AU4876099A - Method and reagent for inhibiting the expression of disease related genes - Google Patents

Description

S F Ref: 349267D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD

PATENT

ORIGINAL

r r s cc r Name an Adrs Name and Address of Applicant: Actual Inventor(s): Ribozyme Pharmaceuticals, Inc.

2950 Wilderness Place Boulder Colorado 80301 UNITED STATES OF AMERICA Dan T Stinchcomb, Bharat Chowrira, Anthony Direnzo, Kenneth G Draper, Lech W Dudycz, Susan Grimm, Alexander Karpeisky, Kevin Kisich, Jasenka Matulic-Adamic, James A McSwiggen, Anil Modak, Pamela Pavco, Leonid Beigelman, Seam M Sullivan, David Sweedler, James D Thompson, Danuta Tracz, Nassim Usman, Francine E Wincott, Tod Woolf Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method and Reagent for Inhibiting the Expression of Disease Related Genes Address for Service: Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 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-a, 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.

O The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. The advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.

In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ration of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to 15 those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site. With their catalytic activity and increased site specificity, ribozymes represent more potent and safe therapeutic molecules than antisense oligonucleotides.

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

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

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

These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the O 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, 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 Watsonl 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 Biochemisty, 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cel, 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.

The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target I CAM-1, IL-5, reLA, TNF-ac, p210 bcr-abl or RSV proteins) encoding mRNA such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required., Alternatively, 20 the ribozymes can be expressed from vectors that are delivered to specific cells. By "vectors" is meant any nucleic acid and/or viral-based technique used to deliver a desired nucleic acid.

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention small enzymatic nucleic acid motifs of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structrure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters Scanion, K.J. et al., 1991, Proc.

Natl. Acad. Sci,. USA, 88, 10591-5; Kashani-Sabet, et al.,1992, Antisense Res. Dev., 2, 3-15; Dropoulic, et al., 1992, J. Virol, 66, 1432- 41; Weerasinghe, et al., 191, J. Virol, 65, 5531-4; Ojwang, et al., 1992, Proc. Natl. Acad. Sci.. USA, 89 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver, et al., 1990 Science, 247, 1222- 1225). Those skilled in the art would realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from the 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.

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

Chem., 269, 25856).

By "inhibit" is meant that the activity or level of ICAM-1,Rel A, TNF-a, p210bcr-abl or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

Such ribozymes are useful for the prevention of the diseases and 15 conditions discussed above, and any other diseases or conditions that are related to the level of ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV protein or activity in a cell or tissue. By "related" is meant that the inhibition of ICAM-1, IL-5, Rel A, TNF-a, p210bcr-abl or RSV mRNA translation, and thus reduction in the level of, ICAM-1, IL-5, Rel A, TNF-a, p210 bc r-abl or 20 RSV proteins will relieve to some extent the symptoms of the disease or condition.

Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.

The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 2,3,6-9, 11, 13, 15-23, 27, 28, 31, 33, 34, 36 and 37.

Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

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.

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

Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huiller et al., 1992 EMBO J. 11,4411-8; Lisziewicz et al., 1993 Proc. Natl.

Acad. Sci. 90 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors

I

(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 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 2 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 O 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 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.

S* 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.

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 65°C 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.

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 is UCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG.

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

S. 20 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 usingTBDMS 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.

-M M

M

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

Figs. 23 25 are diagrammatic representations of hammerhead, hairpin or hepatitis delta virus ribozyme containing self-processing RNA transcript. Solid arrows indicate self-processing sites. Boxes indicate the sites of nucleotide substitution. Solid lines are drawn to show the binding sites of primers used in a primer-extension assay. Lower case letters indicate vector sequence present in the RNA when transcribed from a Hindlll-linearized plasmid. (23) HH Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hammerhead ribozyme. The structure of the hammerhead ribozyme is based on phylogenetic and mutational analysis (reviewed by Symons, 1992 supra).

The trans ribozyme domain extends from nucleotide 1 through 49. After 3'end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides 1. 5 (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 20 hammerhead ribozyme (Ruffner et al., 1990 Biochemistry 29, 10695) and generates the HH(mutant) construct. (24) HP Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hairpin ribozyme. The structure of the hairpin ribozyme is based on phylogenetic and mutational analysis (Berzal-Herranz et al., 1993 EMBO. J 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 coworkers (Been et al., 1992 Biochemistry 31, 11843). The trans-ribozyme domain extends from nucleotides 1 through 48. After 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (AA at positions 49 to 50) at its 3' end. The 3' cis-acting HDV ribozyme is comprised of nucleotides 50 through 114. Roman numerals I, 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 0* enzyme. Transcripts from H templates contain four non-ribozyme 15 nucleotides at the 3' end. N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from N templates contain 220 nonribozyme nucleotides at the 3' end. R, Plasmid templates linearized with Rcal restriction enzyme. Transcripts from R templates contain 450 nonribozyme nucleotides at the 3' end.

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 S29, 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 MgCl2 or with DEPC-treated water prior to being hybridized with 5' end-labeled primers. Cellular lanes contain total cellular RNA from cells transfected with one of the four self-processing constructs. Cellular RNA are probed for ribozyme expression using a sequence specific primerextension assay. Solid arrows indicate the location of primer extension bands corresponding to Full-Length RNA and 3' Cleavage Products.

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

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

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

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

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 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. Northem 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 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 9*999 presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCI 2 RNAs were renatured by cooling the reaction mixture to 370C 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.

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 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 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).

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

Figs. 65a-b is a schematic representation of a method of this invention to synthesize a full length hairpin ribozyme. No splint strand is required for ligation but rather the two fragments hybridize together at helix 4 prior to ligation. The only prerequisite is that the 3' fragment is phosphorylated at its 5' end and that the 3' end of the 5' fragment have a hydroxyl group. The hairpin ribozyme is targeted against site J. H1 and H2 are intermolecular 10 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 *substrate complex as described in the art (Berzal-Herranz et al., 1993 EMBO. J.12, 2567). The ribozyme has been assembled from two fragments (bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent intramolecular helix formation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme). Left panel (HP-K1) indicates 4 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).

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 pl DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1pM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCI2) by :heating to 900C for 2 min. and slow cooling to 370C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37 0 C. Aliquots of 5 pl 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 20 imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale,

CA).

Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary structure of the hairpin ribozyme*substrate 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 proposed secondary structure of the hairpin ribozymesubstrate 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 200C and at 260C.

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

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

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

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

Other abbreviations are standard in the art.

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

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

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

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

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

Figs. 81a-j shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention. R groups are alkyl 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'-Cmethylene and 2'-C-difluoromethylene cytidine.

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

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

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

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

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

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

Ribozymes of this invention block to some extent ICAM-1, IL-5, rel A, TNF-x, p210 b c r ab l, or RSV genes expression and can be used to treat 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 ,p210bcr a bl, 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 O 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 S: incorporated by reference herein. Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from cDNA clones. Labeled RNA *i .o 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, y-interferon, tumor necrosis factor-a, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface (Sringer et. al supra; Dustin et al., supra; and Rothlein et al., 1988 J.

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

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 3095-3099; Dustin and Springer, 1988 J. Cell Bio. 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 supra). The importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J.

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

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

The sequence of human, rat and mouse ICAM-1 mRNA can be screened for accessible sites using a compter folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables 2, 3, and 6-9. (All sequences 10 are 5' to 3' in the tables) While rat, mouse and human sequences can be Sscreened and ribozymes thereafter designed, the human targeted sequences are of most utility.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4 8 and 10. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is 'altered to affect activity and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

o 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 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 Sseveral 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 9 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.

A Phase I clinical trial of a monoclonal anti-ICAM-1 antibody showed significant O 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).

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

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

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

0* 10 Soluble ICAM levels are elevated in Kawasaki disease patients; particularl high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992Arthritis Rheum 35, 672-7; Tsuji, 1992 Arerugi41, 1507-14).

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

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

O 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 10 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 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 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.

S. 10 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. ED. Med. 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. Exp. Med. 172, 1525-8). The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma: Several studies have shown a direct correlation between the number 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.

v..o 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.

4* 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.

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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 (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 supa). 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.

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

Example 3: NF-KB Ribozymes that cleave rel A mRNA represent a novel therapeutic S. 20 approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor o necrosis factor-a (TNF-cx) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion r molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-xB. 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-c( 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 34 proteins) in response to a variety of stimuli phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-KB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-KB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-KB (encoded by the nf-KB2 or nf-KB1 genes, respectively) are generated from the precursors NF-KB1 (p105) or NF-KB2 (p100). The p65 subunit of NF-KB (now termed Rel A is encoded by the rel A locus.

The roles of each specific transcription-activating complex now are being elucidated in cells Perkins, et al., 1992 Proc. Natl Acad. Sci USA 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)).

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Conversely, heterodimers of NF-KB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-iB1/RelA (p50/p65) heterodimers have little effect Liu, N.D. Perkins, R.M. Schmid, G.J.

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

A number of specific inhibitors of NF-KB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-KB binding sites, and over expression of the natural inhibitor MAD-3 (an IkB family member). These agents have been used to show that NF-KB is required for induction of a number of 0 molecules involved in inflammation, as described below.

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

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

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

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

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

RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1, ICAM- 1, IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-re/A ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

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

10 -Rheumatoid arthritis (RA).

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

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

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

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

*Restenosis.

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

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

*Transplantation.

O 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 S-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.

0 *Gene Therapy.

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

Thus, ribozymes of the present invention that cleave 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 O 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-c mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.

Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated leukocytes, that is a potent mediator of inflammatory reactions. Injection of 10 TNFa into experimental animals can simulate the symptoms of systemic d and local inflammatory diseases such as septic shock or rheumatoid arthritis.

*0f> TNF-a was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, 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., ,east 1985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-a have been cloned and found to be related to TNF-B (Shakhov et al., 1990 20 J. Exp. Med. 171, 35-47). Both TNF-a and TNF-B 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 10 expression"- Kisich, Doctoral Dissertation, 1993 University of California, Davis] listing various TNFa targeted ribozymes.

Ribozymes of this invention block to some extent TNF-a expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of septic shock and rheumatoid arthritis. Ribozyme cleavage of TNF-a

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mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

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

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

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

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

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

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

RNA ribozymes and/or genes encoding them will be locally delivered to macrophages by intraperitoneal injection. After a period of ribozyme uptake, the peritoneal macrophages are harvested and induced ex vivo with LPS. The ribozymes that significantly reduce TNF-a secretion are selected. The TNF-a can also be induced after ribozyme treatment with fixed Streptococcus in the peritoneal cavity instead of ex vivo. In this fashion the ability of TNF-a ribozymes to block TNF-a secretion in a 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 -70oC. 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 from septic shock averages 50%, due primarily to multiple organ failure and disseminated vascular coagulation. Septic shock, with an incidence of 10 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-inflammatory cytokines which are not normally detected in the serum, :such as TNF-a, interleukin-1B (IL-18), y-interferon (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 su.ra). 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-113, IL-6, IL-8, PgE 2 acute phase proteins, and TxA 2 in the serum of experimental animals (de Boer et al., 1992 suDra). 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- S(Abney et al., 1991 Imm. Rev. 119, 105-123), some or all of which may contribute to the pathological course of the disease.

Cells cultured from RA joints spontaneously secrete all of the proinflammatory cytokines detected in vivo. Addition of antisera against TNF-(a to these cultures has been shown to reduce IL-la/1 production by these cells to undetectable levels (Abney et al., 1991 Supra). Thus, TNF-a may directly induce the production of other cytokines in the RA joint. Addition of the anti-inflammatory cytokine, TGF-3, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical specimens clearly demonstrate the production of TNF-a, IL-la/3, 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.

Exp. Immunol. 89, 244-250).

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

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

Psoriasis Psoriasis is an inflammatory disorder of the skin characterized by keratinocyte hyperproliferation and immune cell infiltrate (Kupper, 1990 J.

Clin. Invest. 86, 1783-1789). It is a fairly common condition, affecting of the population. The disorder ranges in severity from mild, with small flaky patches of skin, to severe, involving inflammation of the entire epidermis. The cellular infiltrate of psoriasis includes T-lymphocytes, neutrophils, macrophages, and dermal dendrocytes. The majority of Tlymphocytes are activated CD4+ cells of the TH-l 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, i 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 1 15 Semin. Dermatol. 10, 217).

The role of cytokines in the pathogenesis of psoriasis has been Sinvestigated. 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 Dermatol Sci. 6, 127-33) have proposed the following model for the initiation and maintenance of the psoriatic plaque: Tissue damage induces the wound healing response in the skin.

Keratinocytes secrete IL-la, IL-11, IL-6, IL-8, TNF-a. These factors activate the endothelium of dermal capillaries, recruiting PMNs, macrophages, and T-cells into the wound site.

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

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

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

Maintenance of the psoriatic plaque requires continued expression of all of these processes, but attractive points of therapeutic intervention are TNF-a expression by the dermal dendrocyte to maintain activated 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. Alternative 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 keratinocytes to a differentiated state and restoration of normal skin development. Immunosuppressive drugs such as cyclosporine are also in the advanced stages of clinical trials. Due to the nonspecific mechanism of action of corticosteroids, retinoids and immunosuppressives, these treatments exhibit severe side effects and should not be used for extended periods of time unless the condition is life-threatening or disabling. There is a need for a less toxic, effective therapeutic agent in psoriatic patients.

HIV and AIDS The human immunodeficiency virus (HIV) causes several 10 fundamental changes in the human immune system from the time of infection until the development of full-blown acquired immunodeficiency syndrome (AIDS). These changes include a shift in the ratio of CD4+ to CD8+ T-cells, sustained elevation of IL-4 levels, episodic elevation of TNFa and TNF-8 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 nd/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.

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

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

Children with AIDS who were given zidovudine had reduced levels of TNFa compared to those not given zidovudine (Cremoni et al., 1993 AIDS 7, 128). This correlation lends support to the hypothesis that reduced viral 10 replication is physiologically linked to TNF-a levels. Furthermore, recently 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.

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 b.e. 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 Supra).

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

*Gene Therapy.

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

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

Example 5: p210bcr-abl Chronic myelogenous leukemia exhibits a characteristic disease 15 course, presenting initially as a chronic granulocytic hyperplasia, and invariably evolving into an acute leukemia which is caused by the clonal expansion of a cell with a less differentiated phenotype the blast crisis stage of the disease). CML is an unstable disease which ultimately progresses to a terminal stage which resembles acute leukemia. This lethal disease affects approximately 16,000 patients a year.

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

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

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

The b3-a2 and b2-a2 fusion mRNAs encode 210 kd bcr-abl fusion proteins which exhibit oncogenic activity (Daley et al., 1990 Science 247, 824; Heisterkamp et al., 1990 Nature 344, 251). The importance of the bcrabl fusion protein (p210b c r -ab l in the evolution and maintenance of the t10 leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p210 b c r a b l expression. These inhibitory molecules have been shown to inhibit the in 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, 20 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 to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to extemal stimuli.

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

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

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

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

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

The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of CML. Such enzymatic RNA molecules can be delivered exogenously or endogenously to afflicted cells. In the preferred hammerhead motif the small size (less 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 t al., 1992 asura) is an in vitr transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in 20 length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex ivo treatments. This limits the utility of this approach. In this invention, an alternative approach uses smaller 'ribozyme motifs and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, as well as complementary binding of the ribozyme to the mRNA target.

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

Ribozymes of this invention block to some extent p21 0 bcr-abI 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 S. 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 S* provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 30 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

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

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

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

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

RSV is a member of the virus family paramyxoviridae and is classified under the genus Pneumovirus (for a review see Mcintosh 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 18 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 O 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.

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

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract illness during infancy and childhood (Hall, supra) and as such is associated with an estimated 90,000 hospitalizations and 4500 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., 1992 J. Vet. Med. Sci. 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in subsequent natural infections with RSV (Connors et al., 1992 J. Virol. 66, 7444).

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 :i 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.

O While all ten RSV encoded proteins (1C, 1B, N, P, M, SH, 22K, F, G, and L) are essential for viral life cycle and are all potential targets for ribozyme cleavage, certain proteins (mRNAs) are more favorable for ribozyme targeting than the others. For example RSV encoded proteins 1C, 1B, SH and 22K are not found in other members of the family paramyxoviridae and appear to be unique to RSV. In contrast the ectodomain of the G protein and the signal sequence of the F protein show significant sequence divergence at the nucleotide level among various RSV sub-groups (Johnson et 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 G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Hairpin ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography and are resuspended in water.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 32, 34, 36, 37 and 38. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of 10 hammerhead ribozymes listed in Tables 32 and 34(5'-GGCCGAAAGGCCcan be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 37 and 38 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 32, 34, 36, 37 and 38 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

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

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

Optimizing Ribozyme Activity Ribozyme activity can be optimized as described by Draper et al., PCT W093/23569. The details will not be repeated here, but include altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see Eckstein et al., International Publication No.

WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci.

17, 334; Usman et al., International Publication No. WO 93/15187; and 10 Rossi et al., International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic

RNA

molecules. All these publications are hereby incorporated by reference herein.), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Sullivan, et al., PCT W094/02595, incorporated by reference herein, describes the general methods for delivery of enzymatic RNA molecules Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.

Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al., suDra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol RNA polymerase

II

(pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.

Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529- 37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet 10 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 O using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules).

Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA associated with ICAM-1, relA, TNF-oa, p210, bcr-abl or RSV related condition. Such RNA is detected 15 by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second 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=, p210bcr-ab l 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 O 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 protecting groups be used, that the appropriate conditions for the removal of the exocyclic amino protecting groups and the alkylsilyl protecting groups on the 2'-hydroxyl are used, and that the correct work-up and purification procedure of the resulting ribozyme be used.

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

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

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

Applicant has also determined that significant (se bles 394) improvements in the yield of desired full length product (FLP) can be obtained by: 0. 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 S: 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 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 Samino protecting groups (vs 4-20 h 55-65 °C using NH40H/EtOH or NH3/EtOH, vide supra). Other alkylamines, e.g. ethylamine, propylamine, butylamine etc. may also be used.

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

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

The use of anion-exchange resins to purify and/or analyze the 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, the invention features an improved method for the coupling of 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 20 enzymatic activity and be purified without loss of that activity. While specific examples are given herein, those in the art will recognize that equivalent chemical reactions can be performed with the alternative chemicals noted above, which can be optimized and selected by routine experimentation.

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

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

In the case of the 2-step purification procedure, in which the first step 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 pm, preferably 5 gim.

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.

69 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 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 pL of 0.1 M 32.5 gmol) of 20 phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 .L of 0.25 M 100 gmol) 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 gmol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of a 0.25 M 1125 imol) 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 S. 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 OC. Applicant has determined that the use of Smethylamine or NH40H/methylamine for 10-15 min 55-65 OC 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)

The polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or 9 (AMA) 55-65 °C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 4 mL glass screw top vial. NH 4 OH 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 a. 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 Ribozvme Deprotction of2'-Hydroxy Akli Protecting Groups Using Anhydrous TEAoHF To remove the alkylsilyl protecting groups, the ammonia-deprotected oligoribonucleotide was resuspended in 250 pL of 1.4 M anhydrous

HF

solution (1.5 mL N-methylpyrrolidine, 750 gL TEA and 1.0 mL TEA.3HF) and heated to 65 °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.

ExamDle 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 NaC1O 4 at a rate of 0.85 mM/void volume for a Pharmacia Mono Q® anion-exchange column or 100-150 mM NaCIO 4 at a rate of 1.7 mM/void volume for a Dionex NucleoPac® anion-exchange column was used to elute the RNA.

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

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

The eluent was diluted four fold in sterile

H

2 0 to lower the salt concentration and applied to a Pharmacia Mono Q® 16/10 column.

A

gradient from 10-185 mM sodium perchlorate was run over 4 column volumes to elute shorter sequences, the full length product was then eluted in a gradient from 185-214 mM sodium perchlorate in 30 column volumes.

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

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 S. 15 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 C0 3 H/25% CH 3

CN,

buffer A 20 mM NH 4

CO

3 H/10%

CH

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

Example 11 Ribozyme Activity Assay Purified 5'-end labeled RNA substrates (15-25-mers) and purified end labeled ribozymes (-36-mers) were both heated to 95 quenched on ice and equilibrated at 37 o C, separately. Ribozyme stock solutions were 1 jaM, 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 MgCI 2 Reactions were initiated by mixing substrate and ribozyme solutions at t 0. Aliquots of gL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each aliquot was quenched in formamide loading buffer and loaded onto a denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

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

In an attempt to minimize the time required for deprotection and to simplify 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 °C for 15 min and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA.3HF reagent.

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 FELq 13 are tested for their ability to cleave RNA. As shown in the figure 14, ribozymes that are deprotected using one pot protocol have catalytic activity comparable to ribozymes that are deprotected using a two pot protocol.

Example 12a Improved protocol for the synthesis of phosDhorothioate containing RNA and ribozymes using 5-S-Alkvltetrazoles as Activatina 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 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 previous synthesis.

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

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

RNA synthesis is conducted on a 394 (ABI) synthesizer using a modified 2.5 gmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 min coupling step for 2 '-O-methylated RNA. A 6.5-fold excess (162.5 gL of 0.1 M 32.5 gmol) of phosphoramidite M M M- 0 and a 40-fold excess of S--ethyl tetrazole (400 gL of 0.25 M 100 gmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle.

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

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

Average sulfurization efficiency (ASE) is determined using the formula: ASE (PS/Total)l/n-1 15 where, PS integrated 3 1 P 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.

SUsing 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.

ExamDle 13: Protocol for the synthesis of 2 '-N-phtalimidonucleoside phosphoramidite The 2'-amino group of a 2 '-deoxy-2'-amino nucleoside is normally protected with N-( 9 -flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supral 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 derivatives 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.

9 A convenient "one-pot" procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCl/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 p1 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-Cl 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 s:e demonstrated coupling efficiency in 97-98% range. RNA cleavage activity .V6. of ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/or U7 positions (see Figure wherein the 2'-amino positions were either protected with Fmoc or Pht, was identical. Additionally, complete deprotection of 2 '-deoxy-2'-amino-Uridine was confirmed by basecomposition analysis. The coupling efficiency of phosphoramidite 17 was not effected over prolonged storage (1-2 months) at low temperatures.

Protecting 2' Position with a SEM Group e 20 There follows a method using the 2 '-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base deprotection. At the same time, this group should also be readily removed when desired. To that end the t-butyldimethylsilyl group has been efficacious (Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.

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

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

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

There follows a method for synthesis of RNA by protecting the 2'position of a nucleotide during RNA synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group. The method can involve use of standard RNA synthesis conditions as discussed below, or any other equivalent steps. Those in the art are familiar with such steps. The 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., SNature 1990, 344, 565-568, Pieken et al., Science 1991, 253, 314-317, Usman,N.; Cedergren,R.J. Trends in Biochem. Sci. 1992, 17, 334-339, Usman et al., PCT W093/15187, and Sproat,B. European Patent Application 92110298.4.

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

In another aspect, the invention features a method for removal of an SEM group from a nucleoside molecule or an oligonucleotide. The method involves contacting the molecule or oligonucleotide with boron trifluoride etherate

(BF

3 .OEt 2 under SEM removing conditions, in acetonitrile.

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

Otherwise RNA synthesis can be performed by standard methodology.

Referring to Fiqure 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'- 10 protected moiety is converted into a phosphoramidite by standard procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.

Referring to igure 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-((trimethysiletoxmehy-5'- 0- Dimethoxytrityl Uridine (2) Referring to Fiqure 19, 5'-O-dimethoxytrityl uridine 1 (1.0 g, 1.83 mmol) in CH 3 CN (18 mL) was added dibutyltin oxide (1.0 g, 4.03 mmol) and TBAF (1 M, 2.38 mL, 2.38 mmol). The mixture was stirred for 2 h at RT (about 20-25 0 C) at which time (trimethylsilyl)ethoxymethyl chloride

(SEM-

Cl) (487 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.

Examle 15: ynthesis of 2 trimeth ll ethoxymeth I Uridine 4 Nucleoside 2 was detritylated following standard methods, as shown in Egur_9 Example 16: Synthesis of 2 '-O-((trimethvlsilvl)ethoxymethyl)5'.3'-OAcetyi 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 C12) gave 10 20 mg of SEM deprotected nucleoside 6.

Example 18: Synthesis of 2 '-O-((trimethylsiyl)ethoxymethyl)3' 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-((trimethylsilv)ethoxvmethy).5'-O- Dimethoxvtrityl Uridine 3'-(2-Cvanoethvl N. N-diisoproDvlDhosphoramidite) Nucleoside 3 was phosphitylated following standard methods, as shown in Figure 19.

Example 20: Synthesis of RNA Using 2'-Q-SEM Protection Referring to Fiqure 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 mol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 pL of 0.1 M 32.5 pmol) of phosphoramidite and a 80-fold excess of tetrazole (400 gL of 0.5 M 200 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, were 98-99%.

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

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

Referring to Figure 21, the homopolymer was base deprotected with

NH

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

Il. Vectors Expressing Ribozymes There follows a method for expression of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In 15 general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a decreased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful 20 for scaling up production of a ribozyme, which may be either modified or unmodified, in situ or in vitro. Such vector systems can be used to express a desired ribozyme in a specific cell, or can be used in an in vitro system to allow productiuon of large amounts of a desired riboqyne, The vectors of this invention allow a higher yield synthesis of a ribozyme in the form of an RNA transcript which is cleaved in situ or in vitro before or after transcript isolation.

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

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

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

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

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

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

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

83 Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA expression system. The desired second ribozyme may be any desired type of ribozyme, such as a hammerhead, hairpin hepatitis delta virus (HDV) or other catalytic center, and can include group I and group II introns, as discussed above. The first ribozyme is chosen to cleave the encoded cleavage sequence, and may also be any desired ribozyme, for example, a Tetrahymena derived ribozyme, which may, for example, include an imbedded restriction endonuclease site in the center of a self- 10 recognition sequence to aid in vector construction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector.

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

Thus, in one aspect the invention features a vector which includes a 20 first nucleic acid sequence (encoding a first ribozyme having intramolecular cleaving activity), and a second nucleic acid sequence S* (encoding a second ribozyme having intermolecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA transcript encoded by the vector. The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have intermolecular cleaving activity.

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

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

In a second aspect, the invention features a method for formation of a 20 ribozyme expression vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a singlestranded DNA encoding a second ribozyme, as discussed above. The :single-stranded DNA is then allowed to anneal to form a partial duplex DNA which can be filled in by a treatment with an appropriate enzyme, such as a DNA polymerase in the presence of dNTPs, to form a duplex DNA which can then be ligated to the vector. Large vectors resulting from this method can then be selected to insure that a high copy number of the single-stranded DNA encoding the second ribozyme is incorporated into the vector.

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

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

While structural and functional differences exist among the three ribozyme motifs, they self-process efficiently in vivo. All three ribozyme motifs selfprocess to 87-95% completion in the absence of 3' flanking sequences. In vitro, the self-processing constructs described in this invention are significantly more active than those reported by Taira et al., 1990 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.

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 Sribozyme, for example, by hybridization to the region between the flanking arms and the enzymatic RNA. This hybridization will select against the Sshort flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybridization can be accomplished in the presence of a chaotropic agent to prevent nuclease degradation. The oligonucleotides on the matrix can be modified to minimize nuclease activity, for example, by provision of 2 '-O-methyl RNA oligonucleotides Such modifications of the oligonucleotide attached to the column matrix will allow the multiple use of the column with minimal oligo degradation. Many such modifications are known in the art, but a chemically stable nonreducible modification is preferred. For example, phosphorothioate modifications can also be used.

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

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

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

DNA

is placed.

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

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

The vectors include plant viroids, plant viruses which contain single or double-stranded RNA genomes and animal viruses which contain

RNA

genomes, such as the picornaviruses, myxoviruses, paramyxoviruses, hepatitis A virus, reovirus and retroviruses. In many instances cited, use of these viral vectors also results in tissue specific delivery of the ribozymes.

Example 21: Design of self-orocessing 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, 0 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.

S.0 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 0 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 in the art.

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

The cis-cleaving hammerhead ribozyme used in the HH cassette is based on the design of Grosshans and Cech, 1991 supra. As shown in Figure 23, the 3' binding arm of the trans-acting ribozyme is included in the required base-pairing interactions of the cis-cleaving ribozyme to form stem I. Two extra nucleotides, UC, were included at the end of the 3' binding arm to form the self-processing hammerhead ribozyme site (Ruffner et al., 10 1990 spra) 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 15 the two portions and thus allows a minimal five nucleotides to remain at the end of the released trans-acting ribozyme following self-processing. Two variants of HP were designed: HP(GU) and HP(GC). The HP(GU) was constructed with a G-U wobble base pair in helix 2 (A52G substitution; Figure 24). This slight destabilization of helix 2 was intended to improve self-processing activity by promoting product release and preventing the reverse reaction (Berzal-Herranz et al., 1992 Genes Dev. 6, 129; Chowrira et al., 1993 Biochemistry 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing interactiohs 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) 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 (Figure 2).

