAGC

GCAAGCU

GUGUXC

CCGGACC

467 469 473 481 501 502 508 509 512 514 534 556 561 562 585 598 613 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 1167 1168 1169 1182 1183 1184 GC-AGGCU A UCAGUCA AGCMAU C AGUCAGC UUCAGU C AGCGCAU ACGCAIJ c CAGACCA AACCCCU u ccAAGTi-u ACCCC~wV C CAAGUTUC CCAAGU U CCrZMkG CCAAGUU C CUAUAhGA AGtJuCC-J AL UAGAAGA lUUCCaAn A G-AAGAGC GGGGALCy A: CGACCUG UGCGCU- C t3GCUUCC CUCUGC-U U CC-AG,-UGj UCUGC= C CAGGOGA G-AC=CU C AGGCAGG GGCCCCu c cGCCuGc CG-CCUGU,. C CutiCCC CUGUCICU U CCUCATJC UGUCCuJ C CUCAU--CC CCDUCCU C AT-CCCAU LrCC LuCA c ccAuctjUr AUCCCAtT C UUUGUACA CCCALTCU u uGA~CAAu CCAUCUU U GACAAUC UG-ACAAU C GUGCCCC CCGAGCtJ C AAGAUCU UCAAGAU C UGCCGAG CGAAACU C t'GGCACC GCUG;CCtJ C GGUGGG AtJGAGAt7 C UtYCCu.LC G-AGAUCU U CCE2ACUG AGAUCUU C CUACUGU UCTUICCU A CUG=GtT AGGCACAU U GA.GGt3G-, GAGGUGU A UUUCACG GGUGUAU U UCACGGG GGUUU U CA;CGGGAL tJGUAUUE C ACGGGAC CGAGGCU C CTU-tJCG GAUGAGU U LICCCALCC AUGAGUIJ U CCCALccA UGAGUUU C CCACCAU AUtG%.G U UC-JUCrj UG-GUU U CCUUCUG GGLUGUUU C CUUCUGG 229 p 835 845 849 872 883 885 905 906 919 936 937 942 953 962 965 973 986 996 1005 1006 i015 1028 1031 1032 1033 1058 1064 1072 1.082 1083 1092 1097 1098 1102 1125 1127 1131 1132 1133 1137 1140 1153 1158 1680 1681 1683 1686 1690 1704

UUGUGUU

GACCCCEY

GCAGCGCU

TJGCGU

CGUGJCU

AUGGAAU

TJGGAAUU

uu-CAGU

GCCAGAU

AGACGAU

CGALUCG

ACCGGAU

GAAACGU.

AGGACAU.

GAGACC= I AGACCUU I

AGAGCAU

GAAGAGTjU

GAGMUCCI

AGUCCUUI

GUCCUUU

UJCCACC GACGCAUt UGUGCCU t CGCAGCL7 c CUCAGCU u UCAGCUU c Du CUGU C CAGCCCY A GCCCUIAU C UUCCCU U AUJCCCUU U ucccuurj A OU CU C ACGUCAU c GCACCAU c AUCAACU A GAA.GACU UJ AAGACUY C G-ACUUCU C cUUtccLU C C CGGACCC C CCM=G A CGAAC C CUGUO-CG C ucaUGC C CAUGCAG U CCGACCG C CGACCGG C AGOGAGC U CC-AGUAC C CAGLUCC A CCCGCCkL A CAC-ACQ-A C GUCACCG C ACCGGAU U GAGGAGA A~ AAAGGAC A~ UGAGACC U CAAGAGC

AAGALGCA

AUGAAGA

CUOUCAG

J UCAGCGG J CAGCGGA

AGCGGAC

CACCUCG

GACGCAU

IGCOGUWC

TCCCGCAG

CCGCAGC

AGCU3UCU rCUGU~CC UGVCCc

CCCAAGC

CCCUU

UJACGUCA

ACG3CAU

CGUCAUJC

AUCCCUG

CCOGAGC

AACUmJJ

UGAUGAG

UCCUCC

CUCCAU

CALUUGCG

1187 1188 13-98 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 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665

GUUUJCCU

UUTUCCUL7 GGC-A =U

CAGGCCU

TJCGGCCU

GGCCCCU

CCCAAGU

CCAGGCU

CCCUG~tY

CCAUGGU

AtUGMW~ AL7CAGCU

CCCIC=G

UCCCAGU.

CAG=cCU,

AGCCU

cccuccu

ACCUGU

CUGCAGU

tJGCAGEU GGGGCCUt CCUUGC-ut GCUGUGU t CUGUGUU C CUGGCAU C CAUCCGtJ C .GACAACU

C

UCCGAGU U CCGAGUU U CGAGtJUU C AGGGCAU A AUGGAGU

A

UGAGGCU A AGGCUAU A tJAUAACU

C

CUCGCCU A CCCAGCUJ C UCCUIGC C CGGGGCT C AUGGCCrJ C GCCUCCty U CCUCCUU u CUCCUUU c U CUGGGCA C UGGGCAG C AGCCAGG C GG'CUG U GGCCCCG *C CCCAAGtJ C CDGCCC C CAGCCCC C CAGCCAU A UCAGCUC C AGCJCLUG C UGG-CCCA C CCAOUCC C CmGCCC A GCCCCAG C CUCAGGC

AGGCE)GU

AGAGGCC

3UGAtTGAU GAUGAtIG 7GCUUGGC

GGCAACA

CACAGAC

*ACAGACC

*CGUCGAC

*GACAACU

CGAGUUU

UCAGCAG

CAGCAGC

AGCAGCU

CCUGUGG

CCCrJGAG

UAACUCG

ACUCGCC

GICCEPIGU

GUGACAG

CUGCUCC

CACUJGGG

CCCAAUG

CUUUCAG

UCAGGAG

CAGGAGA

AGGAGAU

AUGGACU U Ct3CAGCC 230 1705 LrGGACUU C UCAC= 1707 GCDC= C AGC=CtG 1721 GCOGAGU C AGDAUCA 1726 GCGAD C AGCUCCU 1731 AUCAGCU C CaAGGG 1734 AGCUCCU A AGGGGGU 1754 CUGCCC= C CCCAGAG Table 1,9 Mouse re/ A nt.

Sequence 19 22 26 93 94 100 103 105 106 129 138 148 151 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 467 469 473 481 231 HH -Ribozyrne Sequences HH Ribozyme, Sequence UCTUU COG UGAGCCGAAAGCCGA AGC.kJU CACCACG CGGACCGA AA~ AcGGAGCU UG3CCGC CGA =CGAZ=GCA AL7GGAG GAGGGGA CUAGC-CGAAAGCGI

ACAGAUC

UGAGGG CVGAUGAGGCCGA A.-CGAA

AACAGAU

CaAAGA CUGfGAUG -rCrY1

AGWGGGAA

AGGGAAA COGAflGAGCCGAAACXAA AUGAGcG UGAGGGA CGAGAGCCGAAACGCA

AGAUGAG

C C GAU r"CGAAAW-aCC

AAGAUGA

A==CC aMAGAGCCGAACCA

AAGCCUG.

rDC 'CA CCGAUGAWGCCAAA G,-CGAA

AAGGC~C

GUUCGALU GCOGAAGGC

AUCUCC

GCUGUC COGAWAGCCGi C-,CrAA

AUGAUCU

AUAGCGG CUGAXGAGGCCGAAC CGAA AUCGCAU M= CGA GCCAcGAA~

AAUCGCA

GCAUuua G CG A

AGCGGA

GCCCGCU CUaUG GAGGCCGA cA~ AGCr.CCC CGCCAGG GCCGA c AUaC=G vUGCmGG C aA GG AA cc AflCUGUG UGAU CU UAGCM!GGCC

ADGGOGG

AGCCAUU CUGAUGAGG--CGA W.,r-GAA

AUCOUGA

UCCUUGUJG CUCAUGAGGCCGA -C.CCrAA

AGCCAUU

CCAGGGA CGfGGCr

AUUCGAA

GACCAGG CUGAI GAGGCCGA AGCr.A

AGAUUCG.

"CUU GCCGAAA

ACCAGGG

UCAGGAG L7UGAGGCCGAA cCcAA

AGGGGCC

UGUGGAu arUGGGCCGGCCA

AGGCCGG

UCCCCAC CUAGGCGAGCrA AGtJCAU GCUGUUC CUMG CGAO

AIJGAUCU

CUCAUAG COGAW-AGGCCGAA GGCCGAA

AGCCAUC

CUCCGGA aMUAGCAkGCA

AGACCAU

AGCCUCA CUGAUGAGGCCGA QGGCCGAA AGUkGCC CUGGGCA arUAGCAAGCA

GUA

AGCUAUG COGAUAGCCGGG~rA

AUACOGC

CLUGGA COAUAGCCGAAAGGCCAA

ACUGCGG

GUUCUGG C=GGCCGAArCCG

AGCUAUG

GGUUCUTG CUGMIGAGGCCGXUGGcCCCAA

AAGCUAU

CACACUG CUGAUrGGCCGAA GCGA

AUTCCCCA

CGAACAG COJGAUGAGGCCG AGGCGA AGCCoG GCUGGCtJ CUGAUGAGGCCGAAr.GA AUGGC=i CUGAUCU CUGAUGAGGCCGAAAGCGAcA

ACUCAAA

UGGUCUG CUGAUGAGGC-CGA Ar.GCCGMA

AUUCGCLT

232

S

501 502 508 509 512 514 534 556 561 562 585 598 613 6i6 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 AACGUG CUAUAGCCGAAAC-GCCA

AGGGU

GAACGUG COGAUGAGGCCGAAAG-,-CGPA

AACGGGU

CALM CUAGGCCGAAAG c ACGUGAA uca~iG CGUAGCCGAAGGC=-k

AACGUGA

UCCU=~ CUAGGC

AGGAACG

GCUCCUJC CAUGGCCGAAAGGC~rA

AAGGA

OL'GUC CGAflGAW-CCG ACC AGUCCCC GGAAGCA CGUGA CCGAAAGGCA

AGGCCC

CACCUGG CU GC CGAAAGCCA

AGCAGAG

ucaccuo CMGGGCcGAAACCAcA

AAGCAGA

GCUGG-tY CUCAXUGAr--CCGAAAGCCGAA AtXGGUE UCAGGAG CU urCG-C--uAGCA

AGGGC-

GtJGAGAG CMUA=GCGAAAG~CCC

ACAGGG

GIAUGUGA CUGAGAGCCGAAAGCC

ACGAAG

GGCUGAG COGAflGAGGCC AG,,CGAA

AAGGGAC

CAUGGCU CMALAA Gcc AGGAAGG GAGAUGG CUGAGAGCCGAAG.CCA

AGCG

MUXAA COAU GCCLA<CCr.A

AJCGGAU

GUADU= COArGCCGAA CCC AAALTCGG GUAhfC COUGAGCCGAGCGAA AAAAflCG GGAACAC CUG GAGCCGU CIC

AUGG=C

AGAUCUU C Uc'rACUG CUCGGCA LC% A&UAGCCGAAAGCCA AT3CDGA GCUICCCA ~CcUG GGCGL~rc AGuuccG, CCCCACC CMAU "c

AGGCAG

GCAAGAA CUGGAGCCC-AAGC-AA

AUCUCAU

CAGCAAG CUGAflGAGGCCG AGCCA AGAtJCUC ACAGCAA CUGAUAGGCC~ CGAA AAGATJCU CGCAAUG CU GAUG AGGACCCC,

AGGA

ACACCt7C CUGAUGAGGCCG AGCCA ATGtUCUU cGUGAAP. CMAMG c ACACCUC CCCGUA CMUGfGAWGC c

AUCACC

UCCCGUG CMUGAG~ccC GCCG;A AAt3ACAC GC AMGCCGTJCAA CCr.A AAAUJACA AGAAAAG cGAnGAGGCCGAAGCCGAA

AGCCUCG

UUGAAAaWGV-"AC Cc GAoc AGGAGCC CUUGAGA CUAGAGGCCGkAGGCCA

AAGGAGC

GCUUGAG aCUGAGCCGGCGL

AAAGA

AGCUUGA CUAGG'-

AAAGG

GGAACA.C C GAflGAGGCCAACCAA

AUGGCCA

AGUCCGG CUGAt3AGGAAccCGCCA ACACpAxj GAGUC~r. CVAGGC~U4C

AACACAA

GCGUACG arUGAGGCAAGcc G CGAA AGGAGUc GUCGGC CUGAUGAGCCMAGGCG

ACGGAGG

CGAACAD CUGAr-kWCGAAAWCGAA

AGCCUG

GCAUGGA =UAWZACGAAACGLA

ACUCGIA

=W M.U CUGAXGA~CC7CCG AGCCC-A AGACUCG CGAUCAG CUAGGCGAAGCA

AGGCCGC

GCGAUCA cuAuAGGcc Gcc 233 91i9 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 1.132.

1133 1137 1140 1153 1158 1167 1168 1169 1182 1183 1184 1187 1188 1198 Z.209 1215 2.229 1237 '250 GCEYCkCU CUGAtJGAGGCCGAAGGcGA AGaCG,- G~kCUGG CUGGCCGXGGC

ACUCCAU

AGM=UG CoAGGCCGAAGa AACU=c UGGcaAG CDAGGCXGC'

ACUGA

TJCAUGJG CUGAWG GGCr UGCC AflGG,, CGGUGG CUGAGG CCAJ GCC ATJCAUCrj GUCUGGC C

AUGGCGAGCGAACU

UCUCUUC CUGAflGA GCCGAAA-,C

AUCCGGU

ACL7COG CAGCCGAA~AGCC

AGGUCX

GGOCUCA, CGUAC c -C

AGM=C

=CMOG CUAGGCGAGX AGMXcuc UACUCUU CCAGccGAGCCG

AAGGUCU

L;UUCAU CMDA=-AAGCA

AUACUCU

UUG~aAAG CVGAXGACC 'CGaAA

ACUCUUC

CCAUUGpA CVAA<CL aCC

AMGA=

UCCAUUG aXDA4CAAGCA AAGGACtJ GUCCAIJU CAUGGCCGAC-G~r

AAAGA

CGGGU-FRUG CCWWCMG.CA

AGGCCGG

UUGGAUC CAkGC CMU

AGG

GCACAGC CMMOCMUAGCG AflkCGCC UriGG itTwCMGGCMUC-C

AGGAC

ACUUCGG CUAAGCMG AAGGcMU AGAAGuu aXcGAGCC~kaCG

AGUUC

GGGAMCUWGGCAAGCGAAUG

GGMG c COAUW CGAAAG

AAUG

GCDUUGGG CUAAGCGAG'

ACAGAG

GAAGGU3G C.U CUXGCCGAAGCCGAA

AGGG=U

GLkAGGC CMUAG-=, AMUXGc U==CCAGG CXA;' rA A GG= AUC.= CGAW GG~oMAGC~.AAAAG GM=CUGAMWC'a

AAGM

GCOGAGG CMUAGCAA.CA AnCcuG CUCADCA CUAGGCOAGCA

AGUUGAT

GGGGGAA amcmccwAW= ACUnCAT LrGGGGA CtGMAtUWMMC AWG=M

AACUCALT

AUGGG CWU~=AGCA

AAACUCA,

UGAU= UGAMCWAGCA

CG

CUGAUW= Acc

AACAGCA

UCAC-GAG CUGAUAW4=AAAGGC

AGGGGCC

GGCCUGAG CMA

GGCAAA

UCAGACU CUGAIJG G AMUCC GAAGGUG QA-M AGCrxM CGGUGCU c G U A cc AG C AGGCUG GCUGAGG CJIJAW=CX-UGG=

AGGGACC

GGGGCAG CUAUAGCCGAAAGGCCA

AGCU~C,-

GAGCCEJG MGCU CGAlGGCCGC

AGOCUG

0 .0 234 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 1680 1681 1683 1686 1690 1704 1705 1707 1721 1726 1731 1734 1754 GGGGCAG CMA GCCGAAGGCGAA

AGCUGG

AGGAAGG COAGC--G~A--C-

ACCAUGG

CG-CAGCUT CUG VGAG--CCGA ccr-, AGCC UGGGGGA, CUGAX GAGGCCG ACCGAA AACUCAtJ AGACUM MAGCG AAAcr 'CGAA

ACAGGAG

GGGCG CGAGGCCGM c ACUGG CCGGU COAGGCC'-"AAGaC-,C-

AGAACUG

GACUGGG CUGADGAGGCCG kGCCGA

AGGCC

UCAGC!JLTGU ACCGGG-A

ALGAAAAG

GGC^UtOC UGLccGa, aGAAc ACALGC.rJ AGCAUC?, CUAG GC--AAG,-c..

AM-G=A

CAGCAVC CUGAMAGGC-C-- AAGC AACrUCA GCCAAGC CUGAMGAGGCC CC=~ AGGCC.C OuGU C CUUGGCCAC,,-GA A~ a.

GUCUGOG COGAfGAG-CcGAA -GCCGAA

ACACUCC-

GCUGU CUGAflGAGCCGA AGCCGAA

.AACACUC

GUCCACAL CUA GCc-r Aa.-CG

AIUGCA

AGOUCCCUGUAGCCaUGCCrAAm

ACCGAAG

AA&COCUT CM ccAGCC AA~C-c,-

AGUUGO

CUGCUGA COAGCGA,-CG~AA

ACUCG

GCUGCUG C GAXJGACCGAAGGCCGAA, AACU=tG AGCUGCU CGUGCCGAA,,CGA AAcUu ACACAGG CUGAM AGCC GCGAA

ALGCACC

UUCAGGG COGAUGAGGCC GkN--CCAA

ACUCCALU

CGAGUM CLOG A C CCGA

AGCOUCA

GGCGAGU COGAIGAGCcGOCGAC-A AUAGCrM ACCAGGC COGAUGAGG-CCGA Ck;-CCCGAA AGUU~tah CCCOCric CUGAUGAGGC-CGAA GCCGAA

AGGAGAG

GGGGCAG CUGAAGCCQA,-,C,-A

AGCUGGG

CCUACC CUG-AXGAGGCCGkXXGCCGAA

AGCAGGA

CAUUGGG CWUfGAG-CM kCCAA

AGCCCCG

CUGGGCA CUGAUAGCAAC;C-

AGGUCAG

CACCUGG COGAIJGAGGCCGX A,-CGAA~

AGCAGAG

UCACCUG CUGAUA CGAAr-CA

AAGCAGA

ACCUCCG COGAUGAGGCCGAAG-CCGA

AAGCGAG

GGAGGAG CUGAUGAGG-C AAGGCGA

AGUCUUC

UGGAGGA COGu GGCCG xr.-c~cGAA AAGUuu AAUGGAG CUGAUGAGCCGAAAG,-GCGAA

AGAAGUC

CGCAAUG CUGAUMAGCCG A-GcAA

AGGAA

UGUCCGC CDGAUGAGGCCGAA-GCCA

AUGGAG

AGCAGAG CUGAUAGGCCGXU.GG-AA AGtTCCAU GAGCAGA CUGAUGAGGCCGAAC

AAGUCCA,

AAGAGCA CUGAtK AGGCC-XAAr

AGAAGUC

CUGAUCU COGAUGAGGCCC AC--CC-A

ACUCAAA

AGGAGCU CUG rGGCCGGGCCGA ArCUGAC ACCOUAG CUGAZGAGGCC '-CGC

ACU

AGCACCY CUGAUGACGCCGAA "X AGC U CUCUUrGG CU;LGGCCG AAGCCC-A

AGCACUG

235 Table Human re/ A nt. Position *5 5

S

19 22 26 93 94 100 103 105 106 129 1.38 148 1s1 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 467 469 473 481 501 HH Ribozyme Sequences HH Ribozyme Sequences ~cAGc G a-GGGCC~GX AGCCAfU kcaCkCA

CUAGGC~~~,~ACGAGCC.

CGUCAC CUGU GCCAAA-A ACAjACG GAGGG=GIUAG Gzkc~G

AC,,GUUC

W--GG tc Cc C.CGM AAcA=u GGAAw amLT GCGAGA

AGGGGG

CCCG-u CCGGA C~GCCG c,-cr-

AUC-AGG

UGCCGGG arCG&A MCA AGAUA CL3CC-G IJUaGCGA-4M

AAGAUGA

GGGGCC CDaflGA Gc cG AGG=cG CUCCACA CUGAMJAG3CCGAAGWCC=;A

A.GGGC

GCDCAAU GCCOIGAAAGCC

AUCU=C

GCUGCU a r-GGCCUG=ACC. AflGAVC ~GWGG cm cc AGCccM UGMhGC CMU kGCCM-

AAGCGC-A

GCACUU =U CL

ACGAA

GICCCCG- CUG fG GGC AG CC CG-CCUGGaM a-CCGAA-C AflGxwc UUGGUGGCTGAUA C

AUCUGU

UGAUC*UU CMUvGCACC AflGGUG AGCCAUU CMtUV4CMAA,--.GA

AUCCUGA

UCCUGUG Cr-GAGCC

AGCAU

CaGGGA CAUGAGGCCGAA.-CC

AUGC-GC,

G-ACC.AG

AGUGCG

CCUUGGU CUGWGAG- c1 J ACCAG= CGGUGAkG CWUGW4CGXMrG'CM AGGUcC GG,-CCGU

AGGAW

UGGGG= UGAUGWCG ac

ACCG

uu.cact C MGGCCMG AGCUc; C0UU U CC A ACAAGCtJ CtICAXUAG CGXGGC

AGCCAC~

AGCCCA Ua=UGAWGGCC CL

AGAAGCC

CCGGGCA CUar-GAGG

AGCUCAG

AACUGUG CUM AGGCGACCCGA AI3GCG UCGGA CU JAGC~ACUG7;G.