To prepare DNA inserts that encode self-processing ribozyme cassettes, partially overlapping top- and bottom-strand oligonucleotides (60-90 nucleotides) were designed to include sequences for the T7 promoter, the trans-acting ribozyme, the cis-cleaving ribozyme and appropriate restriction sites for use in cloning (see Fi. 26). The singlestrand portions of annealed oligonucleotides were converted to double- 10 strands using Sequenase® Biochemicals). Insert DNA was ligated S. into EcoR1/Hindlll-digested pucl8 and transformed into E. coli strain S. 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 Su.ra). 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 gM each NTP and 0.5 to 1 jg of linearized plasmid template. The concentration of MgCI 2 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 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 Fi. 26). The resulting transcripts have either non-ribozyme nucleotides at the 3' end (Hindlll-digested template), 220 nucleotides (Ndel digested templates) or 454 nucleotides of downstream sequence (Rcal digested template).

As shown in Fioure 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 supra) have been changed in the HH(mutant) core sequence (see Figure 23) and .so this transcript is unable to undergo self-processing (Fia.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 15 trans-ribozyme (iE.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.

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 pM CTP; 40 pCi [a- 3 2 P]CTP; 12 mM MgCl2; 10 mM DTT. The transcription/self-processing reaction was initiated by the addition of T7 RNA polymerase (15 U/pl). Aliquots of 5 1I 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) (1-e 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 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 suldra; Chowrira Burke, 1991 sulra). 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 sura using a HH that has a different stem I and stem Ill.

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 sup... The reaction mixture was treated with 15 units of 20 ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 p.

DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1M) and internally labeled 622 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris*HCI pH 7.5 and 10 mM MgCl2) by heating to 90°C for 2 min. and slow cooling to 37°C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C. Aliquots of 5 pj were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager® (Molecular Dynamics, Sunnyvale,

CA).

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

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

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

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

Purified cellular RNA (3 gg/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90 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/gI; BRL) in a buffer containing SmM Tris-HCI pH 8.3; 10 mM DTT; 75 mM KCI; 1 mM MgCI 2 1 mM each dNTP. The extension reaction was carried out at 42 0 C for 10 min. The reaction was terminated by adding an equal volume of 2x formamide gel loading buffer and freezing on crushed dry ice. The samples were resolved on a 10% polyacrylamide sequencing gel. The primer sequences are as follows: HH primer, 5'-CTCCAGTTTCGAGCTTT-3'; HDV primer, AAGTAGCCCAGGTCGGACC-3'; HP primer ACCAGGTAATATACCACAAC-3'.

As shown in 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 (Figuare2 "In Vitro +MgCI 2 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 (Eigure_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 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 S. 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 29in vitro "-MgCI 2 control). The predominant RNA detected in all cases corresponds to the primer extension product of full-length precursor RNAs. If, instead, the purified S* RNA containing the full-length precursor is incubated in 10 mM MgCI 2 prior to the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (u in vitro "+MgCl2" control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-processing, even though the RNA in question is inherently able to undergo self-cleavage.

In a second experiment to demonstrate lack of self-processing during work up, internally-labeled precursor RNAs were prepared and added to non-transfected OST7-1 lysates as in the previous control. The internallylabeled precursor RNAs were carried through the RNA purification and primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-processing. By this analysis, the 96 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.

S" 10 In addition, those in the art will recognize that Applicant provides *0 *6 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 S: 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 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).

r 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.

Thus, in one preferred embodiment the invention features a pol III promoter system a type 2 system) used to synthesize a chimeric

RNA

molecule which includes tRNA sequences and a desired RNA a tRNA-based molecule).

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

By "terminus" is meant the terminal bases of an RNA molecule, ending in a 3' hydroxyl or 5' phosphate or 5' cap moiety. By "region" is meant a stretch of bases 5' or 3' from the terminus that are involved in base-paired interactions. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from 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 2 0 nucleotides from the 3' terminus. For example, in the S35 construct described in the present invention (Fig. 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 V techniques generally known in the art. Generally, it is preferred to have the region within 100 bases of the 3' terminus.

By "tRNA molecule" is meant a type 2 pol III 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 .0 20 which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal poJ 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 S99 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 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 15 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 enzymatically to cut the target RNA.

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

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

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

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

In other aspects, the invention includes vectors encoding

RNA

molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this

RNA.

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

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 Ill 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 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- 6344). However, pol III based expression cassettes are theoretically more "attractive for use in expressing antiviral RNAs for the following reasons.

S0Pol 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 *20 III genes encode tRNAs and other functional RNAs necessary for basic "housekeeping" functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types. Finally, pol III transcripts from a given gene accumulate to much greater levels in cells relative to pol II genes.

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

Pol III System The following is just one non-limiting example of the invention. A pol III based genetic element derived from a human tRNAimet gene and termed A3-5 (Eig 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. Bio. 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.

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"; ig. 34). On average, ribozymes were found to accumulate to less than 100 copies per cell in the S. 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 Scontaining enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (Ef.34). One of these modified gene units, termed gave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original A3-5/HHI vector system.

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

The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and antagonist RNAs. Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral 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 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 (Fig35). To try and improve accumulation of the ribozyme, applicant incorporated various RNA structural elements (ELg_4) 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.

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

Referring to Figure 48, there is provided a general structure for a chimeric RNA molecule of this invention. Each N independently represents none or a number of bases which may or may not be base paired. The A S. 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 iM 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 MgCI 2 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 0 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 200C) for 60 min in a buffer .10 containing 66 mM Tris.HCI, pH 7.6, 6.6 mM MgCl2, 10 mM DTT, 0.066 AM ATP and 0.1U/pA 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 min at 370C. The cells were plated on LB agar plates and incubated at 370C for 18 h.

lasmid 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).

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

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

The HHI encoding DNA was cloned into this A3-5-

S

35 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. Bio. 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. 5 In addition, these results demonstrated the relative differences in accumulation among the different constructs (Fi ur35.3). The pattern of V1.1 106 expression seen from the A3-5/HHI ribozyme chimera was similar to 12 other ribozymes cloned into the A3-5 vector (not shown). In MT-2 cell line, ribozyme chimeras accumulated, on average, to less than 100 copies per cell.

Addition of a stem-loop onto the 3' end of A3-5/HHI did not lead to increased A3-5 levels (S3 in Fio. 35.3). The S5 construct containing both and 3' stem-loop structures also did not lead to increased ribozyme levels (Fig. 35. Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original A3-5/HHI vector transcripts (i35,6). This may be due to increased stability of the S35 transcript.

Example 28: Cleavage activity To assay whether ribozymes transcribed in the transduced cells Scontained cleavage activity, total RNA extracted from the transduced MT2 T 15 cells were incubated with a labeled substrate containing the HHI cleavage site (Fiure 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 Mo. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (i38 a n 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 (FigE 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 3-5 vector. Therefore, the S35 gene unit should be much more effective 10 in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.

Example 30: Stability Finally, the bulk S35-transduced line, resistant to G418, was propogated for a period of 3 months (in the absence of G418) to determine 15 if ribozyme expression was stable over extended periods of time. This S"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 (Fure 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 53-54, a hammerhead ribozyme targeted to site I (HHITRZ-A; Fi. 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 0 108 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 o present invention.

Example 32: Ribozyme expression in T cell lines Ribozyme expression in T cell lines stably-transduced with either a retroviral-based or an Adeno-associated virus (AAV)-based ribozyme 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 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. 5) or long (622 nt) RNA (Fio. 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 [y- 3 2 p] ATP and polynucleotide kinase (urr. Protocols Mol.Bi. 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 Cl 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 Fq. -AG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate

RNA

(Turner and Sugimoto, 1988 Supra). RPI A is a HH ribozyme with 6/6 binding arms. This ribozyme was synthesized chemically either as a one piece ribozyme or was synthesized in two fragments followed by ligation to generate a one piece ribozyme. The kcat/KM values for the two ribozymes were comparable.

A template containing T7 RNA polymerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone. The target RNA (containing HH ribozyme cleavage sites B, C and D) was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [o- 3 2 p] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1, following transcription at 37 0 C for 2 hours, to digest away the DNA template used in the transcription. RNA was precipitated with Isopropanol and the pellet was washed two times with 70% ethanol to get rid of salt and nucleotides used in the transcription reaction. RNA is resuspended in DEPC-treated water and stored at 4°C. Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/KM) conditions [Herschlag and Cech 1990 supral. Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris.HCI, pH 7.5 and 10 mM 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 il 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-aired stem II are Scatalytically 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 4 bp stem II) can be shortened without decreasing the catalytic efficiency of the HH ribozyme. The length of stem II was systematically shortened by one base-pair at a time. HH ribozymes with three and two base-paired stem II were chemically synthesized using solid-phase RNA phosphoramidite chemistry (Scaringe et al., 1990 suDra Matched and long substrate RNAs were synthesized and ribozyme assays were carried out as described in example 33. Referring to figures 62, 63 and 64, data shows that shortening stem II of a hammerhead ribozyme does not significantly alter the catalytic efficiency. It is applicant's opinion that hammerhead ribozymes with 2 base-paired stem II region are catalytically active.

xam le nthsis of cat ll ti haiin ribozyme RNA molecules were chemically synthesized having the nucleotide base sequence shown in E!g..5 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 Fiure 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 MgCI 2 and shown to cleave the substrate efficiently (.fig The target and the ribozyme sequences shown in 2 and 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 Hirpin Rbozymes There follows an improved trans-cleaving hairpin ribozyme in which a 15 new hex. g a i r p i n r b o z ym e i n w h (ce 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 provided at the 3' end of the ribozyme after helix 3 as shown in reI In addition, at least two extra bases may be provided in helix 2 and a portion 20 of the substrate corresponding to helix 2 may be either directly linked to the portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atleast one base. By trans-cleaving is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cleavage reaction.

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

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

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

10 Results of experiments suggest that the length of H2 can be 6 bp without significantly reducing the activity of the hairpin ribozyme. The H2 arm length variation does not appear to be sequence dependent.

HP

ribozymes with 6 bp H2 have been designed against five different target RNAs and all five ribozymes efficiently cleaved their cognate target RNA.

15 Additionally, two of these ribozymes were able to successfully inhibit gene I 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 Dliponucleotides with 5'-C-alkvl 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 nucleotide derivatives are shown in Fiure 76. 29-32 and Figure 77, 58- 61 respectively.

This invention relates to the use of 5'-C-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As the term is used in this application, enzymatic nucleic acids are catalytic nucleic molecules that contain alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or 10 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 within the invention are 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides.

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

Thus, in one aspect, the invention features 5'-C-alkylnucleosides, that is a nucleotide base having at the 5'-position on the sugar molecule an alkyl moiety. In a related aspect, the invention also features alkylnucleotides, and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably 0 114 includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above. In preferred embodiments, the sugar of the nucleoside or nucleotide is in an optically pure form, as the talose or allose sugar.

Examples of various alkyl groups useful in this invention are shown in Figure 75, where each R 1 group is any alkyl. These examples are not limiting in the invention. Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More 10 preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 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 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, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or N(CH 3 2 amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated 7 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring

M

atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

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

enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic 15 molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, the invention features triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

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

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

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

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

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

The following are non-limiting examples showing the synthesis of nucleic acids using 5'-C-alkyl-substituted phosphoramidites and the 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.; 20 Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 26-29 and 56-59). These 5'-C-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Examole 38: Methyl-2.3-O-soproyDlidine-6-Deoxy--.D-allofuranoside (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 0H (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. NaHC03 (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 10 chloroform (750 mL), filtered concentrated to 100 mL and purified by flash chromatography in CHCI 3 to yield 45 g of compound 4.

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

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 CH 2

CI

2 to yield 20.0 g of compound Example 40: Methyl-5-Ot-But vdiphenvsilvi-6-Deox D-Allofuranosid 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 CH2C1 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 118 Example 41: Methyl-2.3-di-O-Benzovl-5-O-t-Butvldiphenysilyl-6-Deoxv-p 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 CH 2

CI

2 to yield 9.5 g of compound 7.

9*4* ~Example 42; 1-O-Acetyl-2,3-di-Obenzoyl-5-O-t-Butldiphensill6 *Deoxy-B-D-Allofuranose Dibenzoate 7 (4.7 g, 7.7 mmol) was dissolved in a mixture of AcOH (10.0 mL), Ac20 (20.0 mL) and EtOAc (30 mL) and the reaction mixture was 15 cooled 0 98% H 2

SO

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.

NaHCO 3 and EtOAc (150 mL). After 0.5 h of vigorous stirring the organic phase was separated, washed with brine (2 x 75 mL), dried over MgSO 4 evaporated to dryness and coevaporated with toluene (2 x 50 mL). The 20 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 ca and 0 isomers).

Example 43: 1-(2'3'-diO-Benzovl-5'-tButdihenv 6'-Deox--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 CF3SO 3 SiMe 3 (2.8 g, 12.6 mmol). The reaction mixture was kept at 24 oC for 16 h, concentrated to 1/3 of its original volume, diluted with 100 mL of CH 2

CI

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

CI

2 Yield: 5.7 g Example 44: N-Benzoyl-1-(2'.3'-Di-O-Benzoyl-5'-O-t-Butyldipheniysilyl.6' Deoxv-p--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 C1 2 (100 mL) and extracted with sat. NaHC 3 O (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness.

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

CH

2 C1 2 yielded 1.8 g of compound 15 Example 45: N~-Benzoyl9-2'3'-di- O-Benzoyl-'- t-Butldi Deoxv-B-D-Allofuranosyl)adenine (11).

N

6 -benzoyladenine (2.86 g, 11.86 mmol) was suspended in mixture •of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (7 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH 3 CN (100 mL) followed by 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 ExamDle 4 -lsobutvrv- 2' 3'-di- '-Deox--D-Allfuranosl)auanie (12).

/N

2 -sobutyrylguanine (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 CH 2

CI

2 (100 mL) and extracted with sat. NaHCOs (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: 6 -Benzoyl-9-(2'.3.'di.-benzov.-6. Deoxy.--D-Alofransyl)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 15 Example 48: -Bezovl-9-(2'.3'-di-O-Benzovl5'-Dimethoxvtrivl- Deoxy-B-D-Allofuranosyl)-adenine (19).

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

CI

2 AgN 0 3 (0.34 g, 2.0 mmol), dimethoxytrityl chloride (0.68 g, 2.0 mmol) and sym-collidine (0.48 g) were added under Ar. The reaction mixture was 20 stirred for 2h, diluted with CH 2

CI

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: 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: NS-Benzoyl-9-(-5'-O-Dimethoxytrit-2'- btvdimet iy 6'-Deoxy-B-D-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 AgNOs (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 10 purified by flash chromatography using a hexanes:EtOAc 7:3 gradient.

Yield: 0.7 g Example 51: -Benzovl-9-(-5'-O-Dimethoxtritvl-2'-O-t-butidimethyls-ill 6n'-Deoxv-8-D-Allofuranosv)adenine-3'--Cyanoethv N N-diisooropyl phosphoramidite) (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--Nitrobenzoyl-.2 3-Iso r lidine-eo 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 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 of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.

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

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 MgCI2 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-05). HH-O 1,2,4 and 5 showed almost wild type activity (Figure 79).

However, HH-03 demonstrated low catalytic activity. Ribozymes HH-O1, 2, 3, 4 and 5 are also extremely resistant to degradation by human serum nucleases.

15 Oliaonucleotides with 2'-Deoxy-2'-Alkylnucleotide This invention uses 2 '-deoxy-2'-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or singlestranded DNA, and also as antisense oligonucleotides. As the term is used 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 can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

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

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

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

Examples of various alkyl groups useful in this invention are shown in *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 0 is adjacent the 2 '-position of the sugar molecule.

In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2 '-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2 '-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2'-position an alkyl group. In other related aspects, the invention features 2'dexy2-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

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

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

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

Ribozymes from Figure 80 and Table 45 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17, all of the modified ribozymes retained at lease 1/10 of the wild-type catalytic activity. From Table 45, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 55, data not shown). The order of most agressive nuclease activity was fetal bovine r W 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 1 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 tl/ 2 of the resulting modified ribozymes.

10 However the catalytic activity of these ribozymes was decreased 10-fold.

,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 Sof the amidites, their testing for enzymatic activity and nuclease resistance.

Examle 3: ynthesis of Hammerhead Ribozmes ontai 2'-Deox 2 '-AlkVlnucleotides Other 2'-Modified Nucleotides 0 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

CT-'

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 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.

Examole 56: -(Tetraisoproovl-disiloxane-1 3 -div2'-O-Phenoxtho carbonyl-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 @006

S.

*e

I

S

*6SO 6@ OS 6 S S S. S 0

*OS*

55

S

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(TetraisoproDvl-disiloxane-1 3 -di I-2'-CA-1 -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-Dimethoxtrityl2'-C-AllyI-Uridine (9 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-Dimethoxvtrityl-2'-C-AIIyl-Uridine 3 2 -Cyanoethyl N.Ndiisopropylphosphoramidite) 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 was concentrated in vacuo (40 and purified by flash chromatography on silica gel using a gradient of 10-60% EtOAc in hexanes containing 1% triethylamine mixture as eluent. Yield: 0.78 g white foam.

Example 60: 3 '.5'-O-Tetraisopropyl-disiloxane-1.,3-diyl)-2'--Allyvl.N4 Acetyl-Cvtidine (11) 15 Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile. To the resulting suspension a solution of 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-Dimethoxtrityl-2'-C-Allvl-Acetyl-idine This compound was obtained analogously to the uridine derivative 9 in 55% yield.

Example 62: 5'-O-Dimethoxvtritl-2'-C-allvl- 4 -Acetyl-Ctidine Cvanoethyl N. N-diisooropylphosohoramidite) (12) 2'-O-Dimethoxytrityl-2'-C-allyl-N 4 -acetyl cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichioromethane under argon. NN-Diisopropylethylamine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl NN-diisopropylchlorophosphoramidite (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 chloroform:ethanol 98:2 with 2% triethylamine mixture as eluent. Yield: 0.91 g white foam.

Example 63: 2 '-Deoxy-2'-Methylviene-Uridine

S

2 '-Deoxy-2'-methylene3',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.

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

CI

2 Example 64: 5'-DMT-2'-Deoxv-2'-Meth lene-Uridine (1 2 '-Deoxy-2'-methylene-uridine (0.91 g, 3.79 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

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

Example 65: O-DMT-2'-Deoxy-2'-hMethylene.Uridine 3 2 yanoethyI N.N-diiso-r-opylphosp~horamidite) (17 1 Deoxy-2'-methylene-5'- O-d imethoxytrityl-o-D-ribofu ran 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 vacuo (40 00). The product (0.3 g, 0.4 1 0 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) Examiple 66: 2: x-'Dfurmtyee3 0(erio~o dslx ane-1 .3-diyl)-Uridine 15 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 00. A warm OC) solution of sodium chlorodifluoroacetate in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The *residue was dissolved in 0 H 2

CI

2 and chromatographed over silica gel. 2'- Deoxy-2'difluoromethylene35O(tetraisopropyldisiloxe-,3-diyl)- OVOO uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.

Examle 7: '-Doxy2'-Difluormeth lene-Uridine 2 Deoxy-2'-m ethyle ne3',5'- 0- (tetraisop ropyldisil oxane9 1, 3-d iyl)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'Doy2-ilooehln-rdn (1.1 g, 4.0 mmol, 68%) was eluted with 20% MeCH in CH 2

CI

2 Example 6: 'ODMT2'-Dxyv.2'.DIfluorometylene-uidine(16 2'Doy2-ilooehln-rdn (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 S, w 131 was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2

CI

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

CI

2 (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.36 mL, 2 mmol) *was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.4 mmol). The reaction mixture 15 was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 OC). The product (0.404 g, 0.52 mmol, 52%) was purified by flash chromatography over silica gel using 50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH 2

CI

2 MeOH/ 15:1).

Exam p le 70: 2'-D e ox-2'-Meth en '-O-Tetraisoro- disiloxane-13diyl)-4-N-Acetyl-Cvtidine 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-(tetraisopropyldisiloxane-1,3-diyl) uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h. The mixture was concentrated in vacuo, dissolved in

CH

2

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

SO

4 concentrated in vacuo and the residue chromatographed over silica gel. 23 -Deoxy-2'-methylene..3',5' 0- (tetraisopropyldisiioxanel ,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes.

Example 71: 1-2-ex-'-ehln- DmtoriylO-ribof uran osyl)-4- N-Acetyl-Cytosine 21 2 '-Deoxy-2'-methylene.3','O.(tetraisopropyldisiloxane-1,3-diyl)-4-Nacetyl-cytidine 20 (1.3 g, 2.5 mmol) dissolved in THF (20 ml-) was treated with 1 M TBAF in THF (3 mL) for 20 mn and concentrated in vacua. The 1 0 residue was triturated with petroleum ether and chromatographed on silica gel column. 2 '-Deoxy-2'-methylene4Nacetyl-cytidifle (0.56 g, 1.99 mmol, was eluted with 10% MeOH in CH 2

CI

2 2 '-Deoxy-2'-methylene-4.N acetyl-cytidine (0.56 g, 1.99 mmol) was dissolved in pyridine (10 ml-) and a solution of DM1-Cl (0.81 g, 2.4 mmol) in pyridine (10 ml-) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeQH (2 ml-) was added to quench the reaction. The mixture was C. *concentrated in vacuo and the residue taken up in CH 2

CI

2 (100 ml-) and washed with sat. NaHCO 3 (50 mL), water (50 ml-) and brine (50 mL). The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtQAc:hexanes 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, Examlle 72: 1('Doy2-ehln-r'ODmt io fu ranosyl)-4- N-Akcetyl-Cytosine 3 '-(2-CYanoethl-N. N-diisoroylhosooramidite) (22 1 2 Deoxy-2'-methylene5'- O-di meth oxytrityl~O-Dribof uran osy) 4 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,-ispoycloohshrmdt (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After 10 rn 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: 2 '-DeoY- 21 -Difluoromethylene..3',5..0(TetraisoropyI disiloxane-1 .3-diyl)-4-N-Acetyl-Cytidine (24) Et 3 N (6.9 mL, 50 mmol) was added to a solution Of POC1 3 (0.94 mL, mmol) and 1 ,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 ml-) at 0 00.

A solution of 2'-deoxy-2'-difluoromethylene-3',5'- O-(tetraisop ropyldisi loxane-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 0H 2 01 2 (2 x 100 ml-) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2

SO

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

NaHCO 3 The mixture was concentrated in vacua, dissolved in

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'-difluoromethylene.

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

.Examlle 74: l1( 2 Deoxy-2'- Dif Iuo rom ethylene-5'- -iMetoxytyLBD ribofuranosyl)-4NAcetyl..p~osine 2 '-Deoxy-2'difluoromethylene3,5O.(tetraisoprpydslxne1 diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol) dissolved in THF (20 ml-) was treated with 1 M TBAF in THF (3 ml-) for 20 m and concentrated in vacua.

The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2 '-Deoxy2'difluoromethylene4Nacetyl-cyidine (0.89 g, 2.8 mmol, 72%) was eluted with 10% MeOH in 0H 2 01 2 2'-Deoxy-2'difluoromethyene4.Nacetyl-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 01 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 _011111111111111 0 MW 134 vacua 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 '-DifluoromethyleneS-0Dimehox rityl~.

ribof uran osyfl-4- N-Acetylcytosi ne 3 '-(2-cyanoethyL-N .,N-diisopropXlphos_ phoramidite) (26) 1 2 eoxy-2'-d ifl uo rom ethylen G- mt xtiy-pDrbfurn syl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in dry CH 2

CI

2 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine mL, 2.9 mmol) was added, followed by the dropwise addition of 2cyanoethyl NNdiorplhoohshrmdt (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL).

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

Example 76: 2 '-Keto- 3 '.5'.(Tetraisoroldisloxane-1~.3-diyl)-6-N-(4-t- Butylbenzoyn)-Adenosine (28).

Acetic anhydride (4.6 mL) was added to a solution of 3 propyldisiloxane-1,3dy)6N(4tbtlezol-dnsn (Brown,J.; Christodolou, Jones,S.; Modak,A.; Reese,C.; Sibanda,S.; Ubasawa

A.

Chem .Soc. Perkin Trans. /11989, 1735) (6.2 g, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and washed with water. The organic layer was dried over MgSO 4 and concentrated in vacua. The residue was purified on a silica gel column to yield 2'kt-'5--ttaiorpliioae1 3 -diyl)-6-N(4tbutylben.

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

Example 77: 2 '-Deoxy2'meyene3%'- -(Tetraisoroayisioxnl 3 dil---A enol-dnsn (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 THE (25 mL) cooled at -78 00. The homogeneous orange solution was allowed to warm to -30 00 and a solution of 2'-keto- O-(tetraisopropyldisiloxane l'-il---4tbuybnol-dnsn 135 28 (4.87 g, 7.3 mmol) in THF (25 ml-) was transferred to this mixture under argon pressure. After warming to RT, stirring was continued for 24 h. THF was evaporated and replaced by CH 2

CI

2 (250 mL), water was added mL), and the solution was neutralized with a cooled solution of 2% HCl The organic layer was washed with H 2 0 (20 mL), 5% aqueous NaHCO 3 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacuo to give the crude compound, which was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc 7:3 afforded pure 2 '-deoxy-2'-methylene.3',5'.O-(tetraisopropyldisiloxane-1 3 -diyl)-6-N(4tbutylbenzoyl)adenosine 29 (3.86 g, 5.8 mmol, 79%).

Example 78: 2 '-Deoxy-2'-Methylene6.rN(4-tButylbe 1ZOylAdnosin 2 '-Deoxy-2'-m ethyl ene-', 0-(tetraisop ropyldisi loxane- 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'methyene6N(4-t butylbenzoyl)-adenosine (1.8 g, 4.3 mmol, 74%) was eluted with MeOH in 0H 2 C1 2 Exa ille 79: 5'0DT2-)oya-Mtyln--- ~qX~a~j Adenosie 29 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 01 2 (100 ml-) and washed with sat. NaHCO 3 water and brine. The organic extracts were dried over Mg SO 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%).

Examrole 80: S'O-r)KTV'rI-oX.2'.Met ethy-AALEneN4~l Adeno)sine 2-yanothyL~ N N-diisoprolhshrmdt)(1 4 -t-butylbenzoyl)-adenine 29 dissolved in dry CH 2 01 2 (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 00). The product was purified by flash chromatography over silica gel using 30-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant (0.7 g, 0.76 mmol, Rf 0.45 (0H 2 C1 2 MeOH 20:1) Example 81: 2 '-Deoxy-2'-Difluoroehl -(Tetraiso ropyLdisilox 10 ane- l.

3 -diyl)-r -N.4t-Butbenzoyl)..Adenosine benzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenylphosphine (2.9 g, 11 mmol were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 A warm (60 00) 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 CH 2

CI

2 and chromatographed over silica gel. 2'- Dex-*df *oo ety en-' ~eriorpliioae ,3-diyl)-6- N- (-t-butylbenzoyl)-adenosine (4.1g, 6.4 mo,6% ltdwt hexanes in EtOAc.

Example 82: 2 '-Deoxy-2' D ifl u orom hletheneNtuyl Adenosine 2 '-Deoxy-2'-difluoromethylene-3v,5'O-(tetraisopropyldisiloxane-1,3dil---4tbtlbnol-dnsn (4.1 g, 6.4 mmol) dissolved in THF ml) was treated with 1 M TBAF in THF (10 ml-) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2 '-Deoxy-2'-dif Iu orom ethylen---4tbtlbnol-dnsn (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in 0H 2 C1 2 Example 83: aDMT- 2 'D ox2Difluorometyee 6 ttll benzoyl)-Adenosin 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 vacuo and the residue taken up in 0H 2 C1 2 (100 ml-) and washed with sat. NaHCO 3 water and brine. The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as eluant to yield 30 (2.6 g, 3.41 mmol, 69%).

Example 84: '-O-DMT.2'-Deoxy-2'.Difluoromethylene A(4aUlk benzoyl)-Adenosine 3 -(2-Cyanoethyl N. N-diisopr ~lhos horamidite-) 2 1 l-( 2 '-Deoxy-2'-difl uo rom ethylene.s'- O-dimethoxytrityl-o-D- ribof uranosyl)- 6 -I-(4-t-butylbenzoyl)adenine 30 (2.6 g, 3.4 mmol) dissolved in dry

H

2 C1 2 (25 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 2-cyanoethyl NNdiorplhoohshrmdt (.06 mL, 4.76 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 mn the mixture evaporated to a syrup in vacua (40 00). 32 (2.3 g, 2.4 mmol, 70%) was purified by flash column chromatography over silica gel using 20-50% EtOAc gradient in 20 .ean containing 1% triethylamine, as eluant. Rf 0.52 (CH 2

CI

2 MeOHI 15:1).

Examile 85 O--Deaso opropvldisiloxane- 1 3-diyl)-U idne (33 M ty tihnlhshrnldn~ctt (5.4 16 mmol) was added to a solution of 2 '-keto-3',5'-o-(tetraisopropyl disiloxane-i ,3-diyl)uridine 14 in CH 2

CI

2 under argon. The mixture was left to stir at RT for h. CH 2 01 2 (100 ml-) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCl. The organic layer was washed with H 2 0 (20 mL), 5% aq. NaHCO 3 (20 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

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

Elution with light petroleum ether:EtOAc 7:3 afforded pure 2'-deoxy-2'methoxycarbon ylmethylidine-3' 5 '.O-(tetraisopropyldisiloxane-.1 ,3-diyl)uridine 33 (5.8 g, 10.8 mmol, 67.5%).

138 Example 86: 2 '-Deoxy- 2 '-Methoxycarbonylmethylidineridine (34A% Et 3 N*3 HF (3 mL) was added to a solution of 2 '-deoxy-2'-methoxy.

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

CI

2 (20 mL) and 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'-methoxycarb onylm ethyl id in e uridine 34 (2.4 g, 8 mmol, 86%) with THF:CH 2

CI

2 4:1.

Example 87: 5' M-'Doy2-ehx~abnlehiialrdn 2 '-Deoxy-2'-methoxycarbonylmethylidine.uridine 34 (1.2 g, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-CI (1.5 g, 4.42 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in 0H 2

CI

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

The organic extracts were dried over Mg04 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-'mehoycroymehliineurdi (2.03 g, 3.46 mmol, 86%).

Example 88: 5'OD _-'Doy2 eh ycroy ehldn-rdn 3'-(2-cyanoethyl-V. N-diisopropylohosphoramidfte) (36 D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in dry 0H 2 C1 2 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2cyanoethyl N,N-ispoyclrohshrmdt (0.91 mL, 4.08 mmol).

The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 5'ODT2-ex-'mtoyabnlehldn-rdn (2cya noethyI-N, N-d isop ropylphospho ram idite) 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 (0H2012:MeOH 9.5:0.5).

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

CI

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

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

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

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

The synthesis of 1-O-Ac-2,3-di-O-Bz-D-ribofuranose 5+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-Oisopropylidene-B-D-ribofuranose 5-deoxy-5-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 10 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 triphosphates, 14 Figure 89, for T7 transcription).

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

0 II

O

.(R

3 0) 2

PCX

2 R(R 3 2 B B (R30)2PCX

R

2

R

1

CX

2

R

(R

3 0) 2 P O

(R

3 0) 2 P where

R

1 is H, OH, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, or carbonate; each R 2 is separately H, OH, or R; each R 3 is separately a phosphate protecting group, methyl, ethyl, cyanoethyl, pnitrophenyl, or chlorophenyl; each X is separately any halogen; and each B is any nucleotide base.

The invention in particular features nucleic acid molecules having such modified nucleotides and enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleoside dihalo and/or 3'-deoxy-3'-dihalophosphonates by condensing a f 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) found that a single 3 '-methylenephosphonate linkage had a minor influence on the conformation of a DNA octamer double helix.

1 -0 0- 0- 0- OH OH 1

O

N N.H N] N N

H

(HO)

2

OPCF

2

NH

2 **oo* synthesize nucleoside 5'-deoxy-5'-difluoro-methylphosphonates from

(ETO)

2

POCF

2 Li aldehydes with 3, according to the procedure of Martin et al. (Martin et al Tetrahedron Lett. 1992, 33, 18391842), led to a complex mixture of 3 pro ne common synthetic approach to a,a-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive m substrate y diethy (lithiodifluoromethyl)phosphonate (Obayashi et al., Tetrahedron Lett. 1982, 23, 2323-2326). However, our attempts to synthesisie 5'-deo 5'-eoxy-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-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-difluoromethylphosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon q 143 these examples. These examples demonstrate that it is possible to achieve synthesis of 5'-deoxy-5'-difluoro derivatives in good yield and thus guide those in the art to such equivalent methods. The examples also indicate utility of such synthesis to provide useful oligonucleotides as described above.

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

10 Example 90: Synthesis of Nucleoside difluoromethylphosphonates Referring to Fig. 7, 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 20-25oC), 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) (Ac 2 0, AcOH,

H

2

SO

4 EtOAc, 00C. 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 Vorbroggen conditions (VorbrOggen, Nucleoside Analogs. Chemistry, Biology and Medical Applications, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F 3

CSO

2 OSi(CH 3 3 as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron Lett. 1987, 28, .3623-3626 and references cited therein) (SnCI 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 (HC03-) 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

J

1 ,2 4.5, aH1), 6.42 P H 5.74

J

2 3 4.9, H2), 5.67 (dd, J 3 2 4.9, J 3 4 6.6, OH3), 5.63 (dd, J 3 ,2 6.7, J3,4 3.6, H3), 5.57 (dd, J2,1 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 BAc), 2.12 aAc), 1.39

CH

2

CH

3 3 1 P 5 7.82 (t, P,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, J 5 6 8.1, 1H, H5), 5.79 (dd, *to* J2',1' 4.1, J2',3 6.5, 1H, 5.71 (dd, J3',2' 6.5, J3',4' 6.4, 1H, 4.79 (dd, J4',3' 6.4, J4',F 11.6, 1H, 4.31 4H, CH 2

CH

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

CF

2 1.40 6H, CH 2

CH

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

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

Examle 91:Snthesis of Nucleic Acids ontaininModifed 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-

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 Fia.89, according to known procedures. Nucleic Acid Chem., Leroy B. Townsend, John Wiley Sons, New York 1991, pp. 337-340; i Nucleotide Analogs, Karl Heinz Scheit; John Wiley Sons New York 1980, see* 10 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 15 advantageous as modified nucleotides in any nucleic acid structure, e.g., catalytic or antisense, since they are resistant to exo- and endonucleases that normally degrade unmodified nucleic acids in vivo. They also do not perturb the normal structure of the nucleic acid in which they are incorporated thereby maintaining any activity associated with that structure.

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

Oligonucleotides with Amido or Petido Modificatio This invention replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and portions of the sugar of a nucleotide may be substituted, or the phosphate group may be substituted with amido or peptido moieties. Generally, such a nucleotide has the general structure shown in Formula I below: 146,

B

0 0 N R2 .H

R

1

R

3

I*P-

0 FORMULA I t*o* 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, an amide), an alkyl group, or 10 an amino acid (D or L forms) or peptide containing between 2 and 5 amino acids. The zigzag lines represent hydrogen, or a bond to another base or other chemical moiety known in the art. Preferably, one of R 1 R2 and R 3 is an H, and the other is an amino acid or peptide.

Applicant has recognized that RNA can assume a much more complex structural form than DNA because of the presence of the 2'hydroxyl group in RNA. This group is able to provide additional hydrogen bonding with other hydrogen donors, acceptors and metal ions within the RNA molecule. Applicant now provides molecules which have a modified amine group at the 2' position, such that significantly more complex structures can be formed by the modified oligonucleotide. Such modification with a 2'-amido or peptido group leads to expansion and enrichment of the side-chain hydrogen bonding network. The amide and peptide moieties are responsible for complex structural formation of the oligonucleotide and can form strong complexes with other bases, and interfere with standard base pairing interactions. Such interference will allow the formation of a complex nucleic acid and protein conglomerate.

F,

Oligonucleotides of this invention are significantly more stable than existing oligonucleotides and can potentially form biologically active bioconjugates not previously possible for oligonucleotides. They may also be used for in vitro selection of unique aptamers, that is, randomly generated oligonucleotides which can be folded into an effective ligand for a target protein, nucleic acid or polysaccharide.

Thus, in one aspect, the invention features an oligonucleotide containing the modified base shown in Formula I, above.

In other aspects, the oligonucleotide may include a 3' or 5' nucleotide 10 having a 3' or 5' located amino acid or aminoacyl group. In all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard modifications can be made. For example, an may be replaced with an S, the sugar may lack a base abasic) and the phosphate moiety may be modified to include other substitutions (see Sproat, supra).

Example 93: General procedure for the preparation of 2'-aminoacyl-2' deoxv-2'-aminonucleoside conjugates.

:Referring to Fi...92, to the solution of 2 '-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or amino acid (1 mmol) in methanol [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1ethoxycarbonyl-2-ethoxy-1, 2 -dihydroquinoline (EEDQ) [or 1isobutyloxycarbonyl-2-isobutyloxy- 2 -dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to "C from 3-48 hours. Solvents are removed under reduced pressure and the residual syrup is chromatographed on the column of silica-gel using 1methanol in dichloromethane. Fractions containing the product are concentrated yielding a white foam with yields ranging from 85 to 95 Structures are confirmed by 1 H NMR spectra of conjugates which show correct chemical shifts for nucleoside and aminoacyl part of the molecule.

Further proofs of the structures are obtained by cleaving the aminoacyl protecting groups under appropriate conditions and assigning 1 H NMR resonances for the fully deprotected conjugate.

Partially protected conjugates described above are converted into their 5'-O-dimethoxytrityl derivatives and into 3 '-phosphoramidites using standard procedures (Oligonucleotide Synthesis: A Practical Approach, 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, S. 10 guanosine) and/or abasic moieties.

Example 94: RNA cleavage by hammerhad riboyms contanin 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'-NH- S: alanine 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 20 amount (5 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90C for 2 Smin and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 370C for 10 min in a buffer containing 50 mM Tris-HCI and 10 mM MgCl2. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 370C. Aliquots of 5 1l 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 hammerhead ribozymes containing 2'-NHalanine or 2'-NH-lysine modifications at U4 and U7 positions cleave the target RNA efficiently.

j 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.

I. Preparation of aminoacyl-derivatized solid support A) Synthesis of O-Dimethoxvtrityl (O-DMT) amino acids 15 Referring to Fig. 97, to a solution of L- (or serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4 ,4'-dimethoxytrityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25"C) for S* 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

I.

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 1. Referring to Fij. 9. 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 00 *0 10 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 15 Modification of existing nucleic acid sequences can be achieved by homologous recombination. In this process a transfected sequence recombines with homologous chromosomal sequences and can replace the endogenous cellular sequence. Boggs, 8 International J. Cell Cloning 1990, describes targeted gene modification. It reviews the use of 20 homologous DNA recombination to correct genetic defects. Banga and Boyd, 89 Proc. Natl. Acad. Sci. U.S.A. 1735, 1992, describe a specific example of in vivo site-directed mutagenesis using a 50 base oligonucleotide. In this methodology a gene or gene segment is essentially replaced by the oligonucleotide used.

This invention uses a complementary oligonucleotide to position a nucleotide base changing activity at a particular site on a gene (RNA or genomic DNA), such that the nucleotide modifying activity will change (or revert) a mutation to wild-type, or its equivalent. By reversion or change of a mutation, we refer to reversion in a broad sense, such as when a mutation at a second site which leads to functional reversion to a wild type phenotype. Also, due to the degeneracy of the genetic code, a revertant may be achieved by changing any one of the three codon positions.

Additionally, creation of a stop codon in a deleterious gene (or transcript) is defined here as reverting a mutant phenotype to wild-type. An example of 151 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 10 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 (F-

L

01) and has the advantage that changes may be permanently encoded in the target cell's genetic code. Thus, a single course (or several courses) of treatment may lead to permanent reversion of the genetic disease. If inadvertent chromosomal mutations are introduced this may cause cancer, mutate other genes, or 20 cause genetic changes in the germ-line (in patients of reproductive age).

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 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 Nat Acad Sci ULSA 90:8673-7. Analogously, in one preferred embodiment of this invention a complementary oligomer is used to correct an existiing mutant SRNA, instead of the traditional approach of inhibiting that RNA by 10 antisense.

In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence.

:i 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 which a sequence modifying activity edits a RNA to its proper sequence post-transcriptionally. Higuchi, Single, Kohler, Sommer, and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GluR-B:

A

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

The changes in the base created by the methods of this invention cause a change in the nucleotide sequence, either directly, or after DNA repair by normal cellular mechanisms. These changes functionally correct a genetic defect or introduce a stop codon. Thus, the invention is distinct from techniques in which an active chemical group an alkylator) is attached to an antisense or triple strand oligonucleotide in order to chemically inactivate the target RNA or DNA.

Thus, this invention creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base.

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

Thus, in one aspect, the invention features a method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule. The method includes contacting the nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After formation of the duplex or triplex molecule a base modifying activity chemically or 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 15 methods of correcting defects in DNA, such as homologous recombination, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the transfected nucleic acid. This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.

By "functionally alter" is meant that the ability of the target nucleic acid to perform its normal function transcription or translation control) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.

By "mutant" it is meant a nucleic acid molecule which is altered in some way compared to equivalent molecules present in a normal individual. Such mutants may be well known in the art, and include, molecules present in individuals with known genetic deficiencies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal expression of a gene, such as cancer, thalassemia's and sickle cell anemia, and cystic fibrosis. It allows modulation of lipid metabolism to reduce artery disease, treatment of integrated AIDS genomes, and AIDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a "wild type" phenotype and/or genotype. For deleterious conditions this involves altering a base to allow expression or prevent expression as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant non-human gene) to a wild type no production of a non-human protein). Such modification is performed in trans rather than in cis as in 10 prior methods.

In preferred embodiments, the oligonucleotide is of a length (at least 12 bases, preferably 17 22) sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine; the oligonucleotide is an enzymatic nucleic acid molecule that is active to chemically modify a base (see below); the nucleic acid molecule is DNA or RNA; the oligonucleotide includes a chemical mutagen, the mutagen is nitrous acid; and the oligonucleotide causes deamination of methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to In a most preferred embodiment, the invention features correction of a mutation, rather than inactivation of a target by causing a mutation.

Using in vitro directed evolution, it is possible to screen for ribozymes with catalytic activities different than RNA cleavage. Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences. Science 261:1411-1418. Using these methods of in vitro directed evolution, an enzymatic nucleic acid molecule, or ribozyme that mutates bases, instead of cleaving the phosphodiester backbone can be selected. This is a convenient method of obtaining an enzyme with the appropriate base sequence modifying activities for use in the present invention.

Sequence modifying activities can change one nucleotide to another (or modify a nucleotide so that it will be repaired by the cellular machinery to another nucleotide). Sequence modifying activities could also delete or add one or more nucleotides to a sequence. A specific embodiment of adding sequences is described by Sullenger and Cech, PCT/US94/12976 155 hereby incorporated by reference herein), in which entire exons with wildtype sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 3).

Thus, in another aspect, the invention features ribozymes or enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule. Applicant is the first to determine that such molecules would be useful, and to provide a description of how such molecules might be isolated.

ooo Molecules used to achieve in situ reversion can be delivered using 10 the existing means employed for delivering antisense molecules and ~ribozymes, including liposomes and cationic lipid complexes. If the in situ reverting molecule is composed only of RNA, then expression vectors can be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes expression vectors.

15 There are several advantages of using such an expression vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that S.already express that gene. Furthermore, the corrected gene would be "::"properly regulated by its natural transcriptional promoter. Lastly, reversion can be used when the mutant RNA creates a dominant gain of function protein in sickle cell anemia), where correction of the mutant RNA is necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.

Endogenous Mammalian RNA Editing System 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 rINAs (four-) have been reported to undergo editing in 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-U conversion is catalyzed by cytidine deaminase. The mechanism of conversion of A--G has recently been reported for glutamate receptor

B

subunit (gluR-B) in rat PC12 cells (Higuchi, M. et al. (1993) Cell 75, 1361- 1370). According to Higuchi gluR-B mRNA precursor attains a structure such that intron 11 and exon 11 can form a stable stem-loop structure. This stem-loop structure is a substrate for a nuclear double strand-specific adenosine deaminase enzyme. The deamination will result in the conversion of Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.

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

The following are examples of the invention to illustrate different S: methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific embodiments of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims.

Exam le 7: Exoitin cellular dsRNA dependent Adenine to Inosine Qconverter" An endogenous activity in most mammalian cells and Xenopus oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. Weintraub, H. (1988). An unwinding activity that covalently modifies it double-stranded RNA substrate. Cell, 5, 1089- 1098.). This activity can be used to cause an in situ reversion of a mutation at the RNA level. Referring to Figures 102 and 104, for demonstration purposes a stop codon is incorporated into the coding region of dystrophin, which is fused to the reporter gene luciferase. This stop codon can be reverted by targeting an antisense RNA which is long enough to activate the dsRNA deaminase, which converts Adenines to Inosines. The A to I transition will be read by the ribosome as an A to G transition in some cases and will thereby functionally revert the stop codon.

While other A's in this region may be converted to I's and read as G, converting an A to I cannot create a stop codon. The A to I transitions 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 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 and read as G, converting an A to I cannot create a stop codon, so the :S ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a S: dystrophin protein made from an mRNA reverted by this method should retain full activity.

The following detail specifics of the methodology:

RNA

oligonucleotides were synthesized on a 394 (ABI) synthesizer using phosphoramidite chemistry. The sequence of the synthetic complementary RNA that binds to the mutant dystrophin sequence is as follows to

CCCGCGGTAGATCTTTCTGGAGGCTTACAGTTTTCTACAAACCTCC

CTTCAAA (Seq. ID No. 1) Referring to Figure 104, fifty-nine base pairs of a human dystrophin mutant sequence containing a stop codon was fused in frame to the luciferase coding region using standard cloning technology, into the Hind III and Not I sites of pRC-CMV (Invitrogen, San Diego, CA). The AUG of luciferase was deleted. The sequences of the insert from the Hind III site to the start of the luciferase coding region is to

GCCCCTGAGGAGCGATGGAGGCCTTGAAGGGAGGTTTGTGGAAAA

CTGTAAGCCTCCAGAAAGATCTACCGCGG (Seq ID No. 2) 1 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 700C, and allowing it to slowly cool to 37°C over 30 minutes. Fifty nanograms of :i 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.

supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 25°C. Next, 1.5ul of this mixture was added to a rabbit reticulocyte lysate in vitro translation mixture and translated for two hours according to the manufacturers protocol (Life Technologies, 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.

W 159 Additionally enzymes that modify nucleic acids have been conjugated to oligonucleotides. (Knorre, Valentin, Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993). In the past these conjugated chemical groups or enzymes have been used to inactivate DNA or RNA that is specifically targeted by antisense or triple-strand interactions. Below is a list of useful base changing activities that could be used to change the sequence of DNA or RNA targeted by antisense or triple strand interactions, in order to achieve in situ reversion of mutations, 10 as described herein (see figure 100-104).

1. Deamination of 5-methylcytosine to create thymidine (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

Also, nitrous acid or related compounds promote oxidative deamination of 15 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.) a 2. Deamination of cytosine to create uracil (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993) or by chemical groups similar to nitrous acid that promote oxidative deamination (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.) 3. Deamination of Adenine to be read like G (Inosine) (as done by the adenosine deaminase, AMP deaminase or the dsRNA deaminating activity Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

4. Methylation of cytosine to Transforming thymidine (or uracil) to 0 2 -methyl thymidine (or 0 2 -methyl uracil), to be read as cytosine by alkynitrosoureas (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

160 6. Transforming guanine to 6-0-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 in the present invention. There are a few preferred straightforward chemical modifications that can change one base to another base. Appropriate mutagenic chemicals are placed on the targetting oligonucleotide, nitrous acid, or a suitable protein with such activity. Such chemicals and proteins can be attatched by standard 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 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 0 0 M I 161 A sverson DNA/RNA3 A ransvesion ransversion DNA RNA3 T(U) ITransversion I- I DNA 5

/RNA

7 |Transversion C ITransversion

RNA

2 /DNA6 Transversion G

DNA

6

/RNA

6 Transversion Transversion 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:

2 -methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

0 6 Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978)).

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

In Vitro Selection Strategy Referring to Figure 105, there is provided a schematic describing an approach to selecting for a ribozyme with such base changing activity. An RNA is designed that folds back on itself (this is similar to approaches already used to select for RNA ligases, Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences.

Science 261:1411-1418). A degenerate loop opposing the base to be modified provides for diversity. After incubating this library of molecules in a buffer, the RNA is reverse transcribed into DNA (that is, using standard in vitro evolution protocol. Tuerk and Gold, 249 SciQnce 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.

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 invention, the degenerate domain is flanked by regions containing known sequences (see Figure 105), such that the degenerate domain is placed across from the mutant base (the base that is targeted for 20 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).

Following incubation, nucleic acids are converted into complimentary

DNA

(if the starting pool of nucleic acids is RNA). Nucleic acids with base modification (at the mutant base position) can be separated from rest of the population of nucleic acids by using a variety of methods. For example, a restriction endonuclease cleavage site can either be created or abolished as a result of base modification. If a restriction endonuclease site is created as a result of base modification, then the library can be digested with the restriction endonuclease The fraction of the population that is cleaved by the RE is the population that has been able to catalyze the base modification reaction (active pool). A new piece of DNA (containing oligonucleotide primer binding sites for PCR and RE sites for cloning) is ligated to the termini of the active pool to facilitate PCR amplification and subsequent cycles (if necessary) of selection. The final pool of nucleic acids with the best base modifying activity is cloned in to a plasmid vector 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 10 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.

.JUSA 73, 2294). In addition, applicant has determined that that the RNA 20 portion of the R-loop can be used to target the whole R-loop complex to a desirable intracellular or cellular site, and aid in cellular uptake of the complex. Further, applicant indicates that expression of enzymatically active RNA or ribozymes can be significantly enhanced by use of such Rloop complexes.

Thus, in one aspect, the invention features a method for introduction of enzymatic nucleic acid into a cell or tissue. A complex of a first nucleic acid encoding the enzymatic nucleic acid and a second nucleic acid molecule is provided. The second nucleic acid molecule has sufficient complementarity with the first nucleic acid to be able to form an R-loop base pair structure under physiological conditions. The R-loop is formed in a region of the first nucleic acid molecule which promotes expression of RNA from the first nucleic acid under physiological conditions. The method further includes contacting the complex with a cell or tissue under 164 conditions in which the enzymatic nucleic acid is produced within the cell or tissue.

By "complex" is simply meant that the two nucleic acid molecules interact by intermolecular bond formation (such as by hydrogen bonding) between two complementary base-paired sequences. The complex will generally be stable under physiological condition such that it is able to cause initiation of transcription from the first nucleic acid molecule.

The first and second nucleic acid molecules may be formed from any desired nucleotide bases, either those naturally occurring (such as 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., 15 International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science, 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Sproat,B. European Patent Application 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 with the second nucleic acid. If a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that transcription is enhanced.

10 In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by forming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or transcription termination signal such that once expression is initiated it will continue until the first nucleic acid is degraded.

15 In another related aspect, the invention features a method in which the second nucleic acid is provided with a localization factor, such as a :protein, an antibody, transferin, a nuclear localization peptide, or folate, or other such compounds well known in the art, which will aid in targeting the R-loop complex to a desired cell or tissue.

20 In preferred embodiments, the first nucleic acid is a plasmid, e.g., one without a promoter or a transcription termination signal the second nucleic acid is of length between about 40-200 bases and is formed of ribonucleotides at a majority of positions; and the second nucleic is covalently bonded with a ligand such as a nucleic acid, protein, peptide, lipid, carbohydrate, cellular receptor, nuclear localization factor, or is attached to maleimide or a thiol group: the first nucleic acid is an expression plasmid lacking a promoter able to express a desired gene, it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNA/DNA heteroduplex; no promoter or leader region is provided in the first nucleic acid; and the Rloop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular transcription machinery.

In other preferred embodiments, the first nucleic acid encodes one or more enzymatic nucleic acids, it is formed with a plurality of intramolecular and intermolecular cleaving enzymatic nucleic acids to allow release of therapeutic enzymatic nucleic acid in vivo.

In a further related aspect, the invention features a complex of the above first nucleic acid molecules and second nucleic acid molecules.

R-loop complex An R-loop complex is designed to provide a non-integrating plasmid so that, when an RNA polymerase binds to the plasmid, transcription is continuous until the plasmid is degraded. This is achieved by hybridizing an RNA molecule, 40 to 200 nucleotides in length, to a DNA expression 10 plasmid resulting in an R-loop structure (see figure 106). This RNA, when conjugated with a ligand that binds to a cell surface receptor, triggers Sinternalization 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 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 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 proposed mechanism is that R-loop formation prevents nucleosome assembly, thus making the DNA more accessible for transcription.

Alternatively, 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 invention is to generate R-loops in expression vectors of choice and introduce them into cells to achieve enhanced expression from the expression vector. The presence of an Rloop may aid in the recruitment of cellular transcription machinery. Once an RNA polymerase binds to the plasmid and initiates transcription, the 10 process will continue until a termination signal is reached, or the plasmid is degraded.

2

T

This invention will increase the expression of ribozymes inside a cell. The idea is to construct a plasmid with no transcription termination signal, such that a transcript-containing multiple ribozyme units can be 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.

S. Ligand Targeting Another salient feature of this invention is that the RNA used to 20 generate R-loop structures can be covalently linked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, etc.). Specific ligands can be chosen such that the ligand can bind selectively to a desired cell surface receptor. This ligand-receptor interaction will help internalize a plasmid containing an R-loop. Thus, RNA is used to attach the ligand to the DNA such that localization of the gene to certain regions of the cell is achieved. One of several methods can be used to attach a ligand to RNA.

This includes the incorporation of deoxythymidine containing a 6 carbon spacer having a terminal primary amine into the RNA (see fiure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 BJSol. Qhem 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 15 Proc. Natl. Acad, Sci. USA 88, 8850-8854; Wagner et al., 1992 Proc. Natl.

Acad. Si. 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 R- S. loop forming RNA, an intrinsic thiol can be used to react with the maleimide or the thiols can be introduced into the protein itself using either iminothiolate or succinimidyl acetyl thioacetate (SATA; Duncan et al., 1983 Anal. Biochem 132, 68). The SATA requires an additional deprotection *step using 0.5 M hydroxylamine.

In addition liposomes can be used to cause an R-loop complex to be delivered to an appropriate intracellular cite by techniques well known in the art. For example, pH-sensitive liposomes (Connor and Huang, 1986 Cancer Res. 46, 3431-3435) can be used to facilitate DNA transfection.

Calcium phosphate mediated or electroporation-mediated delivery of the R-loop complex in to desired cells can also be readily acomplished.

In vitro Selection In vitro selection strategies can be used to select nucleic acids that a) can form stable R-loops b) selectively bind to specific cell surface receptors. These nucleic acids can then be covalently linked to each other.

This will help internalize the R-loop-containing plasmid efficiently using receptor-mediated endocytosis. The in vitro selection (evolution) strategy is 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 •eo* 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 15 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 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 suDra). 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.

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.

•C RNAseP RNA (MI RNA) Size: -290 to 400 nucleotides.

RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA.

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

Hammerhead Ribozyme Size: -13 to 40 nucleotides.

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

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

14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent (Figures 1 and 2) Hairpin Ribozyme Size: -50 nucleotides.

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

SBinds 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 O Cleavage of target RNAs recently demonstrated.

Sequence requirements not fully determined.

Binding sites and structural requirements not fully determined. Only 1 known member of this class. Found in Neurospora VS RNA (Figure 172 Table 2 Human ICAM HH Target sequence nt. Position Target Sequences nt. Position Target Sequences 0 *0.0.

00..

.*0 0

CCCCAGU

CUGAGCU

AkGC-UCCt3

CUCOGC-U

UGC-UACU

UC-aGAGU

C-CAACC'J

UCAGCCU

CCUCGCU

UJAUGGCU

CCGCACTJ

UCCQGG~u

TJCCUGCU

CGGGGC-U

GC'JCrJGU CUCtJtJ

CAGACAU

UCU3GU'tU TJCCCCCt

CAAAAGU

AAGUCAU

GGAGGCU

AGCACCU

CCCAAGU

AAG=UG

UGGGCAU

C GACGCLG C CtJCUGC-U C LG-t7AL= A CtCAA C AGAGUUG UJ GCAACC-' C AGCCT3CG C GCMUJGG A TJGGCUCC C CCAGCAQ C CUGGUCC C CUJGCUCG C GGGGCtC C UGUJUCCC U CCCAGGA C CCAGGAC C UGtJGUCC C CC=-tJCA C AAAAGUJC C AUCCUGC C CUGCCCC C CGtJGCTU') C CtJGUGAC U GUUGGGC U GGGCAUJA A GAGACCC 386 394 420 425 427 450 452.

456 495 510 564 592 607 608 609 6"2 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851.