GUUCUGG CUGAGCC

AACUGUG

GGTCUG a U c

A

CACACUG CUGAUGGGCC CA AUrjCA UGACUGA CGUA CC

AGCCUGC

GCUGhCU CUGUGA a c cGA AUAGCCrJ AUGCGCEJ UaMUGAGGCCGA CG-AA ACE7GAJA UGGUCUG CUGAUGAGGCCAAGUCGAA AUGC

CE

AACUUGGar- AGGCCGAAAc

AGGGUTJ

236 502 508 509 512 514 534 556 561 562 585 598 613 616 61.7 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 795 796 797 798 829 834 835 845 849 872 883 885 905 906 919 G-AACUt3G CUGA r;GWCX-C~GAcc

AAGGGG-U

CCG CA GCCGAAAG-CGc

ACOUGG

UCAXG COAG CGAMGGCGcA

AA==UG

UCDu CGA GGGCGGGC)CGcA

AGGAACU

GCUCUC CUGAUGAGGCCGAAG-.CCA AtUWGAA CAGGDCG CDGAUG GCCGAAAGG-GA

AGUCCCC

GCAAGCAL CVAAGCAGCcAM AA AGCCC- CACC=G CUGAGAGCCGGCG

AGCAA

UCACCTJG CCGAUGAG CAGCC GA AACG CCUGCCU CUAUACGCCGAAC-CC

AUGGGUJC

CAGGCG CUGAMAW.=ccGA Ac G1==C GAGGAAG CUGAUMWCCGA_

ACAG

GAUGAGG CUAU GCCGAAC-,.-CA

-AW

GGCAUGzAG CVAM-GCCGAACCA

AAGGAA

AUGGGAUr- CUG GAAGC-- CGkAG M- AGCAAC, AAGALGG C3JGAUGG~G--CAAGCCU

AUGGG

UGUCAAAL CUGAflGAGG-GAGCGA

AUGMW~

AVUGCA COD MG-GAGCGAAGM GAUUGUC CUGAUGAGG-CGAAGCCA

AAGAU=

GGGGAC DG~lG GC CA~.AUUuC, AGAUCUU ~CM GCGAGCCGA

AGCECGG

CtJCGGCA CUGADGAGG-CGAAGCCGAA

AUCDUGA

GCDGCCA, CUAGGCCGAAAGCCG

AGUUUCG

CoCCACC CUAGGCnAGCrA AGc G~3kGGALA CUGA GCC ooc AflCU=A MAGM=h CUGAUGAGGC U CGAA AGAUCUC ACAGUAG CUGAGAG--GAAC<cir

A

CACACAG CGAUG GGCC CCGaU

AGGAAGA

ACACCC CtJGAXGAG,-C AGCGAA AUGUCcu CGVGAAA CtGIX fGAAGCCGAc,

ACCU

CCCGUGA CUAGGCCGAAGCCG

AXUCACC

UCCCGUG CUGAUG.CGAAGGCCrCA AApmCAC GUCCCGTJ

CMUAGCAAGCGAAAA

CGAAAAG CWAGGCC~AGGC AGCCuJCc UUGCQ.AA CUGAGGCCGLA AAGc CUTJGCGA CUCAUGAGGCCWAGCCA

AAGAG

GCGCC~j-G =GMACWAAAGGCCA AAAGGAiG AGCUUGC CUA.GC~AGCA

AAAAGA

C-GAAA CUGAI GAGGCC AAG-C

AUGGCC

GGUCCGG CUGAUGGCCGAAGCCA

ACACAAU

GGGUCCG cuGAt3G cAGG ~ccAA

AACACAA

GCGUAGG CUAGCC GAACCA

AGGG=U

GUCUGCG CUAGGCCAAG=

AGGGAGG

CGCACAG CUGAUGAGGCCGAAGCCGA

AGCCUGC

GCAUGGA CUGAUGAGG CAAG-CGAA

ACACC

CUGCAUG CtrAGAGGCCGAZGCA

AGACACG

CG%-tICGG CUGAUGAGAAGG-CGAA

ACCGC

CCGGUCG CUGAUGAGGCCGAAAGGCCA

MAGGCCG

GCUCACU CUC-AUGAGCCGAAGCCGA

AGCUCCC

237

S

S. S *5 S 936 937 942 953 962 965 973 986 996 1005 1006 1015 1028 1031 1032 1033 1058 1064 1072 3.082 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 CUGAt3GAC-GCCCGAAAGG=CA ;Ur:u GGUACU CMUfGAGGCCGAAAGGCCCAA,

AAUCCA

UGGCAGG CUAGG-CGAAAGGCCG-,A

ACUGGAA

UCGUCUG CUGAUGAGGC-CGAAAGGccGAA, AUCtJGC CCGGUGAC c-GAUGAGG-.CCGAAAGG--cGA

AUCGUCU

AUTCCGGU CUGAUGAGGCCAA=-GCAA AC=G~Xc UCUCCO3C CtJGAXGAGGCCGAAAGCCGAA

AUCCCGGU

GUCCUUUT CUGAI3GAGGCCGAAAGGCC-,AA. AkCUUUC GGUCUCA COGAUGCC-%GAAAGGCCGAA AULUCuI CCUUG CUGAUGAGGCGAAAGGCC,A AG~tTC,,C UG-CUCUU CUGUGC-CGAAAGG-c,-a AAGC,-r UCUUCAL UGA GCCGAAACCGrAA AL-GCUCU CUGAAAG CUGuflGAGGC-CGAAAGGCC-GA ACUCMtX CCGCUGA COGAflGAGGCCGAAAcGGcc,-A

AGG=ACUC

t3CCGCO CUAGCCGAAAGCCA

AAGGACU

GUCCGCU CUr3ktrGAGG%-CCAAAGCC,,A A CGAGGUJG CUGADGAr-GCCGAAAGGCCGAA

AGCGCCGW

AUGCGUC CUuU%.GCC-AAAGGCcA

AC,.-UGG

GCXCAGC CtG UGGGCCGAGCC

AUGCGUC

CUGCGGG uUAJAGCAAGGCCGA

AG~CAC?

GCUG-CGG CUGAflGAGGCCGAAG -CCGJ AAGGCAC AGAAGCU CUGAflGAGGCCGAA GGCCGAA AGCUCGI GGGACAG CUGAUGAGGCCAAACCz AGCt'G GGGG~ACA CC- GCCGAAAGCCA;

AAG%-CG

GCUGGG CUGAGAGCCGAlAAGGCCA

ACAGAG

AAAGGGA CUGAIGAGGC-CA.AAGGCCG--Ak AGGG%'tG G ~AAGG CUGAUGAGGCCAAAGCCA

AZMGGGC

UGACGUA CUGAU GCcCC

AGGGAUJA

AUGACG CUGATJGAGGCCGAAAGGCCGAA AAGG GAUGACG; CUC-XUGAGGCCGA AGCCGAA, AAAGGGA CAGGGAU CUGAGCCGAAAGGCCCGAA

ACGOAA

GCUCAGG COGAUGAGGCCG AAGCCGUL

AUG-ACGU

CAUAG=U UGAUGAGGCCGAAGCc

ACGGUGC

CUCAUCA CUGAUGAGCCGAA C-CCG-AA AGUOGAU GGUGGGA CUGUGAGGCGAGCr.A

ACUCAXJC

UGGUGGG CUG-UAGGCGAGCUA

AACUC;LU

ATJGGUGG CUGAUGAGGCCGAAGCCA

AAACUCA

AGAAGGA CLMGGAGGCCGAAAGGCCG;A. ACACCAt3 CAGAAGG CUGAt GAGGCCCGAA GCCGAA AACACVA CCAGAAG CUGAUGAGGCCGAAAGGCCGAA

AAALCACC

UGCCCAG COGAUGAGGCCGAAG~cA

A-GGAAC

CUGCCCA COGAUGAGGCCGAAAGGCCGALA

AAGGAAA

CCUGGCU CUGAUGA;GGCCGAAGGCGAA

AUCUGCC

CAAGGCC CUGAUGAGGCCGAAGCAA

AGGCCUG

CGGGGCC CrJGAUGAGGCCGAAAGGCC-A6A

AC-GCCGA

ACt3UGGG CUGAt7GAr.GCCGAA -CGAA. AGGGGCC GGGGCAG CUr-AUGAGGCc AAAACOUG;Ic GGGGCUG CUGAUGAGGCCGAAAGGC:CGAA

AGCCUGG

AMJGCtG COGAUGAGGCCGAAG AGCAGGcG 238 9 9 *9*9 9* 9 9* .9 9* 9 9*99 .9 .9 6 1.279 1281 1286 1309 131.5 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 .1577 1579 1583 i588 1622 1628, 1648 1660 1663 1664 1665 1680 1681 1683 1686 1690 1704 1705 1707 1721 1726 1731 1734 1754 GAGCUGA CUGAUCAGGCCGAAAGGCCAA

ACCAUGG

CAGAGCU CUGAGC-CCGAAAGGCCGALA A~jCCAI3 UGGGCCA CU t2GCCGAAAGCGCCAA ACUGAU GGACUGG CUAUAGCCGAAAGcCAA

ACAGGGG

GGCG CUwCCGAAAC,3CcA

ACUGGGA

CDGGGGC CGUGGCCGAAAGGccAA AGGACtjG GCCUGAG CUCAGAGCCGAAGCGc AGGGCnty ACAGCCU CGUAGCC=GAAAGccz

ACGAG

GGCCUCU CO~GAGGCCGAAAG~cCAA

ACAGCGU

AUCAUCA CGAUCCGAAAG~cc

ACUGCALG

CAUCAUC CUUAGCCGAAAGGcccAA

AACUGCAL

GC-CAAGC CUGAUGAGGCCMAAGGccA;

AGGCCCC,

UGUUGC-C CDGAUGAGGCC-AAACCGAA-z

AGCAAC-G

GUCUGUG CtOGAUGC GCCGAAAGcG ACAAG GGUC= UCAGCCGAAAGGCC;A~

AACACAUG

GtJCGACG CUGAUGAGGCCGAAAGGCCA

AZGCCALG

AGUUGUC CUAGCGCCGAAAGGCc

ACGGAMJ

AAACUCG CUACU;GAAGCCCGAA

AGUGU

CUCUGA COArAGCCGAAAGGCCGAA

ACUCCGGA

GCUG=CU trAGCCrGAAAGGcCGCAA AACtJCGG AG7CUGCU CUAGGCGAAAGGCCGAA AAACUCG, CCACAGG CUGAMlAGCCG AGG AA AUJGCCCU CUCAGG CUGAUGA CCGAAAG-.CCGA;L

ACUJCCALU

CGAGUA CUACGCCGAAAGGCCGiA~

AGCCUCAL

GGCGAGZJ COUG CGAAGGCCG AA ALXCU ALA=G CUGAUGAGGCCGAAA

AGUUAUL

C;U.GUCAC CUGA(GAGCC~k kC,CGAA

AGGCGAG

GGAGCAG COAUG GCCGAAGCGAA

AZCUG

CCCAGUG CUGAUGCCGAAAGG-CGAQL AGCAGG A CAflUGGG CVGAUGAGGCCGA GA; AGCCCCG C GAAAG CUGAUGAC-CCGAAAGGCCA

AGGCCAU

CUCCUGA CUGAGCCGU<,L

AGGAGG

UCUCCUG CGUAGCCMGaZAGCCA;

AAGGAGG

AJCCCU COGA GAGGCCGCC-

AAAGGAG

GGAGGAG CcrflAGCC GGCD AGucOUrC UGAGGA CUGAUG CGa~aA=GC

AAGUCEJ

AAUGGAG COGAU GACGCC'Gxa-aCCGAA

AGAAGUC

CGCAAUG COAGAGGCCGMaAGGCCA;

AGGAGAA

UUCCGC COGAUGAGGCGAAAC-G'CA

AUGGAGG

GGCUGAG CUGA! GAGGCCGAX GGCCCAA AGUCCAU GGGCUGA CUAUAGCAAGCA

AAGUTCCA

CAGGGCU CtJGAtIGAGGCCGAA G~rA AAAGUC CUGAUCU COGAUAGGCCGAAGGCCA

ACUCAGC

AGGAGCU CUG-AUGAGGCCGAAGGCCGAA

AUCUGAC

CCCUUAG CUr-AUGAGGCCGArCGAL

AGCUGAU

ACCCCCU CUGAUGAGGCCG AGGCCC-A AGGAGCrJ CUCUGGG CUAGGCGAG-CA

AGCGCALG

9 9 9 9**99@ 9 cc cc cc cc cc Ce.

C

S

Ce.

o c.

C C

*CC

C

C

ec he C CCC CC C CC CC C C CC C C Ce C e eec. e Ce c c C C e ccc e C C C C

C

Table 21 Human rel A ft. Position Hairpin Ribozymefrarget Sequences Hairpin Ribozyme sequence Substrate 156 362 413 606 652 695 853 900 955 1037 1045 1410 1453 1471 UGAGGOG AGAA GUUC

GCUGCUUIG

GCCAUCCC

GLJUCUGGA

GAAGGACA

UUGAGCTJC

CCCACCGA

AGGCUGGG

GGUCGGAA

UGACGAUC

GUCGGIJGG;

GGCCGGGG

A GAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCUC

GLJCC

GUG

GCAG

GUGU

GCUG

GCGU

GCCG

GtJAU

GCUG

GUGG

GCAG

GUGC

GUGA

ACCAGAGAAACACACGUUGUGG(JACUUACCUGuA

ACCAGAGAAACACACGUUIGUGUAAUACCJGGUA

ACCAGAGAAMCACACGUUGUGGUACAUACCJGGUA

ACCAGAGACACACGUTJJ1JACAUACCJGGUA

ACCAGAGAAMCACACGUEGUGUAJAUUACCUGUA

ACCAGAGAAACACACGUUGAAUUAUGGUA

ACCAG-AGAAACACACGUUrGLJGU1ACLAUACCGGUA ACCAGAGAAMCACACGUUGGGuTAAUUACCT1SGUA ACCAGAGAAACACAcGUJuGGuTJAUUTACCUGGUA

ACC-AGAGAAACACUUGUGGUAUUACUGGUA

ACCAGAGAAACACACGUUGUGU1AAUUACCUGGUA ACCAGAGAACACACGUGGGU1AQ1AUUACCGUA GAACIJ GEI GAGCA GCC GGACU GCC CCACA GUU CUGCC GCC ACACU GCC CAGCUJ GCC ACGCA GAC CGGCG GCC AUACA GAC CAGCG GAC CCACC GAC

CCCCCUCA

CAAGCAGC

GGGAUGGC

UCCAGAAC

UGUCCUUC

GAGCUCAA

UCGGUGGG

CCCAGCCLJ

UUCCGACC

GAUCGUCA

CCACCGAC

CCCCGGCC CAUCAUCA AGAA ACAGCUGG; AGAA GAUGCCAG AGAA GCACA GAC CCAGCUGU UCACA GAC CUGGCAUC 0 0 Table 22 Mouse re/A Hairpin Ribozymefrarget Sequences nt. Position Hairpin Ribozyme sequence Substrate 137 273 343 366 633 676 834 881 1100 1205 1361 1385 1431 1449 1802 2009 2124 2233 2354 GUUGCJUc AGAA GAGAUUCG AGAA GCCAUCCC AGAA

GGGCAGAG'AGAA

UUGAGCUC AGAA CCCACCGA AGAA AGGC(JGGG AGAA CAUCACAh

ACMA

AGGUCUAG ACMA CCCCACAG AGAA GGGCUUCC AGAA CAGLCAUCA ACMA ACUICCUGG

AGAA

GAUCCCAG AGAA AAGUCGGG AGAA UGCCUCCA

ACMA

UCCUCUCG AGAA AUUCUGAA AGAA UCACUAAA AGAA

GUUC

GLIUC

CLJCC

GCCU

CIJGU

GCUC

GCGU

CCC

GCCC

GUIGC

GCGU

GCAG

GUJGC

GUCGA

CUG

GUCC

GCAC

GCCA

GUCrJ ACCAGAAAACACACGUUGUA1VACCIGUA ACCAGAGAAACAC~cGUGIUACTAcYACCUGGUA ACCAGAGAAACACACGUUGUJrJIUAAUACCJGGUA

ACCACAGAAACACACGUTJGUCAUACCAUGGUA

ACCAGAGAAACACACGUU1GUGG1JAAUUACCW)GGUA ACCAGAGAAACACACGU1JG1GGTJACAUUACCUGGUA

ACCAGAGAMCCACGUUGUGGUAUACUGJA

ACCAGAGAACCACUUGGACAUJJCCUGGU~TA

ACCAGAGAAACACACGUUTG1GG1AAUACCJGGUA

ACCAAAMACACACGGGGACAACCJGGUA

ACCAGAGAAACACACGUGLGGUACAUUACCJGGUA

ACCAGAGAAACACACGU1J()G(JGUACAUACCJGGUA

ACCAGAGAAACACACGUTGUGUAAUUACCGGJA

ACCAGAGAAACIACACGU1JGUGrJUACJTJACC1GGUA

ACAAAAAAGUGGUCUACGU

CAACA CCC GAAGCAAC CAACA CUU CGAAUCC GGACLj GCC GGCAuGC AGGCU CAC CUCUccC ACACU CCC CAGCUCAA GAGCU GCC ucGGG ACCCC GAC CCCACCT CGC CCC UUCUCAUC CCGCA CCC CUACACCU

GCACC

ACGCU

CUIGCA

GCACA

UCACA

CAGCU

GCACA

GUGCU

UGGCC

AGACA

GUC

GUC

GUU

GAC

CAC

CC

CAC

CC

CC

CC

CUCUCCC

GGAACCC

UCAUCCUG

CCAGGAGU

CUGGCAUC

CCcCACUU

UGGACCCA

CGACACCA

UUCACAAU

UACUIGA

241 Table 23: Huma-sn TNF-a EH Ribozyme Target Sequence 0 0 at.

Position 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 HE Target Sequience Po t.

Psition

GGCAGWU

GCAGC3U

AGG-UCU

GUtJU-U I UUCcU

GCOUCCU

UuCCOCU

CUCACAU

CA.CGC-=

CCACLT-

ACCCDCU C

CCUCU

CUAU

AGC-GCU C CAGGGCtJ C CGGUGCU L tJGCUUGU Uj GCUUGUY C LUGUUCU C UCGCCU C AGjCCUC U GCCrUCULJ C CUCUtJCD C UUCUCCE U UCUCC=t c UCCUGAXJ C CCACGCTJ C ACGCUCU= U CG7CUCU C CU3GCACU U UGCACUJU U GAGUGAU C GZAAGAGU C GGGACCt3 C GACCUCtJ C cCUCUCU C UCt7CUCU A CUC~kAU C *AGCCCtJ c

UCUCUUCC

J CCUCUCA CUCoCAC k CU

CUACCCC

cacc-Cu

CCCDGGA

ICGGGACG

CCCAAGA

CAGGCCG

CUCAGC

AGCCUCU

UUCUCCU

cuccuuc

UCCMUC

CUUCCUG

CCUGAflC COGAflCG

GUGGCAG

UJUCUGCC

CUG-CCUG

UGCCOGC

UGGAGUG

GGAGUGA

GGCcCCC

CCCCAGG

ucucaA

UCUAAUC

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

GUCAGAU

AGAUCAU

AUCAUCU

UCAiCUU

AUCUUCU

AGCCUGU

CCCAUGT

AUMGUG

AAACCCU

GGCAGC-U

AUGCCCU

GAGAGAU

GUGCCU

GGCCUGU

UGVACCtJ ACCUCAu

CU=ATCU

AUCtUhCu

CCCAGGUC

AGGLJCCU

GUCCUCUt

UCCUCUUC

UIGCCCCEJ C AUGUGCU C UCUCCu C ACACCAU C GCCGCAU C UCGCCCtJ C GCCGUCU C GUCUCCU A CCAAGGU C UCAACCU C ACCUCCU C CLUCCL3CL C CUGCCAU C CCCUG.GU A AGCCCAU c CCCAUCU A C AUCM=~ C tUcCCA U- CUCGAAC C UCG-AACC C GAACCCC A GCCC-aUG U GCGCAA ;L GCAAACC C A.AG-CU

CUGGCCA

kACCAGCU

AGAGGGC

CCE3CAUC

AUCUACU

UACUCCC

CUJCCCAG

*CCAGGC

*CUCUUCA.