ACCrjUGU A CUGGCA7i C

CACCC=CU

cUCC-C~J C CCCC-TJCJ U AGAACCU U GAACCt2U A LTJ1=CCU A CCAACCU C UGCMUt C CUCGAGGU C GAGAW C AGCCAJAU U GCCAAUrJ U CCAALJLUU C AAU-74J'CU C GAGCUGU U AGCUGUU UJ AACACCU C GCCCCCU A a C C-a= C CAGACCU U AGACCUU U CCUtJUGU C AGCG-ACt C CACAACtj U AACUUGU C CC-CGGGtJ C C-GG%-UCCtJ A CCGUGU C GGUCUGU U GtJCUGrUr C C-CG%-tJGU U GGCUGUU C UCCCAGU C CCAGtCtJ C CCCAG,-;U C CAGAGGJ U Cr-CACGtJ C GUCACCU A

CUGGACU

CACGAACG

CzC-UCU UUGrCAG

ACCCUAC

CUACG

CGCUGCC-

CGUJC-GC-G

ACCGACCA

ACCAUGG

UCUCUG

CUCGUGC

UCGUGCC

GUGCCGC

UGAGAAC

GAGAACA

GGCCCCC

CCAG-CUC

CAGACCU

UGUCCUG

GUCCUGC

CrUGCCAG

CCCC-ACA

Gt7CAGCC

AG-CCCCC

CUMAGAGG

G-AC-GUGG

UTJ=CCC

CCCtJGGA

CCUGGAC

CCCAGUC

CCAGUCU

TJCC-GAGG

C-G-AGGCC

CACCUCG

WAACCCC

ACCUATJG

UC-C-CAAkC ACCCCGU UJ GCCLUA GUtJGCCUJ A AAAAGGA AAC-GAGU U GCUCCUG AGUUGC C AAGG%-UGU A AGAAGAU- A AUGUGCtJ A GUGCUAU U TJGCUAULTU C GGGCAGU C AACAGCTJ A AAAACCU U AAACCUJU C CCUECCU C Ct7GCCrUG

UGAACUG

GCCAACC

TUUCAAAC

CAAACUG

AAACUGC

AACAGCU

AAAC=tJ

CCUCACC

CUCACCG

ACCGUGU

173 863 866 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 1092 1093 1125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1,295 1306 1321 1334 1344 1351 1353 1366 1367 1368 1380 1388 1398 1402 UJACAGCJ IC ACAGCUU 1C CAGCUUU C ACGLGAU U CGV3GATJU C CAGAGGU C G-A=U C CCACCC!J A ADGGGTGU U UGG=G3 C CCCAGC C CGCAGC-U U GCA~J C AC7IUU C UCCUGCUu C G-CCAGCU U CCAGCU-TU A AGCUUAUJ A C-C-GAGCU U GGCAGCUU C UUCGUGU C GUCCUGU A G-AGGGAU U GGAUUGU C AGAAAAUT U G-AAAAUU C GCAGA.CU C CCAGGCU U AACCCAU U CCGAGCU C CAAGUGU C AGUGUCTJ A UGGCACU U GGC-ACU U GCACL'TJU C UGCCCAU C C-CGGAAU C UG-ACUGU C UGUCACU C

UCCGGCG

CC,-GGC

CGGC-GCC

UGACGA

UCAGAAG

*AGAAGGG

GAGCCAA

*CCAGCCC

CAGCCCA

CUCtGAL

CUCCUGC

UCCUGCt7

C=JCU

UGCAACC

AU~.ACA

MACAA

CACAAGA

CGUGUCC

GUGUCCU

Ct3GUAUTG

UGGCCCC

GUCCGGG

CGGGAAA

CCCAGCA

CCAGCAG

CAAUGUG

GGGGGAA

GCCCGAG

AAGUGUC

LMAAGGA

AAGGAUG

TUCCCACtJ

CCCACUG

CCACGC

GGGGAAU

AGUGACU

ACUCGAG

GAGAUCU

ALCGALCU C CUUJCUCG GACUCCt3 U CUCGGCC ACUCCtJU C UCGGCCA UJC=tCU C GGCCAAG AAGGCCU C AGUCAGU CCUCAGU C AGTJGG GUGCAGU A ATJAZUGG CAGMJAU A CUGGGGA TJGACCAU C UACAGCU ACCAUT-CU A CAXGCUUU 1408 1410 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 151.8 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 TCGAC-AU C TUGAGGG GAGAUCU UJ GAGGGCA GGCACCU A CCUCtJGU CCtJACCtJ C UGUCGGG CCUCUGU C GGGCCAG GAGCACU C AAGGGCGA GGGAGG-U C ZLCC:CGCG AUJGUI C UCCCCCC GUJGC-UCTJ C CCCCCG CCCCC-GU A UGAGAUU AUGAGAU U GUCAUCA AGATUU C AUrCAUCAk UUGtJCAU C AT-CACUG UCAXUCA!U C ACt3GUGG CUGUG,-tJ A GCAGccG CCGCAG;U C AUAAUGG CAGUCAU A AUGGGCA CAGGCCtJ C AGCACGtJ AGCACG;U A CCUCUAU CGtJACCtI C UJAUAACC UACCUCU A UAACCGC CCUCUAU A ACCGCCA GGAAGAU C AAGAAAtJ AAGAAAU A CAGACUA ACAGACEJ A CAACAGG CACGCCt7 C CCUGAAC UGAACCU A UCCCGGG JAACCUAU C CCGGGAC AGGGCCtJ C UUCCUCG GGCCUTCU U CCUCGC GCCUCUU C CUCGGCC U=ttCCU C GGCC UJCGGCCtJ U CCCAUAU CGGCCU C CCALTAUU UJUCCCAU A tJUGGUGG CCCAUATJ U GGUGGC:A AAGACAU A UGCCAUG UGCAGCU A CACCUAC tJACACCU A CCGGCCC AGGGCAUJ U GUCCUCA GCATUGU C CUCAGUC UUGUCCU C AGUCAGA CCUCAGU C AGAUACA GUCAGAU A CAACAGC ACAGCAU U UGGGGCC CACA~TjJ U GGGSGCCA CCAUGGU A CCUGCAC CACACCU A AAACACU AAACACU A GGCCACG 174 1856 i861 1865 1868 1877 1901 1912 1922 1923 1928 1930 1964 1983 1996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2115 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173 2174 2175 2176 2183 2185 2186 2187 CACGCAU C UGAUCUG AUCUGAU C UG%.DGUC G-AUCtJGU A GUCACAU CUGL=~G C ACAUGAC CAUGACU A AGCCAAG CAAGACU C AAGACAU ACNtGAU U GIAUGGAU UGGAUGU U AAAGUCU GrGAUGUU A3 AAGUCOA UUAAAGU C UAGCCtJG AAAGUCU A C-C='LGCAU GAGACAU A GCCCCAC AGGZACAU A CAACUGG GGGAAAU A CUGAAAC UGAAACTJ U G C LUGC tC C-C-UGCr-tJ A UUC-GGUJA UJGCCUAU U GGGUTAUG AkUUGGGU A UGCtJGAG ACAGACU U ACA~vXAG CACUU A CAGAAGA UGGCCCt3 C CAM~GAC CCUCCAU A GACAUGU CA6UGUGU A GCAULCAA GUAGCAU C AAAACAC CCACACtJ U CCUGCG CACACUU C C,;GACGG GCCAGCU U GGGCACU CUGCUGU C UACUGAC GC-UGUCU A CUGACCC CAACCCU. U GAt3GAtJA UGAUGAtI A TJGUMUU G,.ALAU A UEJUATUC UAUGUAU U TALUCAU AUGUAUU U AUUCAUtJ UGUAUUU A TUUCAtJ UAtUUAU U CAUUEJGU AUUUA~Tj' C AUDTGU UAUUCAU U UGUUJAUU AUJUCAUJU U GUUAUWE CAUUEJGU U AUtJUUAC A=UGUU A UUULTACC UUGUUAU U UUACCAG TJGUtJAUE U UACCAGC GUUAUUTU U ACCAGCtJ UUAUE3UU A CCAGCUA ACCAGCU A UtUtiLUG CAGCMJU U UAUUGAG AGCUAUU UJ AUUGAGU CtIAMUJU A UUGAGUG 2189 2196 2198 2199 2200 2201 2205 2210 2220 2224 2226 2233 2 242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 237-9 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 UAUUUAU U GAGUGUjC LGAGUGO C UUU~G AGUG=C U UUAUIGUk GUGUCU U UAL-GLIAG UGuCUUUU AUGtJAGG GUCM=EJ A VGTJAGGC UUUAUGU A C-GCUAA GUAGGC-U A AAUGAAC UGAA-A~U A GaOcuc'j CAUAGGU C UCUC-GCC UA G G ,CU C uGGCCUC CUGCCU-- C ACGGAGC CGGAGCU C CAGUjCC UCCCAGtJ C CAUJGUCA UCCAUGU c AC.AmrUC GUCACAU U cAGu~C UCACAUJU c AAGGUCA UJCAAGGU C ACCAGGU ACCAGGU A CAGDUGU GtUhCAGU U GUACAGG CAGUUGU A C-GGEMUG UACAGGU U GUACACU AGG,-UUGU A CACUGCA AkaAAGAu c AAAIJGGG UGGC-ACU U CUCAZUG GGGACUU C UTCAUUGG G-ACtJUCU C AUUGGCC UJUCUCAt U GGcccAAC CCUGCCU U UCCCCAG CtJGCCU U CCCCAGA UGCCUUU C CCCAGAA GAGMUGA U UUUCE-mU AGUGAUEJU uucu;LUC GUGAUUU U UCCAUCG UGAUUUU U CUAUCGG GAUUUUtj C UAUCGGC UUUUEJCU A UCGGCAC UUUCUJAU c GGCACA AAGCACtJ A UAU.-GGAC GCACUAUJ A UGGACUG GACUGGU A AUGGUtJC UAAUGGU U CACAGGU AAUGGUU C ACAGGUU CACAGGU U CAGAGAU ACAGGtJU C AGAGAUU CAGAGAU U ACCCAGU AGAGAUU A CCCAGUG GAGGCCU u ADiuCcu AGGCCUU A UUCCUCC 5** 2451 2452 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2 545 2568 2579 2585 2588 2591 2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2633 2659 2689 2691 2700 2704 2711 2712 2721 2724 2744

GCCUUTAUI

C CU L7U (J JATJCCU CtJCTU I GACACCU I ACACCUE7 t CCTJTJGU Z

GCCACCUC

CCCACAU

CAULACAU L AUACAUU L UACAUUUt C CCAGE7Gt t CA.GUGUU C UGACACtI C CAGC'GGU C GUCAtJGt C AGGGAAU A CCAAG-Ct7 A UAUGCCU TJ GCCUUGU C tJUGUCCtJ C GUCCUCtI T CUCUUGU C GUCC7GL U UCCUGUE U uuUjGCALU U TUUGCAUE7 U UGCAUJTU C GGGAGCU U UUGCACt A GC:ACtJATJU UGCAGCU C Ct7CCAGU U UCCAGUJU U CCAGtJUU C CAkGLJGAU C UCAGGGU C CCAAGGU A AAGGUAU U GAGGACt7 C ACUCCCU C CCCAGCt7 U CCAGCUUW U GAAGGGU C GGGUCAU C .7 CCtJCCC 3 CCCCCCA

"CCCCCAA

7 UGUUAGC 7 GVUCCC I AGCCACC GCCACCtJ

CCCACCC

LCAUULCU

J UCUGCCA

TCUGCCAG

'UGCCAGU

rCACAAUG ACA6AUGA

AGCGGUJC

AUGUCt7G tJGGACAU tGCCCAA

*UGCCUUG

*GUCCTJCU

CtJCU=G UUGUCCt7 GL7CCt7Gt CUjGUUUG

UGCAUU

GCAUUUC

UCACUGG

CA6CUGGG ACT2GGGA

GCACMU

UUGCAGC

GCAGCt7C CAGtUUC

UJCCUGCA

CCUGCAG

CUGCAGTJ

AGGGtJCC

CUGCAAG

UUGGAGG

GGAGGAC

CCtJCCCA

CCAGCUU

UJGGA6AGG

GGAAGGG

AUCCGCG

CCGU

2750 2759 2761 2765 2769 2797 2803 2804 281.3 2815 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 29213 2914 2915 2916 2917 2918 292.9 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 UJAUG%'TtJ A GACAAGC ACAAiGCtJ C UCGCUCU AAGCUCU7 C C-UCUGEJ TJUCG%-t C TUGUCACC GCUCtJGt C ACCCAGG GUGCAAU C AUGGUUC UCAUGGO U CAkCUGCAk CAUGGUU C -ACUGCAG C'JGCAXGt C UUGACCU GCAGt7CU U G-ACCUUUE Ut7GACCVJ UJ UUGGCtJ UGACCUU U UGGGCt7C GACCtUUU U Gc-GC-UCAk UUGGGCU c AAGuGAU AAGUGAU C CUCCCAC UGAUCCU C CCACCtJC CCCACCU C AGCCUCC t7CAGCCU C CUGAGUA CCUGA~TJ A GCUGGGA GGACCAU A GCUCAC AUTAGGCTJ C ACAACAC GGCAAAU u uGAuturu GCAAATJU U GALuUU AMtJUGAU U UUTUUUUJ UUUGAUU U uuuuuuu Ut7GAUU'U U UUUUUU TUGAUUJTU U M-UUULuU GAUUUUU u L~uuurUU AtJEUUUUUUUU u= UUUUEIJU U UtUU!XJU UUrtuu UUUUUU ttuutut7U u UE7UUUUC UUUUUEJE u ut7uuuct UUUtUUU U UUU7CAG UUtUtUUU U UUUCAC-A UUUUUUtj U UUCAGAG UtUUUUtj U UCAGAGA UUEJUUUU U CAGAGAC UEUUUt7 C AGZAGACG ACGGGG-u c ucGC:AAC GGGGUCU C GCAACAU C-CAACAU U GCCCAGA CCAGACU U CCUUGU CAGACtJU C CUUjUGUG ACUUCCU U UGtIGUEJA CUUCCUU U GUGUUAG UUUGE7GU U AGUUAAU UUGUGUTU A GUUTAAUTA UGUUAGU U AAUTAAAG UGUGUGU A UGUGUAG 176 2966 G~T~rM A AULZU=G 2969 AGtULMAU A AAGCr-UU 2975 UAAAGCU U tTCM-AAC 2976 AAAG'tJU U CUCAACU 2977 AkG%'-M'U C UCAACtJG 2979 GCUUUCU C AACUGCC *.0 177 Table 3 Mouse IOAM HH Target Sequence nt. Position Target Sequence nt. Position Target Sequence en.

C

S.

C

en.

C.

C

C.

C

31 34 48 54 58 64 96 102 108 115 119 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 785 786 792 794 807 833 846 851 CCCUg-GU CaGuGgU uGgt~uCtJ

CUCUGCU

tuCtjcaU gCAcAcU aggACCU tTggGCCU CaTugccu CACCCCtJ Cu cugCU UgCcaGU

CUCUGC

uGGuuCUT C-gaaTUGU Ct3CUGCEJ CAGuCgtJ UTJrJGLTGU UCCugU C;LgAAGU i AAGcCuUC GGuGGgU C gcCACut7 C CagAAGU Z AAGtUrGu L U.GuIGCuJ lu AaCCCaU c ccUGCCU A AgGGuuU c AGggGCtJ C AAGct7GU u AGgAGAU A cUGUGCtJ u GUcCaAU U aGcUgUrJ u GuGCAGU C GGcCJGT U GcC.GUU u UggagG;U C CugGgCt U CUCgGaU a CAaAGcU c CCcugGTJ C GagACCU c C acC-GutG u Ct-'CTGCU C UGCUCtJ CUCcaca a AC-gGUcG U GuAgCCtJ C AGC-CUgG C GugAUGG u UaG%-tCC C CCAGCAG C CUGGcCC a CUGCt~gG C cuGGCcC C tUGCUCCU aaCCAGGA ZCugGccC agCCaCu 1AAAAacC .1 gt~uuUGC

UG-CCC

CGLTGCaG CLTcUGgC TGUUUUrC ruuGCucc GAGAaCu

UCCUAAA

AggAaGA txCTaCrJG CUGCCUa TUGAgCUrG UgagAAC CAcACtJG gAgCrJGa gUCcGCU

UCCUGC

CCuGcCU UJCWL-aG GGAGaCu uAC=tGG GAcaCCC ACCuUG UacCAgC 367 374 375 378 386 394 420 425 427 450 451 456 495 51i0 564 592 607 608 609 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 1353 1366 1367 1368 1380 1388 1398 1402 ~AuGCU U CAACCcg gAAgCCUj U CCt~gC AAgCCUt C CLTgCCCC CuacCaU C ACCGUG;U ACCSGUGU A UtUcGuU CCGGACU u ucGC-L- CACaCuU C CCCcCcg CaCCCCtJ C ccaG:C-G Ca9CtjCU c aGCAGug AGgACCtJ c ACCCT~gC GAAaCcU u ucctuuuG UUACCCEJ c aGCCaCu CUAcCaU C ACCGGu UGC-UGCEJ C CGUGGGG CUCAGGJ a UCCAU-CC GAaA-GAU C ACaugGG AGCCAAU U UtJCaUG GCCAATUE U CEJCaUGC CCAAUUU~ C UCaUjGCC AAUUCrJ C aEGCCGC aAGCE7GU U UGA;LZcug AG,-UGUUj U GAGcugA cgagCCU a GGCCaCC GaCCUCtJ A CCAGCcu UUCAGC-.U C CgGuCCU CCGAcuu U cGauCE~u AGgaCcU c acCCtJGC CCUgUuU C CtJGCCuc gGCGgCU C CaCCuC-A uACAACEJ U uucAGcu AA~tJuu C AGCuCCg aCCaGaU C CUgGAGa iUGC-gCCU c GuGaUGG CaGUcGtJ C cGcUuCC! GGCUtGU U uCCUGcc uJuUGcU C CCUGGAa AGUC-ggU c gAaGgJG UaaCAgtj c uacaACtj aGCAkCcU c CCCACcu GUACUgU a CCACtJcu UGCCCAU C GGGGugg GGaG-AcU C AGUGgCU UGgCOGCU C ACagaAc UG~Jgc-U u GAG-AaCU **see 6 178 863 866 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 1092 1093 1125 1163 1164 1166 1172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 .306 1321 1334 1344 1351 1793 1797 1802 1812 1813 1825 1837 1845 AgCcACU u G;LagCCU TU AUUCgtJU U TUCUUCCJ C AUGGCUt C CCUugGU a ct~auAaU c uAat~cAU u UaACagU C ACagtCtJ A TJACAaCJ U ACAaCUU U CAaCMtjt u ACcaGA.U c uGaGAgT C ugC-AGUT C GAGGUCU C CCACuCJ c AcuGGaU c UGGaccU u CCCAaC7 C CGaAGC7.U GaAGCUU C AGCUt3CU u UCCUGuU u cuCuGCJ c gCuGCUU u AcuTJUuU u GGuAcaJ a GaAGCUU C Ut7CGUuU C GUgCUGU A G;LaGGgU c uGAgaG3 C AGgAgAU a GAggggT C GCAGACJ C gaAGGCUc AACCCAUc auGAGCU C ugAaUGU a UgGUCCtJ C CacCAGU C acCAGAU c ACuGgAU c CAGCAJU U CCAcGcU A CAugCCU u cgAgcCU A CcUCJgG

CUGCCC

cCGGagA augCAAG AacCcGJ gagGUGA ALuCUGG CUG.-uGc TJA.AaCU C~aCUUU U~uCaG%-u uCaGCuC CaGCuCC CUGgaGA UGggGAA UCgGAAG gGAAGGG aAaauAA uCA~gCC CAGCCaA utCUUG-A CUuuUGC UuuUGCU

UUCU

aaaAACC cr.cCA.CA6 UgaACAg CACcAGu CGUGt~gC uuugCU CgGagaG UGGuCCu GUgCaaG uGGGgAA CugAGCc uCAGCAG ugAaaUG aGGaGgA UCCuaAa gAGaGUg UAAguuA gGcugGA AcAUAaA CuggAGa UcaGGCC acccuCA CCtucugC uAgCuc 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 gCGAGAY( GAGgU=Jc ccCACC I aCUgCCti uCJCt~at, GAaggCUC GGaAuGY C AguUGuU cUGuE7CU L Cu1-guC-ctj L AUGAaAU c gGAcUAU a UUaUguU u cuAcCAt7 C ucaUGG.-U c CuauAal C ugGUCALU u CAUGCCJ u AGCACcU c CUtUhngU u UAugTuUU A UgUuUAI A GaAAGAU C AgGAUAU A ACAaguU A CcCaC=t C gaAACCJ u AACCE~uU C AGGaCCU C aGCCaCU U GCCaCUU C aCtutCCU C cCGGaCU U CGGaCJU u UgCCCAU c CggAUAU a gAG-ACcJ c gGCgGCU c gAagCCU u gaGaCAU u GCAUJUGU u UJUagagU U7 UagagUU U agagUUJ U gagtJUUU A ACCAGCtJ A CAGCUAU- U AGCUAUU U GCTJAtUU A 99gGgaGG GgaaGgg LCuUUUGJ I gGt~aGaG iGccCCuG AgGaGGA ACCaGga LUgOCCC LCCuCauG LUGAGAac aliggUJCc X UCAUuc LAUaACcG ACCGUGu *cCAGgCG AUuctJGG *gtJGGGCc AGCAgcU CCcaccU UAtMACC UAA~fCGC

ACCGCCA

AgGAuAU CAaguUA CAgaAGG CCTJGAgC UCCuuuG CuuuGAa a9CCUgG CCEJCuGg CUCuGgC uGgCT~gu uCgAUcU CgAUcUU ggGGUGG ccUGag UaCCAgc CACCUca CCuGCCC GUCCcCA CUCuaau

UTJACCAG

UJACCAGC

ACCAGCU

CCAGCUA

UUUAUJG

UAUUGAG

ATJUGAGU

TJUGAGUa 179 0 *0 i856 1861 i865 1868 1877 1901 191 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 2417 2418 2425 2426 2433 2434 2448 2449 CggaCuU u cGAXJCUu AcaJGAU a UccAGUa cAcuUGU A GcCuCAg CaccAGU C ACAflaAa CAUGcCtU u AGCagcu uAAaACU C AAGggAc AuAI~agU a GAUcagri TJGaAtIGU a uAMtiua uGAUGctJ c AgGUaUc UUAgAGU u UuaCCaG AgAGUutJ u aCCaGcU CAGC-AU u GuCCCca AGGAuAU A CALAgE~ua aGGSAgAU A CtG-AgcC UGgAgCU a GCgGaC-c Gctauu3 A UUaaUA UGCCcAtJ c GGGgugG ggUGGuU c UuCUGG gCuGgCU a gCAGAgG CuGACcU c CuGgAGg TJ-,cuCC'J C CAcAucC- CuaCCALU c acCg3GU CAcuTGt A GCcLCAg GUAGCcU C AgAgCua CaACuCEJ U CuUGAuG CACAXC=t C Cccc-CG GCCANG=t c GGaggaU CaGCUaU u tJLuUGAg cCUGUuU c Ct3GcCuC C-uCtJu U cuUG.Iug Uaut~aAU u UagAgUU uugATUTtJ A UUtkUUa gAU~JAU U UAUTaAtJ 2189 21.96 2198 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 TJAUtUUAU U G-AGUacC caACtUct u CUUgAIJG gcaGcCU c L"UAUG,,u GccUCUu a UguuAu Uct~uccU. c AUGCAaG aagUJUU A UGUcGGC UULJAUGLI c GGCcugA GgA~aCtJ c AgUGgcu.

cugg9CAU u O GUECiC Cuic-AG,.t a UCcauCC UgGaUCtJ C aC-OcCgC CtJGaCC'J C cuGGAGg uC-C-G-U a gCgGaCC tUauCcaU C CA7UccCA UCCMaUU C ACAcUgA a7CACAUJ U C~.cGG-Ug Ur-CACkUU C ACGUgc 9gAAUGU C ACCAGGa ACCAGaIJ c CtiGgaGa GaAggGtJ c GTUgCAaG aAGcUGU u ugaGcuG UAuAaGu u allggcCU caGUgGU ui CUCUGCu gAAAGAU C AcAIJGGG UGaGACU c CUgccLJG GaaACcU uU tCcUUuG GACCt3CU a ccaGcCu UUuc9AU c uuCCAgC CCcagCtJ c UCagC-AG CUCUUU U gaaCAGA aaCCUU C CuuuGAA agGUGgU U cTEJCtga gGUGgUEJ c UUCUgag agGgJUU c UCtJACUG UGctUtJU c ucAuaaG aAgJUUU a ugucGGC alUUCU A UuGcCcC allcCagu a GaCACAA AAaCACU A UgtJGGAC aagCtjgU u UGagCTUG uACtJGUt c AgGaugC AAuGt~cu c cG-AGGcc GAaGcctT u ccugCc gacCuCU a cCAGCcU CCC-AGCU c UcagcaG gagGucU c GGAAGGG GAAGCGt, C gUgCaaG GGuaCAU a CGuGUGc gG;UGgGU c cGtJGcAG AUGtUdJU U UJGLTAUU A UAMUUUAU U A~TUtU u acuucAU U AU9UATUU U UAUUUaU U AgUUGtJU u gAAUGGU a AcUGGaU C CAugGGU c AkuuaaUtI u uAC-AGuU U AG-AGutJU u CGAaGCCU U AaGCCtJU c AUUaAU UUaAUU aAtJUUag AUUaaTU cuclAtU aUQAaTU AaUULUAg UgcUcCC CAuAcGEJ uCAGGcc gAGgGuty AGAGuTU uaCCAGc aCCA.Gcu CCUgcC cUgcCC 180 2451 Gc~ugut7 u CCUgCCU 2750 UAUtuUau u GAguAcc 2452 CCEuguuT C CugCCtJc 2759 ccggaCtJ u UC-aJCt 2455 gAagCCU u CCUgCCC 2761 AgGaccri C ~C~ 2459 CCa~aC3 U CCCCCc 2765 UuUuGCtJ C UGcCgCu 2460 CaCaCUU C CCCCCcg 2769 agJCUGtT C AaaCAGG 2479 GAgACCU c UaccAGC 2797 allGaAAU C AUGGt~cC 2480 uCACCgE7 U GUgAuCC 2803 UCAUGGtJ c CcagGCg 2483 CCaaJGu c AGCCACC 2804 ggUGC~gU C cgUGC.AG 2484 Ctutuu c aCcAguc 2813 CUcCgC-U C CUiGACCc 2492 agCACCt3 C CCC-ACCu 28i5 aCAGtJCU a ckaCUtJ 2504 CCCACcEI A CUUUgU 2821 cCGAC-tJ c cU-GGagg 2508 uAUcCAtJ c caUcCCA 2822 gGAgCcU a cGGaCJu 2509 uUAgkgt U uUaCCAG 2823 ugC(JJ a GcaCcCAk 2510 UJAgAgUU u UaCCA~c 2829 cUGGaCU a uAatcAti 2520 CuuuUGU U CcCAAUG 2837 AgGtJGgU u Ctuouga 2521 cAGcaUtJ u ACccUcA 2840 tJGAgaCU C CugCCt~g 2533 UG~ugCU C AGguaUC 2847 CCaAugU C AGCCaCC .2540 CAXGCaGU C cgcUgUG 2853 gCAGCC-3 C uUauGUu 2545 GL~gcUGt7 a UGGuCcU 2860 gCcaAGU A aCt~uGA 2568 guGaAgU c UGuCaAA 2872 GGAC~u3 c aGCca.Ag 2579 auAA~ut3 A UGgCcTG 2877 uUccC-U a cC uCAC *2585 cugGCaU U GUuCt7CU 2899 cGgAcuU U CAUcUU 2588 GCaUUGU u CUCr~aaU 2900 uuAAuUU a G~gUUU 2591 UgGUuCU C UgcUCC 2904 AcUUc.AU U cUctlaUtl 2593 cTuCUuJ U GcuCUGC 2905 ctUUU c U~cUaUjUg .2596 CUuUUGU u CccaaUG 2906 tJUGAUgU a UL JaUi a *2601 acCgUGU a UuCg9UU 2907 UJGuaUUTU a TUUaaUUJ 2602 T-CCaGc3 a cCAUccC 2908 GAagctjU c UUU-UgcU 2607 cUcGgAJ a UacCUGG 2909 AgcUUcU U UUgcUcU .*:2608 caGCAgU c CgCUGuG 2910 UgUaUJU a UUaaUU 002609 gGaAI~gU C ACcaGGA 2911 UgUa~Ut a TJUaaUUU 2620 aGCGAcCU c aCcCUgc 291-2 UUgUUcU c UaaUgUC 2626 UUuCgaU c UJUcCAGC 2913 UUUcUcU a cr-ggUCA *2628 GCACacU U GuAGCcu 2914 UgctUUUU c UcaUaAG 2635 UuCAGCU C CgGUccu 2915 allUUaUUt a a~Tj-uAGA 2640 ggC~uGU U UCCUGCc 2916 UatitcgU U UcCgGAG 2641 cCCAGcU c uCaGCAG 2917 aUlUcgUU U cCgGG 2642 CCuGUUUJ C CUGCcuc 2918 UjTcgUTj'U c CgGAGAg 2653 uAcUGgU C AGGaUgC 2919 tJUcUcaU a AGgGuCG 2659 gaAGGGU C gUGCAAG 2931 ugGaGGU C UCGgAAg 2689 CuAAuGJ c UccGAGG 2933 GaGGUCU C GgAAggg 2941 GagACAU U GuCCccA 2951 CCAcgCU a CCUcUGC 2952 C-AGcagU C CgcIGUJG 2955 AgUgaCJ c UGUGUcA 2956 uTjUCCtJU U GaaUcAa 2961 UcUGUGU c AGccAcU 2962 allGUaUU u alUAAUu 2965 UuUgAaU c AAUAAAG 2966 GcUgGCTJ A gCAgAGg 2969 A-a~cAAU A AAGuUjU 2975 MkgAGUU UJ UacCAgC 2976 gAgGgUU U CUCuACtJ 2977 AAGCUgU u UgAgCtJG 2979 uCaUtJCt C uAut3GCC Sof 00 182 Table 4 Human IOAM HH Ribozyme Sequences nt. Position Ribozyme Sequence 11 CA=WctC cMUGAG GCGAAAGGc;cGAA ACUGcOOG 23 AGCAAG CUGAUGAGAAGGCCGA

AGCUCAG

26 AGUAGCA CCCAUGAGGCCAAAGC-CCA AGGAGCtJ 31 CtJCUGAG CUC-AGAGCCCGAAACGCCGAA

AGCAGAG

34 CAACTJCU CUGAUGAGGCCAAAGGCCGA A~Az AGU CUCGAUGAGGCCA.uGfAAA :uc UCA 48 CGAGGCY CUJGAUGAGGCMAACCCGAA AG7tIUC-C 54 CCAt3AGC CUG-AUGAGGC-CGA.AGG-CCG;L AGG-u-GA 58 GGAGCCA CUGAUGAGGCCGAAAC-CCGAA

AGCGAGG

64 CM7CUIGG CLTGA!3GAGGCCGAA G~C-GAA AGCCr_-JA 96 GGACCAG CUGAUGAGGCCGAAAGCcG--A

AGUCG

102 CGAAGCAG CUGAUGAGGCC G- 'CGAA ACCAGGA, *108 GAGCCCC CLMAUGGCCGAAAGGCCGAA

AGCAGGA

119 UCCUGGG CLM-AUGAGCCAAGCCA

ACAGC

4120 GUCCt7GG CU GAUGAGGCCGAAGGCCA

ACAG

146 GGACACA CrUA=G-~-3CA

AUGUCUG

152 T7G-AGGGG, CLUGAUGAGGCCG-AA ,--CCAA ACACAGA 158 GACtUtUrJ CUGAUGxAGCCGC-AACGCG

AGGGGGA

*165 GCAGGAU CLMUGAGGCCGAAAGCG.