UUCAAGG

CAAGGGC

AAGGGCC

CACCC;LT

CUCACCC

ACCCACA

AGCCGCA

GCCG=C

UCCUACC

CTJACCAG

CCAGACC

AACCt7CC

CUCUCUG

UCUC-CCA

VGCCAUC

AAGAGCC

UGAGCCC

UAUCEIGG

UCUGGGA

E Target Sequence 242 CAUCEMhU C T3GGGAGG GAGGGU C UUCCAGC 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

GGGGUCU

GGGU=U

ACCGACEI

CUGAGAU

GAUCAAU

CCCGACU

CGACAU

ACLUCU

CUCGC

UCQG.CU

GCCGAGUJ

GGCAGGU

CAGGUCU

G7CMCU UCtACUU1

UGAU

CGAACALU(

CCAACCU I

CAACCU

AACGCCUC

CCCCAAU C AAUCCCt3 t AUCCCUU t TucCCOuu ccuCua~ V CUUr.I.UU A ACCCCCU c CCCU=c u ccuccou C .ACACCCU c UCAACCU c AACCUCU U ACCUCUU C UCUGGCU C AGAGAAU U GGGGGCU U GGGGCtJU A UtUAGGGtJ c CCAAGCU U CAAGCUU3 A UGAACU U AGAA.CtU

U

GAACUtMt A CA.CCACU U ACCACUU C Ct3GGGAT U U CCAGCUTG C CAGCEJGG C AGCGCU C AAI3CGGC C GGCCCGA A UCUCGAC C UTCGACU C GACUUU U UG-CCG U GCCGAGU C T3GGGCAG C MicuOuG A~ CUUUGG LJ UGGGADC U GGGWUCA

AUUGCCC

J GCCCUGU

CAACCUU

3 CCCAAAC

:CCAAACG

CCCOGCc CcUUUAU.

TUAUCACC

ADCCC

6TUACCcC rACCCCCU *cccccuc

*CAUGA

~AGACAC

AACCUCU

UUCUGGC-

CUGGICUC

UGGCUCAL

AAAAAGAL

GGGGGCU

AGGGUCG

CGGG-UCGG

GGAACCC

AGAACU

GAACUUU

MLXGCAA

AAGCAAC

AGCAACA

CGAAAcc

GAAACCU

CAGGAAU

960 1001 1007 1008 102i 1029 1040 1046 1047 1051 1060 1067 1085 i086 1091 1124 115 1151 1152 11598 ill59 U162 1164 1166 1174 1175 1176 1183 1184 1187 1208 1224 1228 1230 1232 1233 1234 1238 1239 1245 1251 1252 1254 1255 1256 1258 U7GGGAUU C AGGAAUG AACCACU A AGAUC UAAGAAU U CAAACU AAGAAUU C AAACE2GG GGC-GCCui C CAGAACUT CAQ-AAC C ACUGGG GGGGCCU A CAGCrUU UaCAGCU U UGAUCCCC ACAGC-t 'U G-ATJCC-U CUUUGALU c C-t3GAC:A Cj'GACALJ C VGGAAUC ty C UGGAGAC GGAGCCEYU tGGurTCrJ GAGCCUU U G,,tiEJG L7CUUG U C!GGCCAL UUTJGG-Ut C UGGCC-)AG CAGGACU U GAGAAGA AAGMACCU C ACCt~hGA CUCACCUT A GAAAtJU tAGAAAT U GACACAA.

UGGACCU u aGc-CCUU GGACCUU A GGCCUUC- MkGGCU U CCUCUCU AGGCCUE C Ctcua CU CCUUC=t C UJCUCCAG tJUCCUCU C UCCAGAU CCUCtjCt C CAG3AU-GU CAC-AT.GU U ULCCAGAC AGAtjUC. U CcaAC tJ GAUGt~r= c CAGhCL7 CCAGACU U_ CCUUGAG CAGAC=r c CtJGAGA ACDUCCU U G-AG-CAC CAC-CCUT c CCCAUGG GCCAG-tJ C CCUjCL7LU GCUCCCu c UiAuuuAu UTCCCUTCU A UUTJ~.t= CCUCLmu U UUGUUU CU=tUU UJ AUGUUUG UCUAULI A UGUUUGc UUUAUGU u UGCACUU UMU)UL UT C*CArjrG UUGCACEJ U GUGAUUA VUGMAU U ATJUUAUU UGUGAUU A UUUA UC-AUTJAU U T-ULUAUU GAUUMT-u U AUUUUU AUUAUrU A UjUAUUTUA TJAULT~u U AUtIUAUU 243

S*

S

1259 1261 1.262 1263 1265 1266 1267 1269 1270 1272 1273 1274 1276 1277 1278 1280 1281 1282 1294 1296 1297 1298 1300 1301 1.315 1317 1334 2.345 2.3S0 1359 1360 1361 1362 1386 1393 1394 1401 1414 1422 1423 1425 1426 1427 1431 1 432 1436 1437 1438 AUUTJ.U A UUUU7 UaAEUCU U UAUUAU MUA U AUUM AUMUU A UUUM UAUML~U U UAUCATU AUUCAUU UJ AfUA=U UUMIlU A UtAUnD;L tmfltUrCAU U AUua.UU AATJU A UUOALUU tJkuu~U u UUUC.U LVTXflf U AUUflU AflCVUU A UUUflt3U tUMMU U TJAU~C AUU~kUU U AUUMWCA UUM=U A UMCAG muaflwhU U tkCAGALU AUU~kUU U ACAGAUG U~kUU A CAGAUGA UGAA=G A UaM AAUGMU U UMu=G AflG~luu U. ADuuGGG UGtMhlU A UUOGGA UA CU u GGGaAA AUUMflU U GGGAGAC CCG-GGU A TUCCUGG GGG=tU C CUGGG CCA A GGAGCUTG 1440 1441 1446 1448 1449 1451 1456 1457 1461 1464 1466 1479 1480 1494 1498 1501 1512 1517 1528 1533 1537 1540 1546 1549 1551 1552 1566 1572 1576 1577 UGUUUUUT UJ AAAAtUXj GUUUUU A AAALJIU UUAAAAU A UUALTCUG AAAAU U :kucvrAU AAAUAUU A UC-,MuUj AUAtUtJhD C U-ArMJA AUCUGWtj U AA~1GU UCUGAUU A AGUGC AUAAGU U GULt-hAAA AAGUUGU C L7AACAA GMMGCU A AACAAUG LCtGWAU U UGCG-.Cr C-CMLG-tX U GGAr-;CC CAAtJG C ACUCADLJ UGUCX'TJ C AUCG CACUCAU U GCUGAG GAGGCCJ C UGCUCCC CUCOGCU C CCCA=G AGGGAGU U GUCu, GUUGUGU C VUAUC UGUCUGU A AtJCGGCC CJGtMAU C GGCCUAC LUCGGCCU A CEJAIUCA GCCEUhCU A UUCAGL-G CUACUAU U CAGUGGC t3ACMUU C Arc GAGAAAU A AAGGU.-,L-G VU.AAGCGU U GCEJUUAGG GMtjtGCU U AGGAAAG GUUMMt-M A GGAAAA GC-UGC-cu

CUUG-GCU

GAXCAflGU

ACAUOU

CAtJGUUU

AUGUUM

GAACAAU

AGGCUGU

C-CCULU

CCCAUGU

CUGGCCUT

UJGUMCCU

GUGCCUU

GCCUUJCU1 CCE3UCUU

CUUCULMU

TJUUTJGAtY UUTUGAUJU I AUL1fUUC t UtkUG'Ur t tLMXGLUEJU 1: U GGCDCAG C AGAAG U UUCCGUG U UCCGUGA U CCGUGAA C CGUGAAA A G.GUQ U CCCA.U= C CCPJ3GU A GCCU C UGUGCC u CUUMMG C ~uuu~UG UJ UUGAUL= 3 UGAUUIAU 3 GAUOAUG 3 AUGUt7=

UGUUULU

MiUUA

UTUMA

ULAAAAU

244 Table 24: Human TNF-a Hammerhead Ribozyme Sequences t.

Posi~.ti±on HE R:Lbozyme Sequence

S

*5 9 9 a.

a *5 0@*S S S S. S a t a. S 28 29 31 33 34 37 39 44 58 65 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 GGAAGAG CU xGG~cGAAAGGccGAA

ACCUGC

AGGAAGA CUA CG%-CrAAACGcCGAA

AACCUGC

AGAGGAA CVr-AUGAGGCC,-AAAGGC-GA A~ujACCU UGAGAGG CUGAUGAGr.=GAAAGGCCAA

AGAGAAC

GUJGAGAG CUGAUGACCCrXAAGGCCGAA

AAGACAA

UATGUGS CCGG AAGGCCGAA

AG

AGEM=G CUG CCCGAAGGCCAA AG1AGA GGGCAG CUA~a GCAAAGCC AUGUGAr GAGCGUG CUA~XGCGAAAC-A

AGCCGUG

GGGGAGA CUAG cGCCAAACCGc AGGrG= CAGGGGA CUAGG-CGGCCA

AGAGG=

UCCAGGG CUAUAGCC-.AAC<CC

AGAGAGG

CGUCCCG CUGAflGAGCCGAA :CCGAA AUCAflGC VCUUGGG CUAr-GCCGCGGA

AGCGCCU

CC-GCCUG CUA GOGAAAGGCCGA AGC=cUG GAGGAAC ~CUGMCGa-aj=

AGCACCG

GCDUGAGG CUAGCCGAAAGC

ACAAC

GGCUGAG CUAU GCCGAAAGG C AACAAGC AGALGGCU CUGAUGAGGCG AACCG-AA

AGGAACA

AGGAGAA ~CcUG GGCCJ

AGGCUMGA

GAAGGAG CUACCGAAAGGCC

AGAGGCU

GGAAGGA CUGAUGAGCCGAAAGGCCGA

AAGAGGC

CAGGAAG CUGADGAGCcGAAAGCGA

AGAAGAG

GAUCAGG CUAGAGCCrtAAA=GAA

AGGAGAA

CGAUCAG ~CUGMGC cCG

AAGGAA

CUGCCAC CUGAUGAGGCCGAAAGrCCGAA

AUCAGGA,

GGCAGAA CUGCAUGAGGCCGAAr-CGAA

AGCGOG

CAGGCAG CUAL GCCGAAGccr-A AGAGCGu GCAGGCA CUAUGGGaAAGCCA

AAGAGCG

CACUCCA CUGAUGAGGCCGAAAGCCA

AGUGCAG

UJCACUCC C~.UACCCAAGCA

AAZUGCA

GGGGGCC CUGAUGAGGCGAAGCCGAA

AUCACYC

CCUGGGG COGAUGAGrGCCGAAGGccG.AA Acucuuc TUtkGAGA CUGATOA} cG

AGGUCCC

GAIMUAGA Lr AGAG_2LTJC CUGAUCA CUGAUGAGGCCGAAGOCCWA,

AGAGAGG

GG%-t3GAU CUGAUC-AGCCGAAGGCCCAA

AG.AGAGA

GAGGGCU CUGAUC-AGGCCG AAGCCGA

AUUAG

tJGGGCCA Ct fGAGCCGAAAGCGcA

AGGGCUJG

a.

S

245 321 AGAAGAU CUAGGCCCAAGC- AUCrJGC 324 UCGAGA6A CUGAtMAGG-iCG .AAG Lc AU~C 326 GUUCGAG CUGAflGAGGCCGAA 3C-,.L AGUA 327 GGSUUCGAL CUGAVGA~rCGccG GCCGC.p A.AGUG 329 GGGC CUGA GGGCC

AGAA

352 CAIJGGGC CUGAUGAGGCCG AAGGCUCA

ACAC,'-U

361 0 SUACth CUG AG%--G~CAA GCC-AA ACAXUGGG 364 w.7UUGC CUGvX rz~Gcc,-AA

ACAACAIJ

374 UCAGCUU CUAUAGGCCCCAA

AGGWUUU

391 GCCACUG CUUGAAG~CZC,-AAAGCrU~ AGUrCC 421 UGG7r-A G CJvUACCAAGCP

AGG

449 AGCUGGU CUCGACGGCCGG CG- AflCUCUC 468 GCCCUCU CUGA GG=CaC.GGCc: kUGC,3A;C 480 GAWNAGG CJC- I--AAGGC -,AA ACAGGCC 484 AGU.GAU CUGAUAGGCCGAA, o-Cc,:L AGUcrA 487 GGGAGA Ct1GAV~kGGCCGA A CGA6A ArGAWGU 489 CUGG;CGAG CUGAUJUGAGGCC,-AA GCCG'-AA

AGAIJGAG

*492 GACU COGAXGAGGCCGAA GC=GA AG~Gk= *.499 UGAAGAG CUGAUGAGGCCGAAAGC.AA

ACCUGG

504 GCCCUEG CUGAUGAGGCCG GCU--,)AA AGAGGAC 505 GGCCCUU COAG=GAG,,-

AAGAGGA

*525 AUGGGUG CUGGflGAGGCCGAA=,CA

AGGGGCA

538 GGGUGAG COGAUGAGGCCGAAGGA AG=CAc 541 GGU CUGAWZGCCGAAZC

AGGAGCA

0 0553 GCUG-GCU COAGr-CGAGCGA AflG= 0 562 AGACGGC CUAMGCGAAGCA

AUGCGGC

0.0. 0 568 GGSUAGGA CUC-;tJGAGGCCC X GrC-.AA ACGGCC-A 570 CUGGEIhG. CUGALWMGAcGACGC-C-

AGACGGC

0.573 G-CUG CUGAUAG cGA CU AGGAGAC *.586 GGAGGME CUAGGCGAGCGA

ACCUMG

592 CAGAGAG CUGAIJGAGC CGUAA AGGUUGA 595 UGGCAGA CUGAUGAGGCCGAG CC,- AAGMCU 597 GAt3GGCA CUGAUAGGCCGAA GGCC'GaA

AGAGGA.G

0.0604 GGCUCUU CUG;LAGGCCAGC C -A AUGGCAG -a637 GGCUC;A CUGAUGAG~cQGCCGAA.CA

ACC=G

667 CCAGATUA CUGA GAGGCCGACCG6

AUGGGCEJ

669 UTCCCAGA CUGAUGAGGCC AAGCCGAA

AGU

671 CCt3CCCA CUGAWGAGGCCC- CCGAA AUAGAUG 682 GCUGGAA Ct3GAUGAGGCCGAAAG.^CGAA

ACCCCUC

684 CAGCUGG CUGAUGAGCCGAAGGCCGAA

AGACCCC

685 CCAGCUG CUAGGC

AAGACCU

709 CACCGCUJ CTGA.,;G GAGCGA

AGUCGGU

721 GCCGAT3U CUGA~kWCX-AGCC- A UCUCAG 725 UJCCGGCC CL-GACZ,,-AAGG CCGGAcc ArJUGAUc 735 GtCGAGA CUGAflGAGGCCGAAGGCCC-.A

AGUCGGG

737 AAGUCGA CUG-AUG flGCCU AGCGAA AUAGrJCG 739 CAAAGUC CUGAtJ GGCCGAAGGCGAA

AGAUAGU

744 CUCGGCA CUGAUGAGGcCCU AGCGAA

AGUCGAG

246 4%.

4%.

4% 4% 4% 745 753 763 765 768 769 775 778 801 808 809 820 833 837 838 839 841 842 849 852 853 863 869 871 872 878 890 898 899 904 917 918 924 925 926 945 946 959 960 1001 1007 1008 1021 1029 1040 1046 1047 1051 1060 ACUCGGC COGAUAGGCcGAAAGrcGAA AaZtTCGA, CUGCCCA CNUGACCrAA=,:LA ACtCGGC CAAAC CUGAflGAGC-CCCAA GCCG-AA ACcEJGCC CCCAAAG CUG AGAGCCGAAAG-GC-C-,, AGACCUG GAIU=C;CUGUG G AAAGCCGAA AGUACAC UGPAVCCC CUACAGCAAGcA AAGThnGG, GGGCAAU CUGAGAAGGCCGAlAAGGcccGA

AL-CCC.:A

ACAGGGC CUAGCCGAAAr--rA

ALMAUCC

AAGGUCG CUGAUCAGGCCCG GCCGA,;A AL-GUUCC GOUGGG CUGAUGA CGA~AAGGCCG_,AA AGG,-UUG CGUOUGG CLL~A3CGAGCCY r-_cA AAC.GGUUG GCGCAZZG CUGAflGAGGCCGAAAGGCC,.A

AGGG;MU

AUAAAGG CMAUGA~vCCrAAAG~Cc-GAA

AL-LGGGG

GGtUAUXA CUAGAGCCGAAAGGCCCGAA AGC.CaUL GGGA CUGP AGGA AAAGGCCC AAGGGU GGGaM CUGA 3GGCC XcC,;, AAAGW,;, AGGGGGLTGU G GGCGCGAACC-A

AUAAAGG;

GAGGGG G UAGCCGAAGG-CcG;A~ AAaA UCUGAAG CGAAAAGGCCGA

AGGGGGU

t-UCE1G CUACGCCCAGGCCGA

AGGAGGG

RJCU CrAGGCCGAAAGGCCCAA

AAG

AC-AGGJU CUAGA cGAAAGGCCGA! A AGGGUG GCCAGAA CUArAGCCGAGCCG AGG3UUrA GAGCCAG CUGAUGAGCCGAAAGG.CCGAA

AGAGGUU

UGAGCCA CUG rGCCGAAGCC:

AXAA.GU

UCUUUU CGUAGCCGUGCCA

AGCCAGA

AGCC C GAGGCcrrCGA A LTJCrJC CGACCCU CrUGArAGGCGAa-CCGA;A

;C=CCC

CCGACCC CUGAUGAGCGCCGAAGGC-CG

AAGCCCC

GGGUU C GMGGCAAGCC

ACCCUAA

AAG= COG GAGCCGAAAGGCCGAA

AGCUUIGG

AAAGUUC CUAGGCCGAAAG-C-.U AAGCUrJG UUGCUA CGUAGCUGAAAGGCCGA

AGUUCUA

UUUU CUGACAGCCAAGCCGAA

AAGUUCU

UGOUGCtJ CLMALGAGGCCGAAGCGAA~

AAAGUUC

GGtJUUCG CUGAfGAGGCCGAAGCCGAA AGUGGUG.Lr AGGUUUC CUGAUGAGGC CGAA AAGGGGU AUUCCUG CUGAUGAGGCCG AGCCA

AUCCCAG

CAUUCCU CUGAUGAGGCCGXZUCCGAA

AAUCCCA

GAAUUCU CUGAUGrAGGCCGAA GGCCGL AGuJGuu CAGUUUG CUGAGGCCAAGCCA AtUCUCA CCAGUUU CUGAUGAGGCCGc GGA AAyUCrJr AGUUCUG CUG UAGGCC .MGCCccc AGGCCCC CCCCAGU CUAGGCGAC-CA

AGUUCUG

AAAGCUG CUGAUGAGGCCGAAAGCCG-;.A

AGGCCCC

GGGAUCA CUGAUG-AGGCCG LAGGCCGAA

AGCEJGUA

AGGGAUC CUGAtJGAC-GCCGAAAGGCCGA

AAGCUGU

UGUCAGG CtJGAUAGGCCGAA GCCGAA A7CA6AAG GAUUCCAL CUGAGAGCGcA GCGAA AX-GUCAG 247 1067 GUCU3CCA CJuGADGAGCCGAAAG,-CCGAA AUUCaG 1085 AGAACCA C=AWGccaAAG- CGA AGrGUCC 1086 CAGAACC CUGAIJGAGGCCGAAAGGCCGAA AAGGvCUC 1090 UGGCCAG COGAMAGGCGAGGCAA

ACCAAAG

1091 CUGGCCA CUGAUGAGGCCGAAGCC .A AACCAAA 1113 UCrjC CVGA-%GGCCG -CGA AGUCCt3G 1124 uCGU N ci--u UGAAGGC-CG UGrccrAA AGGc,- rj 1129 CAAUJUC CUG~AGG--C--AAGC,,A

AGGTJGAG

1135 OUUGUfc Cu ~GCGG=AACC r ;LUULTCJA 1151 AAGGCCU CUGAUGAGGCCGA~rG,-CCA

AGGUCCA

1152 GAAGGCC CUGAUAGG-CGAAAGCCCAX

AAGGUCC

1158 AGAGAGG CUZ GCGXA- -r.k AGG-CCUA 1159 GAGAGAG CUGAGAG=ccGUAGGCGAA AAGGCCtJ 1162 ACGAGA CUGACAG3LCGAA AGGAGG 1164 AGGA ~r~rcAA

AGAGGAA

1166 ACAUCCG CUC-AMAGGCCCGACCC-A

AGAGAGG

1174 MUCGMA CUGA1~kGGCCGAAAG CGAA ACAUCU 1175 AGUCUGG CTJGAflGAGGCCGAA GCCA AAOWMc *1176 AAGUCUG CUMUXlAGGCCGAAACCCA

AAACAUC

*1183 CUCAAGG CUGAU~k GGCCGAAAGGCCMA

AGUCUGG

1184 UCUCAAG CUGAUGAGCCG~a krCGAA

AAUCUG

1187 Liu UCUC CUAGGCGAG-CGL AGGAAGrJ 1208 CCADGGG COGAflGAGGCCGAAAGGCCGAA-

AGGG.CUG

1224 AMBuGG COGAMAUG XAGGCC,CAA

AGCUGGC

1228 AM"At CUAAGC~ArCGL

AGCGAGC

1230 ACAMAA CrJGAUGAGGCCGAXGG CA AGAGG-A *1232 AAACAXM. CUAGWCGAMC

AUW.AGG

1233 CAAACA CUGAUGAGGCCGAAAGGCCGAA

AXVUAGA

1238 AAGUGCA CUGAUGAGGCMCCGAAAA ACAtkAA 1239 CAAGUGC am-UGAGGCCGAXGGLCGA 9* 145 UAA cA O CUGXAGGCCAAA ccGAA AGuGCAA 1251 AAMULAAU CUGAflGAGGCCGAAOCGL

AUCACAA

1252 tMAUAA COGAL3GAGOCCGAA GGCGAA AAtJCA~CA 1254 AAX3AAIA CUGA CCGAAC-G

AUAAUJCA

*1255 AAAU1AAU CUGAXXAIGGCCGAA GCCCGAA AAU~Auc 1256 UAAAILhA

CUGAUGAGGCCGA."GGCCAAAAUAAU

1258 APJAAAI CUAGGCC-AGCCA

ALZAAAMT

1259 AAAUAAA CUGAUGAGGCCGAAAGCGAAD

AAUAAAU

1261 ALAAUA CUGAGAGGCC;GccAA AUmALV 1262 AAtMAAnJ CUGAUGAGr-CCGAA GGCCGAA AAtAt3A 1263 UAADAAA CUGAUGAGGCCGAGCG AAAU.AAj 1265 AAUAA CTJGAtflAGGC CCGA-Q~AA

AIAAAUA,

1266 AAALALtJ aXUGAGGCCGUGGCGAA

AALMAAU

1267 UAAAUAA CUGAUAGWCCGAAG-CCCA6A

AAAUAAA

1269 AAtDUAU CUGAUGAGCCG A,-C

AUAAAUA

1.272 AtMUL CUGAUGAGCCGAAAGCCCI

AAUAAAU

1272 MtAUATJ CUGAUGA cG 248 1274 AAAVAAA CUGAUGAGCCCCAAAGcc-AA. AAADfltT 1276 GAA CUGAflGAGC-CCGAGCCGAA

AULA~UM

1277 UGMAAU CUvGACAGGCCGAAAGGCCG

AIAMU

1278 CUGMUA CUGAUGAGGccGAAAGCCAA AAAUXlA~ 1280 AtJCUGUA CU~rGCCC-A--CAA AUThA 1281 C-ALCUGU CUUA~cAA~CA AAfLAAXj 1282 UCAUCUG CUGAflGAGCCGAAGCCGAA,

AAAVAAA

1294 A.AALVhAA COGAUGAC-CCAAAGCcA ACAflUCk 1296 CCAAAUA CUGA UGAGGCCGAAAGCCCGAA AflI.CAU 1297 CCCAAA CUGAUAGCCGAAGCC-;

AACAU

1298 UCCCAA CUGAUGAGG-ccAAAGG_-CA AAAt~kCA.

1300 UCL=CA C3GwAflGAGGCC G C GCA AA 1301 GCUCCC CUArA-CGAAGC-A

AA~UXA

1315 CCCAGGA CUC-AIrAGG-CcGAA--GGGA AccccGG 1317 CCCC CUCAr=GA--GAACcGCGA BAcCC 1334 CAGCUCC CUGAICG-CAGGCCCcc

ACAUU.G

1345 CUGA=C CUGAt GAGCCG CGA-CA

AGGCAGC-

1350 CAUGUCTU CUGAUGAGGCCGAAC,-GAA

AGCCAAG

*1359 CACGGAA CULrAGGCcC-A G-CCCA.A ACALTGUC :.1360 UCACGGA CUGAflAGGAAAGGCG

AACALUW.

1361 TJLCACGG CUGAUGAGGCGaAAC-C

AAAAL

1362 UUCACG CUGACA--AAGG~CCGA

AAAACAU

**1386 AACAGCC CUGAflrAGGCCGAGCC3A

AUL-<UUC

*1393 ACAMW CUGAVG3AGGCA.cGCC-A

ACAGCCU

1394 UAAU G AUG;GGCCGkkNGCGA

AACAGC:C

1401 AGGGGGC CUGALGAGGCC c-Gr. ACUG *1414 AGGCACA CUTGAUGAGGcGCCGAA

AGGCCAG

**.1422 UCAAAAG CUGA~I GcaAAGGCC C-AA AGCA *1423 AUCAAAA CUGAUGAGGCCG-AAGCCC AGCACA 1425 UAAUCAA CUG AGGASCC aCGAA

AGAAGGC

*1426 ALTAMCA CUGAI GAGGCCGXUfGCGA AkG :.1427 CALAAAUC CUUAC~,;,C-C

AAAGA.AG

1431 AAAACAU CUGAIMXGGCCGA GCGA AUCAAAA 1432 uaAAAAA CUGAXGAGG-CGAA -GCCGAA AkCAUj 1436 UUMUAA CUGA AGGCC AA CCGAA ACA7AA 1438 AUUUUEAA CUGAUGAGGC.AA C AA AAACAI3A 1439 UAUUUA7 CUGAUAGG-CcGAA GCCGAA, AAAACAU 1440 AtUUU CUGAmt-tGGccrGAG G-CGA AAAAACAL 1441 AAI3LIU CUGAUGAGGC-CGAAGCCCGA~

AAAAAAC

1446 CAGAUAA CUGAIY-AGCCGAAGGCGAA AUoUUrm 1448 AUCAGAU CUGAUGAGGCCGAA G~rA AUAUU=r 1449 AAUCAGA AAZLUrj 1451 UEIAUCA CUAucGGGACcGMA

AUAAXUMU

1456 ACAACUU CUGAuGAGGCCC,--u GCGAA AU U 1457 GACAC CETGA=-AGGCCG AcC-CGAA

AATJCA

1461 UUUAGAC CUGAUGAGGCCC-AG,-xGCCA ACUtUAA 1464 UULU CUC-AUGAGGCCC.G GGCCGA ACAACUL7 1466 CAUUGUU CUGAGGGCCAZGCCA,

AGACAAC

249 1479 GUCACCA CUGAUGAGGCCGA .AGGCCGAA AUCAGCA.

1480 GGUCACC CLCuGUGAGGCCGAAAGGcGAA

AAC

1494 AAUGA.GU CUGAUGAGGCCr-A~~,CA

ACAGGU

1498 CAGCAAU CUGAGAGGCCGAAAGGcGAA

AGUGACAL

1501 CCUCAGC CUGADGAG~CCGAAM GCCGAA AflGAGUG is=2 GCAA CU vUAGGCCGAAAGGcCGAA

AGGCCUC

1517 CCCUGGG CUGAI GAGCLCGAA GCCG ACA .1528 CAGACAC CUGAflQAGGccGAA cCGA ACUCCCU 1533 ClACA CU ANUAGG-CcGAAA(;GcCG

ACACAAC

1537 GGCCGAU CUGAG~AGGcc,-Cz ACAr,:CA 1540 GtAGGCC CUGAUAC-GAAGGCCGAA AIL7CV 1546 UGAAt~.G CVGAtr-AGC trAGCCA AGGCC 1549 CACOGAA CUzAfLrAGGCCGAAGcCGA AGtLhGGC 151 GCCACUG CUGAUGAGG-CCAGG-c.AA 1552 CGCCACU CV AUAGGCCMAlCC AAUNGoM 1566 CAACCUU CUCQWGAGGCC ALItU=Cu 1572 CCcaXIGC CUGAGAGGCCGAAGCCGAA ACatI 1576 CUOU~c COGUGAGGGAGCCC AA AGCktAC 1577 UCUOUCc CUGAUAGGCCGAAG=CG

AAGCAAC

250 Table 25: Mouse TNF-a HETarget Sequences nt. HM Target Sequence Ut. H Target Sequence Position Position 66 UgGAAIJ a GcucCck 324 G9GUAU C GGuC 101 GGCAGU U Ct~gUcCC 347 GAGAagU u CCCAaaU 101 GGCAGgU u CuGUccC 364 CCUCCCU C ~w 102 GMGGUU C UgUcCCU 366 UCc-=CU c ArCAuu 102 gCAGgUU c ugUcCu 366 UcCCUCU C auC;LuU i0 rUCUgti c CCUUUC 369 CUCrJCAI C AazuCUa 110 UgUccctJU u UC~AucA 376 CA~uuCU a UGG-ccA 11gtJCcCUU u CaCUAC 390 AgACCCU C AcaCUcAN 1"guCCCuU u CAO.Cc 396 uc-aCALcU C AGAUCAU 112 UcCCUUU C ACizcACU 401 cUCAGAU C AucOucUy 116 DUCACU C AcUgcc 404 AGA~U C aucUCaA 137 G~CaCAU C uCCcUCc 406 AUCAflCL U cucaAAa 139 caCAiiCU C CCCcg 406 AUcA~cU U cucAA 177 GCAUGAT C CGCGACG 407 UCAI3U= C UCaAAau 207 AGCGCaCU C CCCcAaA 409 AIJCUUC C aAAauuC 228 GGGGCuU C CAGAACU 409 AuUuuCU c AaAAWUC 228 CGGGCuU c CA~aactT 409 alUUcU c AAAauUc 236 CA~aaCU C CAGG=G 432 AGCUU A Gcc-kCG *236 CAZ~aACU c cAg~ 249 GGugCCU a UgUCUcA 249 GGUGCCU a UGucUCa 444 ACGU~cGU A GCAAACC 501 AcGCCCtI C CUGGCCA 261 VCAGCCU C UUCUCaU 5.60 gGgUUGU a CCUuguc 261 UCAgCCU C UU~cau 560 GGguuGU A CCUuguC 263 ACCCUCU U CL~afUC 564 UGtrj u gUCUAC 263 AgCCUCU U CUcauUC 567 ACCEJUgu C UACUCCC 264 GCCtJCUU C UCAUUCC 569 Ct~ugucu A CUCCCAG 264 gCCUCUU C UcaUUCc 572 gUCtU&= C CCAGGUu *.266 CUCUUCU C alUOCCG 572 GUCUaCU c CCAGguu 269 UUCUCaU U CCUGcUU 572 GuCUacU C CCAgGUu 270 UCUCAUUE C CUGcUuG 579 CCCAGG u CUCUCJCA 276 TJCCUGCU u GUGGCAO 580 CCAGguU C UCUrcAa 297 CCACGCU C UUCEuC 580 CCaGGuU c UCuUcaa 299 ACGCUCU U CUGUCLa 582 AGGUUCU C UUCaagg 300 CGCUCU C UGuCUaC 582 AGGUuCU C UUCAAGG 304 CUuCUgU c uAcUGaa 584 GUuCUCt3 U CAAGGGa 306 UctJGUcU a cUgAAcU 585 UuCUCUY C AAGGGaC 314 CtJGaACU U cGGgUG 608 CCCGaCU a CgugCuC 315 UGaACEJU c GGgGUGA 615 aCgUGcU C CUC;LCC 315 uGaaCUU c GGGguGa 615 AcGU C CUCACC 324 gGGtJGaU c GgUCCcC 618 UGCUCCU C ACCCACA V 251

S.

S

S.

630 630 638 643 645 647 663 669 669 672 674 681 681 681 734 734 744 746 759 759 761 762 786 798 802 812 816 821 822 830 840 842 842 842 845 846 852 855 887 891 905 905 905 914 915 919 928 928 932 ACACC9U C AGCC~au ACACCgU C AgCCgaU agcCgAUl u uGCt~aUc alUUGcU a uCUcAit2A UuGCuaU C UCaM=C GCuaJCU C aLMCCAG agAAaGt7 C AACCUCC UCAACCU C CUCUCUG UcAAccrJ c cUd.o=t ACCrJCCEJ C UCUGCCg CtJCCouC C uGCCguc cUGCCgU C Aaga~cC CUGC~gU C AAGAGCC CL3GcCgU C aaGAgcC CCCoUGG A UGAG-,CC CcCUGUt a ugaGCCc AGCCCAI7 a UAccOG CCAaU A cCUGGGAL GAgGAGU C UUCCAGc GAGGaGu c UucGccr GGaGtICU U CCAGCUG GaGUC3U C CAGCUGG ACCaACU c AGCG=U CtJGAGgU C AAUCL1GC- GgUCAAu c uGcccaA CCCaA9U A cuUaGALC AgUACuU a GACU~uLG UUaGA=r U tJGCgGAG UaGACUU U GCgGArU GCgGAGU C cGGGCALG GCGCA=G C tIUo CAGGEYCU A CUUtMGa CAGgucu a CrjuugGA cagGuC3 a CDUUgGA GUCUACU U UGGagUC UCUACUU U GGagUCA UUGGagU C AIJUGCuC GagUCAU U GCuCUGU AUCCaUrJ c UCUACC AuucuCrJ a CCCaGCC CCcCaCU C UgaCCCC CCCCacU c UgAcCC CcCCACU c UGAc.CC GAcCCcU U uacUicUG ACCCCuU u acUCuGA CtJUUAcU c ugaCCcC GA~ccu ua uauuguc gAcCCCU U UTJUguc CCOUUAU U gucuacU 940 943 972 972 973 984 984 985 997 1010o 1017 1018 1019 1073 1096 1106 i1107 1108 1115s 1 113 1164 1180 1203 1210 121 1214 1218 12128 128 1218 1219 3.219 1226 1226 1227 1227 1228 1238 1262 1283 1283 1285 1287 1287 1.288 1289 1293 1293 1294 GUCt.J.CU c CUJCAGaG t3ACcCU C AGaGcCc UCLaaCU u AgAAAGg LucUaaCU u AaAaAgG CUaACuU A GAAAggG AGgGgAU U auGGcuc AG-GGgaU UJ allGgcUc GGGGauU a tUGcUCa UcA'%GAgU c CAAcucu CUguGCEJ c AGAgCJT C-AZAgCU U UcAaCAA AGAgCurJ U cAaC-AAhC G-agctu-uu c AaCAACU UgGGCCty c ucAI~gCA AAGgAcU C .AA1gGG aUGGGcU U uccGA UGC-CUU u CCGAAjuU G-GgCuuu e cGaauUiC CC-GAAuU C ACUGGaG CGA.AugU C CA~uCCU gagU;GgU c AgGUUGc UcUgUcU c agaAUGA aaGALUCU c AGGCCU cAGCGCCU U CCUacCU AGCCUU C CUacCUu CCULCCU a cCjucAG CCUACCU u CaGACCu CC~aaCCU U C-AGACcu CCUACcU u cAgAccTJ ccuaccuy u cAG.Accu CuaCCUU C AGACcuu CuAcCUU c agACcUU CagACCU U uCCAgAC CAG-AcCU U UCCAGAC agACctUu CCAgACu AG-AccUU U CCAGA~CU GAccUUJU C CGACUC gACUCuu c CUGAGG CAGCCuU C CUCACaG CCC-CcUt C uaUUUAU CCcCCCUT C UAUUUAU CCCCUCU A UrLrMUaU CcU.CUAU u UauAuUU C,--JCE]AJ U TJAUaUUU Ct7C!U U AUaUUUG UCUATjUU A UaUUUMGC UUUAUaU U CC-CACtUU UUUaUaU ua UGcAcUu UtU.UaU U GCACUa 252 1300 1303 1304 1306 1307 1307 1308 1310 1310 1310 3.311 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 ANuuUAUU

UUADM~U

uMUX mwDuu A UUUCUU A UuUauUU

UUAU

VAT.U=

u UauUUAu U AUUM=U U AUUCATU UUCACU U aluUhUu C.cuUaU u AuUuAUlE acUuAtlU A UUtAUM, uuAmwh u mu~fltmu UAUMfl U AULTAI=J UaOUaUJU U AuuAXuU AXUAUUU A UUMDUA UauUuAU U AUUUAX= URflU U MXUfLU MfUD U AUL1U AUUU A UUMUUU 1462 1470 1472 1473 1474 1478 1479 1479 1484 1498 1511 1514 1516 i529 1529 1530 1530 1563 1563 1568 1589 1592 1617 1623 1633 25 aCcuuau GcuCcuC UUUUGCU I UUUGcUUa UUUtGUU AAAuauU t AcccAay C cAatltGT c allUGUCU u CgcugAU u cGCUOGAU Ui gC3GAtJU u GCt3GALU U Ug.aAcCU c ugaaCCU C CUCUGCU C UGaCUGu A CtJGtAAU u GAGAAAU A tMhAAat c UUAaaaU a AgGgaCU a .1 GCCUCCU ttUUGCU 3 UUGCtJUA 3 UGCUUAU 7 GcUrJAUG I AUMt~Ua LUGUUUaa TaaaAcAA TAUCUaAc UuAAuAA AAuAAcG UGuGAC

U=~GAC

gGUgacC

GGUJIGACC

UGcUCCC

UGCUCCC

CCCAcGG AUuGcCC GcCCUAC AkAGaUcG GC-uLahaa aaAAaCC gCCagGA AUAU A UUtMhUU fUUMAU U mwmULtU AUUM.VU U AUU UUaAWUU A VUXUrD.

AUUAWW A UUUD muaLuthU u u~I3UgC AtUUU U AXUUgC ~UUUU A lUUUgCuu aWUEIrLU U UgCUUAU AUUUAUU U gCUUAT3G auUUGCU U Au.GA~uG UUUGCUU A uGAkiGu !LrAA=~ A UUaUUU AAGMU U M~IU= AUGL~UM U AUUWGGa UGUlUU A UDUGGaLA uAUutMU u UGGaAGG tJAUUtMhU U UC.GaAGg AUUEMU U GGaAGgC GGGGUgU C CE7GGaGG gCUgUCEU U cAGACAg GCUGuCT U cagacAr, GACAUGU U UU~cuGUG ACAUGUU U UCuGUGA CA.DG=U U CuGUGAA AUGtUUu c uGJGAAA AUGUUTJ c ugugAaA gaGCUGU c CCCAccU CtUGGCCEJ c ucuaccE ggCCUCU C UaCCuuG, 253 Table 26: Mouse TINF- Hammerhead RibozYme Sequences nt. Mouse BERibozyme Sequence Posi-tion UCCUGSC CUGAflGALGGCGAA CC=AA AGUCCCUJ 66 UGGGAGC CUAGCCGAG-CA AULM=c 101 GGGACAG CUG GANGGCCr3AAAGGC-CGAA .AcctuG_ 102 AGGGACA CUAXMG GC=-UAGGC-CG

A;ACC

102 ACZGAC;L CUGAX-IGG-CGAA GG CGAA AAcCUC i06 UGAAAGG CUGUGGG=-k G.CCGAA ACAGAC 2.10 UGAGUGA C=GAG- CGAAAGCCGAA

AGGA;

GCA=~ GCC--GkAAGGCCA

AA=C~

I~ GC~GG CUkGGCCAIC- AAGG2AC *12AGA~'GV UG CGAAAGCCG

AAG

2.16GGCCAGU CUGAUA=CwGCCGAA AGUGaAA 137 GGAGGGA CUGAt2GAGC-CGAAAGGCCGAA AuGcGGC 2.39 CWr.GAG COGAtGACGCCGAG)GCCCAA

AGAUGUG

177 CGUCGCG CUG;LUGAGGCCGX. GCGA AV= 207 U=C-GG GAUA=CGAGGC

AG=

228 AGM=UCUG UGA=GAAGGCCGAC L AAGCCCC 228 AGMUCUG CUGAL AGGCCGA~AGCCA

AAGCCC-

*236 CCGCCUG CUGA=AGGCCUGGCG

AG.UCUG

*236 CC,.ccUG CUGAUG3AG.GC-'Ca,- -cGAA AGUUCtJG *249 LUGNGACA CLUGAUGAGG~CGXGGCCG.-

AGGCACC

249 UGAGACA COAGG--CGAG~G;

AGCCACC

.261 AUGAGA COACG-CAAr'-rA Ar.-UGA *26- AUrGAA CUAGCC--,AA~c AGCG-jGA 263 GAUGAG CUAGGCGaAG-c;

AGAGGCU

263 GAADGAG CUGAVGAGGCCGC kr AGAG~c 26-GAG CUGAUGAGGC-CGAAAGr.CCGAA

AAG

:264 GGALAUGA CUGA.2AGGCCGAUAAGGCCGAA

AAGAGGC

266 CAGGAAU CUGAU'GAGGAA~ccA

AGAA

269 AAGCAGG CUAGG-CG~aCA

AUGAGAA

270 CAAGCG CUGAUGAGGCCG Ga,-CCGAA AAUGAGA 276 CUGCCAC CUG ~rAGGCC AAGc

ACCAGA

297 GACAGAA CUAGG--GAcGcA

AC-CGUG

299 tLhGACAG C GAUGGCAAA GGrrCA AGAGCGUj 300 GCGCA MXCwJ'kGA-CC -b~ccc AAC,-CCG 304 UUCAG CUGAVCAGZ--GcAAGccccA AC~aAAG 306 AGJUCAG CUGAUmAGGcc~G--rCAA

AGACAGA

314 CACCCCG CUGAUGAGG--cCGAGC AGUUrA 31.5 tJCACCCC CtUGAGGCAACCG-GAAc

AAM-UCA

254 315 UCAccC

AAGUUCA

324 GGGGACC CUAGG-CGAGCGL Auccc 324 GGGGACC CUGAUGA GCCAAG-AA AuCcC 347 AUUUGGG CUGGflGAGGCCGAAAGG-CGAA AcuUTCUC 364 CG. CUUGAGCCAAAGCCGAA AGGAG 366 AACUGAV CUGAUGGGCICGAAAGGCGAA AcGAG 369 MAACU CUGAGGCCvAAG~C-CGAA Avc,-cu-t 376 UGGC vJGCGCCG Gc-,A AcAACM 390 UvGGU CUG AGAGCCGAAAGC-GAA AG-Q-,-CrJ 396 AUGA=COGU GCCGAAGCGAA AUG 401 AGAAGAU Cr GAUGGCCGAA CG Aflc-, 404 UOCUNGA CUGAUGAGGCO -AGcaAA A~r-AUCrj 406 UUUGAG CUG UGAGGCCGAAAGCMCA

AAUW

406 UUUGAG C rnUAGCCGAAA1f-CcAA AG?.U 407 AUUUG CUGAUGAGCL-,wAAGGCGNA AAGALMG 409 GAA=U CUGAUAGGCCuGAAGGCCGAA Ar~AAA 409 GAAfUUU CUG NGAGGC-CGAA.AGCCAA ACA=Q 409 GAAU= UU CAGCGAGG-, AGAAGAU 432 CGG C~~GcGAGc~ A A~G= 444CGUC CUG~u-arX CcGAAAGGCcWAA ACG;cUy ***501 UGGCCALG CUCAUGAGCCC~vAAGCCGAA

AGGU=

*560 GACAG CUGAX3JGG--a~aAG~Cc-GAA

ACAACCC-

560 GACAAGG CUAGA MAG~CA ACAACCC 564 AGUAAC CUAGG-CAA:G--A

AC-CA

567 CGGGAG CUGA wXGCCruna-ZGGCCGAA AGCAAGt ***572 AACCUGG LGUNGCC- GAA 572 AACCUGG CUGGflGAGGCCGAAAGGccAA

AUACA

*.79 UGAAQAG CUA~AGCGAGcc.