ACUUUUG

i 68 GGGGCAG CUGALtGAGGCCG-AA G.CCGAA AUGACtit *185 CAGCACG CUGAUGAGGCCGAAAGCCAA

AGCCUCC

209 GUCACAC CUGAUGAGGCCGAA GGCCGAA AGGUGCtJ 227 GCCCAAC C 3GAUGAGGCCGAAGOCGAA

ACUUEGGG

230 tJAUGCCC CEJGAEGGGCCAAAGGCCA

ACAACUUE

*237 GGGUCYC CUGAUIGAGGCC-AAACCCGA

AUGCCCA

248 EUUAGGC CUGAU jGGCCAAAGCC

ACGGGGU

253 UC-UUUT CUGAUGAGGCcCAAGCC-A

AGGCAAC

263 CAGSAGC CUGAUGAGGCCG-AAAGGC-CGAA

ACEJCCUUE

267 CAGGCAG CLGAIUGAGGccGAAGGCGAA AGCAACrJ *a293 CAGEJUCA C G-AuGAGGccG AA GCA ACACCLTrj 319 GGUuGGC Ct3GAuGAGGCCGAA -GcC-G AucEJucr 335 GUUJEGAA. CUGAUGAGGCC-GAAAGGCCG-A

AGCACAU

337 CAGUMJG Ct7G-AUGALGGCCGA .GGCCC-AA

AUAGCAC

338 GCAGUUU CUGAUGAGGCCG A.CCGAA

AAUAGCA

359 AGcuGuu cuGAtJGAGGccGAAGGc-GAA ACrjGCCC 367 AAGGUUU CUGALUGAGGCCGAAGCGAA

AGCEJGUU

374 GGEJGAGG CtYGAGAGGCCGAA CCA AGtJEJUjr 375 CGGEJGAG CUG-AUGAGr.CCCGAAG.GCCGAA

AAGGUEU

378 ACACGE CUGAL-GAGG.CCC-.A GrCCGAA AGGAAGG 386 AGUCCAG CUG:-ATUGAGGCCGAAAGCCGA

ACACGGU

394 CGUUCEJG CUC-AUGAGGCCGAAAGGCCGAA

AGUCCAG

420 AAGAGGG CUE AUGAGGCC .ALGGCCGAA

AGGGGUG

425 CEJGCCAA CUGAUGAGGCCC AGGCCCGAA

AGGGGAG

qW 427 GCuUGC- CUGAUAGGCCGAAAGGCCGAA AGAGGGG 450 GTkGGGU CUAGGCA AGGCCA ~UTCU 451 CGLMZ.GG CUCGAIr.AGGCCGAAAGGCCGAA AAGGUUC 456 GGCAGCG CU.GAUGAGGCCGAAAGGCCGAA AGGGTJA 495 CCACGGt7 CUGAtGAC-GCCGAAAGGCCGALA AGGUUGG 510 CCCCACG CUGATJGAGGCCGAAAGCGAA AGCGC.

564 UG UGAGGCGAAGCA ACCMG 592 CCAUGG-U CUGAUGAGGcCGAAAGGCCGA.A AUCEICUC 607 CACGAGA CUAGGCGA~C AM UGGCtJ 608 GCACGAG CUGA6UGAGGCCGA.AAGGCC,'"AA AAUUGGC 609 C-GCACr-A CMAGGCCAAC%7-G AAAUUGG 611 GC~GCA CUGAL7CV'%=CGAGGCCGA A~-AATU 656 GUtJCUCA C~';UAGCAAGCA ACAGCtJC 657 UGUUC CUGALI GCCGAAAGGCCGAA AACA=C 668 GGGGGCC CUG;tLAGGCCGAAAGGCCCGAA AGGUGUU 677 GAGCtJGG CUGAUGAGGCCGAAAGGCCGAA AGGGGGC 684 AGGU=tG CTGAUGAGGGAAAGGCCCGAA ACUGGU ~.692 CAGGACA CM3AUGAGGCCGAAAGGCCGAA AGGCUtG 693 GCAGGAC CUGA.UGA=GCGAAAGGCCGAA AAGGUCU *696 CCGGCAG CtJGAUGAGGCCGAAAGGCCGAA ACAAAGG *709 UGUGGGG CTJGAUGAGGCCGALAAGGCCGAA AGUCGCU 720 GGCUGAC CMUG!GAGGCCGAAAGGCCGAA AGUUGUG 723 GGGGGCU CUGAUGAGGCCGAAAGGCCGAA ACAAGUtJ 735 CCUCUAG C3GAIGAGGCCGAAAGGCCGAA ACCCGGG *738 CCACCtJC CUGAUGAGGCCGAAAGGCCGAA AGGACC 765 GGCAACA CtGALGAGGC-CGAAAGGCCGA6A ACC-A=G 769 UCCAGGG CUGAUG=GCGAAAGGCCGAA ACAGACC 770 Gt7CCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGAC 785 GACUGGG CUGAUGAGGCCGAAAGGCCGAA AcAGccc 786 AGACtJGG CUAGGCGAGCGAAACAGCC 792 CCUCCGA CUGAUGGGCGAAAGGCCGAA ACUGGGA 794 GGCCUCC CfJGAtIAGGCCGAAAGGCCGAA AGACUGG 807 CCAGGUG Ct3GALUGAGCGAAAGGCCGAA ACCUGGG ~833 GGGGUtJC CUGAUGAGGCCGAAAGGCCGAA AcctJctG *846 CAUAGGU CUGAUJGAGGCCGAAAGGCCGAA ACLUG3GG *851 GVUGCCA Ct3GAUGAGGCCGAAAGGCCGAA AGGtJGAC 863 CGAGAAG CtJGAt3GAGGCCGAAAGGCCGAA AGtJcGUU 866 GGCCGAG CUGAUGAGGCGAAAGGCCGAA AGGAGUC 867 UGGCCG-A Ct3GAUGAGGCGAAAGGCCGAA AAGGAGU 869 CUUGGCC CUGAUGAGGCCGAAAGGCCGAA AGAAGGA, 881 ACUGACtI CUGA6UGAGGCCGAAAGGCCGAA AGGCCUU 885 UCACA6CU CUTGAUIGAGCGAAAGGCCGAA ACOGAGG 933 CCAGUAU CUGAUGAGGCCGAAAGGCCGAA ACUGCAC 936 TUCCCC-AG CUGAUGAGGCCGAAAGGCCGAA AUTjALCrG 978 AGCtJGTA CtJGAUGAGGCCGAAAGGCCGAA AUGGt3CA 980 AAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGAUGGU 986 CGCCGG-A CUGAUGAC-GCCGAAAGGCCGAA AGCUGUA 987 GCGCCGG CUGAUGAGGCCGAAAGGCCGAA AAGCTJGU 988 GGCGCCG CUGAUGAGGCCGAAAGGCCGAA AAAGCTJG 184 w1005 UCGt3CAG CUGrAGGCCGAAAGGCCGAA A;urcACGtJ 1006 UU=GECA CUGAUGAGGCCGAAAGGCCGAA

AA.UCACG

1023 CUUCUTGA Ct3GAfGAGGCCGAAAGGCCGAA

ACCUCUG

1025 CCCUUCU CUGAUGAGGCCGAAAGGCCGAA AGACCtIC 1066 UTJGGCUC CLtGAUGAGGCCGAA GGCCGAA AGGGUGG 1092 GGGCtJGG CUGATJGAGGCCGAAAGGCCGA.

ACCCCAUJ

1093 TGGCCUG Ct3GAtGAGGCCG-AAAGGCCGAA

AACCCCA

1125 UJCAGCAG CUGA~raAGCCGAAAGGCCGA~

AC-C-UGGG

1163 GCAGGAG CUGGAGCCGAAAGGCCGAA

ACU-YGCG

1164 AGCAGGA CGAUGACGCCGAAAGCCGCAA

AAG%--JGC

1166 AGA.GCAG C=GAGGCCGkGCCA

AGAAGZUJ

1172 GGUUGC- CUGAAGcGAGGCCGA 2.ACAGC-; 1200Uo CtYGLUAGGCCGAAGGCcG AGCtJGGC 1201 UUGUGUA CUGAUAGGCCGAAAGGCGAA

AAG--UGG

1203 uU~uGUG CUGAUAGGCCGA.AAGc-ccAA,

AUAA~CJ

1227 GGACACG Ct3GAAGGCCGAAAGGCCGAA AGCtccoo:1228 AGGACAC CUGAUGrAGGCCGAAAGGCCGAA ;ULC=CC ::.1233 CAUACAG CUGALrAGGCCGAAAGGCCGAA ACACGA16.

1238 GGGGCCA CUGAUGAGGCCGAAAGGCCGAA

ACAGGAC

*1264 CCCGGAC Ct7GAUGAGGCCGAAAGGCCGAA At3CCCEJC 1267 Ut3UCCCG CtJGAI2GAGGCCGXAAGGCCGAA ACA6AUJCC 1294 UCJGGG CUGAUGAGGCCG AAGCCCA AUUU 1295 CtC1G CUGAUGAGGCCGAAAGGCCGA AAUUrUC 1306 CACATUG aUGAtdX=AAGGCcGA Gc AGE3UGC *1321 TUuCCCCC CUGAUGAGGCCGAAAGGCCGA

AGCCUGG

*1334 CUCGGGC CUGAUAGGCCGAMJGGCCCGA6 kt;GGrj-U *1344 GACACUU CGUAGCAAGCA

GUG

1351 UCCUUtUA CUGAUGAGGCCGAAAGGCCGAA ACACUtjG 1353 CAUCCtJU CUGAUGAGGCCGAAAGGCCGAA

AGACACU

*~1366 AGQGGGA CU GA GGCCGAAGGCCGA AGtJGCCA 1367 CAGUGGG CUGAUGAGGCCGAAAGGCCGA

AAGUGCC

1368 GCAGUGG CUGA!trAGGCCGAAGGCCGA

AAAGUG~C

1380 AUUCCCC CUGAUGAGGCCGAAAGGCCA AtJGGGA 1388 AGtJCACU CUGAUGAGGCCGAAAGGCCGA6

AUUCCCC

1398 CUCGAGU CtJGAtGAGGCCGAA GCGAA ACAGUCA6 **1402 AGA.UCUC CUGAUGAGGCCGAAGGCCA AGUGAcA 1408 CCCtJCAA CUGAUGAGG;CCGAAGGCCGA AUCrICGA 1410 UGCCCUC CUGALtJGAGGCCGAAGGCCGAA A-AtIcuc 1421 ACAGAGG CUGAUGAGGCCGAG~CA AGCGcc 1425 CCCG-ACA CUGAUGAGGCCGAAAGGCCQAA

AGGUAGG

1429 Ct3GGCCC CUGAUGAGGcGAAAGGCCA

ACAGAGG

1444 UCCCCtJU CUGAUGAGGCCAA GCcGAA AGUGCUC 1455 CGCGGGU cuGAU-GAGGCCGA GGrcCGAA AcctJccc 1482 GGGGGGA CUGALGAGGCCGAA GGCCGAA .AGCACAU 1484 CCGGGGG cUGAuGAGGCCGAAALGGccG.AA

AGAGG-AC

1493 A.UCUCA CUGAGAGGCCGAAGGCCA

ACCGGGG

1500 UGAUGAC CUGAUGAGGCCAGCG AUCUCAtJ 1503 UGAUGAU CUr-AUAGGCCGAAGGC

ACAAUCU

1506 CAGUGAU CtJGAUGAGGCCGAAAGGCCGAA AUGACAA6 185 4 9*

S.

S..

K.

1509 1518 1530 1533 1551 i35;9 1563 1565 1567 1584 1592 i599 i651 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 i922 1923 1928 1930 1964 1983 ccACAGtJ CDGPAUGAGGCCGuAAAGGCCWA CGGCUGC MUGA CGAAAGGCCGAA CCAflrAUI CUGAUGAAGGCCGAA UGCCCAX3 CUGAGGCCGAAAGCCGA ACGUGCU UAAGCAACGA AL~iGAGG CGtAGCAAGCA GGuUA CT3GAUGAGGCCGAAGGCGAA GCGGUUTA G!AGCAACGA LUG-C-G7 CLGAUGAGGCCGAAAGGCCGA ALUUtU CMAUAGGCCGAAAGCCGAA UAGUCUG CUGA~rGAGGCCGAAAGGCCGAA

CCUGUUTG

GUtJCAGG

CCCGGGA

GtJCCCGG

CGAGGAA

GCCGAGG

GGCCGAG

GAAGGCC

ATJAUGGG

AA!T.UGG

CCACCAA

UGCCACC

CAUGGCA

GU=.GU

GGGCCGG

UGAGGAC

GACUGAG

UCUGACtJ

GUGUUGU

GGCCCCA

tTGGCCCC

GUGCAGG

AGUGUUU

CGUGGCC

CAGAUCA

GACUACA

AUGUGAC

GUCAUGU

CUUGGCU

AUIGUCU

AUJCCAUC

AGACUUU

UAGACUU

CAGGCUA

AUCAGGC

GUGGGGC

CCAGUUG

CtJGAUGAGGCCAGGCGA AGUCUGU CUGAUGzAGGCCGAAAGGCCGAA AGCGUG CUGArGAGGCCG-AAAGGCCGAA AkGGUUCA CUGAUGAGGCCGAAAGGCCGAA AUA$GTJU CtJGAUGAGGCCGAAAGGCCGAA AGGCCCU CUGzAUGAGGCCGAAAGGCCGAA AGAGGCC CUGAUGAGGCCGAAAGGCCGAA AAGAGGC CUGALGAGGCCGAAAGGCCGAA6 AGGAAGA CUGAUGAGGCCGAAAGGCCGAA AGGCCGA CUGGAGGCCGAAAGCCGAA AAGGCCG CtJGAUGAGGCCGAAAGGCCGAA AUCGGAA CUGAUGGGGGCCGAA AUTAUGG CUGAVGAGGCCGAAAGGCCCGAA AUGUCUEJ COGA~AGGCCGAAAGGCCGA6A AGCUGCA CUGAUGAGGCCGAAAGGCCGAA AGGUGUA CUGAUGAG-GCCGAAAGGCCGA.A AUGCCCU CUGAulGAGGCCGAAAGGCCGAA ACAAUGC CUGADGAGGCCGAAAGGCCGAvA AGGACAA CtC4GAUGAGGCCGAAAGGCCGAA ACUGAGG CUGaAUGAGGCCGAAAGGCCGAA AUCUGAC CtrGAUGAGGCCGAAAGGCCGAA AuGCt3Gu CUGATJGAGGCCGAAAGGCCGAA AAIJGCuG CUGATJGAGGCCGAAAGGCCGAA6 ACCAUGG CUGAUGAGGCCGAAAGGCCGAA AGGJGUG CrJGAUGAGGCCGAAAGGCCGAA AGuGtuuu CUGAUGAGGCCGAAAGGCC G AUGCGUG CtJGAUGAGGCCGAAAGGCCGAA AUCAGAU CUGAUAGGCCGAAAGGCCGAAL ACAGAUiC CUGAUGAGGCCGAAAGGCCGAA ACUACAG CGAUGAGGCCGAAAGGCCGAA AGucAUjG Ct3GAWAGGCCGAAAGGCCGAA AGUCtJUG CUGAIJGAGGCCGAAAGGCCGAA AUCAUGU CUGAUGAGGCCGAAAGG.CCGAA

ACAUCCA

CUGAUGAGGCCGAAAGGCCGAA AACAUCC CUGA~ruXGGCCGAAAGGCCGAA ACUUUAA CUGAUGAGGCCGAAAGGCCCGAA AGAQUUU CUGAUGAGGCCGAAAGGCCGAA AUGUC CUGAUGAGGCCGAAAGGCCGAA6 AUGtJCCU

AUGAUGA

ACCACAG

ACUGCGG

AUJGACUG

AGGCCUG

ACGUJGC-U

AGGUACG

AGAGGUjEA

AUCUCCC

ALTUUCrjtX 186 W i996 GUUUCAG CtJGAL3AGGCCGAAGGCCGAA AtJCUCCC 2005 AGGCAGC CUAGC,-G -CA AGUCA 2013 UACCCAA CtUGAfGAGGCC--GAAAGGC-CGAA

AGGCAGC

2015 CAUIACCC CUGAUGGC-CGAAAGGCCGAA AtIJAGCA 2020 CUCAGCA CUGAVGAC-C-CCGZAACGCC-AA

ACCCAAU

2039 CUUCUGU C GrAGGCCGAAAGCGAA AGUCUGrJ 2040 U~3CGCUGAZtGAGGCCGAAAGGCCGAA AAGUCtJG 2057 GtJCUAUG CUGNAACGCCGAAGGCGAA

AGGGCCZA

2061 ACAUGC CUGAUGCAG%-C-CGAAAGGCCCGAA

AUGGAGG

2071 UUGAEJGC CUGAUGACGCCGXAAGCCGAA ACACAtG 2076 GuGUJUUU CUGAUC-AGGCCCAAAGGCCC-A. AUGtIAC 2097 Cr--JCAGQ CUGAMCAGGCCG--AAAC-GCCC-AA

AGUGCGG

2098 C--GUCAG CUGAUAGGCGA AAGw~~UGUG 2115 A~TUGCCC CUGA;UGAGGCCGAAAGGCCGA.A

AGCUGGC

2128 TJGUA CUGALIGAGGc-CGAAAGGCGAA AcA6GcAG 2130 GGGUCAG CUGAUGAGGCCGAA GCCGAA AACAGC 2145 UAUCAUC CUG AGCCGAAAGGCCGAA

AGGG%-UUG

.2152 AAAIUACA Ct3GAUG A-GCCGAAAGCCGAA

AUCAUCA

2156 GAAUAAA C! GA;GAGCCC-AA C-C%-CGAA ACAAUAUC 2158 AUGAAUA CUJGAZGAGG-CGAACGCCGAA

ALMCAUJA

2159 AAUGAAU CUGAUGAGGCCGAAGc-cccGA AAUjAcAt 2160 AAAUGAA CUAxCG-"C CA AAAUACA 2162 ACAA6AUG CUGAUGGC-CCAAAGGCCGAA,

AUAAAUJA

2163 AACAAATU CUGAL'GAGGCCGA AGGCCGAA AAUAAAUr 2166 AAI3AACA CUC-AVGAGGCCAAGCG

AUGAAU.A

2167 AAAM~AC CUGAtGACGCCGAAAGCCGAA. AAt7CAA6U 9:2170 G7UAAAALU M GAUGAGGCCCGAAAGG-cCGAA ACAA6AUG 2171 GGGAA CUGAUGAGGCcG-AAAc-CCA. AAA6AU 2173 CtrGUAA CUC-AUC-AGGCCCAA C-GCCCGAA AUiAACAA *2174 GCUGGCA CLTG'tr.C=GA-------A

AUAC

2175 AGCUGGU CUGAW-NGGCCGAAAGC-CCG~

AAAIUAAC

2176 UALGCUGG CUGAUGAGGCCAAGAC

AAAALTAA

2183 CAAUAAA CUGAUGAGGCCGAA Zccc-AA AGC-UGGtJ ~2185 CUCAAUJA C A tJAGCCAACCCA AUjAGCUG 9..2186 ACUCAAU CUG GAC-CC^.AGGCCGA AAUTAGCt 2187 CAC13CAA CUGAXJGAGCCAAAG CCGAA AAAJAC 2189 GACACUC Ct3GATMAGGCCGAA GCCCGA AUAAAUA 2196 CAUAAAA CUGAUGAGGCCCAAG-CCGA

ACACUCAL

2198 UACA6ThAA CUGAL7GAGGCCGA CCW.A AGACACU 2199 CUJACAUA CUGAUGAGGCC ,GGCCGAA

AAAA

2200 CCUALCAU CUGAUGAGGccGAAAGCGA AACrACA 2201 GCCUACA. CTJGAUGAGGCCGAAAGG-CCGAA

AAAAGAC

2205 UUUAGCC CUGAUrAGC-AGGCGAA~~

ACAUTAAA

2210 GUUCAUU CtJGAUGAGGCCCAA GGCCGAA AGCCUAC 2220 AGAGACC CUGAUGAXGGCC, AAGCCGAA

AUJGUUCA

2224 C-GCCAGAL CUGAUAGGCCC-A.-GAA L ACCUAUG 2226 GAGGCCA CUGA-GAcGCCGACA

AGACCEIA

2233 GC-UCCGU CUGALUGAGGCC .AAGCGAA AGGCCAG 2242 C-G-ACUGG' CUGAW-Ar-GCCGACCCGA

AGCUCCG

187 w2248 UGACAtJG CUGA GA-GCCGAAAGCGc ALCUGGGA 2254 UGAA=~t CUGAGAG~CCGAAAGGCCCAA

ACAUCGA

2259 GACCUG CUGAGCCGAAAGOCCGL

AUGEJGAC

2260 UGAC=tt CtGAI]GAGGAAA CCGAA AAUGtJGA 2266 ACCUGGU CUGAUGAGGc~Cc tAGCGJ ACCL3UA 2274 AC.ACUG CLUGAUGAGGCCGAAAG-GCCGAA

ALCCUGG;U

2279 CCUGCAC CUGAUGAGGCCGAAG CG ACtGM~C 2282 CAACCt3G CU-DAGCAAGCA ACALCtJG 2288 AGU.GAC CUGAGGC-AGGCcGA ACCJG-LgL 2291 UGCA=UG CUGAM GGCCGXUMCGA

ACAACCU

2321 CCCAUUU CUjGAflGAG:7,CCAAAGGCCG-AA AEJCUuuEJ 2338 CAAUGAG CrUr AGCCCAAGGCCGA AGtJCC.

2 33 9 CCAA CUC-AUAG--CG AGGCCGA;LA

GUCCC-

2341 GGCCAAU CUC-ALUAGGCCG flG~cCGAA AGAGJC 2344 GUt3GGCC CUGAUGAGGCCGAAGCGrAA 2358 CUGGGGA M-AUGAGGC-CGA AGCCGAA tAG **2359 UCUGGGG CUGAUGAGGCCGAGGCCA;

AAGGCAG

:2360 TUCUGGG CE3GAUAZGCCGAA rcrA AAAGGcc 2376 AtU.GAAA Ct3GAUGAcrCC aUGCGAA

AUC.CTUC

**..2377 GAEMCAA CUGALX AGGCCG AGCCGAA AAUCACrJ 2378 CGAEJAGA COGAX GAGrGcCGAA CCGAA AAAUC:AC 0:.2379 CCG.AUAG CUGAVGAGGC CG CAA AAAAUCA *002380 GCCGPJ3A CUCGA GGCCGAAGCGAA

AAAAAUC

2382 GUTGCCCGA CUGAUGAGGCCGAA GG~-A AGAAAAA *2384 UUGt3GCC CUAGGCGAAGCA

AUACA

2399 Gt7CCAA CJGA6UG GGCCGAAAGGCGA

AGUGCUU

2401 CAGUCCA CUGAUGAGGCCGAAAG.CGLA~ AtUhGUGC 2411 GAACCAU CUGLUAUGGA AAGGCUG

ACCAGEJC

2417 ACCUGUG CLGAUGAGGcCGAGccA ACCAuLTA *:.2418 AACCUGTJ cuGGAGGCCGAAACCCA

AACCATU

ow.2425 AUCtJCtG Ct3GA~A GGAAA G CGAA ACCtJGUG ow.2426 AAUCUCtJ CUGALGAGCAGCC CGA AACCUGU 2433 ACUGGG CUGAD AGGCCGAAGCCGAA

AUCUCUG

*2434 CACUGGG CU'UAGCAAGCA AAUCtJCr 2448 GAGAAU CLTGAUGAGGCCGAAAGG CGAA AGGCCtJC *:.:2449 GGAGGA. curGAuGAGGcC cGAcrA AAGG-CCt 2451 AGGGAGG CUGAUGAGGCCGAAGGCCGAA AtJAAGGC 2452 AAGGGAG CUGAtJGAGrGCCGAAAGGCCQAA AAtAG 2455 GGGAAGG CUGAUGAGGCCGAAGCCA

AGGAAUA

2459 UGGGGGG CUGAUGAGGCCGAA GGCCGAA

AGGGAGG

2460 UUGGGGG CUGAUGAGGCCGAAAGGCCA

AAGGGAG

2479 GCUAACA CUGAUGAGGCCGAAAGCCA AGGtJGUC 2480 GGCLAAC CUG-AIGAGGCCGAA GGCCGAA AAGGtGTJ 2483 GGutJGm cuGAuGAGcGAACGArccG

ACAAAGG

2484 AGGUGGC CUG-AUGAGGCCGAAAGGCCGAA

AACAAAG

2492 GGGUGGG CUG-t7 AGGCCG AGGCCGAA, AGGtJGr 2504 AGAAAUJG CUGA6UGAGr.CCGAA GGCCGAA At7GUGGG 2508 UGGCAGA CUJGAUGAGCCGAA.CC

AUGAJG

2509 CUGCCAG CUGAUr=GGCCGAAG CGAA AAUGUAU 188 U2510 ACUGGCA CUG;LLGAGGCCGAAACC-GccGA~ AAAUGaA 2520 CAUUGtJG CUr.AUAGGCCCGaAAGGCcGAA AcAC=G 2521 TJC-AflUt CUUGAGGCCAAAGGCcGAA AAA~CrG 2533 GACCGCU CtMGAfGAGGCCGA cCGAA, AGUUCA 2540 CAGACAU CUGAt3GAC-GCCGAAAGG-CCG:AA

ACCGCUGM

2545 AUGUCCA CUGAUGAGGC-CGAAAGCCGAA

ACAUGAC

2568 UUGaGGCA CUGAUGAGG-CCGAAAGGC-CGAA ALULCc 2579 CAAGGCA CUGAUG-AGGCCGAAAGC-C-,:k AGCj=G 2585 AGAGGAC CUGAUAGCCAAGCGAA AC-GC-AtTA 2588 ACAAGAG CUCGAVGAGGCCGvjA GGAA ACAAGGC 2591. AGACA CUC-ArA-GG-CGAAAGGc--_rA