ACCUGGG

Sao8 UUGAAGA CUGAU~A0GCCG AGC.CCrU AAC 580 UUGAAGP,. CUGAGGCCAAAG~C--

AACCUGG;

Ba2 CCUU=A CUG~LALGCCCGAAAGGCCCA

AGAACCU

:582 CCOUGAA CUGAL AGGCCGAAAGGCCGAA

ACAC

5a4 uJCCCUw CUG UAGCCGAAAGG;CCAA

AGAGNAAC

*585 GUCCCUU CUAGGC-,A;~cC, AAGAG= 608 GAGCACG CUGuJCaX.AG--GAAA(ccC=

AGUG

615 GGGWGAG CUG~A~ACCCCG GcCGQA Ar,=A=~ 615 GGGUGAG CUGAtraAGGCCGAAGcCAA AGCAcCrU 618 UG3GGGU CVUA GGCCGA

AC,,C

630 AUCGGCY CUG;LUGAGG-CCIAAGGCCGAA AC-Ct t 630 AXVCGGCU CUGAUGAGGCCGMAGC

ACGGU

638 GAmkhGaf" COGAGGAAGGCCG AAC~ AL1CW.CU 643 UAUGAGA, CUGAX GAGGCCCGAGCCGCAA

AGA.U

645 GGUAUC-A Ct' AUGAGGCC

AUGC

647 CUGGrJAU CUGAU;GAG=C-C-CCAA

ACXAC;C

255 663 GGAGGUU CUGAUI GCCG.A

XCUU=

669 C-AGAGA. CUrmAXGACGCCCAAA 669 CAGAGAG CoUAcGAGCCGAAAGC,-C

,,GO

672 CGGCA CUCAIrmGGCCAAAGCCA

CA

674 GACGGCA CUrxAfGAGGCCGAGG-Cr.A pACAG 681 GULCUU CU GAA-G-CGAA C GAA ALC,,G 681 GGCCuu CUMAUMAGGCCGAAA<,C=,A ACXGc= 681 GGUu CU GCCCAAGCCA 734 GGC-CUCA CUGA~AC~rGX-C;

ICAC~G

734 GGCUCAL CUGVGAGGCCGAAAGGCQA

ACCA-C

746 UCCCAGG 'UAGGCCAAAG--- Aa;=GG- 759 GCUGGAA CUG~Tv cGACCAAG,-cGCL AcCC 759 G-%-UGGAA CUAGG CAAG-C,,

ACCC

761 CAGC=G CUGAUAGGCCGAA GCMM. AGAC-,aCC 762 CCAGCCUGA aGCrAMCA

AAGCVC

786 CAGCGC-U CUGAUTlGGCCAtGCC-l

AGI

798 CGM~J Ct7GAUGAGGCC AcCCCAG 802 TUCGCGCA COUrtAGGCCGAAAGCCG1L AUG 812GU~AAGCOGAUGAGGCCGXAAGG-CMGA ACtU= .816 CAAAGUC CW-A-'G-CGAAGCGA AAiax= 821 CUCCGCA CUGAUGAGGCCG AAAGCCC-X AGOGa~ 822 ACUCCGC CUAMGCAAG-G;

AAUCD

830 CUGCC-=CGAU C

ACC

840 CAAAGUA COGA-AGa=rAGCCGA ACCUG-c 842 UCCAAAG COGAUGAGGCCGA AGCGA

AGACCU

842 UCCAAAG CUaflGA CCG

AGACCCIG

842 UCCAAAG CUGGX3GAGGCCCGA GGCAA

AGACCU

*845 GACUCCA CUGAXGAGGCCGAAA CCGAA AGAA 846 UGAC C WkGCAAGCa

AAGUG,

852 GAGCAAXJ COAGGCGAAG-G;

ACC

855 ACGAGC CUGAXXA3CC AflGA=U *887 GGGCAMP CWMGCGXAGCA AAGGA7 891 GCUGGG CUGAM~ALGCXAc GCC

AGAL:

905 GGGGUc COGAUGAG=CG GCCGAA AGOG=G 905 GGGGUCA CMUGAGGCCGAAGCC

AGM

~905 G30CUCA CUGAWA=~CGAAAGCGA;

AGG=

914 CAGAGUA CUGAnGGCGccc~ ccGA AG=GGGUc 919 GGGVtCA, C~-UAGCGA Grc A AGGAAAz 928 GACAAUA CUGAUGAGG-C UGCL-AA

AGGG=C

928 GACAAUA CUGAGGCCAGCGA

AGG=GC

932 AGUAGAC CUcGx7GAGGCCGAAGCCA

AUUA

940 CUCUGAG CUAGGCGU-CG;

AGAA

943 GGCUC CUGXAGGCCGAAG.CGA AGGAGtA 972 CCUUUCLT CUGAflGAGGCCCtJAG<CGj A~uaAm; 972 CCUUUCtI CMUGAGCUGGic L rt AZU r 973 CCCUUUC CMUWCCAWCA AA==r 984 GAGCCAU CUAGGCGAA,-CA AuJCCCC 256 984 GAGCAU CUGAUGAGG-CCAAGCCGAA AuCCCCU 985 UAGCCL CUGAUAGG~.C7-cmu AAU=Cc 997 A~mGUG COGUGA~~G--Cu1 GCG ACUCUGA 1010 AAGCUCU CUCA AG~CCGAAGCCCA 1017 UUUU; CUGAflGAGGCCCAAAGCCGAA ArG-CrCU 1018 GCGUM CUGAUGAGCCGAAAGGA.;A A;CUCrj 1019 AOUGU CUGAUCGGCCGAA GCcGAA AAAG=U 1073 UGCAUGAL CUCGAUAC-GCCCAAGGccGAA AGC4-C-CA 1106 CA CVAVAGG-CCAAAGC-ccsAA AGCCUU 1106 AUUCCGG CUGAUAGGCC~Ua.GGcC U AC-CCCAL 108 AAU= G AUAGGCCGAAAGGca;GL -,,CC 1115 CUCCAGU CUGAJJGAGG-CCG GCCA AAUCC,-G 11.33 AGGAAWG CUGAUGAGc GCCGGCAA ACAM-CG 1164 GCAACCU CU XGAGCCGAAAGCCAA ACCACU 1180 ~UCU CtUGAUGAGGCCCAAAGGcCGAA

ACGA~

1203 AAGC-CCU CVAGGC~UAccA AGACU 1210 AGGG CUGAUGAGGCCGAAAGC-CCAA AGCC 1211 AAGG~kG CVGAXJGAGGCCGAAA aCGa3. AAGGCCUa 1214 CUGAAGG CUGADCAGGCCrAAAGGCCGAA AGGAAGG *1218 AGGUCUTG CUGAX GAGGCCGNLUA GCCCGAG 1218 AGGUCUG CUvVAGCXAG~,AAMG i 218 AGGUCU CUG AGCGCCrvuAGCCGA ACGMG 1218 AGGUCU LCUGAUMAGGC~a-aa. CCAA AGG= 1219 AAGGU CU ulUCGC1nAAG-_ ,,aGG= 1219 AAGG=C CUC GAGGCCGAAAGGcCG=AA AAGA 1226 GUCUGGA CUCA~vGGCCGAAGLCGcQ AG,-UCU *1226 GUCUGGA CUGAUAGGCUG.AAGUCC-,

AGGUCUG

1227 AGUCUGG CUGAXraAGC.CCGAAAGG,-CQ

AAGGUCU

1.227 AGCUGG CUG.ALM3AGGCCr.AAAGCCQA

AAGGUCTJ

1228 GAGUCUG CUGAUGAGCCCGa-AcGCCAA

AAAGGUC

1238 CCUCAGG CUGAflMGCCAAG C AAGA=~ 262 CUGUGAG COAGGCGAG-CA

AAGGCUG

*1283 AXV"AA CUGAXrACGCCCGAA Cr.Cxrk ACGGGG 1283 AIMAAXM CUrmAXrX'tGGCCGA.A3GcCrA

AGGG=G

1285 AMMUTh.A CUGAUGAGGCCCAAAGCAA

AGMG

*1287 AAM UAGOCGAGCCA

U.G

1287 AAAUA3A CUGAflGAGGCCGAAGCGAA

AURAGG

*1288 CAAAMU CUGAUGAGGCC

AAG

1289 GCAALA CUGAVflAGGCCMGA G=CCChA AAAZMGA 2.293 AAGUGCA CUAGGCGAGCCA

AU~AZA

1293 AAGUGCA COGAAGAGGCCGAAAGGCCGA;

AGMU..AA

1294 UAG=G CVMAUMAGGCCA GG;A AAM 1300 AAAAAD CTGAUGCGAGGC CGA L AUCAA 1.303 AAMULZLU CUr3AUGAGCCMAG))~UCAA

AUAMG

1304 MAUAM CUGAXJGAGGCCGAAACCGL

AAA

1306 AA~kAXA CUAGG-CGAGCGA

AUVA

1307 2AC.U.CG~GcC

AAMAUM

i307 AAALMhAU CVGAtrAGGCcGAA G,-CGAA A;L=.~UA.

257 1308 0AMUA CDGAXUGAG---CGAAAGGCCGAA AAAtMhA .L310 A;MAAAU COGAUGAGC-GXIlaG-CCAA

AGA.A

1310 AArLWAA CU rUGCGCCGA AW-CCA AX7AA,j :310 AAMLVAA CUGAUA- AAGGCC GA AMAA 1311. AA~fAL CUGAMJAGCCGAA -CCGAA

~-AAMAU

1- i AAALVAA C~3~l CGAAAG=C-AA

A

1311 AAAA CU AUAGGCCGA acccGr 213 AE~AAM CC-GG-CG~kAGCCG AUAA=k 11213 AtLAAM COUGAGCCGAA ,-CGAA AawA~f~ 2.313 AMAWV, CUG UAr-CMXzZCA AXAM61 2.314 AAUAAU CJGAGCC ljc.CGA

AAUIIAM

214 AAAALT CUG~AM--GGAAGG-,A;

AAMAL

1 ~L 5 ~CUGAGAG-CcGXVC-Ar,

AULGAA

117AAXLhAU CUG~tflAGGCCGA GC-GA ALAAMJ 1318 AAAUM= CGUGAGC -A -rCGA ;AAA i219 MAMMI~A CUGAUGAGCCaG CGAA AUAAAA 1326 AAAL. CUGGflGAGC CG-CA~ AAAIVA 1.328 GCAAAML CUGAI GAGGCGA

AUAIA

1329 AGCAAAD CUGAGAGCCGAAAGCAA

AMAAU

1330 AACAAA COAGGCGAAG-GA

AAAAA

:41332 .AVACA CUGArGAGWCCGAAGCCGAA

AMPAUM

1333 CAVI.h.AC CUGAXImaGGC_-G AGG-CCAA

ALMAAUJ

1.337 CAUDCAU CUGAMGAGGCCGA. CCM AGAAA 1338 ACflUUCP.. =CAGCCA

AAGCAAA

1346 AAALA CU-GAGGGAA~ CGA ACAUUM~ .4.1348 CCAAAI CMAUGG-CMG4G AA AMC=~ 1349 MCCAAA CMUGA=GCGAGCG

AMC

2352 CUUCCAA CMUG~AGGCC GCC-aA

AAAMM~

13152 CCMuCCA CUGAUG G--cc GA ALAA *3 53 G11 U2CC CUG flGAG=GCC a aCGAAAUA 1369 CCUCCACUGAUGAGGCCGAAAGCCGAA

CCC

41398 CUGUCLI CU G C CGA AGACAMC 38CocUCUG COGAXM GAA ,CGAA AGACAGC 1412 CACAkGAA CUG~UAVGGCCQ

ACAUGEIC

14134~~~

CJG

i*i UUAA CUGUGLGCGAAZ. C AAACAG 145UUUCACA CUGAXGAC-CGAAAC..AA

AAAACAU

1415 UULTCACA CGUACGAr-CCAAAAACAUy 1438 AGMUGG CUCAXJAGGAX4~GCCG ACAGCrCU 1451 AGG-aLG CUGAXr-acGCC,-AAC, CaGC 1453 CAAGGV C~.~IGC,

AGAGC

145AACAGG CXAVAGCCGAAG G AGAGAGG 1462 AGGAGGC CUGAL-AG-CcXUGCCG

ACAAGGJ

1470 AGCAA COGUG~~Co-Cck G= AGGA=G 1.472 tNAGCAA CUAGGCMAGC

AGGAG,

1473 A~kACA C7JMUGAC-C

CGPAAAM

1474 C;LAGC CUGALUA Gc -CCGAA AJ~AG 1478A M-AAGCAi-CA

AGCAAA

258 147I9 UMAC CUGAUGAGGCCGAA cG.A AGCA 1479 UUAAUA CUGAGGCCGAAAGGc.c~AA

AGX;

1484 U~UGUU CUGUAG~CCGAAAGGA AACA~ 1498 GOLM=I CrJG7,GAGCCvGAAGGcG AM 1511 UUAGAC CUC%1EGAGGCCGAAZAGGCCGAA AVDGGU 15i4 UMUMAA CUGAUJGGGCCGAAAGCCA ACAAnUU 1516 CGUflU CUGAVGGCCAAAGGCCmU AGCA 1529 GUC-ACCA CUGAUGACGCCGAAAGGCCGA AUCWG 1529 GUCACCA CJw~mGG CAAGCGA AUCACCG 1530 GGUCACIC CCUGAGGCCMGAAA~G AA AAUCAGC 1530 GGUCACC CUAUAG~CCGAAAGGCCrAA AAC 1563 GGGAGCA C AAGGCC-AAAG~C--CGa-

,AGGUUCA

1563 GGAGCAL CUGAUGAGC-CGAAAGOCCGA;A AGGUUCAL 1568 CCGUGG CUuU 'CCCu'ACC GAA AGCAAG 1589 GGGCAAU C GUAGC~UG---,A

ACAUCA

1592 G~kGGC CGAUAG~CCGAAAGCCGAA ALtL~k= 1617 CGAflcU Ctr-AUAG~CCGAAAC,-c,AA AUUC=U 1623 UUUAAGC CUAUAGGCCCGAGCcCA

AUCM

1633 GG~U= CUGAXAGCAAGGcGAA AflUU.A *so* *00 9*000

S.

Sb p. 5

S.

S 55 55 5

S

5** S S P

S

0*

S

S*

*4*S S

S

5* 55 S 550 05 0 so *5 *S* S. S S *S 5 S 5 5.5 S S S S ~5* S S S

S

''Tble 27: hlumaW I'NF-a flairpi RiI)ozyII Scqucmcces Pos nt. Hlairpin Ribozyme Sequence it ion 46 AGCCGUGG AQAA GLJAUGU ACCAGAGAACACACGUGUGGUAJAACUUA 254 296 317 387 404 453 518 554 565 576 607 704 726 730 824 1042 1168 1178 1202 1.220 1284 1340 1390 GAGGGUGG AGAA GGAGAAGA AGAA CUGCCACG AGAA GUGCACCA AGAA CAAAGIJGC AGAA CCUICUGGG AGAA GGCCAGAG AGAA AGAAGAUG AGAA GCCACUGG AGAA AIJUGGCCC AGAA GCACCACC AGAA GGUGGAGG AGAA GGGAUIGC AGAA UGGUAGGA AGAA

UGACCUUG

CCUUCUC

AGCGCUGA

GAUAGUCG

UCGAGAUA

GGGAUUGG

GGGAUCAA

CUGGAAAC

UCAAGGAA

AUGGGGAG

AUAGAGGG

AUACAUUC

UGAGCCAA

UACAUGGG

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGMA

GUGGGU ACCAGAGAAACACAGUGUGGUAAUUACCLJGGUA GAGGAA ACCAGAGAAACACACGUJGUGGWJACAUUACC1JGGUA GGAAGG ACCAGAGAAACACAC0UIGUGIAIAUUACGGUA GAAGAG ACCAGAGAAACACACGUU1GUGGUAAUUACC1JJGUA GGCAGA ACCAGAGAAACACACGUUIJGGUAAUACCIJGGUA GAUCAC ACCAGAGAAACACACGUTJGD(GUACAUUACCUGGUA GAUUAG ACCAGAGAAACACACGKJUGUGGIJAC1AUACCUGUA GACUGC ACAAAAAAGUGGUCUACGU GCCCCU ACCAGAGAAACAACGUUGLU0GUAAUUACCUGGUA GUUCAG ACCAGAGAAACACAGGIJ1GGUQACAUUGUA GGUUAU ACAAAAAAGUGGUCUACGU GCCUUG ACCAGAGAAACACACGUU)GUGGUACAIJUACCtJGGUA GAUGGU ACAAAAAAGEGGUCUACGU GOGAUG ACCAGAGAAACACACQGJrGUACAUACCUGJGUA GGUAGG ACAAAAAAGUGGUCUACGU GGAAGA ACCAGAGAAACACACGUJGTJGGUAAu1ACCIJGGUA GIJCACC ACAAAAAAGLGGUCUACGU GAUUIGA ACCAGAGAAACACACGUJGUGUAAUUACCJGGUA GGCCGA ACAAAAAAGUUMC~ACGU GGGGAG ACCAGAGAAACAcACGUU!GTJGGUACAUUACCJGGUA GUAGGC ACCAGAGAACACACrUIrGGUAAUUACCW 3

-GUA

GGAGAG ACCAGAGAAACACUUGEJrGUACIJACCGUA GGAAAC ACAAAAAAGUGGUCUACGU GCUC ACCAGAGAMACACACGUUGUGUACAUUACCUGGUA GGCUCC ACCAGAGAACACACJUGUIGGUACAUJIACCJIGGUA GUAAAU ACCAGAGMCAAGUGGUACAUAcGuA GCUCCU ACCAGAGAAACACACGLTGGUACAUUACaUGUA GCCUAU ACAAAAAAGUGGUCUAa