ACGAC

2593 ACAGGAC CUG.AUGAGGCCGAAA G CCAA, aGAGGAC 2596 CAAACAG CUGAruGGAcAAGGC-crA

ACAA

2601 APAAUGCA CUGvAMAG~CCGAAAGCzcGtr

ACAGGAC

2602 GAAADGC CUGAflGAGGcGAAAGG,-CC-A

AACAGGA,

2607 CCAGUGA CUGAUG-AGGCCGAAAGCCGA

AUGCAAA

2608 CCCAGUG CtYGAUGAGGCCCGAa-aG CGAA AAUCAA *2609 UCCCAGU CUGAflGAGGCCGAAGCC

AAAUGCA

2620 AIMhG=.G CUGAUGAkGGCCGXUAAGGCc.-A

ACUC

262 GCUGC. A CUGA.UGAGGCCGAAAGCCGCAA AGA6A ***2628 GAGCUGC CtJGA~rzACGCCG AGC,CGA6A ALVGUGrC 2635 GAAACUG CUJGAUGAGGCGAAGGCCQA

ACCUGC

2640 UGCAGGA CUGAUGAGGCCGAAAGCCA

ACUGGAG

2641 CtJGCAGG CUCGAUCAG.-CCGA AACCCA AACtEGA *2642 ACUGCAG CUIGAGAGGCCGAAGGCCCAA

AAACUGG

2653 GGXCCCU CUGA MGGCCGAAAGGCCGAA

AUCACUG

2659 CUUGCAG CUGAt GAGGCCG-AAGCCGAA

ACCCUGA

2689 CCUCCAA CLUGAtr3GGCCGAA -CcGAA ACUUG 2691 GUCCYCC CUGAUGANGGCCGAAGGCC-rGA AMCr *2700 UGGGAGG CUAGGCGA~GCA AGtJCCUC 2704 AAGCUG CUGAUGAGG=cGAAAGGCCGAA AGCSAGTj 2711 CCUUCCA C rAUAGGCCGAAAGGCCGAA

AGCUGG

2712 CCCUUCC CUGACAGGCCGAAGCG6

AAGCUG

2721 CGCGGAI7 CtJGAUGAGGCCGXUGCCGAA6

ACCCUUC

2724 ACACGCG CUGADGAGGCCGAAAGGCccGAA

AIGACCC

**27144 CU.AcA CUGA rGGCCGAAGGCCG

ACACC

2750 GCUUJGUC CUGAUGAGGCCGXUGr-CCGAA

ACACAUJA

2759 AGAGCGA Ct3GAUGAGGCCGAA G GAA ACUG= 2761 ACAGAGC CUGAUG-AGr.CCGAAGGCGA.A

AGAGCM~

2765 GGUGACA CtJGAUGAGGCCGAAAGGCCGA

AGCGAA

2769 CCUGGGU CUGAUGAGGCCGA A. ACAGAGC 2797 GAACCAU CUGAUGAGGCCGAMGGCGAA

AUQGCAC

2803 UGCAGUG CUGAGAGGCCGAGGA

ACCAUA

2804 CUGCAGU CUGAUGAGGCCGAAG CGAA AACCUG 2813 AGGUCAA CUGAUGAGGCCGAAAGGCCGAA ACE7GCAG 2815 AAAGGUC Cr7GAUGAGGCCGXUGCCGAA A-ACrJGC 2821 AGCCCAA CUJGAUGAGGCCGAArCGAA

AGGUC

2822 GAGCCCA CUGAUGAGGCC G ~AAGG.ACUCA 2823 UGAGCCC CUr-AUGAGGCCGAAG CGAA AAAG-GUC 189 w 282.9 ATOCA.tJ COGAUGAGGCCGAAAGCCCAA AGCCCAA 2837 GUGGGAG CUGAUGAGGCCGA AGCCGAA AUCACUU 2840 GAGGUJGG AAGCA.AGGAflCA 2847 GGAGGCU CtJG UGAGGCCGAAGGCCGAA AGGUGGG 2853 UACUCAG CUGAUGAGGCCGA.A=CGAA AGGCA 2860 TUCCCA.GC CG!AGGPAGCA ACUCAGG 2872 Gt3GAGCC CUCA3GAGGCCG-rAAAGG-CCAA AUGGUCC 2877 GUGUUGU CtGUAGCAAGC A CCU 2899 AAAAUCA Ct3GuflGAGG%-CrGAAAGC;CCAA WUUGCC 2900 AAAAAUC CUG GPmAGGCCCAAAGGCCGALA AAU=X-C 2904 AAAAAAA CUGAUGAGGCCGAAAGGCC-,AA AUCAAAU 2905 AAAAAAA CUGAtUGAGGCC GACC AAUTCAAA 2906 AAAAAAA CUAGGCG.AGC AAAYUCA 2907 AAAAAAA CUvG AGGCCGAAAGGcCGA AAAAUCA 2908 AAAAAAA CUGAUGAGGCCGAAAC-CGAA AAAAArc 2909 AAAAAAA CGAGC Ct~ AAAAAAU 2910 AAAAAA6A CUGAUAGGCCGAAAGC7,CCAA AAAAAAPA *2911 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA *b*o 2912 GAAAAAA CUGAUGAGCGCCrzAAAGGCCGAA AAAAAAA *2913 UGAAAAA CUGAflGAGGC-CGAAAGGCCGAA JAAAAAA 2914 CUGAAAA CtGAlUGAGGCCGAAAGGCCc-AA6 ?AAA 2915 UCUGAA6A CLt AUGAGGCCGAAAGGCCCGAA AAAAAAA6 2916 CUCUGAA CUGAUGAGGCGAAAGGCCG-AA AAAAAAA 2917 UTCUGA, CUGAGAGGCCGAIAAGGCCGA AAAAAAA6 *2918 UCCUG. CUGAUGAGGCCGAAA.GGCCGAA AA)AAA 2919 CGUCUCU CUC-AUGAGGCCGAAAGGCCGAA AAAAAAA 2933 AGUTGC CUGAtJGAGGCCGAAAGGCCGA6A ACCCC 2941 UCUGGG CUGAfLGAGGCCGAAAGGCCGAA AGtJUC 2951 ACAAGG CGAUGAGGCCGAAAGGCCGA6A AGUCG *2952 ACAAAG CUGAUGAGGCCGAAAGGCCGAA AGUCGG 2955 UTAACACA CEJGAUGAGGCCGAAAGGCCGAA AGGAAGU 2956 C~ACAC CUGAUGAGGCCGAAAGGCCGALA AAGGAAG 2961 AUUAACt3 CUGAUG-AGGCGAAAGGCCGAA ACACAAA6 2962 tMhUtUAC CUG-AUGAGGCCGAAAGGCC.AA A.ACACA6A **2965 cuuuATuU cuGAuGAGGcCGAAAGGccrGA AcaAAr-A 2966 GCUUUJAU CUGAUGAGGCCGAA GCCGA AACUAAC 2969 AAAG=U CUGAUGaAGGrCCGAAGGC--6 AUCAhLCU 2975 GUGAGA CUGAUGAGGCCGAAAGGCCGAA AGCUUUA 2976 AGEJUGAG CUGAUGAGGCCGAAAGGCC-AA. A-AZCUrU 2977 CAGtTUGA CUGAUGAGGCCGAAAGGCCGL AAAGCU 2979 GGCAGtJU CUGAUGAGGCCGAAAGGCCG-

AGAAAGC

190 Table Mouse ICAM HH Ribozyme Sequence nt. Position Ribozyme Sequence ill CACGI CUGA.UGAG-GCCGAAAx~GCCA ACCAGGG 23 A=CAW.G CUGACUGC-CC,-AAAG-,cCG zCC:ACtUG 26 AGGAGCA CLt.GAGGGCCGAAAGCCGAA AGAACCA1 3i UGUGGAG CUJGAGGCCGAAAGGCCGAA AGCAGAG 34 CGACCCJ Ct2GAUC GCCGAACGCCGAA. ALUGAGAA AGGCAC CUGAUGAGGCCGAAAGGCCGAA AGtJGtGC 0.*48 CCYAGGCU CDGA-GAGGCCGAAAG~ccGAA AGGEJCCrJ 54 CCAUC-AC CUGAflGAGGCCGAAAGCGAA AGGCCCAL 58 GGAct= CUGAGAGGCCGAAGGCCGA AGGCAUG .964 CUGCDGG CDGAruGAGGCCGAAAGGCCGAA. AGGGGtJG 6 GGCCAG CUGAUGAGGCCG-AAAGGCAA AGCAGAG *102 CCAGCAG CUGAflGACGCCGPAAGGCC-AA ACUGGCIL 108 GGGCCAG CUGAGAGGCCGAA-AZCCGAA AGCAGAG 115 AGGAGC-A. CrJGAnGAGGCCGAAACGCCGAA AGAACCA 119 UJCCOGGt CUGPAVGAGCCGJaAAGCCGAA ACAUUCC 120 GGGCCAG CUGAX3GAGGCCGAAAGGcCAA AGCAGAG 146 GGAAGCG CUGAUGAGLCGAAAGGCCGA AC^GACUG 152 AGUGC3 CUGAUGAGr.CCGAAAGGCCGA

ACACA

158 GG-UUUUU CUG AGAGGCCGAAAGGCCGAA

AACAGGA

165 GCAAAAC CUGAUGAGGCCGAAAGGCCGAA ACUUCUG :168 GGGCA CUGAUGAGGCCGAAAGr.CCGAA AAGG=tE *185 CUGCACG CGAUGAGGCCGAAAr.GCCGA

ACCCACC

209 GCCAGAG CUG= GAGGCCGAAAGGCCGA

AAGTJGGC

227 GCAAAC CUG~AGGCGAAAGGCCAA A=tECUG too.. 230 GGAGCAA C GAUGAGGCCGAAAGGCCGAA CAU 237 AGUUCUC CUGAUGAGGCCGAAGGCCG AaCACA 248 UUUTAGGA CLUGALTGAGCCGAAAGGCCA

AUGGGUU

253 UCtJUCCU C 7GALTGAGGCCGAAAGGCCCAA

AGGCAGG

263 CAGt3AGA CUGAUJGAGGCCGAAAGGCCGAA AAACCCtJ 267 UAGGCAG CUGALUGAGGCCGAAAMGA

AGCCCCU

293 CAGCUCA CUr-AUGAGGCCGAGrCCGA ACAGCtU 319 GGCtJCAG CUGAUGAWGCCGAAGGCCGA AUCUCCu 335 GUUCUCA CEJGAUGAGGCCGAAAGGCCGAA

AOCACAG

337 CAGUGUG CJGAUGA.GGCCGAAAGGCCr.AA AUJGGiAC 338 UCAGCtJC Ct3GAUGAGGCCGAAAGGCCA

AACAGCU

359 AGCGGAC CU 7 GAUGAGGCCGAAAGGCCGAA ACtJGCAC 367 CGGGUt3G CUGAUGAGGCCG Lb.GGCCGA AGCCAU 374 GGGCAGG CUGA.UGAGGCGAAAGGCCA AGGCt2UC 375 GGGGCAG CUGAUGAGGCCGAAAGrCCGM

AAGGCUU

378 ACACGGU CUGAUC-AGGCCG aAGCCGAA AUGGUAG 386 AAACGAA Ct3GAUGAGGCCGAAAGGCCGA

ACACGGU

394 AGAUJCCA CUGA.UG-AG-GCGAAGGCCG.

AGUCCGG

420 CGGGGGG C'UGALGAC-GCCGAAACGCCA

A-GUCG

425 CUjGC--G cuGA.uGAGGcCGAAAG~cCCCA3

AGGGG

427 CACUGCtJ CUGAUGAGGCCGAAAG=CGA6A kcAGCEJG 450 GCAGGGU Ct3GAUJGAGGCGAAAGGCcc ACGt3U 451 CAAAGA Ct7GAtGAGGCCGAGCCG6

AG-UU=C

456 AGUGGCY Ct3GAUGGCCG GCGAA AL.G-hA 495 ACACGGU CUGAGAGCCGAAGGCCM AVLGMt~L 510 CCCCACG CUGAUGAGGCCGAAACGCCGAA AkGCAGC-.

564 GGAUGGA CUGAULGAC-GCCGAAAGGCCGA

ACCJGAG

592 C--rAkLGU CL-UGAGC;AAGGCA-Gc WtUtJUC 607 CAUCIAGA CUGAUGAGGCCGAAAGGCCGAA AUUGjGCtJ 608 GCAUGAG CUGALUGAGGCCG=AAGGCCGAA

-AIGGC

609 GCAUGA CUGAUGAGGCCGAAAGGCCG AtAUJG 611 GCGGCAU CUGALTGAGGCCGAAGGCCGA

AC-AA)ATJU

*00 65 ACC UAGAGGCCGAAAGGCCGAA ACAGCUUt se*657 UCAGCUC CUGAtr=AGGCCGuNAGGCCGA Ac~AGC oo. 668 GGUGGCC CtUGAUGAGGCCGAGCCA

AGGCUCG

V00677 AGGCtGG CUGAUGAGGCCGAAGGCCA

AGAGGUC

es684 AGGACCG CUGAUC-AGGCCAAAGG%-CcA

AGCUGAA

692 AACAUCG Ct3GAUGAGGCCGAAAGGCCGAA AAGtJCCG 69 C9G UAGGCGAGCCA

GUC

693 GAGGCG CtJGAUGAGGCCGAAAGGCCGAA

AGAGGC

609 GAGG CUG'AUG-AGGCCGAAAGGCCGAA

AACAG

720 AGGtJ' CUTGAUC-ACv-CGAAAGGCCC-A

AGCCGCC

723 CGGAGC-U Cu-GAUGAGGCCGAAAGGCCGAA AAAAGtU '*735 UCtICCAG CUGAUGAGGCCGAAGGCCGA

AUCLG

738 CCAT-CAC CUGAUGAGGCCGAAGGCCA6

AGGCCCA

*765 GGAAGCG Ct7GAUG-AGGCCGAAAGGCCGAA

ACGACUG,

769 GGCAGGA CUGAUGAGGCCGAAAGGCCGA

ACAGGCC

oooo*770 UUCCAGG CUGAUGJCAGC-ccGAAAGGCCG-Az AGcAAAA 785 C-GCAGGA CUGAUGAGGCCGAAAGCC AA GGCC 786 AGGCAGG CUGAUGAGCCGAAAGGCCGA

AACAGGC

CtJUCCGA CUGAUGAGGCcGAAAGGCCGA ACCUrCCA 794 AGUCtTCC CUGAUGAGGCCGAAAC-GCCGAA

AGCCCAG

807 CCAGGUA C~r-AUGAGGCCG AA.%-CCGAA AtICCGAG 833 GC-G-GUC CUGAUGAC-GCCGAAGCCGAA

AGCUUUG

846 C.ACGGU CUGAUGAGGCCGAAGGCCGA

ACCAGGG

851 GCUGGUA CUGAtJGAGGCCGAAAGGCCA

AGGUCEJC

863 CCAGAGG CUGAUGAGGCCGAAAGGCCGALA AGUGGCrJ 866 GGGCAGG CUGAUC-AGGCCGAAAGGCCA

AGGCEJUC

867 tJCtCCC-G CGAUGAGGCCGAAAGGCCGA

AACGAAU_

869 CUUGCAU CUGAUGC-GCCGAAAGGCCGA

AC-GAAGA

881 ACGGGtJ CUGAUG ZGCCGAAAGGCCGA6A

AAGCCAUJ

885 UCACCUC CUGAUGAC-GCCGAAAGGCCA

ACCAAGG

933 CCAGAAU CUGAUGAGGCCGAAAGGCCA AUtJAUAG 936 GCACCAG CLGAUGALGC-CCGAAAGGCCGA AuGAuEJA 978 AGUUGUA CUGAUC-AGGCCGAAAGGCCGA

ACUGULJA

980 AAAGUrUG CUGAUGAGGCCGAAAGGCCGA AGCtJCGU 986 AGCUJGAA CUGAUGAC-GCCGAAGGCCCA AzG~TJGA 987 C.c-CUGCA CUGAUAGC-C-AAGGCCGAA AAGUUtGTJ 988 GAGC,:G C.UGAUG,; C- CCGAAAGGCCG AAA.GUbUG 192 1005 U-TCUCAG CUGAUGAGGCCGAAAGGCCGA A7cuGG~u 1006 UUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACEJCTCA 1023 CtJUCCGA6 CUGA6UGAGGCCGAAAGC-CGAA

ACCUCA

1025 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCtJC 1066 Ut3ALUUU CUGAUGAGGCCGAAAGGCCGAA

AGAGUGG

1092 GGCCUGA Ct3GAI3GAGGCCGAAAGGCCGAA AUCCAGrJ 1093 UUGGCUG CUGALIGAGGCCGAAAGGCCGAA

AGG%-UCCA

1.125 UC-AAGAA Ct3GAL'GAGGCCGAAGccA

AGUUGGG

1163 c-CAAAAG CUGAUGAGGCcGA.AGMcGAA

AGCUUCG

1164 AGCAAAA CUGAUGAGGCCGAAGGCCGAA

AAGCUUC

1166 AG-AGCA6A CtJGA6UGAG7GCCGAAAGGCCGAA AGAAGCtJ 1172 -GUUUU CtJGAt3GAGGCCGAAAGGCCGAA,

AACAGGA

1200 UGUGGAG CUGAXMGGCCGAGCCGAA

AGCAGAG

1201 CtGU3CA CtJGAUGAGGCCGAAAGGCCGA6A

AAGCAGC

1203 ACUGGTJG Ct3GAr AGGCCGAAAGGCCGA AAAAAGU 1.227 GCACrACG CUGAt3GAGGCCGAAGGCCGA AUGtJACC 6****1228 AGCAAAA CUGAUGAGG-CGAAAGGCCGA6A AAGCUUtC 0:.1233 CUCUCCG CUGAUGAGGCCGAAAGGCcGAA AAAcGA 1238 AGGACCA CtJGAUGAGGCCGAAAGGCCGAA AcAGCAC 1264 CUUGCAC Ct2GAiGA GGCCGAAAGGCCGAA ACCCUUC 61267 TJUCCCCA CUCAUGAGGCCGAAAGGCCGAA

ACEICUCA

1294 GGCt3CAG CUGAUGAGG.CCGAAAGGC-CGAA AUCtJCCTJ 1295 CUGCUGA CtJGAUGAGGCCGAAGGCCGA

ACCC

1306 CATUUCA CUGAUG=ACGCCGAA~AGGCCGA AGUCtiGC 1321 UCCUCCU CUAUGAGGCCGAAAGGCCGAA AGCCUtJC 1334 UMM3r.A CUGAUGAGGCCGAAAGGCCGA

AUGGGUU

1344 CACtJCUC Ct3GAUGAGGCCGAAAGGCCGA AGCtJCAU 1351 tJAACUUA CUGAUJGAGGCCGAAAGGCCGAA A-AuutcA 1353 CAC=tJC cUGAUGAGGcCGAAAGGc AccC-ACT *se1366 AGrJUGUA CUGAUGAGGCCGAAAGGCCGAA ACUGUUrA 1367 AGGtJGGG CUGAUGAGGCCGAAAGGCCGAA AGGtJGCtJ *61368 AGAGUGG CUGAt3GAGGCCGAAAGGCCGAA ACAGuAC 1380 CCACCCC CUGAUGAGGCCGAAAGGCCGAA

AUGGGCA

1388 AGCCA6Ct CUGAUGAGGCCGAAAGGCCGAA

AGUCUCC

1398 GUUCtJGI CUGAUGAGGCCGAAAGGC ACAGcC-A 1402 AGUt7CtC CUGAUGAGGCCGAAAGCCG AAGCAcA 1408 CCt7CCCC CUGAUG.AGGCCGAAAGGcCA AuCUCGC 1410 CCCtJUCC CtJGAUGAGGCCGAAAGGCCGAA

AGACCUC

1421 ACAAAAG CUGAUGAGGCCGAMAGGCCGAA AGGuGGG 1425 CtJCUACC CUGAUGAGGCCGAAAGGCCA AGG CAGU 1429 CAGGGGC CUGAUGAGGCCGAAAGGCCGAA AuAGAGA 1444 UCCUCCU CUGAUGAGGCCGAAAGGCCGA AGCCUEJc 1455 UCCUGGU CtJGAUGAGGCCGAAAGGCCGA AcAUtJCC 1482 GGGAGCA CUGAUGAGGCCGAAAGGCCGA

AACAACU

1484 CAUGAGG CUGAUGAGGCCGAAAGGccGA

AGAACAG

1493 GUUCUCA Ct7GAUJGAGGCCGAAAGGCCGAA

AGCACAG

1500 GGACCAU CUGAUGAGGCCGAAAGGCCGU

AUUUCAU

1503 GAUGAU CUGAtJ-AGGCCGA GGCCG-AA, AUAGUCC 1506 CGGUU CUGAUJGAGGCCGAAGGCC'A

AACAUA

193 1509 ACACGGt3 CIGAUGAIGGCCGAAAGGCCGAA AUGGUAG 1518 CGCCUGG CGUGCAAGCG ACCAtJGA 1530 CCAGAAU CGGAC.AGCA AUAUA 1533 GGCCCAC ~UA ~ALMCCA 1551 AG--L..CU CUt3AGXAGCcGGGCCGAA AGGCAIJG 1559 AGGJGGG -WGAAcGc%-,UCG ~GCU 1563 GG-UE]AUA COAGGG= =A ACVIAG 1565 GCGGUU;Lc AAAAUA 1567 UGGCGGU CUGAUGAGGCCGAAAGGCCG- AMACA, 1584 AtTh.UCCU ~UA ~AUCUUEJC 1592 tTh~U GGcAACULGA AIDAUcC 1599 CCUtG CU=AGAGGCCGAAAGCGAA AACUUGU A. 1651 GCUCAG G UG cGA GcG AGGUGGG :0 1661 CAAAGGA CUAGGCA AGGCCA ~UUUC 1663 UUCAAAG CGfGGCG ACCGAAAGU 1678 CCAGGCU CJ~~GCGAGCGAAGGUCCtJ 1680 CCAGAGG CUAG~CGA~CGAAGUGGCEJ i681 GCCAGAG CUUGAGGCCG-AAAGGCCGAA AAGGC 1684 ACAGCCPA CGUACGAGCC A GAAGU ~1690 AGAUCGA CU~GGCGAGCGAAGUCCGG 1691 AAGA6UCG CGIGGCGAGCA AAGUCCG 1696 CC.-=CC CEG=AUGAGGCCGAAACGCCG-AA AUGGGCA 1698 CUCCAGG cUAG~~GAI~C AAU=~ 0* 1737 GCUGGJA cUA3AGCAAGc A GUCUC 1.750 UGAGG UGAGGGAAGc AGCCGCC AAA1756 GGGCAGG CtJGAUGAGGCCGAAAGGCCGAA AGGCtUUC 1787 UGGGGAC CL7GAUG-AGGCCGAAAGGCCGAA AUGCC 1790 AUUAGAG CUGAUGAGGCCGAAAGGCCGAA ACAAUGC 1793 UCCAGCC C!7GAUAGGCCGAAAGGCCGA AGGACCA 0***1797 UjUUAUGU CtGAUGAGGCCGAAAGGCCGAA ACUGGUG 1802 UCUCCAG CtJGAUGAGGCCGAAAGGCCGAA AUCtJGGU 1812 GGCCUGA CGGAcGAACC) AUCCGJ 1813 UGAGGGU CUGAUGAGGCCGAAAGGCCG-AA AAUGCEJG 1825 GCAGAGG CUGAUGAGGCCGAAAGGCCG-AA AGCGUGG 1837 GGAGCUA CUGAUGAGGCCGAAAC-GCCGAL AGGCAUG 1845 GGUGGCC CUGAUGAGGCCGAALAGGCCGAA AGGCUCG 1856 AAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCG 1861 UACUGGA CUGAULGAGC-CCGAAAGGCCG-AA AUCAUGU 1865 CUGAGGC CtJGAIJGAGGCCGAAAGGCCGAA ACAAGUG 1868 UUUAUGU CUGAUGAGGCCGAAAC-GCCCAA ACUGGtJG 1877 AGCUGCTJ CUGAUGAGGCCGAAAGCCGAA AGGCAUG 1901 GUCCCU CUGAUGAGGCCGAAAG-GCCGAA AGUULUA 1912 ACUGAUC CGAUGAC-GCCGAAAGGCCGAA ACUAUAU 1922 UAACUUA CUGAUGAGGCGAAGGCCG ACAUUCA 1923 GAUACCU CUG ALGAGGCCGAAAGGCCGAA AGCAUCA 1928 CUGGDAA CtJGAUGAGC-CCGAAG~CCGAA ACUCUAA 1930 AGCUGGU CUGAUC-AGGCCCGAACGCCCGAA AAA7CU 1964 UGGGGAC CUGAUG'C-;C-GCCGCAC-GCCGA.A AUGUCUC 1983 UAACUUG CUGA6UGAC-GCCGAAc C-GCCaA-k XU-t.CCJ 194 1996 GGCtJCAG CtJGAUGAGGCCCAAAGGC-GAA ALiuctcut 2005 G%-zUCCGC CUGAUGAGGCCCGAAACGMGAA AGCCCA 2013 UACUCAA CUAA CGAAGCA AAAtJAGC 2015 CCACCCC CUGAUJGAGGCCAAGGCC'A

AUGGGCA

2020 Ct.7AGAA CUGAUG-ACZ-AAAGG-r-CAA

AACCACC

2039 CCUCt3GC CUGAUGAGCCC-AAAGGCCAA AGCCAC 2040 CCtJCCAG CUGAUCAGGCcLmAAC-GCGA AGGtJCAG 2057 GGAt3GUG CtJGAUGAGGCCCAAAGGCCAA

AC-GAGCA

2061 ACGC~T3 Ct3GAUGAGGCZ-CAAAC-GCC-AA

AUGGIAG

2071 CtJGAGGC CUGAUGAGGCCGAAAGGCC ACAAtG 2076 UAGCUCtI CUGAUGAGGCCGAAAC-GCcGr= AkGGC-UAC *2097 CAUCAAG CUGAUGAGGCCGlAAGGCCGA

AGALG%.R;G

.2098 CGGGGGG CtYGAUGAC-GCCGAAAGCCC-AA AAGtIGUG 2115 AUCCtTCC CUGAUGAGGCCGAAAGGCCGAA AG%-t3GGC 2128 Ct7CAAUA CUGAUGAGGCCGAAAGGCCGAA AT1ALG%-IG **2130 GAGGCAG Cr3GAUGAGGCAAAGC-CCGAA

AAACAGG

2145 CAUCAJAG CUGALTGAGGCCC-AAAG-GCCC;Aa

AGAGWJG

2152 AACtJCUA Ct7GAUGAGGCCGAAAGCCCGA

AUUAAUA

2156 UAAUAAA CUGAvGGCCGAAAGCCG

ACAUCAA

.:*2158 AUUAAtJA CUGAUGAGGCCGAAAGGCCGAA

AUJACAUC

2 159 AALTUAAU Ct3GAUGAGGCCGAAAGCCvAX

AAUACAY

*2160 AAAUCAA CUGALUGAGGCCC-AAGCC-A AAtJAC-A 2162 CTJAAAUU CUGAUGAG=CGAAAGGCCGA

AUAAAUA

2163 AAUtAAU CUGAUGGGCCGAAGcGCAA

AAT-ACAU

*2166 AtUAGAG CUGAUGAGGCCG-AAAGGCCGAA AuGAAGu 2167 AAUUAAIJ Ct3GAUGAGGCCC-AAGGCCGAA AAUJACAtJ 2170 CtJAAAUU CUGA6UC-G%-CCGAAAGC-CCGA.A

AUAAAUTA

2171 GGGAGCA CUGAUGAGGCCCGAAAGGC-CGAM AACAACtJ *2173 CUGGt3AA CUGAUGAGGCCGAAA-kAGCCG;, ACtJCL.A 2174 GCUGGUA CUGAUGAGGCCGAAAGCcCGAA AACtJCUA 2175 AGCUGGU CUGAJGAGGCCGAAAGCCCA

AACUCU

2176 tU.GCUGG CUGAUGAGGCCGAGGCCGC-A

AAACUJC

2183 CA.UAAA CUGAUGAGGCCGAACGCCCAA AG-tJGGU 2185 CtJCAAUA CUGAUGAGGCC-AAAC-GCCC-AA

AUAGC!JG

2186 ACUCAAU CGAUGAGGCCGAAAC-CCGAA AAIJAGCtJ 2187 UACUCAA CUGAUGAGGCCGAAGGCCGA

AAAUAGC

2189 GGUACtJC CUGALUrAGGCCGAA=GAAC

AUAAAUJA

2196 CAUCAAG CUG;LUGAGGCCGA G~ccGAA AGAGutJG 2198 AACA6UAA CtJGAUGACGCCCA AGCCCAA AGGCtJGC 2199 AUAAACA CUGAUGCAGGCCGAAAGGCCG-AA

AAGAGGC

2200 CUtJGCAU CUGATJG-AGGcrC-AAAGGcG-A AGmGA 2201 GCCGACA CUGAtYGAGGCCAAAGCcr_,A

AAAACULT

2205 UCAGGCC CUGAUGAGCcAAGGcC.A

ACAUAAA

2210 AGCCACtJ CLTC.AUGAGGCCGAAAGGCCGA AGUCUcc 2220 AGAGAAC cUGAuGAGGccGAAGGcG

AUGCCAG

2224 GGAUJGGA CUGAUC-AGGCCCGAAAGGCCGAA ACCUGA6G 2226 GCGGCCU CUC-AUGAGGCCGAAA-GCCG.A AGAUCC-k 2233 CCUCCAG CUAUGAC-CG-26AGc-C: AGuCA 2242 GTJCCGC CUGAU-AGGCCG ACGc-c~z-. AGcCtCCA 195 2248 UGCGGAUG CUGAUGAXGGCCGAAAGGCGAA AUGGAIJA 2254 UJCAGtJG CUGAX3GAGGCCGAAAGGCCGAA AAUUGGA 2259 CACCGMJ CUIGAUGAGGCCGAAAGGCCGAA AUGtJGAU 2260 GCACC.GU CL II'GAGGCCGAAAGGCCGAA AAUGUGA 2266 UCCUJC-CTJ CrGALt~uIGGCCGAAAGGCaGAA ACAjuUCC 2274 UJCUCCAG CUGAGGCCGAAAGGCCGAA AUCt3GUt 2279 Ct2UGCAC CtUGAUGAGGCCGAAAGGCCGA ACCIUC 2282 CACU*ICA CUGLCATvAGGCCGAAAGGCcGAA ACAGCUU 2288 ACGCCB.U Ct3GAt3GAGGCCGAAAGGCCGAA ACtJUAt2A 2291. AG CAZ-rA G CUG-AfLrGGGCCGAAAGGCCGAA ACCACtG 2321 CCCATJG CUG-AUG-AGGCCGAAAGGCCGAA AUCU7EJC *2338 CAGGCAG CUGAUGAGGCCGAAAGGccGAA AGUCTJCA **2339 CAAAGGA CtJGAtIGAGGCCGAAAGGCCGAA AGGUTUC 2341 AGGCtJGG CUGAUGAGGCCGA.AAGGCCGAA AGAGGUC 2344 GCTJGGAA CtrGAflGAGGCCGAAAGGCcGAA AUCGAAA 2358 CtJGCUGA CUAUGGCCGAAAGCcGAA AGCtJGGG 2359 UGUCCUGAUGAGGCCGAAAGG-CCAA

AAAGCAG

2360 UUCAAAG CUGAUGAGGCCGAAAGGccGAA AAAGGUU 2376 UCAGAAG CUJGAUGAGGCCGAAAGGCCGAA ACCACCtJ 2377 CtJCAGAA, CUGALUGAGGCCGAAAGGCCGAA

AACCACC

2378 CAGUAGA CUGAUGAGGCCGAAAGGCCGAA AAACCC *2379 CUUtAUGA CtJGADTGAGGCCGAAAGGCCGAAU AAAAGCA 2380 GCCGACA CtJGAUGAXGGCCGAA.AGGCC,,,AA AAAACUJE 2382 GGGGCAA CUGAUGAGGCCGAAAGGCCG-AA AGAGA AU *2384 UUGUGUC CUGA GAGCCCGAAAGGCCGAA AcuGGAu 2399 GTJCCACA CUGAUGAGGCCGAAAGGCCG-AA AGUGU 2401 CAGCUCA CUGAUGAGGCCGAAAGGccGAA AkCAGCUEJ 2411 GCAUCCU CUUGAGAGCXGCCC-AAAGGccGAA6 ACCAGUA .2417 ACGUJAUG CUGAUJGAGGCCGAAAGGCCGAA

ACCADUUC

2418 GGCCUGA6 CIGAUGAGGCCGAAAGGCCA

AUCCAGU

2425 AACCCUC CUGAIJGAGGC-CGA.AAGGCCGAA

ACCCAUG

2426 AAACtJCU CUGAUJGAGGCCGAAAGGCCGAA6

AAUUAATU

2433 GCt7GGEIA CtJGAUGAGGCCGAAAGGCCGAA AACtJCUA 2434 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA

AAACEJCU

2448 GGGCAGG CUGAUGAGGCC AAAGGCCGAA AGGCUUC 2449 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA

AAGGCU

2451 AGGCAGG CUGAUGAGGCCGAAAGGCCGAA

AACAGGC

2452 GAGGCAG CUGAUGAGGCCGAAAGGCGA

AAACAGG

2455 GGGCAGG CUGAUGAGGCCGAAAGGCCGA

AGGC

2459 GGGGGGG CUGAUGAGGCC^"AAAGGCCGA

AGUGUGG

2460 CGGGGGG CUGAULGAGGCCGAAAGGCCGAA6

AAGUGUG

2479 GCUGGUA CtJGAUTGAGGCCGAAAGGCCGAA

AGGUCUC

2480 GCAUCAC CUGAUGAGGCCGAAAGGCCGAA

ACCGUGA

2483 GGUGGCU CtJGAUGAGGCCGAAAGGCCGA

ACAUUGG

2484 GACUGGtJ CUGAUGAGGCCGAAAGGCCGAA

AAAAAAG

2492 AGGUGGG CLTGAUGAGGCCGAAGGCCGA AGG;UGCt 2504 ACAAAAG CUC-AUGAC-GCCGAA--ACGCCC-A.

AGGUGGC-

2508 UC-GGAUT-G C--AL'AGCCCC AC-GCCG AUGGAUA 2509 CU.tA CGUC-G.A GGCCGA ACtJCEAA 196 2510 GCEJGGMJ CUC-AUGAGGCCCGAAAGGCCGAA AACUCUA 2520 CAUUGGG CTJGAUGAGGCCGAAAGGCCGAA ACAAAG 2521 UGALGGU CUA3AGCP.AGCA IUGMt~G 2533 GAUACCU CtGUAGCAAGC A ia=C~f 2540 CXCAGCG7 CUGflGGCGG CCGAA ACLGCUG 2545 AGGACCA CUUGAGCCAAAC-GCCGAA ACAGCAC 2568 UTJEGACAN CUG-AUGAG.-CCGruAA~Cr-CG-AA ACtUCAC 2579 CAGGCCAk C!GALGAGGCCGAAGGCGAA AACUUAU 2585 AGAGAAC CUC-Atr-GACGCCGAAAC-C-CCGAA AUGCCAG 2588 AUUAGAG C UAGGCCGAAGGCCWAA ACAAUGC 2591 AGGAGCA CUGjAtrJAGGCCGAAAGG-CcGAA AGAACCA 2593 GCA-ACCUG!AGCAAGCA AAAGAAG *2596 CAtJTGG-G CUGALIAGGCCCAAAGGCCGAA ACAAAAS3 2601 AAACGAA CUGAUGAGGCCCGAAAJGGCCGAJA AC-ACGGU *2602 GGGAUC-G CUGAUGAGGCCGAAAGGCCGAA AGCUGGA 2607 CCAGMU CUGAUGAGGCCGAAAGGCCCAA AUCCGAG 2608 CACAGC-G CUC-;LJGAGGCCGAAAGGCCGAA ACUGUCUG 2609 U-CCUGGU CUGAUGAGGCCGAAAC-GCCGAA ACAiucc 2620 GCAGC7%-Z CUC-AUGAGGCCGXAAGGCCGAA AGrGUCCU *2626 GYCUGGAA CUGAt]GAGGCCGAAuAC-GCCGAA AUCGA)A ~2628 AC-GCUAC CUAGGCGAGCGAAGUGUGC 2635 AGGACCG CUGA;UGAGGCCGAAAGGCCGAA AGCtJGAA 2640 GGCAGGA CUGAGGCGAAAGGCCGAA ACAGGCC 2641 CJGCtJC-A CUGAUGAC7GCCGA.AAkGCCCGAA6 AGCUGGG 2642 GAGGCAG Ct7GALMAGGCCGAAAC-GCCGAA6 AAACAG 2653 GCAUCCU CtJGAUGANGGCCGAAAC-GC-CGAA ACCAGUA~ 2659 CUUGCAC CUGAUGAGGCCGAALAGGCCGAA ACCCUUC 2689 CCUCGGA CUG-A6UGAGGCCGAAAC-GCCGAA ACALTUAG 2691 GGCCUCG CGUAGCAA-CGAAGACAUU 2700 GGGCACG CUGA6UGAGGCCGAAAC-GCCGAA AGGCUEJC *2704 AGGCUGG CUGAUGAGGCCGAAAGGCCGAA AGAGGt3C 2711 CUGCUGCA CUCAUGAGGCCGAAAZ-GCCGAA AGCULrG 2712 CCCUUCC CUGAUGACGCCGiAAAGGCCGAA AGACCUC 2721 CUEJGCAC CEJGAUGAGC-CCGu'AACGGCCGAA ACCCUUC 2724 GCACA7CG CUGATU"GAGGCCGAAAGGCCGAA AUGtJACC 2744 CUGCACG CUGATJuGGGCCGAAAGC-CCGAA ACCCACC 2750 GGUACtJC CUGAUGAGGCCGAAGCGCCGAA AUAAAUJA 2759 AG-AUCGA CUGA6UGAGGCCGAAAGGCCGAA AGUCCGG 2761 GCAGCGGU CUGAUJGAGGCCG-AAAGGCCGAA AGGUCCU 2765 AGCGGC:A CUGAUGAGGCCGAAAGCCGAA AZCAAAA 2769 CCUGUUtJ CTJGAUGAGGCCGAAAGGCCGAA ACAGACTJ 2797 GGACCAU CUG-AUGAGGCCCGAAA.C-GfCCGAA ALUUCAU 2803 CGCCUGG CUAUGAGC-CCGAAA.C-GCCGAA ACCAtJGA 2804 Ct3GCACG CUGALUGAGGCCGAAAGGCCGAA ACCCACC 2813 GGGucAG cuGAuGAGrccGAAAGGccGA ACCGGAG 2815 AAGUUG CUCAUGAGC-CCGAA;L-GCCGAA AGACUGtJ 2821 CCUCCAG CUGA;UC-ACGCCC-AAAC-CCCGAA AGGtCAG 2822 AAGUCC3 CUG G;,'C--CCGAAAx'-;C-CCGAA AGC-=CC 2823 UGC--AC-C CUCA CC-GCCGA -CGA-A AMAGC-CA 197 S2829 AUGAUUA CUGAJGAGGCCGAAGCCGAA

ACUCCAG

2837 UCAGAAG CUGAUGALGGCCG-AAAr.GCCGAA ACCACCtJ 2840 CAGGCAG CUGA.UGAGGCCGAAAGGccG-aA AGUCUCA 2847 GGUGGCU CUGA.UGAGGCCGAAAGGCCGAA

ACAUUGG

2853 1 ACAUAA CE7GAUGAGGCCGa-aAGGCcG-AA AC-CJGC 2860 UCACAGU CUGALUGAGGCCGAAAGGCCGAA ACU3EGGC 2872 CUUGCGCU CUJGAUGGGAGGu GA AGGUCC 2877 GGAUGG CtJGAMGGGCGAGG,- AG-,CGGA 2899 AAGAUCG aUGAUGAGCCGAAAGGCCGALA

AAGUCC-.

2900 AkAAACtJC CUGAUGAGCCGAXAAGGCCMA

AAAUUAA

2904 AAUAGAG CUGAUJGAGGCCGAAAGGCCG-AA

AUGAAGTJ

2905 CAAUAGA CUGAUGAGGC:CGAAAGGccakA AAuGAAG 2906 UAAtUhAA CtGAUGAGGCCGAAAGGCCGALA

ACAUCAA

2907 AAAUUAA CUG;LUGAGGCCGAAA1GGCCGAA AAA~uAC 2908 AGCAAAA CUGAULGAGGCCGAAAGGCCGAA

AAGCEIUC

2909 AGAGCAA CUGAUGAGGCCGAAAGGCCG-AA

AGAAGCU

2910 AAAUUJAA aUGALUGAGGCCGAAAGGCCGA

AAAUACA

2911 AAAUUAA CUGAUGAGGCCGAA GCCGAA AAAUjACA 2912 GACAUUA CUGAUGA GGCCGAAAGGCCA

AGAACAA

2913 UGACCAG CUGAUGAGGCCGAAAGGCCGAA

AGAGAAA

2914 CUUAUTGA CUGAUGAGGCCGAAAGGCCGAA

AAAAGCA

2915 UCtAAAU CUGAUGAGGccGAAAGcCCG

AALUAAAU

*2916 CUCCGGA Ct3UGJ CAAGCCA ACGAUA 2917

UC

7 JCCGG CUGAUGAGGCCGAAAGGCCGAA

AACGAAU

2918 CUCUCCG CtJGAUGAGGCCGAAAGGCCGAA

AAACGAA

2919 CGACCCU CtJGAUGAGGCCGAAAGGCCGA

AUGAGAA

*2931 CUCCGA C 3GAUGAGGCCGAAAGCCGAA

ACCUCCA

2933 CCCUUCC CUGAUGAGGCCGAAAGGCCGA

AGACCUC

2941 UGGGGAC CGALIGAGGCCGAAAGGCCGAA,

AUGUCUC

2951 GCAGAGG CUGAUGAGGCCGAAAGGCCAA

AGCGUGG

2952 CACAGCG CEJGAUGAGGCCCAAAGGCCGA

ACUGCUG

2955 UGACACA CUGAt3GAGGCCGAAAGGCCGA

AGUCACU

2956 UUGAtJUC CUGAtYGAGGCCGAAAGGCCGA

AAGGAAA

2961 AGUGGCU CUGAUGAGGCCGAAAGGCA

ACACAGA

2962 A3AtUAA CUGAUGAGGCCGAAAGGCCGA

AAUACAU

2965 CUUTJAUU CUGAUGAGGCCGAAAGGCCGA

AUUCAAA

2966 CCUCUGC CUGAUGAGGCCGAAGGCCGAA

ACCCAGC

2969 AAAACUU CUGAUGAGGCCGAAAGGA;

AUUGAUU

2975 GCUGGUA CUGAUGAGGCCGAAAGGCCGA

AACUCUA

2976 AGUAGAG CUGAUGAGGCCGAAGCGA

AACCCUC

2977 CAGCUCA CUGAUGAGGCCGAAAGGCCGA ACAGCtUU 2979 GGCAAUA CUGAUGAGGCCGAAAGGCCGA

AGAAUGA

Trable 6 Huftman ICAM Hairpin Ribozyme/Suibstrate Sequences nt.Hari ioyeSqec PositionHariRioyeSqnc Substrate 86 343 635 3 782 920 1301 1373 1521 1594 2008 2034 2125 2132 2276 2810

GGGCCGCG

GGAGUGCG

CCCAUCAG

GCCCUUGG

LJGUUCUCA

AGACUGGG

CUGCACAC

ACAUUGGA

CCCCGAUG

AUJQACUGC

CUGUUGUA

ACCCAALJA

UTJCLGUAA

GGUCAGIJA

GGGUUGGG

ACCUGUAC

AAGGLJCAA

AGAA GCUG AGAA GCGC AGAA GUUU AGAA GOAG AGAA GCUC AGAA GCCC AGAA GCCc AGAA GCUG AGAA GUGG AGAA GCUA AGAA GUAU AGAA GCAA AGAA GUGG AGAA GCAG AGAA GUAG AGAA GUAC AGAA GCAG ACCAGAGAAACACACGUUGUGGcUACAU1ACCUGGUA ACCAGAGAAACACACGIJIGI)cJGI

JACAIACCUIGG(JA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGACACACGUUGUGGUACAIJJACC1JGGUA

ACCGAGAAACCACGUGUGGUACWYJACCUGGUA

ACCAAGAAACACACGUUGIJGGUACAUUACCJGGUA

ACCAGAGACACGUUGUGGUACAUACCJGGUA

ACCAGAGAAACACACGUUGGGUACAUJTACCJGGUA

ACCAGAGAAAACACGUUGUGGUACAUUIACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUIACUGGUA

ACCAGAGAAACACACGUUGUIGGUAAUUACCUGGUA

ACCAGAGAAACACACGIJUGUGGUACAUUACCTJGJA

ACCAGAGAAACACACGUUGUGGUAJACCGGUA

ACCAGAGAAACACACGUUGUGGUACTJ1JACC1JGTJA ACCAGAGAAACACACGUUGUGGUACAUU1ACC1J(GUA

ACCAGAGAAACACACGUUGUGGUACAUUACCWJ(GJA

CAGCA

GCGCIJ

AAACIJ

CUGCG

GAGCU

GGGCU

CGGCU

CAGCA

CCACU

UAGCA

AUACA

UUGCU

CCACA

CUGCUJ

CUACII

GUACA

CUGCA

GCC CCCGGCCC CCC CGCACUCC GCC CUGAUGGG GCC CCAAGGGC GUI) UGAGAACA GUU CCCAGUCU GAC GUGUGCAG GAC UCCAALJGU GCC CAUCGGGG CCC GCAGEJCAU GAC UACAACAG

CC

GAC

GUC

GAC

GUI)

GUC

UAUUGGGU

UUACAGAA

UACUGACC

CCCAACCC

GUACAGGiJ

UUIGACCUEJ

I

9.

9* 9* 9 9..

9* 9 9 .9 9** 9** 9* *9 9 9 9. *9 *9 *9 9*9 9 .9 9 .9 9 9* 9 9 9** 0 t Table 7 Mouse ICAM Hairpin Ribozymne/S ubst rate Sequences nt. Hairpin Ribozyme Sequence Position Substrate 164 252 284 318 447 804 847 913 946 1234 1275 1325 1350 1534 1851 1880

GGGAUCAC

UGAGGAAG

UCAGCUCA

GCACAGCG

AAGCGGAC

AGAGCUGG

UCUCCUGG

UCIJACCAA

AGGAUCUG

AAGEJUGUA

CCCAAGCA

AUUUCAGA

UGCCEJUCC

CCCCGAUG

ACAUAAGA

GUCCACCG

AGAAUIGAA

AGAA GUGA ACCAGAGAAACACACGUUGUGGIJACAUUACCUGGUA AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAA GCUU ACCAGAGAAACACACGUUGUGGUACAIJUACCUGGUA AGAA GCUG ACCAGAGAAACACACGUU(GUGGUACAUUACCJGGUA AGAA GCAC ACCAGAAAAACGUUGUGGUACAUACCYJGGUA AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCGGUA AGAA GCAU ACCAGAGAACACACGUUGUGGUACAUUIACCJGGUA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUJACCJGGUA AGAA GCUA ACCAGAGAAACACACGUUGUGGUACAUUACCJGGUA AGAA GUUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGYUA AGAA GUCU ACCAGAGAACACACGUUGUGGUACAUUACCJGGJA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUTJACCUGGUA AGAA GCAG ACCAGAGAAACACACGUJGUGGUACWIJACCJGGUA AGAA GCAG ACCAGAGAAMCACACGUUGUIGGUACAUUACCJJ(GUA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCTJ(GUA AGAA GUAG ACCAGAGAAACACACGluGIJGGUACAUIJAccIGGuA AGAA GCGU ACC-AGAGAACACACGUUGUGGUACAUUACCJGGUA

UCACC

GAACU

AAGCU

CAGCA

GUGCA

CCGCG

AUGCC

CCACU

UAGCG

UAACA

AGACG

CAGCA

CUGCA

CUGCU

UGGCA

GUt'

GLUU

GUt'

GUC

GUC

GAC

GAC

GCC

GAC

GUC

GAC

GAC

GAC

GCC

GCC

GUGAUCCC

UGAGCUGA

CGCUJGUGC

GUCCGCUU

CCAGCUCU

CCAGGAGA

LJUGGUAGA

CAGAUCCU

UACAACUU

UGCUUGGG

UCUGAAAU

GGAAGGCA

CAUCGGGG

UCUUAUGU

ACGCU GAC UUICAUUCU 0 *0 0* 0 0.* 0~* 0 0** 0* 00 0 0 90 0 0* 0 0 0*O 0 0 0 0* 0 0 0 0* 0 *9 00 0 ~0 0** 01 Table 8 Rat ICAM Hairpin R ibozyme/Subst rate Sequences nt. Hairpin Ribozyme Sequence Substrate Position 59 84 295 329 433 626 806 849 915 1182 1307 1357 1382 1858 1887 2012 2303 2539

AAAGUGCA

GGAGCAGA

GGGAUCAC

GCACAGUG

AAGCCGAG

UUCCACCA

CAUUCUUG

UCUCCAGG

UCCACUGA

AGGGUCUG

ACCUCCAA

AUGUAAGA

UGCUUIJCC

UCCCGAUA

GCCCACCA

AGAAGGAA

GAGUUGGG

AGACUCCA

AGAA GCAG

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCAU

GCGA

GCUG

GCGU

GCGC

GUGA

GCAU

GUGG

GCCA

GCAG

GCUG

GCAG

GCGG

GUAG

GCCU

GUGI)

CUG

GCUU

ACCAGAGAAACACACGUUGUGGUACAJUACCUGGTJA

ACCAGAGAAACACACGULJGUGGUAC-AUUACCIJGGIJA

ACCAGAGAAACACACGUUGUGGUACATJUACCUIGGUA

ACCAGAGAAACACACGUUIGUGGUACAUUACCUGGUA

ACC-AGAGAAACACACGUIJGtGGUACAUUACCUGGUA

ACCAGAGAAACACACGUJLGUGGUAC-AUUACCJGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGIJA

ACCAGAGAAACACACGUUGUGGUACAUUACCIJGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGIJA

ACCAGAGAAACACACGUUGUGGUACAUTJACCIGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUtGUGGUACAUUACCUGGIJA ACCAGAGAAACACACGUUtGUGGUACAUTJACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCJGGTJA

ACCAGAGAAACACACGUUGUGGUACAUUACCIJGGUA

ACCAGAGAAACACACGUUG(JGGUACAUUACCUGGUA

ACCAGAGAACACACGUUGUGGUACAUUACCIGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCIJGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGIJA

CUJGCIJ

AUGCU

UCGCC

CAGCA

ACGCA

GCGCU

UCACI)

AUGCU

CCACU

UGGCG

CUGCG

CAGCA

CUGCA

CCGCU

CUACA

AGGCUJ

ACACI)

CCACA

AAGCU

GCC

GCC

GUI)

GAC

GUC

GCC

GUI)

GAC

GC

GAC

GCC

GAC

GCC

GCC

GCC

GAC

GUC

GCC

GUI)

UGCACUJU

UCUGCUCC

GUGAUCCC

CACUG;UGC

CUCGGCUIJ

UGGUGGAA

CAAGAAUG

COUGGAGA

UCAGUGGA

CAGACCCU

UUGGAGGU

UCUUACAU

GGAAAGCA

UAUCGGGA

UGGUGGGC

UUCCUUCLJ

CCCAACUC

UGGAGUCI)

GUGGGAGG

CCUCCCAC AGAA 201 Table 9: Rat ICATM HH Ribozyme Target Sequence Ut Position U1 23 26 31 34 48 54 58 64 96 102 108 -115 ,IQ9 120 146 152 158 168 185 209 227 230 237 248 253 263 257 293 319 335 337 338 359 367 374 375 378 386

GAUCCAAC

GCUGACUU.

GAACUG=t

CCUCUGCU

CUGAACCU

CtJCAAGGU

GAGAACCU

CCCCGCCtJ

CCGUCCU

CAAUGGCU

CCUCU=C

CUCCUGGU

GGACUGCU

UCCUACC

GACACUU

GUUGUC-AU

CCAGACC

ACCCGGCUJ

AU=U

UGAACAGU

GAAGCCUU

GGGTJGGAU

CAGCCCCU

GACCAAGU

CAAGCDU

CUGAAGCU

GGCCCCCU

CACUGCCUT

GAGCCAAU

GAAGCCLTt3

GAAGCUCU

CGGAGGAUJ

ACUGU=C

vUGUCtC.U AAGCtrJD

CACGCAGU

CAAUGGCtJ UtU.CCcc

AGAAGCC

ACCCACCU

CGCUGUGE7 U CACACUGA c cuucucm c UUCCUcur C CUGUCU C AGAUTIACW A CAAGCCCC C GGCCUGGG C cCr.GAGCC U LIAGCUCCC U CAACCCGU C CUGGU=t C CUGG-U=G U GGGGAACU U tJGUUCCCA C CCCAACU C CCCGGGCC U GGAAC C CACCTJCAA C ACGAGUCA A CUUCCCCC C CUGCCUCG C CGUGCAGG A AUCUGACC A ACUGUGAA U GrJGGGAGG C GIACACCCC A CCUtAGGA C AGUGGAGG U UJCUCAUGC C CUGCCUCG U CAAGCUGA C ACAAACGA U UGAGAACLJ A tJGGUCCt3C C AAGCtGAG C CUCGGCUEJ Tj CAACCCGtJ C ACCCACCtJ UCCYGCCtTC CACWGGUtA J UUGGAGCU HE Target sequence nt.

Position 394 420 425 427 450 451 456 495 510 564 592 607 608 609 631 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 1

GCACCCCU

CCU6-CGGCU1 U C AkAGAACC-U GGtCt= CUCGCuUI

GCCACCU

GQGC -GC GAAAAL-Gut

GGGAGUAU

C-AGCCAAU Z AGCCAAtJ iC CCr-AUUU C CAAtJUUCU C GUCACUGT U UCAM't7GU C GAACt3GCU C GCACCCt7 c AGGCCAGC-U C CCAGAC=t V CGGACUUu c GCCUr C CAGCAUUU A Ct3AACt7 U.

CAACUt3jU C CUCCUGGE7u C UCCUGCCU C ACUGUGCrJ U UCtUGU U CUUUU C AGGCCUGU Uj GGCCLJGUUj U CUJCCrJGGU C UCCLJGCCU C GCUCAGAU A CCUGGG.-tJ U CU7GACAGU U GCUCACcu u CAUGGCLT U CCATJGCt3U C U COM)IACAG CCAG-rCA :7 C--GCCACC

JAAAAACCA

:AUC-tJGc-,

:CCCCAGC-C

U GCrCA, ACOGtJGUA I CCAACCAC

ACCAGGGA

=CA~GC

AL-GCUUCA

CAUAGAAUG

UIUCCU7CMJ

CCAMGCGCA

C~GCDU

C-GAACtJCC

QC.UU=CC

CCCUCU

CCCCUCAC

UUCrAGCUC AGtTCCCA 7CUGUCGC

GC-GGUGGA

UG-AGAACU

CCCtJGGAA

CCUGGAAG

UCCUGCCtj

CCUGCCTJC

CUGGL7CGC

UGAAGC-UC

UACCUGGA

GGAZACUA

AUUUAUJUG

JAGCAGCU

CAACCCGU

CUCUcGALCA HETarget Sequence 202 ccc.

4 c ccc.

ce c 9 ccc.

*c.c cc c c b.c.

cc..

c cc 'ccc c cc cc c 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 1066 1092 1093 1125 1163 2164 1166 1200 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 1334 1344 1351 1353 1366 1367 1368 1380 1388 1398 1402 1408 1410

GACCACCU

cucuuccu

AAUGGCUU

GACCAAG

UGUGUT

GCAGAGAU

UUGAGAAU

IGAAUCU

CUACAACU

TUACAACCtU

ACAACUULT

tJUCGULT GtJGGGAGU

CCGGAGG;U

GGAGGUCUT

CCLUhCCUU

AGAGGGGU

AGGGAU

C CCCACC C UUGCGAAG C AACCCGUG A ACUGUGAA C GUUCCCAG U UUGUGUCA.