U

Substrate ACAUACU GAC CCACGGCLJ ACCCACG GCU CCACCCUC UUCC~UCA 0CC UCIJUCUCC CCUUCCU GAU CGUGGCAG CLJCULJCU GCC UGCUGCAC UCUGCCU OWU GCACUUUG GUGAUCG GCC CCCAGAGG CUAAUCA 0CC CUICUGGCC GCAGUCA GAU CAUCUU)CU AGGOCA GCU CCAGUGGC CUGAACC 0CC GGGCCAAU AUAACCA GCU GGUGGUGC CAAGGCU 0CC CCUCCACC ACCAUCA 0CC GCPAJCGCC CAUCGCC GUC UCCUACCA CCLJACCA GAC CAAGGUCA UCUUCCA GCU GGAGAAfG GGUGACC GAC UCAGCGCIJ UCAALJCG 0CC CGACUAUC UCGGCCC GAC UAUCLJCGA CUCCCCU 0CC CCAAUCCC GCCUACA GCU IJUGAUCCC CUCUCCA GAU GUUUCCAG GUUUCCA GAC UUCCUU)GA GAOCCCA 0CC CUCCCCAU GGAGCCA Gai CCCIJCUAU AUUUACA GAU GAAUGLJAU AG0GAGCU 0CC UUGGCUCA kUAGGCU GUU CCCAUGUA GAU UAAGUIJGI GAUl UUIGGUGAC GCU CCCCAGGG GCC UACUAUUIC 1452 1475 1513 1541 ACAACUUA AGAA GAUAAU ACAAAhAAGUGGUCUACGU

AUUAUCU

GUCACCAA AGAA GCAIJUG ACCAGAGAAACACAGUGIJGGTAUUACVCrMUA

~JCIJGC

CCCLJGGGG AGAA GAGGCC ACCAGAGAAACACACUUGIJGGUACAUUA-CCUGGUA

GGCC'JC'

GAAUAGUA AGMA GAUUAC ACCAGAGAAACACACGuGMuACAUUACCUGGUA~

GUAAUCG

S S S S S* S S S S S S S

S

Table 28: Mouse TNF-a Hairpin Ribozyme Sequences Hairpin Ribozyme Sequence Substrate 103 256 272 301 325 370 383 397 467 546 549 598 603 631 634 675 691 764 803 895 906- 920 953 1175 1220 1230 1256 1274 fLAAAQ3 AGAA GAACCU A CGAAACCCJXUUCUaCUG AGGUhC GLC r AJ MAGAAtGA AGAA GAGACA A~AWACAMn~WXMG IU~AJ~rAG~aX CUGCACA MGAA GGAM~ CAUC wLAxA W~uA~uA AGAA GAAGAG ~AMAJAWAAM"rWw r QCUL GUC UAUAA CtUXXX3 PL3AA GAUCAC ACAGAAWAa~~MCMOWG G~.AC GU QXAAG GOCCAUAG AGAA GALUMA ~tA~rAAAAaaCXA nilLC GUUChTGO GUGLfAM AGAA GOCC X CA"AWlaUWMpCUG UGOCCA GA rA AGAAAU AGA GAG=ACWAcwaXnmxmxx UCr GUCLux GCXACUXO PI3AA QtttCU AQWAWCC~aaCXDGLJ GAC AACCCALJC ALAA GCAO2 A~WAWCCGaAAM -GGW GO Gi1GU UACAACCC AGAA Gafl99 A~r-AWAjAMUWXA GCAC

GGU

GUGUOG AAA GCMtU _CWChAAQEaAMWMCAGUGCOG AG2AC~uA AGAA GooXAG AWm~-~XU~WWMGI a

M

Mxt GAC U~oa3C ANCAAUC AMA GALWU A~rA GCx G~AUJUG GALWUA AM OUGCW.r GUAC GAU ULUC~ QUUUAC AGAA G~fAOJWGA-r ,MMGCGrAZ GUCAQJOG WLAA GOGOCU AGADjCU GC CA AGUACUUG AGAA GAULU AXWAAACX UCAAC GmCkB AG GL X3 .A A GN U XIA ACr-AGAGAAC A aX G a AU M a LA M M G CC OCAC IC GLAUAAl AAA GAGUGA AXW-WAAM mmBWAW=M CA GAC CCLAC fA A A AGMA A GU3~ A An o a AGGXJMAG AA GooMC CAMW"_AMjWXM Mrsm MNir GAC MUUIJc ~AN3A~AA 3AA~GACC~ICAu GUC Uautttu WCAULAA AGAA QXA 2AU ACV N A uiCr C NAWAAA GAMW ML~AAA _U AG 262

I

LI)uI I nL C4- (3 rII ALn Ln 263 Table 29: Human bcr/abl HCH Target Sequence =D NO.

IMTarget Sequence GAAAGOX CUJ CG33Mi S S S. S 9* S *5*5*5 ULA~r- Ur AAAA=U UUAAAGzZ a3u OAGxx 264 Table 30: Humann bcr-abl HIH Ribozyme Sequences Sequence ZlNO.

26 27 28 EmRibozyme sequence GGCUUCUUU COGAUGAC-CCCGAA.AGGCCG-AA AUCUGc ACtUGC^-GC-",G CUGiAUGAGGCCG-r3AAGCca ACGCrj C LTACU=GCG- C UWX GCAAAGCCG-AA AAGGCuucu GAAGGCUUUU CDAGAGIr.-L- C r AACUCUGC=U ACUGGCCGCUG GGCCGAAGGCC ACSCcUU= UACUG-GCCGCU CUAGGCGAA-,CA AAC-G=cuuuu 265 Table 31: RSV (1B) BE Target Sequence at Position EM Tazget Sequence at.

Position 14 18 1.9 54 57 77 94 97 101 110 1.13 118 122 134 3.37 148 149 152 1.54 157 161 165 176 188 208 209 210 214 215 221 226 239 241 242 251 261 265 267 274

GGCAAAU

AAtLh.AAU

AAUCAAU

AUCAfU

UAUGAU

ALCCGU

ccUGUC A AAUCAAU C AADUCAG U CAGCCAA C ACCAC A AU C A CACcCAM C A CA ACA U GUCACUU C ACUUGAG AGACCAU A AUACAU CCMA A ACAUCAC AMACA. C ACMLACC CAflCACU A ACCAGAG GAGACAXJ C AMA=CA ACAflCAL A ACACACA CACAAAU U MUMEC ACAAAUU

U

AAOUU A AAU. A UUAMW A AtMhD=r U ACUVGAU A GAMAAU C AADGCAU A

AMUM=

CUCCNAM

GAMULAU

AUGAAUG

GGAGAA

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 EMTaxget Sequence AAAAEAU A CUGAAUA ACtJGAAU A CAAC;AC-A ACAAAAU A LCC-CACIU UGfCACU U UCZcrAU GGCACtJU U CCCUAVG GCACUUU C CCr.mAUrC UUUCC"ty A UG-CC-AWJ LyGCCAAU A OUCAUCAk CCAAXIAU U CAUCAAIJ CAAV=u C AUCA-AU MUUCAU C AAUCAUG CAUCAA~U C AflGAflG GAUG=G U CUCAGAA AUGGG=r C ULUhGAAU GGGttTCU U AGAAUGC GGUCU A GAALTGCA AAUGrCA'U U GCAjU UCC-C-= U AAGCCUA UGGCMMj A AGCCUAC MAGCCU A CAAAGCIA AAAGCAU A CUCCCA GCAtXCU C CCALUX;J C-JCCCArj A AUL7CA CCAAj A UACAAGU At1AMU A CAAGOAU UACAAGU A UGA.UCUC GUAtMAZ C UCALAUCC AUGAUCrJ C AAUCCAU TUCUCALAU C CAUXAAATJ AAUCCAD A AAUUtUCA CAtUhAAU U UCAACAC AUAAIW U CAACACA UAAAMrMt C AACACAA ACACAAU A UUCACAC ACAAI3AU U CACAC;A CAAAUU C ACACAAU ACACAAU C UAAA~CA ACAAUCU A AAACAAC AACAAtj C U~t3CAU CAACUCT A UGCAU-AA M~.UGCAU A ACEIUAC GCCACAU U mtc~fuc CCAAUU U ACAIUC CACA=U A CAflVCCUT UU~CAU U CCUGGUC UUACAUU c cuGGLTCA UCCUGG C AACTUflG GCAACU A UGAAAflG UAAACU A U~kCA=; AAACah.U U ACACAAA AACOATJU A CACAAAG ACAAAGU A GGXAGcA AAGCACU A AAUAUfAA ACUAAM5 A MAAAA UAPLhLW A AAA.AAIJA AAAAAAU A MkCUG.AA 266 458 CAXMACU A MCU=c~ 460 MQCmu A cuccA= 463 CUD= C CAM=~7 467 ACtUCCU A GUC=A 470 CAG C CAGA=G 489 UAAAAU U AMU=L& 490 GAAAATJU A MGAM 492 AAAUWD A GAAMU 495 UMVD-GU A AVUaXAA 267 Table 32: RSV-(1B) HERibozyme Sequence nt. EM Ribazyme 3eqZUOce Position AUUGAUU CUGAUGALGCCGAAAGGccGAA 1AUMjGCC 14 COGAATU CUGAlAGCCGAAAGCCC. AfUU=li 18 UUG-tUG CUGAUGAGGCCGAAAGGCCA AflGAUU 19 GUUGC%-U COAG GGCCGcA AAU3GAUx 54 GGU= CUGAUGAGGCCGAA -C~A AtUCA= 57 UGGC- CUGAUGAGC-CCGAGGCM AUtUhrcM 77 UGUCU CGr-XGcGGoCCGc AUCMjjc 94 AAGUGAC CUGAUGAGGCCGAAAGQC

ACGGU=

97 CUCAAGU CLAGGCGAAGCA ACAc=c 101 UGGUCCC CUGAUGAGGCCGA AGcm c 1.10 AGUM COGAU~CAGCi C'r,-AA AUlG==c 113 UAUGU GccGAAAG cc ADUlG GGM CGUAGC Gcc

AGI

122 CVCOGGU Ct7GAtVGAGCC AI3UGAUG *134 GUGRMU COG-flGAOGGCCGAAAG rA AUMUC *137 UGUGUCtGAUGAGGCCGAAAGC,-CCALA AUGA=~r 148 GaMZuA CUGGXJGAGGCCG AGCCX A.rUG 149 AGAX CTJGAGAGCGXQAGGtCCCA

AAUU=~

150 AAUXM COUGAGGGAAGcCCGAA

AAAXUM

**152 UCAAGM~ COXfGAUCNCCAAGC

AUAA

154 UAUCAAG CMUGXGAGGCcG~CGAAAG- A AMIZ~hAA 157 AUWUAUC CUGAfGA3GCCGAAGGCG

AGMUW'

161 CAUGAWU CUGALr7GaGCCGAACTA AUC a 165 CUGAGCCGA )G-G

AUGAM

188 UUCAC CUGAtM~r-GCc

AGUUUUC

208 GXVJMMU Cr3GAUMV.GCc GAU fGrIGGC 209 G~aAUU CrUM33GAoG GAGGCA AAUGrj,- ::*210 AGQA.ADG CUMGAWGCc

AAAU

214 GACCAGG COMUMAGCCGQkG

AUUA

215 UGACCAG CUaUGAGGCGA JGCA

AAXWGUA

221 CADAGUU anGAGAGCCAAAGGCCGA ACCkGGA.

226 CAfUUCA COGALGAGGCCGAA AG MUGcAC 239 UGUGM~A C~mMAGCGAGCaL

AGUUUCA,

241 UUUGUGU CMUMV4CGAAAGCCG ALMM=u 242 CUUUGUG CUAGG-CAACC-

AAM=GU

251 UGCUUCC CUMWW CCXAGC~CaAA,

ACUUUC;U

261 UUM CUGAnGGAAGGcc .A AGTJGCUU? 265 UUUUUUA CUMGAXW4AGkUGGCM

ADUUAGFJ

267 WUUU CUAG AU7WXtXJ 274 UUCAtMM. CUGArAGCAA;CGAAULU 276 MU=CG~.GCGAGcALAAUUUU 283 268 295 AGG= CUGAUGAGGCCCAAA=-oCcAA

ADEMAGU

303 ATMGGGA CUCAUMAGGCCMGAA~an~cAA AGocc= 304 C.AMkGGG CUGAUAGGCCGAAAGOGAAj

AAGM=C

305 GCAZUDG CCMVJAGGCCGAACCG AAA=rC 309 AUUGCv= CUGAUGAGGCCG kGCC.-AA

AGGGAAA

317 %GA& CU~iArAGGCCAAAGcCCA AXXXGc= 319 AVUGAUG CUGAXGAGGCCMGAARG~cr.A Amw=j~ 320 GADfUGAU CLUAGGCCC-GG GA AAflX-r, 323 OAUMAUU COGAUMVX-Cc GC-.AA

AUU

327 CCA Cr GAUGG IAAG CGM AUG 337 0 -MLAG CCrU~kGCMAAW,-GA AC L 338 AU~CCk CW-IGA~GCXcAAG

AACCCAU

340 GCAIlUCO CCU3M4CMLGGCG

~AACCC

341 UGCA13UC CUGGA~AGcc AAGXcC 350 M.AU=c CUGAGGGccQ AGA Auc 356 UGGCU CUGAXrAGGMAGGCC

AUGCA

357 GW4 CUAGGCCAGCM AAUc 363 LAUo UU=GCAAXG

AGGMM

:372 AUGGGAG COGAMAGGC CG-Ccpj

AD==U

375 AUtUA=G CVUGAAGCCMcU~c;

AMU=

380 UGMMI CUGAGG-AAGG CCG1t A Ar MM **383 ACUUGtMk CUGAXXAGCC AAGC

ADU=G

385 GALCA CUGADGUAGrcC ACUU~~a 396 GGAfUMA Ct7GAXMGGCCMGGA

AUCAMC

*398 AUGA13U Cu GAGCAAG AA GU 402 AfUUATG CUG ATOX- c

AM

406 UGXAU CUAMnrI

AU=

4io UGACCM~-AGCCGAAMCGAAAUG~U 410 UGGUU CrJUM=GxAAGcc

CGAA

421 GUGAA CUGU~MAXI3cC )rfGGAM AAAUtUA~ 424 AUUG=U CCDGWG GCC kGGC

AAM=~T

:432 UGU MCVMVJMWGCCQG-L

AUG

434 GO CUG C EJGAGGCCGXIAGGccGAA

AMMMM

446 AUGCAACGUCGAG-L

AGUUGUU

448 UMfGCA CCGAXXAGc- jk

AGAG

454 GMU CUGAUMLGCMUAG~ r.A AUGAM 458 UGGAGUa COGAXJGAGGCCGAGCGAA

AGM

460 UkUG-GAG CMUAGcWGCA

AMGM

463 GACM= G UAGCGX CCA AGWLIuA 467 UCUGGAC CUGAAGGCCGAAGGCG;L AUflAG 470 CCWUCUG CVGAW1AGCCMUAWJj Acam 489 UCCL CUAMXGCMAGCjAAUOC 490 AUtMhCU COGAUMAGGCMXGCrA A A)UrUrC 492 AAAXWAC amCUGAW'AGCLr AIAUUr 495 UUAAU Ct 3 GUAGt AA GGO~CG

A=~M

269 Table 33: RSV (10) HEtarget Sequence Ut.

Posi±tion~ 16 i7 21 31 32 36 37 38.

42 46 so 51 67 68 71 76 81 87 88 92 93 100 101 104 105 120 1.25 128 129 135 143 145 151 155 156 159 163 164 Target Sequen~ce GGCAAAU A AGAZf=r UAAA U VGIALAAG AAGAAU U GAMAGU AfVUUGAL1 A AGMCCA GNIMAZG A CCACUtM UCCACLT U AAAfUUA ACCA=~ A AAUA COU.AAAU U MALC UtBAXWU U AACDCCC tUAAAUU A ACOCCCU UUUAACU C CCOUGU ACUCCCU U GGUtmGCA CCUUGG U AGAGAUG CUDG%-UU A GAG~AfGG CAGCAAU U CAUXUGAG AGCAAUU C AUtUGAGU AAWCAU U GAUVG AUUGAGU A UGALU"A GtfLrAU= A AAAGUEIA tULAAGU U AGAUIAC AAAAGUU A GAUk GUa~AMU U'ACLAAA DaGAflU A CAA)AVU ACAAAAD u uGuum&G CAXW~fU U GUUMAC AAUUUGU U UGACAI AUCDGUU U GACAALJG AUGXXGU A GCAfUU Gt7AGCAtI U GUUAAAJI GCAUAIG U AAAAAWA CAlUUGU A AAAAflAA t~aAAAU A ACAUC~u ACADUGCU A t1kcmwA AU%.=thL A CUGAIWhA UAU A AAULULAU GA~kAAU U AAUACAU AUAAJ A AX~hCAUU AAUUAAU A CIX3UUAA AAUACAU U UAACTAA AUACAJE U AXM;LAc 165 169 175 176 1.81 192 196 201 206 216 221 222 231 232 234 235 241 247 249 250 256 259 262 265 267 270 273 278 283 284 285 300 303 316 317 319 321 338 339 346 p ait.1 Target Sequience M~CA=U A ACUAAC- UUUAAC A AC,-C-,-M UkAcC=UT U GGC-,AA, ACGCUU U GCt3IWG UO iG,-cUr A ;kCZAG.U CAGCMAU AL CtAc.-A GAMUu A CAAUC,-a AUACAT3 C AAALJUGA AUCAAAU U GAAflGGC AUGG=A U GUGotM AUUGU=r U UGUG=A UUUGGUU U GtVGCAUG UGCArU= U AUL1ACX; GCAU~Gu AL uahcAAG ALMUUu U AC-AAGUA ULIAMU A CAAGtMG M~AW~ A GLUGAtkU MUAU A UUCGC--c G--GAM~U U U3CCCUAM MGAUAUUt U G,--LTAk UUGCCCEJ A AMAAA CCCTLAy A ALM~AT MUUA A AtM;M=~ tMAWAAU A UUGUAkt) AUAAUAU u GimGaAA AAUmflGu A GMAAAAU uuGaTi A AAAflCCA GLAAAU C CA.AUUUC AUCCAAJJ U UCACAAC UCCAAME U CACAACA CCAAt=r C ACAA.A VGCCAGU A CUACAAA CA=t~Ct A CAAAAE7G UGA=G U AUAUjG G-GAGGUU A TJUAUGG AGGU~x A UAUGGGA GO AVAUj A UGGGAAA AUGGAAU U AACACAU UC-GAAIJU A ACACAxJU AACAOAU U GCUCUCA 270 350 C UCA.AC 352 umGcmu c AAccaA 358 UCAACCU A AUGGU 364 U.AU C UCMMA 366 AXGUU A CCM~vVM 369 GUCMCU A GACAi 379 UtACAA U GUAAAU 387 GUAAAU U AAAflUCUT 388 UGAAAUU A AAUUCC 392 AUUAAMU U COCOCAM 393 UM.AUflU C UCCAAAA 395 AAP~flUC C C.AAAAM 405 AAAAACU A AGUAU 412 AAGGAY U CAACAAU 413 AGUflU C AACAAflG 427 GACJCCAU U AUGtAA 428 ACCAIU A M~AAU 430 CAAUUM A UGAAUCA 436 MLGA C AA~g= *.440 AAiUCPaD U AUCUGAA 441 AflCAAIJU A UCUGAAU 443 CADW~ C UGAAUAM 0 449 WUM U ALVOGA *450 LMAADU A CVUGA 453 AADUMM U G~flUM 458 CUGGA U 0GnCU 459 UUGADU U GAflCOM .463 AUUUGAID C UML.WCC 465 UUGflC U AIAnCCAU 466 UGA13CD A AfC~ 469 UCU~kAU C CAU 473 AAflC=f A AADUaM 477 CAMAAU U AUAUML 478 AULUMAA MDUA 480 AAAMX A AUCAML .483 UatUJ 1 D U AAk= 484 MUM A .ADUA 487 A&AJAAU AUCAA=U 489 uta.Amu C AACAC 494 )iflC.)CU A GCAAAUC 501 AGC 4 AAUL C AAGU 507 UCAA13U C ACMAcA 511 LTGUCCU A ACACA 519 AcCC= U AGUMAU 520 CCCADnU A GUTChAM 523 C~XuaAW U AAMUMA 524 AULVLGU A AU~tMAA

F

271 Table 34: RSV (10) ERRibozyme Sequence nt.

Position 16 17 21 31 32 36 37 38 42 46 51 67 68 71 76 81 87 88 92 93 100 101 104 105 120 .125 128 129 135 143 145 1=i 155 156 i59 1.63 164 i63 AAAUUCU CUGUGGGCCGAAAGGcCGAA ma~ut,- CO AUCA caVC-caAC,-CCCA

AUU

ACmUC cuAVAGCCGAGCCG AAflMCU TGGUAMr COGAUGAGGCCAAACC-..A AZCkA~u UAAAU GULI G GCCAAAG~r..

XAMU

UUAAAUU CMAU Gr-AG,--CA

AAGUGGU

GGAGMtM CCU AGCc A-_CGA ADUkA GGGAGUU CUAGGCCGXGCCA

AAUUA

AGGGA'GU CGUACCGAXUXCc

AAAUU

ACCAAGG CUAG~-CXAGCA

GMP

UCt6-kcc CvmG a c L AGWG.J= C==CU 1 ?7MeC-

ACCAJ

CVCAAM UQ ZGAGCCI-

AMG=

AcDkUA U AMWCCC.AG -CW ACDCAALu tIAACUUU CLAMGCGXAGCA AUA~c tGAUCU COGAGAGGCGX"GG,-L-.A; MCUUrJA AUU-tUGU CU AGG--AGG=iCA

AUCUAAC

AALUUUJG CUGAGAC--GM.. GG

AACM

UMAACA, CUVkCCGAAGC AfUU=~ GUCAAAc CAGCrGAA=G AAflUUUG AUUGUMCA U~,-CkAGCA

AZAAUU

CAUU UGA GCCGXAGGQCCA

AACAAADJ

ACAA7GC CMflGGCCUA AXoU= MULAC CUAGW-G"3CA AJGCUAc UkUUUUU CUA GGCGACWA

ACA=

L~aluUU CUG GGrr-cG1I=CGAA

AACAU

ACCA= CU MGCGAGCA

AUUUUUA

ATCAGtIA CUGAtUGCGCCGX"GCCAA AGM=~r UMUCAG CUGLAWCGcAJC, ALaGcAu AUMAUU CMUWZGCCAAGG CZ AUU AUGMOU ar-U~aUMC CAGGC AUUMUc AAUGA UG GCCGXG-CM AA3JU=A UMWMAU CMUAGCCAOC

AUAUE

UUAGUCA CUAGGCkCCGAA~

AUGUAUU

GUMMMU aMUAGC~AGCA

AAGM

CGtUAGU CL-Uu-3GAAACZCGCCAA AAA j HE Ribozyme Sequence 272 169 AAAG=G CMGAUGAGGCC 1 Ca 'Gr- A AGUaAA 175 UAGCC CUC;XAAC-CNAGccG ,-tccG -jGUM 176 CUCAGCC CUCArA GGGCCAA~,-CrGUA

,GC

181 ACUGCCT CUGAUGAGGLCc GUArcC,-AA AGCCAAA 192 UUGUAG C*Ut3G -AGCCGA GCCA AyCUG i96 UOGAfUM COUGA .GG-AAGcc C-cc UaAU= 201 UCAAflOU C 1ArACran a aGG'-CCAA

AUG

206 GCCADUC CUG AGCCGCcG CCC, AUUUGMU 216 CAAACAC CUGAAUAGGCCG AGLCCAA AUlCc_-Mu 221 JACA CUGArAGCCrAG,--CGAA

ACCA

222 CMC, Ct7GAru-G~CCGAAGAA AAc=XCA 231 UUGCAAM UGAAC-CAAGC- ACAuCCA 232 CUUGIA CUAAC-CCGAAG -GA AACA=~ 234 UCUUGU CVGAAGAGGCCG CCG AAU 235 ctahcuU CUGAXrAGGCCGA. AGCCCGA AAVC 241 AXCAC CUCOGACCcGAccG C-_CA ACUUGUA 247 GGGCAAA CCGAUGAGGCCG AG-CGA AUCALcuA 249 T.TGGGCA CUMGAGGCA AAGGCCGAA

AUIIC;LC

250 UMGGGC COG~ADGccGAA cc. AAmUCA .256 0U=~X CUGAUCAgGG GAGGt

AGGGCA

259 AM3 COGAUGAGGCCG AGCC AUM=,- 262 ACAAMO CUGCX GAGGCCMX. GCC AUMUM 265 AC~kCAA CUGA! GAGGCCGAGGC

AUMU~

267 UUACEMC CUGAUGAGGC.VUW AMW=~x 270 AUUCC COUGflAGCCGA;GCC

ACAAZU

273 UUVAUDlU CUGAGAGGCUa-GGA

ACUAA

*278 GAAADMU COGAMAGG-CG AGCCA AUUC 283 GOUGUGA CUGAGAGCC;AACrA AflUGGAU 284 UGOuGU CUGAXGAGC GCC AAUUGGA 285 UUGUUGU3 COAGG--GAA C AAAM-GG 300 uu PG= CuGAXJGAGGcC AGGcrAA ACUGccA .303 CAUUUG COGAUGAGGCCGAAAGUCCGAA AGEMh.CG 317 CAMflA

CG~G

317 VCCA= COGAUnaGG-CCAAAccrA AAccUi ::.321 UOUCCA CUGAUflAGCCtAACCA

AXI

338 ADGUU COGAGAGCCGAGG=G ArUc=u 346 UGAGAGC CUGAGAGG,-CG AG-CGAA

AUGUG=

350 AGGOUGA CUGAX7GAGGCC

AGCAAUG

352 UmGGUU CUGAflGAGGCCGAAGCCA

AGA~C-AA

358 AGACCAU CUGAUAGCCGAAAGCGAA

AGWMUA

364 UCUAGMA CGGAM~AGCr-t rtC

CA

366 CAflCG CUGAUGAGGCCG -GGCA

AGACCAU

369 UGUCAUJC CUGA GAGGCc CG ,CAA AGkG 379 AUUUCAC COGMUCGAflGAC,-cc A UUGUCA 387 AGAAUUU CUGAfGAGCGGGCC~GAA AUUrUCAC 388 GAGAATnU CUArGCG~XGCA AAUrUU 392 UUUGGAG LUAGAGCGCC~CcA

AUUUAAU

273 393 UUUUGGA CLTUGGCG

A

3 GCGAA AA3UUM.A 395 uuuuuuG CMAGtAGGCCGAaZGG.GM

AG.A.WUU

405 AAflCACU COAA--~AZCG

AGUUUD

412 AflUGOUG COGUAC, cAGGCA

AUCJ

413 CAfDUGDU CMUAGCCAGCC AAkACU 427 UOCMU CUGGflGAGGCCGAAG=CA AUtMMC 428 AflUCAM CD AtXGAGGCCG AAfl=G 430 DUaMMMCAUAGCAGC A UMMr 436 GAflU CUGAXAGGCCGAAXQA

AUUC

440 U AGAU CUGAUGAGGCC G'GCGAA

AUGGAUU

441 AfDCAGA COAGZCGAAGCA AAur6W 443 MADUc CVGUAG

AMW

449 UccaAGU CUGUGA~r-=UAGCC

AUCG

450 AUCCAG CUGAUGAGMG

AMUUCAG

453 CAAATJCC CMAGAMGAAGG- GAA AGULAu 458 AAGAlCA CDUGAGCCGAAGCA

AUCA

459 akAGAVC CGGALGAGGCcc

AUCA

463 GGWMt~A COGDG GOCCGAAGQ

AUCAAAU

465 AGGAU CVGAtXGAGWCMUcr CA AG C ,*466 MUX CUWWG AGOCA

AAGAM

46 U M MU4..G AA M G A U W.

473 MUWMD CMUGAGCCGX CCA AnVaGA 477 UAV CUGDGAA

AUUAM

478 UMAUM COGAXUGAGGCCGXUGCCGAA

AAUM

480 LmuaAAU CUAGGCGAGCG AflU'ju' 483 UGAM<fl Cr3GAxMAGCCMAAG AuarmmA :.*484 UUGAMD CMDGAAGC-CAGCC GA A ~frm 487 LUO Ct7GAfGAGGCCGAAC CA AUrM~ar 48 GCk. CUAMZCGAG=AU~am 494 GAUU=G CUGAUWM GC

AGM

501 UGACnUU CtXGAtxGAGCCGAAGGCGr A UiUGr 07 DUGUM aMUG GCCGAAGCA

ACAUG

**511 AUGu= CDMGXAGCCGXGCC.A Gj 519 AUMALV CMGAWCCGAAX

AUGG

520 MD.AC CWTF

AUG

523 ru *524 Uk=CMAGAGCCAGGCcr.A

ACM=

1" 274 Table 35: RSV HE Target Sequence nt.

Position E Target Sequence POSItion S S

S

S

9 21 23 24 32 37 65 66 70 .73 82 89 108 31i1 I113 3.17 120 123 126 127 146 150 154 166 167 169 170 173 186 189 192 196 1.97 205 206 209 213

GGCAAAU

GAVUGCU

GGCUCUU

GC-AAAGU

GOCAAGU

GAAUGAU

AMLCACU

CAAAGAU

AUCAACT

UCAACU

COGU

AGCAAAU3

ACACC-.U

AGGAGAU

AGAMGU

UGAMCU

MUCCT

CCM.AU

A CAAAA C UCGCAA V A~CrAAG A GCAAAGU C AAGUGM U GAAUGALU A CACUCAA C AACAAAG C AACt3CCU U CCGUCMU C UGUC C AUCCAGC C CAGCAAA A CACCAUC A GLUmnUGA, A VGALCU k CUCCA

:CLAUA

kAUMI I AJGAtTU

UGAZUG

217 21.8 220 229 231 235 236 254 260 263 27.7 279 284 299 305 315 318 326 327 346 347 355 356 361 370 371 383 384 389 395 401 406 408 415 418 431 449 453 460 472 474 E Target Sequence GUEww U AMtrym GOM~U= A muccZGCA AUGOUA. A UCC,-,r GCGZG C Mkac C-AUGC= A C-Gr-T3ACG LUCUAGGTJ U AGGAAZA CUAGGMr A GGIAGAG ACAC=A A AAAAXUAC UAAAAAU A Cr-cAG AA.A~CU C AGAGAVGM GC-GGGAU A UCAflGrM GGGALz C AfLMUAA -AIUCA=G A AAAGCAA AMl~C-= A GAGAA UAG-A=G A ACAACAC AA~CACAU C GUCAAGA ACACau C AAQc.U AAGACAU U AAraAA AGACAflU A AUGG=AAA AUGAAAx)U TJGAAGUG UC-AAAUU U GA.AG= GAAGJGU U AACALIU AAGUMU A ACAUG UtM.ACAU U GGMAcC GCAAGC U AACAACU CAAGCUU A ACAACUG CUGAAAU U CAAAUcA UGAAAuU3 C AAAUCAA UUCAAAUJ C AACAnUG UCAACAU U GAGAL7G UGAGAU A GAAUCUA AUA3AAXJ C UkAAAA AGAADC A GAAAAUC AGAAAAu C Ct~ACAAA AAAUCCU A CAAAAAA AAAM=C A AAAGAAA GAGAGrJ A GCUC-CAG GG-GU C CAG-AAUJA CCAGALAU A CAGGCAu CAtr=GA-v C UCCUGAU UGACUCU C Ct3AIMUUG CAflCAA A AGO= AA.M.AG U AYUGUGC ~AU A UGUGGCA =AMU U AUMADC GkAUGU A UUAAUCA, AUGUMU U AAUCACA L'UUAUJU A ANJCACAG tUThAU C ACAGAAG AGAUGCU A AucAM C~caAU. C AWAA=U UUfl A AAUcAC CAMLAAU U cAcUrG AtMh.AU c AcuGG,-u ACtOGGGU U AAI.Th=U CCGGU A AUaGGUA.

GG---VCAU A GGtatI=t AAX~hGG A UGUA 275 4, 0* .480 491 494 496 497 501 503 511 '512 515 51B 522 526 527 544 549 531 552 563 564 573 576 581 584 603 604 613 614 617 629 640 641 643 652 653 663 670 671 672 674 680 681 682 683 686 687 690 691 692 UCCUGAU U GUGGGMU GCAflrAU A AXMflThf UGAflAAD A UaW AGUNM~U U AGUA ULM A UGM AWaM A UAGCAGC U&DG=D A GCAG=A GCGCU U 1IGMAM3 CAGC~rU A GaAMLA CAMG A AUACML UAML A ACMULNU AAU)ACU A AAl ACTMAA U AGCAGCA CWLA=D A GCAGCA GACAGAV C VUC= AUC~rU C U~kCAGC CUGUCU U ACAGCCG UGGuC~ A CAGCCG;U CGUGU U AGGAGAG CMAVU A GGAGAGC GAIGAGCU A AUPAflGU AGCLD. A AUGUCCLT AULWGU C CMLA 6 AUUCU A AAAAAtI GAAAG U ACAAAGG AAAMU A CAAAGGC AAAGGCLT u Acmkccc AAGGCUU A CUACCCA GCUMLCU A CCCAAGG AGGACAD A GCCAACA AACAGCU U CUUAA ACAGCU C MOfGAAG .AGCUDCU A UAA= GXA= U tGAAAAA AAGGU U GAAAAAC AAAAAU C CCCACUU CCCOCU U MUG CCCACD U ADAGAUG CCACrUU A. MMUMr ACUUMO A GAUflJUU MG= U UUU AGAUGUU U DuGOcA GAUG=U U UGULUcAX AUCUUUU U GUUCAUU UUUUUGU U CAUUUU UUUUUU C AUUU=G UGUUCAIJ U T3UGGUTAU GUUCTU U DCG=UA UUCAUU U GGJJJG 696 698 706 708 709 711 726 731 740 741 742 743 751 754 755 756 766 787 788 800 802 803 831 81.5 816 822 824 825 829 830 840 866 869 875 876 877 883 895 913 914 916 921 923 925 943 946 947 949 950 OuuuUG A UAGCACA UGMWA A GCACAAU GCACAAU c UUCACC ACAAUCT U CUACCAG CAAUCDU C t!hC,-CA AUCUUCU A CCAGAGC; VGGCAGU A GAGLTUGA GMTu--.u-U U GAGGGA AAGGGAU U UUUGCAG.

AGGGAtUU U UUGCAGG GGGAUUU U UGCAGGAl GGAUuur U GCAGGAU GCAGGAU U GJUUAUUG GGAUU U UAtJGAAU GAUQ=u U AX3GAAfG AUUUUU A UGAA=G AAtUGCCU A UGGUGCA GUGAUGU U ACGGUGG UGWMWU A CGUGG GGGGAGU C Ut~hGCAA GGAGUCU U AGCAAAA GAGUCU A GC.AAAAT GCAAAAU C AGUtUAA.

AAUCAGU U AAAAAUJA AUCAGUU A AAAAMIU UAAA A UtM1UU AAAAJAX U AUGULVAG AAAMTJU A t3GUAW AtJMflW U AGGACAU UCZU= A OC,:AUG ACAt7CCU A GUGUGCAL AACAAGU U GUUGAGG AAGUGU U GAGGU=r UUGAGGU Uj MuAA UGAG=U U AUGAI GAGG.UUU A UGAALVLU UaWCGAU A UGCCCAA C.AAAAAU U GC.GUGG-U GCAGGAU U CU~CAXJ CAGGAUU C UACCAMl GGAMUC A CCALMMJ COA=cU A M~fUGAA ACCAI;AU A UUGAACA CAUAUAU U GAACAAC AAAGCAU C AUtMhVU GCAUCAtj U ALTIJCU CAUCAUU A UUAUC-UU UCAUUAU U AUCUOUG CAUT-U A UCUUUGA

S.

S

p 276 952 UCUTAU C UUUGACU 954 AiflA=lU U GACUCA 955 UfLVUU U GACUCAA 960 UUUM=C C AAVUU= 964 ACOCA)D U UCCA 965 CUCAAWU U CCCCU 966 ~~UCAAU C CUCCUU 969 AUUU C AcCrUc 973 CCUCACU U CUCCAU 974 CrCA= C CAGUG 976 CACU=u C CAU 983 CCGU A GCU 986 GU A UaAGCk 988 G~kYJ U AGGAU 989 MkGMU A GCGCAAG 1007 CUGC-CU A GGCAL19A 1013 UAGGCAU A AUflGG sose1024 GGAGGU A CAGAGU 2032 CAAG A CACA *:.*1044 GAGGAAD C AAGAVCU .9 150 UCA C UAAX 052 AAGMflC A UAUGAUG *1054 GAUCIJ A UGUC 1072 AAGGAU A UG~A 1085 MAACAL C MAAAA 1103 GUUA U AAa~c 1.104 UGGAVU A A~AA l108 A~akACLY A CArT 1115ACAG= A CUkGACU ii2.18 GUGACU A GACUUGA, 1123 C~kGAC U GACAGCAL .*119 AAGu-ACU A GAGGC= .146 AGGGC7 A tJCMAMCM 1148 AGGCA C AAAC 1155 CAA).CAD C A~cua;L *16 SUAC T AATJCCAA 1161 UCAGCUU A AflCCA 164 G--UUAU C cAAAG 1.173 AAAGD A AGAU 1181 AUAM A GAC-UU 11.87 LMGAGC U UGG~J 1188 AGAGCUU U GAUA 1,193 UUUGAGU U AAMfLAA 1.1.94 UUGAGUU A AMLAAAA 277 Table 36: RSV EH Bibozyme Sequence nt Position HE Rtibozyme Sequen~ce 73 82 89 108 117 120 123 126 127 146 150 154 1.55 166 167 i69 170 173 186 189 192 i96 197 205 206 209 213 AUItJUD CUMAGGCCGAAAG3-cccGA A==Cc UUGCLUA CUAGGC--AC-CA

AG-_CAC

CUUUGCU COAGGCCGAGoC,-cr- AGAGCrA ACOUIUC CUGAW-AGCCGAAAG-CC-A~

AAGGC

UCAACUU COGAUGA GGM~AAGGC- ACrUrtJ= AUTCAUrjC CGAUGtCCGAAAGGC.C- AM7rGAC UUGAGUG COGAVAG GGCCGAA AUCAM-c CUUUGU3U CUGAUGAGGccrA AG'CC,%A

AC.,,GMU

AGAA=U CUAG CCG-AAAGGCC,A ,,UcLMM AUGACAG CUAGCCGAAAG-GccA AGUUGAn GAUGACA arGA ccAGCAA

AAGUUGA

GCT3GGAU COAGGCGAAGCA

ACAAA

UUUGCtJG CUAUACCCGh 1 1 ,CGAA AUGACAG GAUGGG CUAUAGCCGAAGGCCC,; AUUUGIt7 UCCGUMCGWa-GXUGcaC;-CG;

=GG'U

U~aAUAC CUG GGCCGGCC4r-.A

ADCCCU

GMUCAA CGUGGCCGACA ACMMC~j GAGUAUCOAGCCGAA-CGcA AU;C=u ULV4GAG CUA AGCCGAA.-CGC

AUCAAUA

tUhAUUAG COAU GCCGAAC,-C; AG=CArJ UaUMAU CUAGCCGAACCG~

AGGAM

ACWUAC.UGALMGCCGAACCGA AMMtG CACAUJCA CUGAUGt-"AAGG CGA AAUUAGG ACUmkUU CUGAaA'c

AUU

CAAU COAUAGCCG ACC AAJ GCCACAU CUGAXXGAGGCCG QkGCCCA

ACUU

UGCCACA CUGAUGAWGCCGAA ,'CGAA AACCUAU GAUL1AAU CUGA AGGCCAG.CCcGA ACM=Cc UGAUEDLA CUGAGGCCGC<CL

AACAUTGC

UGGAUJU CUGAUGAGCAAAGG CCG;L AGAAAU CUGOGAU C GUGAGG-C kGC'-.AA AAkACA CUUCUGU CGUAGCCGAACCA

AMMTALIA

UEIAMAU CUGAUGG;-AAGGCCGL

AGAU

AAUUEXhII CUGAIGAGGCCGAAGA AflUA~CA GUGaAAUU ~CUGUAGCGACCG;L AUGAXrJ CCCAGUG CUGAT GAGGCCAAGCC;L AUtUMUG ACCCAGY CUGATGAGGCCGAAr.CCGAA

AAMUU=

AC=tAUU CUGAGCCGAGCpG;L

ACCCAGU

TGACCUAU CUGUGAGGCCGAACG,-CGAA AAcCAG ACAUACC CUfLrGAG cCc A AUGALACC MLGLC CtUGAGCCGAAAGXCGA

A;CCLTATUEI

217 CGMAGCCAAUoM 278M~ 218 U C X3A xAUAG~C<=AAGGccGAA XaUAC 220 CAUCCCA CUMGLAGGccGAAAG=ccGAA AAMIUh., 229 MACCML COGAUGAGOCCGAAAGcCCGAA AtCAM 231 CC~kACC COGAUGAGGCCGAAAGCCGAL AGCAXICc 235 UCOUCCt7 COGAUMGGCCAAAGGCCGAA AAC-ca;G 236 cucuuccCOUGAGGCCGAAGCCCGAA

AACCLUL

254 GMlUMU CUGAIMGOGAAGOA

AUG

260 CUCUGAG CWUMtXGCGcGGCG;

AUUM

2-63 CUUW CUGAU G=CQAGCCA AGMnU= 277 M GAX uwGmc~x==.A AflCcc 279 UOUACAU COGI~AUWGCCMLZGC AM==cc 284 uuocuU CUGAKAWGc AC GA 299 uahcvDC UGAU~GGCCG AAC.-C2AA

ACUCC;AU

305 GUGUG CVUGA~GGCOGAAG GA ACAU= 315 UCCUWAC COAAGCMAOCA

AU

318 AUU= CUGAL7GAGCcaAGCC

AC

326 UUCCAUU MWZCtxA AAccr U AfU= 327 UUUCAU CMU'C=UGCM AAMrj *346 CA3UC; CUMC-

U

347 ACACUUC Ct7GAMXAGCCMUGGG

AAUUCA

C.U MUWC

ACACUUC

:9356 CCAAflGU CUGXAGCCGAAG

AACA=U

361 GCOMCC CtXMfMWZcCCMGCCA

AUUA

370 AGOOUGU ca 3AGGCc AAOCW

AGCU=G

371 CkG= ct~zAc WCG

AC

383 UADU=UG UMWC ACCr

AUUU=A

384 UOAUMu COGAUWGCAAGCCA

AAUUUCA

389 CAAUGMu CMAMAtxz C=X-UAGzrGA

AUUAA

:.o395 CkU=Cu COGAUM% C~CGXZG rA AUGUEJGA 0 9 9-UWA=LI CUGAIJCGGCCMUAGCCCGAA AflCUCAA 406 UOUUCA COGAMAGGC'CGAXJ CCWA AUCM GAUUUUC CU&MGCGUG~ AGAUU=r 415 UUA CGAXXAGCCM QGGCCMU AUUUU= *418 uuumUU arU =CCXGGActGJ)Cr

AGGUU

431 UOUcuOU CUMOGCGTAGCrA A~a= 449 CUGGC arAGtXAGCC GAGGCOGA

ACUU

~453 M==UCOMMGGUGGGC AGCr C 60AUGCCU CVGAXGAGGCCGAAA GCCGAA AUUCUGG 480 AIXCCAC CMUALGAGCCAAGCCA;

AUCG~.

491 ALIhAU CUGADMGJ GAGCCGM

AUAC

494 UkCADAA CMUGXAGGCGAAGC=,A AU JC 497 c~Uc arUAGCAAGCCA

AAU

501 GCUGCUA COGUGflGC-CCGAAr CG ACAMAU 503 AUGCUG CMUGG=CAGM

AMWAYJ

511 LWJUAM3 CUGAGAGCCG~k CCA AUGCUGC 279 512 uaku= CUGAUGAGGCCAAAGGMC.AA AAtUG-CM 515 akom CUGAUGG-4AGGc CC-GAA. AC~kNM 518 A~uuaGU COGADMGGAAG CG A Ava;Lcm ON 522 GCMLWU CCGAUMXGCCCAAG~.

AGOU.WU

526 VGCWUCU CUAGGCGAAGCA

AUUUAGU

527 CUGCMGC CUGAAGG-CAcc -CCG AAfUrA 544 AAGACCA COGA! aGAGCCGAAGCC

AUCOGUC

549 GC-UG3A COMAG GCCG CCrA ACCAGAU 551 CGGCUGU CUGUGAGCGAAGC-

AGACA

552 ACGCG C GAUAGGCCGAAC-CA

AAGACCA

563 CUcCU COGAGGccGAGCCC.

AUCACGG

564 GC-UCMcC CUGAUXAGGCCGA CCC. AJCACCG 573 ACAUWt CUGAUG-CC.GAAAGGCCC.AA~

AGCUCUC

576 AGGACA CUG~A-Gr-CGAAMGGCCQA AUkC 581 UUUUG CUGALGAGGCC--AAGGCC-U ACAUrJ= 584 CflUUUU CUGAUGAGC-CCGAAAGGCCGAA

AGGACAU

603 CCUUOGU COUGAGAGGcc~xaGGcrAA ACUucC 604 GCCUUM UCAMG CAAGCA

AACGU

613 GGG~kGU COGADCAGGCCMG CCGAA AGCCUUU 614 UG CUGADXkGGCCGAAAGX

AAGCCU

617 CCUCGG CMUACC

AGUAC

640 UGUC=~ Cr3GAIGAG~C-crCCGCCA AUW=~r 64UUMWh fCUGAMWG CkGCGAA

AGCUU

.643 CAUUCU CU--GCXAX--- CG.uA AGAGCUG *652 UUUUUCA CUGAUGW=Gr CCGAA

GAC

653 GUUUUC COGAixGAGCCGAAGCC

AACACOU

663 AAGOUG CUGA3MAC<GG AACc

AU

*670 AUCtUTM CUGAMG GCCAGGCCGAA

AGUG=G

671 CAUC~kU CMUGAG-CMUAGCGAA, AAGOGr.

***672 ACAUCLIA CUGALIGAGGCCGAAAGGCCGAA

AAAG=U

674 AAACAXJC CUGAUAGGCCMGXCC 'AJ AUAAGT 680 GAACAAA CUVAGCGAGCG

ACAUCA

681 TJGAACAA COGxAU GCCGAA)GCCA

AACAUCU

682 AUGAACA CMAVAGGCGAAA-GL

CAYACAX.C

683 AADGAAC COUGAMAGGCCGAXGC-CGA

AAAACAU

686 CAAAAUG CUrGAGGCCMAAGC

ACAAAA

::.687 CCAAAAD CUCUG3ACC

AAAAA

*690 ALMCCAAu kUGAC-CC J~kGCC GA AUA 691 tUAkcm CtXIGAZCMCCGAffA

AA

T

WAAC

692 CtACC CUGAXX GCCGAArQCQC7GAA

AGAA.

696 UGUGCDL CL7GAMW4~CCAAGGCGA

ACCAMA.

698 ALIUGUGC aCuGAAcGccaArYGGCQA

AXJACCAA

706 G-GUAGAA CUMVJMlGA-GAAAGGCCGA

AUUGUW

708 CUGCGth CUGAUMWZCCGAA CCGAA

AGAUUGU

709 UCUGGU CUGAUGAcGC I fV,, AAGAUUG 711 CCUC= GGA A~rAAW-

AXW;J

726 UCAACUC CUGAfGAGCCGAtGCAA

ACUGCCA

731 t7CCCUrJC CU~vUCCG AAACC CGAA ACUCUAC 280 740 CUGC.AA CUGA~r(GAAGGC C A GAA~ ADCCCr 741 CCUGCAA CUGPGAGCCCCAAGCCG.A

AA

742 VCCOG= CDGAXUGAGGCCGAA scaA AAAX3CCZ 743 AUCCUGC CUGAGAGCCGAAA~CCA&

AAAACC,

751 C1fAAMAC CUGAX.GAG~CCG a-ZGcCGA AUCCUc 754 AUUCAI CUMGAGGCCGAAGCM ACAAU-c 755 CAUCCAU COGAflGAGCXA GCCG AACAMC 756 GCCflUCA CUGAXGAGG-GAGCCG AA AAAAA 766 UGCACCA CUArNGCAAGCv

AGCAUU

787 CCACCGJ CUGAGAGGCCGaAGC,-,

ACADCAC

788 CCCACCG COG~lr-AGGCX:G AGc~a

AAC=

800 UtUCA CUCAEAGGCCrjGGCr; ACr;Ccc 802 UUUU CUGAUGAGG~C MkGGG;

AGACUCC

803 AXIUUUGC CrJ AtGAGGCCAAG-Cr.U

AAGCUC

83.1 UUtMhLU CUGALUAGCCGAAAGGCCAA

AUUU=~

81.5 DuUU CUGAIUAGGCCGA AGCCGAA X-GU 816 AMfU= CUGAUGAGGCCGAAGCCC-

AX-JGAD

822 AAAIA COGAX AGGCCGAGcrA Amu 824 COAGGCGAG=A AaDUUU 825 AcA aWMAG=C aMXM AAMAflUU 829 AU= CCGAUMMt CAAGG C=AA ACUMUU 840 VGCAAC CUG~LrAGGCCCAAXCA AG=flG 866 C-COCAAC CTJG~AUGCCGXZG Acuumu *869 AAACCUC CUAGG-

ACAACD=

875 AUUOAC Gcc

ACCUCAA

876 DUA COUGAGGCGcXU.GCG A~rUC;.

877 AfLMUCA. CUGAW-AGGCCCAAC4G; AAACCt3C *883 UUGGCA Cr3GnAUAGGCC GAA-CcGAA ADUCpAz ::895 ACCACCC CUGUGAGCCUAC)CGMA AnUUU= ***913 AUGGLUhG C~AGGCGAr-C

ADACCGC

914 UAUGM LATYI wMCCU 916 =A~DG C GGGCAZZCGL

AGAAWCC

923 U AIA CUG~vXAaGCCG )UGC1

AUGU

925 LGU~UC COGAUGAGGCCMGA CCC. AMR 943 OAEAU COGAflGAG~C-C

AUC

::.946

AGAW

1 .AX CMUGAGAGGCCGAAGCA

UAG

9 4 7A A G A D A c G A a iAt U G3 r U v 950 UCAAAGA CUGAflGAGCCG UM ALhU 952 AGUCAAM CUAMC GA

AUX

054 UGAGUCA CUGrflGAGGCCGAXrL

AGAM

955 UUGAGUJC CUGAU.GAGGCCGAAQQCCGQA AAGAtTA 960 GGAAM UU GOCAGCGA AG AA 964 GUGAGGA. CUAGGCGAG4CA

AUGAG

965 AGUGAGG CUGAM3AGGCGXUGCCG

AAUAG

966 AAUAG CUG;LtJAGGCCGAAGCCA AAA~rA 969 GAGAAGU CUGALUGAGCAGc CCA AGGAAAU 281 973 ACUGGAG CUGAUAGGCCGAAAGCC--'.AA

AGUMAGG

974 CACUGGA CUGAGAGCCGXUir;-CGA

AAUAG

976 MCACrJG CUGAUGAGG-c GCG AAGCC. AGAGU 983 CUA~C CUGAACAGCCGA-A

ACXCUG

986 UGCCUAA CUAGGccAA CA AC~kc= 988 AUUGCCU CUGALTvUGG Gcc C3AA A~gLCt;, 989 CAUUGCC CUGAW-AG-CcGAAAGCGM AA~cr= 1007 UrCAUGc CUAGCCGAC-CA

NGGCCAG,

1013 CJCCCAU CUGAI GAGGCCG AACCGAA ALTc,-C 1024 ACCUCUG CUGAIr-AGG-CC--UGCGAA

ACCUC-

1032 CvCrr= COAGGCGAA,,C. Ac==c 1044 AGAflCUU CUGAGAMG-C.-AAAGC- AMr, 1050 UCAUM CUCLGACG-CCGAAC,-C- AVC-, i052 Cm=A Ct3 AUG=GXAAG L AGAUMU 1054 UG-CAIJCA CUGAUGAGGCGAA WCG AMAUC 1072 UUCAGCA CUGALUGAGCCGAAGGCGAA

AUG,--CU

1085 UUUCUVU CUGAGAGGCGAGCG

AGUU

1103 UUG COGADMIGGCCGXACCGAA

M

1104 CGMAG CVGAUGGGCCGAGCGA

AAU~CAM

U108 GA=CUG XA4GCCrkAG C. AGu 1115 AGUCtAG CUGAUGAGCZCCA C ACAC~UG U118 UCAAGUC CUGAUGAGr-G AGGCG

AGA=

2.123 UCUCC CUGAWlAGGCCGAGGC.

AGC=G

11139 LMGCCUC CUGAUGACCC GCCGA A G AGUCUrJ *1146 UGUUCGA CUGAXGAC7GCCGXUG,CGAA

AGCCUC

1148 GAUG=U cu-UAG-CAAGrA AaG=CT 1135 ULMAGCU CUGAfGr-AAGGCCGAc.A

AULUG

1160 UUGGAU CUGAGAGGCCGAAGCCG

AGCUMAU

1164 UCUUUG CUUGAGAGGCCGAAAGCCG,' Ar~aXGC 1173 ACAUCAU CGX o cGoCCG AUC=u 1181 AAGCUC CUGAGAGc GGCCGACA AcAr=~ *1187 tkC(CA CUGAUAGGCCGXUGCGA AGLl 1188 UtMACUC CVAAGCAAGCA

AAGCUCU

uuutmh.U aUGAGGCCGAA G=A ACUCAA 1194 ULULUUAU ClArAG'CAAGCA

ACUCA

nt. HP *ioz m SequenceS S S Sus tr t Position0 a"GU AGA GUUU ACAAAAAAGUGGUCUA.GU AAAAC GAU

***AUCACAG*

CUGGAC AGAA GUCUUUA ACCAGAGAAACACA(Y.JtUrUACAUU1AccuaGUA AAAGACIJ GU) GUCACUG 472 CAGGCUjCC AGAA GGACUA ACCAGAGAAACACACMG-Unn.~1A~?A~IAUAUc GAl) GGAGCCUrJ Table 88: [RSV lP ilibozyme/Substrnte Sequence nt. Hairpin Bioym equence substrate Position Rbzm 476 AUCCCAC7A AGAA GGAGAG ACCAGAGAAACACA(TIJJQJIAAACCUGUA CUCUCCU GAU UGGGU 540 AAGACCAG AGAA GUCCCC ACAAAAAAGUGGUCUACGU GGGGACA GAU cuGGUCUU 554 CIJAALJCAC AGAA GUAAGA ACAAAAAAM)GGUCUACGU UCULJACA GCC GUGAIJUAG 636 UUCAIJAGA AGAA GUUGGC ACAAAAAAGUUGAAUMGu GCCAACh GCU UCUAUGAA 998 CCUAGGCC AGMA GCAUUG ACAAAAAAGUU(UcUACGU CAhUGCrJ GCU GGCCUAGG 1156 UUGGAtJUA AGAA GAMMU ACAAAAAAG~UGAAUMOU AMCAucA GCU UAAUCCAAI 284 Table 39:' Large-Scale Synthesis Sequence A9T

A

9

T

(GGU)

3

GGT

(GG U) 3

GGT

C

9

T

CgT

U

9

T

U

9 A (36-mer) A (36-mer) A (36-mer) A (36-mer) A (36-mer) Activator [Added/Final] (min) T [0.5010.331 S [0.25/0.17] T [0.50/0.33] S [0.25/0.17] T (0.50/0.331 S (0.2510.17] 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] Amidite [Added/Final] (min) [0.1/0.02] (0.1/0.02] (0.110.02] (0.1/0.02] (0.1/0.02] [0.1/0.02] (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 15 M 15 M 15 M 15

M

89 78 81 97 Time* Full Length Product 15/1iSm 15/15 M 15/15 mn 15/15 m 10/5

M

*Where two coupling times are indicated the first .~?IIa refers to RNA e-" 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 Au cu 3 wnere lowerecase represents 2 -O-methylnucleotides.

285 Table 40: Base Deprotection Sequence iBu(GGU) 4 iPrP(GGU) 4

C.

C

Deprotection Reagent NH4OH/EtOH

MA

AMA

MA

AMA

NH4OH/EtOH

MA

AMA

MA

AMA

NH

4 OHIEtOH

MA

AMA

MA

AMA

NH4OH/EtOH Time (min) 16 h 10 m 10 m 10 m 10 m 4 h 10 m 10 m 10 m 10 m T C 55 65 65 55 55 65 65 65 55 55 Full Length Product 62.5 62.7 74.8 75.0 77.2 44.8 65.9 59.8 61.3 60.1 cgu 4 h 10 m loin 10 m loin 4 h 75.2 79.1 77.1 79.8 75.5 22.7 28.9 A (36-mer) 286 Table 41: 2'-C-Alkylsilyl Deprotection

S.

S

S

S

.SSSS*

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

TBAF

1.4 M HF 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 1.5 h Time (min) T C Full Length Product 20 84.5 65 81.0 20 60.9 65 67.8 20 86.2 65 86.1 20 84.8 65 84.5 20 25.2 65 30.6 TBAM F 245 h 20 29.7 B is UCU CCA UCU GAU GAG GCC GAA AGG CCG AAA AUC CCU

V

Table 42 NMJL Data for UC Dimers containuing Phosphorothioate Linkage Synthesis It 3524 3525 3530 3526 3578 3529 Typ e ri bo ribo ribo ribo ribo ribo Delivery 2 x 3s 2 x3 s 2 x3 s I x 5s I x 15 s 1x 6s Eq.

10.4 10.4 10.4 08.6 08.6 08.6 Wait 2 x 100 s 2 x75 s 2 x75 s 1 x 300 s 1 x 250 s I x 150 s

ASE(%

95.9 92.6 92.1 100.0 100.0 73.7 9. 9 9.

*99 9 9..

9 9 99 9.

9* 9. 9.

99 9 9 99 9 9 999 9 9 9 9 9 9 9 9* 9 *999 9 9 9 9 Tabl e 4 3: NMR Data for l 5 -iner RNA containing Phosphoroth-ioate Linkages Syn tesis 0 3581 3663 'lyrpe ribo ribo Delivery I x5S 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 '-0-Me 2 -0-Me 2 '-O-Me Ix orS 2 x4 s I x5S 08.6 13.8 08.6 I x 250s 2 x 300 1 x 300 s 99.7 99.8 99.8 289 Table 44. Kinetics of Self-Processing In Vitro determ efroaeon-inea lestu s ki Ka min' Sye1 :5 Thpesu tio deries tecu extet o st o ribozymssgine relfevaeo S ongoing transcription (Long tUblenbeck, 1994 Proc. Nat]. Acad. Sci. TSA 91, S. 6977) as a function of time and the unimolecular rate constant for cleavage Each value of k represents the average range) of values determined from two experiments.

290 Table Entry Modification tiM t/ Activity Stability t (ts) 1 U4& U7=U 1 0.1 1 2 U4 U7 2'-OMe-U 4 260 650 3 U4 2=CH2-U 6.5 120 180 4 U7 2-=CH 2 -U 8 280 350

U

4 U7 2'--H 2 -U 9.5 120 130 *gos 6 U4 2'=CF 2 -U 5 320 640 7 U7 2'=CF 2 -U 4 220 640 8 U4 U7 =2'=CF2U 20 550 S*3 2 0 160 *9 U4 2'-F-U 4 320 800 10 U7 2'-F-U 8 400 500 11 U4 U7 2'.p.U 4 300 750 12 U4 2-C-Allyl-U 3 >500 >1700 13 U7 2'-C-AII(I-U 3 2200 14 3 220730 14 U4 U72= 2'-CAIIyI-U 3 120 400 *fee 15 U4 2'-araF-U 5 >500 >1000 16 U7 2'-araF-j 4 350 875 17

U

4 U7=2u-arp..U 15 500 330 18 U4 2'-NH 2 -U 10 500 500 19 U7 2Z-NH 2 -U 5 500 1000

U

4 U7 2'NH 2 -U 2 300 1500 21 U4 dU 6 100 170 22 U4 U7 dU 4 240 600

Claims (23)

1. An enzymatic nucleic acid molecule which cleaves respiratory syncytial virus (RSV) mRNA or RSV genomic RNA in a gene region selected from the group consisting of 1C, 1B, and N gene regions.

2. The enzymatic nucleic acid molecule of claim 1, the binding arms of which comprise sequences complementary to any one of the sequences in Tables 31, 33, 37 and 38.

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

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

The enzymatic nucleic acid molecule of claim 1 or claim 2, comprising between 12 and 100 bases complementary to said mRNA or genomic RNA. 15

6. The enzymatic nucleic molecule of claim 5 comprising between 14 and oelo 24 bases complementary to said mRNA or genomic RNA.

7. The enzymatic nucleic acid molecule of claim 1 or claim 2, comprising a between 5 and 23 bases complementary to said mRNA or genomic RNA. go*:

8. The enzymatic nucleic acid molecule of claim 7 comprising between 20 and 18 bases complementary to said mRNA or genomic RNA.

9. An enzymatic nucleic acid molecule consisting essentially ofa sequence selected from the group of sequences in Tables 32, 34 and 36-38.

10. A mammalian cell comprising an enzymatic nucleic acid molecule of S0:any one of claims 1-9. *00°0 25

11. The cell of claim 10, wherein said cell is a human cell.

12. An expression vector including nucleic acid encoding at least one enzymatic nucleic acid molecule of any one of claims 1-9 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

13. A mammalian cell including an expression vector claim 12.

14. The cell of claim 13, wherein said cell is a human cell.

A method for treatment of a pathological condition related to the level of RSV by administering to a patient an enzymatic nucleic acid molecule of any one of claims 1-9.

16. A method for treatment of a pathological condition related to the level of RSV by administering to a patient an expression vector of claim 12.

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

18. An enzymatic nucleic acid molecule according to any one of claims 1-9 when used for the treatment of a pathological condition related to the level of RSV in a patient.

19. An expression vector according to claim 12 when used for the treatment of a pathological condition related to the level of RSV in a patient.

An enzymatic nucleic acid or an expression vector when used according o0 to claim 18 or claim 19, wherein said patient is a human.

21. Use of an enzymatic nucleic acid molecule according to any one of claims 1-9 for the manufacture of a medicament for the treatment of a pathological condition related to the level of RSV in a patient.

22. Use of an expression vector according to claim 12 for the manufacture is5 of a medicament for the treatment of a pathological condition related to the level of RSV in a patient.

23. Use according to claim 21 or claim 22 wherein said patient is a human. SS 040" Dated 9 November, 2001 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person S" SPRUSON FERGUSON 0 [R:\LIBZ]05575.doc:lam i:"i i