C UACAACU A CAACVUU U DUCA=u U UCAGCDC U CAGCUCC C GUGGCGUC A T3CACCAGG C UCAGAAGG C AGAAGGG U GUUCCCAA C UCAGCAGAL C CAGCCCCU c c* 9 9 cccc c ccc...

c CCCCAACU C TJUGUGATJ ACGACG U CUUWUCU CGACGCU C UUUUGCUiC ACGCUVC U UUGCUCUG CUCUUGCUT C TUGCGG=C AUCCAAU C ACACUGAA UUGGGCUU C UCCACAG GGGCDUCU C CACAGGC TUUGGAACU C CAUGGCUT GCGGGCUU C GUGAIrU CUCCDGGU C CUGGUCGC UGUGCLVhJJ A UGGVCCL3C G-GAAAGA.U C ALVCGGGU GUCACUGU U CAAGAA13G CAGAGADJU U UGUGUCAG AGAGGGG;U C UCAGCAGA AGCAGACE c t.1tACAUGC AACAGAGU C UGGGGAAA GEIAUUCGU U CCCAGAGC UCGGUGCU C. AGGUAXJCC UCAGG=C A AGAGGACU UAGCAGCU C AACAAUIGG AGGGUACU U CCCCCAG CGtk=CU C CCCCAGGC GAUGGUGU C CCGCUGCC CUTGCCLIAU C GGGAUGGU t3GGar 3 A ACUGGAUG3 Ct7GGCUGU C ACAGGACA CUGUTGCU U GAGAACUG UUCGUGAU C GUGGCGUC' CGAACUTAU C CZAGUGGAC 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1.506 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

GGGLMCU

ACCCXCCU

ALACUUGU

AkGAAGG%-U

GGGAGUAU

AGGGMAC

ACUGCUCU

CCUGGGGU

CGUGAAAU

GAAAAUGU'

LUGGGUC;LU

GCCACCAU

GUCCUGGU

ACCUGGGU

CUGAUCXUI

GO GCCCU

UGGGAAGU

uccaA=Ci tJGACACUI ACAccUAu t

AGGAAGAUC

CAGGADAUI

UACAAGUU

CCC~cGc CUMCACtU r GAACAGAU C GAGAACCU C GGGCUOUCU C GGCCUGUU U CUGCUCGU A CCCCACCU A CCGGACUU V CUCCDGGU C UCAGAUAU A GAUCACAU U- GUCCAUUU A CCUCUGCUT C GAGAACCU c GACACUGrJ C AUGGU=C C UCCCU u GCUCAGAU A AACAGAGU c GCGGGCTJU c GCCACCAU C ACCCACCu c AGAGGACU c CCCCEMhA C CAUGUGCtJ A C CCCC-AGGC C CtJCtGGCtJ A GCCUCAGG C AGGAGGAG C ACCAGGGA U CCCCCAGG U CC-UCUUG U GGAGACEJA U AUGGUCAA U CCAACCAC PAUUG~xrJG

GCC-GUUGU

3 CGGGCUU

UGCUCGCA

CCUGUUUA

I UGUCCCA

UGACCGCC

I ACCGCCAG

AGGAUILUA

CA-AGUtAC

CAGAAGGC

CCOGAGCC

rGCCCUGGU

AAUGGAC-A

GGCCUGGG

CCLUGCCLTC

GACCUCUJC

CAUACAUU

CGAJCUUTC

CUGG;UCGC

CCUGGAGA

CACGGUGC

CACCUATJU

CUGGUCCU

GGCCUGGG

CCCAACUC

ACCUGGAC

AAAAACCA

UACCUGGA

UGGGGAAA

GUGAUCGtJ

ACUGUGUA

ACAGGGUA

GGAGGGGC

UGACCUGC

UAUGGUCC

203 5 S .9 0 9~ S 9 *0SS 0*00 *0 00 S. 0 S *0S* 09 9 *000 0S0* *0

S

*SSSS@

5 i.865 1868 1877 1901 1912 1922 1923 1928 1930 1964 1983 :!996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2U125 2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173 2174 2175 2176 2183 22.85 2186 2187 2189 2196 UUCCII-2J

TJCACCGAGU

ACA.GUAC

CUAAAC7

GAACAGAU

AUGUEAU

t2GGACGat3 G--c AG Au LrOGAWAcU -AGAGPZLku

GAGAACCU

MLGGAAG-Cu ALtGJAGU

CCE--GC~J.

CUGCCMLU'

MUJUCGU

CGGAGGAU 4

CCUGACO"U

C=GUCZ

GCG;UCCA6UI AMCUr=t VGUAGCUc CClAACUCPJ T C-_cwGAC-t trjccGAcU AGUGCCGt3 GCCt7GUUU

C

CCAA.CtUCt !UGAUGAAU C tCGACAUU .A MUGAU 10 GAUGIDAU Uj AU.GaA7UU A kAAUUCJt A tM.UUUtTU A UC-AUGt;LU U G-AUGTJAUU U GUTLIJT. U CAGULAUU u UGUGCAU A UJCUCCAUU A AUUUCUEJU C GAAAAUGU

U

UC-ACAG~rj- A ACAGUUAU U C~GA= U AGULM=~u A UMJUUEMU U CUGACAGU

U

A GACACAAG C AUMUAAAU Uj CCCCCAGG C AAC-XU2CCA C AAUGGACAL U AUCCICCUA C ACCUUUAG A UCCUGGA A AC=GXMU U G"%=UCAGC C GC-CCrt-GG C UUCAAGCU."r U AULTGCCA A. VCG GA C GC-CvXCGU AL CCCUUC

:ACAAACGA

:UGAG

CAAUGGCtJ LM~hC;CCLtL k GCCUCAGG

:AGGCCUAA

I GULGAUGU

CUGGAGGU

LGGGUCCCG

Cr-AtGAT.C *CUGCuct

GUUGAUG

'UACAACUU

6 jumuA~ r JiJUAA~jrAUTAt3U

UUAAUUA

CCUUuUTU

AUCCAGAG

UAUMLIU

AUM-AUUC

AAUUCAGAL

AUUGAGEIA

UGGUCCUC

CCCCUGcrU

ACGAGUA

CCAACCAC

UULTAUUGA

TJAT'UUGAGU

AIM-C-AGUA

UUGAGLIIC

GAGUACCC

AUUUAUrJG 2198 2199 2200 2201 2205 2210 2220 2224 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 GAA=ltCU C CGAG..tjCA AC-ACrtCJU A CAUGCCAG GGGmkC= c ccccAGC-C GGGCEJCtC CACAGGTJrc E3UU00=U C AGCCACUG C ;GAACU A ACUGGAUG GAGAACCU C GGCUG~C-, ACAtXh.CAU Ui C=tCC-uU LC3GACCU C AGLGCCAC: UCALGCUU C ACAGAACJ Ct.CCLTG-U C tM'G-UC-c AUCCAAUU C A CACXUG; A GAUCACAU U CACr AUCAO.LUU C ACGOGcU AXJCAGGAU A MkhCAAU GAGCAGG;U U AACAUGUA GGAAAGALU C AUACGG=t ACAGUUAU U MAUUGAGU GCClCGu c ciUcC-,AU CAGGAU A CAAGUt~hC GGAAAGAU C AUACGC-GU UE3GGCUu c ucrCAGG GG-%tZmkmu c CCCCGGC GGGCCE;GU C GGr,-'GC;cL Ct'GC-UCCGU A GACCUCUC CCCrUC-CCU C CUCCAcA CCAUCCAu c ccAcAGAA, CUGU=j c CCt3GAAG GAACUGCU C UUCCUC~u GACUUCC-U U CUCmkUUA CUMGAUU C UUUCACGA, Ct7GC-UCUEJ C CUCUUGCG UGAU-UUCU U UCACGAGEJ AUE7UCULTU C ACG-AGUCA.

UAUJCCC-GU A GACACAAr, UAAIJACU A UGUGGACG UGUGCUAtJ A UGGUCCUC CAATJUUCU C AUGCUCA AUfCAGGAU A tUhCA.GUU UCAUGCErJ C ACAGAACU ULJAUCAAU U CAGAGUUC CCUGGGGtU U GGAGACUA UCAGAGtU C UGACAGUT) CGGAGG;LU C ACAAACGC, UGAACAGU A CUUCCCCC: GAAGCCUU C CUGCCt7CG GGCCUGUUj U CCUC-CCUC GCCEJGUUUj C CUC-CCUC'J 204 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2521 2533 2540 2545 2568 2579 2585 2588 2591 2593 2596 2601 2602 2607 2608 2609 2620 2626 2628 2635 2640 2641 2642 2653 2659 2689 2691 2700 2704 2711 2712 2721 2724 2744 2750 2759

ACAUUCC

CCCOGCCU

GCGCCGU

CCUtGU

G-ACCACC

ACCLt~AU

ACALCU

GIUCA=U

Gt.CCUU

CCUUGU

ACAGCAUU

UCGG"GC-U

CAGAGAUU

CCUGCACU I

CUGCUCGU

cuUCCU tCUCUU= Ct3CCU=GU tJGUGCMU I GUCCUG C GUGGGAGU CUULGCU C UGGAG.AC

TJCAGAGOUC

CtJCUCAGU A UCACAGA.U

C

GCL1CAGGU

A

CCCCACCU A GC CUGUU C CCACAGGU C AGAAGGGU

C

ACUAGGGU

C

UCAGGCCU

A

ACGGUCU U GACCACCU

C

ccEcC U CCUCCUU A GGAAAGAU

C

AAGAUJCAU A GGGtJGGAU

C

A CCU~U= C CUCCCACA U GUUCCCAA A UtUhCCGCC U GUGAUCC U CCCAIGU C CCAAZC C CCCACCUA A CAUU3CCUA U CCL.CCUU C CJAhCCUt A CACCCAUU UJ CCCAAUGY C CCAAUGtJC U ACCCCUICA C AGGtChDCC C: CGGA.CUUU U UGOGUCAG JUT GCCCUGG k GALCCDUt

:CCACAGCC

UUGCGAAG

kCCCCUGCU

:CUGTCGC

k UGGUCCUC

GCCGUUGU

LUCACCAGG

'CCGUGGGA

LACDGG&UG

UGACA=U

6GUGCUG=r r UCAC

UCCAUCC

CCAUU

Ct3GCCrEc

AGGUGC

CtJGCAAGC

CUGAACU

AGAGGACU

CCCCCAGG

CCCACCUA

AGGAAGGU

GGAAGGUG

AtUhCG=G CGtJGCAG 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 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969

CGGACV

CGUGAA

CUCAUJG

UCAUGC

GCUCCC

CGIGACU

CCUGAO

UACAAC

CAACMt

UCGGMG

CACA=G

GCACCCC

UEIACCCC

UUJCGAUC

uG.GCUG

UGGAGUC

AGGCAGC

GGCUGAC

GAACUGC

GGCUGAC

GUUGAU(G

CUGCUCU

UGAUGUMl

GAACUC-C

AUCUUC!

AUGUA

UUUUUX

UAUUAL

CUtJCCULCt

AUUUCUUU.

UtJUUGUGt)

GAUGGUGU

UGGAGUCU

CAGUACUU

ACCAUGCtJ CCGGAC UU tJG'-tICCt3 cruJuCCUU

TUAUUGUGU

UGUGJATU

CUUEIGAAU

UGGAAGCtj

GAAUCAAU

rujC GATUCtjUCc CUC UGCGGCCTJ AUU ACCCCUGC AUU AUGGUCA CU CACAGAAC QUC ACAGAAcrJ XU C CtGACCCU 3UC GAULtJUCc CU C CLGGAGGU JU U CAGCUCCc UC AG-CULCCC:A UC AG7JAUCC UA CTJUCCCCC "U C CCAGCGCA .UC ACCCACCty uu CCGAcMG UU CCCUGGAA ~C GGGCcA UC CCAGCACC UC CGGALCtUUU Ui UCCUUCUCU UC UUTCCUCtU IU CCtUCUCU UA UUUAtTUAA UT CCUCourCG UU UUMUU T UCCUCLU 3C UCULC 3C TUAUUACCC 3A UUAAUUcA IC GOCCCAG UJ AAUUCAGA A AUUCAGAG C UUCGAAG U GCGAAGAC C ACGAGUCA C AGCCaTCUG C CCGCUGCC C CCAGCACC C CCCCAGGC U CCUCUGAC U CGAUCUUC C UGACAUGG U GAAUCAAU C AGCC-ACEJG C GU-UCCCAG C AAUAAAGU C TJUCAAGCU A AAGUUUUA GACGAACUT A UJCGAGUGG 205 2975 UGGAAGCU C UUCAAGCUT 2976 MUAWUC CUCA.CCUG 2977 GAAG-CUCU U CAAGCMGA 206 Table 10: Rat ICAMT EH Ribozyme Sequences to.

00 00 C nt.

Position 23 26 31 34 48 54 58 64 96 102 108 115 U19 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 Rat E Ribozym. Sequence UC-AGUtG C13GATJGAGGCGAAAGGGAA AX7CCGAtC tUIGACIAAG CUG 3LGAGGCCGA-.AAGG CCGA AA17JCAGC AAGAGGAA c GAL~xAGGCCGAAAGC'-G

ACGUUC

AGGACCAG C GAUC-A=GGCAAAGGCCG;.A AG7CAGAGG GO~AATCU CUGAIJGAGGCCGAAAG.7CCGALA AGt-=cAG GGGGCUUG CUGAUGAGGCCGAAAGGCCGAA

ACCUUGAG

CCCAGGCC CUG UGAGGCCGAAAGGCCGAA

AGGTUUC

GGCTJCAGG CUGAUGAGCCGAAAGGCCCAA

AGGCGGG

GGGAGCUA CUGUGGGCC-GAAAGC-CGAA

AGCAC

ACCGGG CUGAUGAGGCCGAAAGGCCGAA

AGCCAXUG

AGGACCAG CUGAU G%-CCAAAGGCCGAA

ACA'GAGG

GCGACCAG CUGAGAGGCCGAAAGGCCGAA

ACCAG

AGJUCCC CU'GAUGAGGCCGAAAGG;CCGA) AGCAGuCC UGGGAACA C~-U GCCGAAAGGCCCGAA kG,-.MGGA GAGUUGGG CUGAUGAGGCCGMaAGGCCGA

ACAGUGUC

GGCCCGG CUGAUGAGGCCGAAAGG-CCGA

AUCACAAC

GGAGUUCCQVGGCGr.AAAGGCCGAA AGG-JtJGG TJUGAGGUG CCGAUG GCCGAAGGCGAA\ Ar.C-GGGU UGACUCGU CUGAUGAGGCCGA~aGGCCC-

AAAGAAAU

GGGGGAAG CUGAflGAGGCCAAGGCCA AMtJGUUCA CGAGGCAG CUGAX3GAGGCCG-AAAGG-cCGAA

AAGCO_UC

CCUGCACG CUr-AUGAGGCCGAAAGGCCGA;.

AT-TCCACCC

GGt3CAGAU CUGACGAGGCCGAAAGGCCGA

AGGGGCU

UUCACAGU CUGAUGAGCCGAAAGGCCGAA AcCGGUC CCUCCCAC CCGVGACfC

ACAGCUUG

GGGGUGUJC LUAGGCGAAAGGCCG-aA

AGCUUTCAQ,

UCCUAAGG CUGAUGAGGCCGAAAGCCA

AGGGGGCC

CCUCCACTJ CUG GGCGAAAGGCCGAA AG.GCAGUTG C-3AGAGA CUGAUGAGGCCGAArCGAA

AUUGGC-UC

CGAGCAG CtUGAGGCGAAGCC

AAGGCUEUC

tICAGCUUJG CUGAUGAGGCCGAAAGGCCA

AG-AGCUUC

UCGUUTJG? CUAGGCGAAGCA

AUJCCUCG

AGUUCE3CA CLGUA CGA1LAGGCCGAA AGCACAGU GAGGACCA CUGAUGAGGCCGAAAGGCCGAA

AUAGCACA

CMCAGCUU CUGAUGGC-CGAAAGG.CCGAA

AAGAGCUU

AAGCCGAG CUGAt3GAGGCCGAAAGGccGAA

ACUGCGUG

ACGGGUtJG CUGAUGAGGCCGACCA AGCCAUjUG AGGUGGGUJ CUGAUGAGGCCGAAAGGCCGAA,

AGGGG-LT;A

GAGGCAGG CUGAUGAGGCCAAAGGCCAA

AGGCUCU

UACCCUGU CUGAUGAGGCCAAAGGCCGA

AGGUGGG,-U

AGCtJCCAA CUGAUGAGGCCGAAAGGCCA

ACACAGC:G

207 394 CVGUtUCAG CVGAGAGGCCCAAACGCCGAA AGCACCAC 420 UGCGCUGG CUGAflGAGGCCGAAAGGCCGAA AGGGGUGC 425 GGUGGCAG CAGGCAAGCAAAGCCGAGG 427 TUC-GU=U CUGAUfGACGCCGAAAGGCCGAA AAC-AGGGA 450 CGCAGGAU J AGGUUCUU 451 GCGGGG CVAG=GAG-CA AAGUCCC 456 UGGGA

AAGCGAG

4905 UACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGtiGGC 510 UUCCCACG COGGAGOCCGAAAGGCcGAA ACGCAC 564 GOCCUUGG CUGAGAGCcGAAAGGCCGAA AtkUUUJUC 592 UCCCUGGU CUGGflGAGGCCGAAAZGCCGAA, A~CJCC 607 GCAMMAGA CMX UGAGGCCGAAGC a AUUGtC 608 AG-CAUGAG COGAUGAGGCCGAAAGGCCGAA AAUUGGCUT 609 AAGCMMlG CUGAUGAGGCCGAAAGGCCGAA AAAU(GG 611 UGAAGCAIU CUGAflGAGGCCGAAAGGccGAA AAAAU.UG 656 CAUCUG COGAflGAGGCCrGAAAGGCC-AA, AcAGUGAC 657 AflCU CUGAUGAGGCCGAAAGGCCGAA ACAGG 668 AAAGGAk COGccAGGAAAGGccGAA ACGUC 684 AAAGUCCG CTJGAUGAGGCCGAAAGGCCGAA

AGCUGCCEJ

.692 GGAGUUCC CUGAUGAGGCCGAAAG~ccGAA AGGUCUGG 693 GGAAGAUC CO GP AAGCCGAAAGCcGAA AAAGUjCCG 696 AGAGGCAG COMAUGAGGCCCGAAAGCCcGAA AAACAGGC 709 GUGAGGGG CUGAUGAGGCCGAAAGGccGA AAALUGCUjG 720 GAGCUGAA CCGAflGAGGCCGAAAG-GCCGAA

AGUUGUAG

723 UGG-GAGC CUGAX3GAGGCCGAAAGGcCGAA AAAAGUUG 9735 GCGACCAG CUGACNGGGLcGaAG ACCAGG 738 UCCALCCCC CUCflGAGGCCGAAAGGCCGAA AGCAGGA 765 AGUUCUCA CUCA3GAGGCCGAGGCU ACCACAGM 769 UUCCAkGG CUC7AUGAGCCGAAACGccAA ACACAAGA ***770 CUUCCAGG CUGAUGAG-GCCCAAAGCCGA AACACAAG 785 AGGCAGGA CEXNUAGGCCGAAAGGCCGAA. ACACG=C 786 GAGGCAGG CGALTCAGGCCGAAAGGCCGA.L AACAGCC .792 GCGALCCAG CUGAUGAAGGCCGAAAGCCA ACCAGGAD 794 G-AGCU CUGAUGAGGCCGAAAGCCGAA

AGGCAGGA

*807 TJCCAGGUA CUGAUGaAGGCCGAAAGGc AUiCUGAGC 833 UAGUCUCC L"CGADAGGCCGAAGCGAA ACCCCAGG 846 CAAUAAU CUGAUGAGGCCGAAAGc ACUGUCAG 852. AGCUCU CUGA rA=GCGAAAGGCCGAA AGGUGAGC 863 ACGGGUUG CUCGAUGAGGCCUAAG~cCGAA AGCCAT=tj 866 UGCAG CUAGGCGAAC A AAGCALIGG 867 UAGGUGGG CUGAfGAGCCGCAAAGCcGA AGGtjGGUC 869 CMTCGcAA CUGAVGAGGCCGAA==U~

AGGAAGAG

881 CACGGGUU CUCAlGAGCCCAAAG~CCCC AAG,=UU 885 UUCACAGU CUG JLGAGGCCGAAAGGC-CGAA ACUUGG;UC 933 CUGvGGAAC CUGAUCACCCCGAA GC~CGAA AAUjACACA 936 UGACACAA CUGAUGAGGCCGAAAGGCCGAA

AUCUCUGC

978 AAGUUGUA CUCAUGAGCCGAAGGCCGA

AUUCUCAA

980 AAAAGUU CUGAWGAGGCCGAAAGGCCGA AGAjxUCUC 208 0* 986 987 988 1005 1006 1023 1 025 i066 1092 1093 i115 1163 1164 i2166 i2172 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1-306 1321 1334 1344 1351i 1353 1366 1367 1368 1380 1388 1398 1402 1408 1410 1421 1425 1429 1444 14 5 5 1482 1484 1493 GAGCUGA CUGAUG-AGCrGAAAGG.r-CCAA AGUAL GGAGCUGA COAGAGGCCGAAACG-CGAA AAGuuGUA GGAGCETG GAAAGGCCGAA AAAGu= G-ACGCCAC CUGAflGAGGCCGAAAGGCCGAA Aur-AcGAA CCDGGDGA CUCIAGCCGAAAGGC-CGAA ACDCC,-AC CCUUCUGA CUCGAU~vGGCCCQAAAGCGcAA AcCucCG CM=CU CUGAUGAGCCGAAAGG%-LcCGAA A-aCC=CC UWGGAAC CUArAGAAAGCCG,-AA AAGG LMCGCUG CUGAMAGG cGAa-Cc.A. ACCCCU -IGGGG^=COGUAGtQAAC~c% AVCAACAA CUGCiLUGAGGc-cmAAAGcCGAA AGUUCG= AGCAAAAG CUGGrv= AGG-CC,-AA ACGLCGU GAGCAAAA CUAGAGCCGAAAGGCCGAA AAGCGUCr, CAC-AGCAA CMU GC-CGAAAGGCC,-aA AC-AAGC~r AGGCCX-k CUG AuGGCCGAAAGG-_CGA AGCAAAAG UUCAGUGU CUAJAG-GAAAGGc-CGA~ AAULGGAU CCUGUGGA CUCAUGACCCGAArGccGA

AACCCAA,

GACCUGUG C GMAU AGGCCGCA AGAACCC ACACAUG CUGAtGAGCCCGAAAZGCCGA

AGUUCCA

ACGA!UCAC CUGAVGA=GCGAAAGGcC.A

AACCCGC

GCC-ACCAG CUAU GCCGAGc-_CGAA ACCCCG GAGSACCA CUG CGAGGCC GA,-C:A AUACCACA ACCCGEIAU CUGAXUGAGCCAAAGC-,. AUCUUCC= CATUCUG CUGAUGAGGCCAAGCC,--

ACAGUGAC

LCGAC-ACA CUGA GGCCGAAAGG=cCGXA A~IJCUCUG UCUGCUC-A CUOG AG~CCGAAGGCCGAA a=CCCLT GCmUGU CUGAUGGCCGAAAGGCCGAA AGUCUGCU UrW.CCCCA CUGmADuICCAGGCCGAACQ ACUCUGU GCUCUGGG CUGAUGAGGCCGAAAGCGAc ACCAAIJAC CGGALA=C CUGAG GCCCAAAGCCLz AC,-%CCGp AGCCU=CU vU% GCCAAAGG-_CGAA, AG=CUGA CCAUUGUU CUGAUAGCCGZ-aGGCCC~

AGCU-CUA

CCOGGGGG CDtGLIGAGCCGAAACGCCAA A~tCC GCCUGGGG CUGAMGAGGCCrAAAGCCGA

AAGMAC

GGCAGCGG CUGAUJGAGGCCGAAA-GCC

ACCAUC

ACCAUCC CUGAUGAGGCCG aAGCGAA A~GGCALG CAUCCAGU CUGAUGAGGcCAAAGcrGA AGCCc UGUCCUGU CUGAUGAGGCCGaAAGCC

ACAGCCAG

CAGOUUCUJC CUGAUGAGGCCGAAAGC,LGAA AAGCACAG GACGCCAC CUGAUTCGGCCGuAAGGCCGAA

AUCACGAA

GOCCACUC CUGAXJGAGGCCGAAAGGCCGAA

AMUGUUCG

GCCUJGGGG CUC-nAGAGCCGAAGCCCGA

AAGUA=C

AGCCAGAG CUGAX3GAGG-CCAAAGCCG AGGGG CCUG'CAGGC CUG=AUGAGG-CCGAAAGCCGzA

ACAAGUAU

CUCCE7CCU CUGAX3GAGGCCGA GGCakA AGCCUUCU UCCCUGGUJ CUGAUCACGGCCGAAAGCCA

AMACCCC

CCUGGGGG CUGGGCCGA AGGCCG-AA AGUACCCUT GCAAGAGG COGAUGAGGC-C AAGCCG- AGAGCAZU UAGUCUCC CUGAUGAGGCCGX;A-GCCCG-

ACCCC-AGG

209 1.500 UUGACCU CUGAUGGGCCGAAAGGCCGAA AUUrUCAc 1503 Gt3GGDUGG CUG~XAGGCCGAAGGGAA

ACAUU=JC

1506 CCAACAAU CUGAUGAGGC-CGAAAGGCCGA AtJGACCCA 1509 TACACAGU CUMAGA GCGAAAGCCGAA AL-GG-UGGC 1518 ACAACGGC CUGAUGAG-GCCGmAAAGGCCGAA

ACCAGGAC

1530 ACAAUUAUJ CUGIJGAGGCCraAa=GCCAA

ACCCAGG-U

1533 AAGC-="GC CCuI~LTGAGGCCGAAAccGA

AUGAIJCAG

1551 MGAGCA CtJGAflGAGGCC-.XAGCGcG

AGCGGCC-X

1559 MACGG CUGAGAGGCCGAAGG--L

ACDUCCA

1563 UG-" C GrAGGCC.UGGCGAA

AGGUGGA

1565 GGGCA CUGAXACG-- GCCCGA AGGUUA 1567 CUGGCGC;U LLG1GGccAA~CA

ATGGUGU

1584 U~tUTCCtJ CVGAGAGGCCGAAGCcCGAA AflCUU3CCU 1592 GL-MACUUG CUGAtJGAGCC-rGAAAGGcCQGAA A~tkcCUrG i599 GCCUOUC ulUC% AGGCAACGccA AACUiA 1651 GGCOCAGG CU GAGGCCGA GCCAA AGGCGGGG *1661 ACCAGGG CUGAUGAGGCCGAAAGGCCAA

AAGUGCAG

1~ 1663 ttC=CAjflU CUGAIGAC-GCCG.AAGGCCGA& ADfCUGUUC 1678 C~CAS=c CtGAGAGGCCGAAGGCCGAA AG3GUrUCtC 1680 GACCUGUG CUGAUGAGGCCGAAGGcCGAA

AGAAGCC'C

1681 GAGGCAGG CUGALUCAGGCCGAAAGCCGAA

AACAGGCC

*1684 GAGAGGUC cV AUGAGGCCGAAAGGCCGA

ACGAGCAG

1691 GAAGAfl= CUGADAGGCCGCAAGCCGA AAGtUCCGG *1696 GCGACCAG CUGAUGAGGCCGAAAGGC;

ACCAGGAG

i698 UCUCCGG CU GUG GCCGAACGGc

AUUCUGA

1737 GCACCGTJG CUAGGCGAAGCA AnjGUncA 1750 AAUAGGUG Ct3GAUGAGCGcAA CCGAA, AAAUGGAC 1756 AGGACCAG CUGACGAGCGCCG a-aaGCGA

AGCAGAGG

1787 CCCAGGCC CUaAflrACGGCCGAAC CC-A AGGUUCUC *1790 GAGOUGGG COGUGA=AAGGCCGAAJ ACAGrJGUC 1793 GUCCAGCT-. CU AUGACGCCGAA ~CCCQ ACGACCMU 1797 UGGUUDJUU CUCAUGAGGCCGAA GCCL AACAGGGA 1802 UCCAGLI CUGAflGAGGCCGAAGCCGAA

AUCUGALGC

1812 UUt3CCCCA CUGAflGAGGCcGAAGCCAA

ACUCJGU

1813 ACGAUCAC CUGAflGAGGCCGAA .CCA AAGCCCCC 1825 UACACAGU CUGAI GGGCc:GAAGGCA AtJGtJGGC 1837 UCCCUGU LirGAI3GAGGCL-GA zkCCCGAA

AGGUGGGU

1845 GCCCUC CUGaAUGAGGCCGAAGC~A

AGUCCUCU

1856 GCAGGUCA. CIGALTGAGGCCC, CGAA .AfUAGGG 1861 GGACCAUAh CtrzAUGAGGCCGAAAGCCQA

AGCACAUG

1865 CUUGUGUC CUGADGAcGGcGAA GCCGAAL ACCGGAr 1868 AIUUUM CUGATJCAGGzcGAAAG,-cCGAA ACtUCGUGA 1877 CCUGG CUGAtUGAGGrCcGAA CCCCAA AGtrACUGU 1901 GUACuu CUGAUJGAGGCCGUAAGGCCGAA

AGUUUUAG

1912 UGC3CCAtUU CUGAUGAGGCCGAA GG!CCGAA AUCUGUrJC 1922 UAGGCAAU CDGAflGACC-GGCC CCGAA ACUUACAU 1923 CaAAAGGU CUGAXUGAGGccG AGCGAA

AGCGUCCA

1928 UCCAGGUA CUGA 7GAGGCCGAA C,'CGAA AUCUGAC 210 a a a a.

a 1930 2.964 1983 1.996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2115 2128 21.30 2145 2152 21.56 2158 2159 2150.

2162 2 163 2166 2167 2170 2171 2173 2174 2175 2176 2183 2185 2186 2187 2189 21-96 21.98 2199 2200 2201 2205 2210 2220 2224 CAUCCPAGU COuUACCCGAAAGCCCGAA

AZUCU=~

GCLM'ACAC CUGPArGAGGCCGAAAGGCCcAA AAAflUC CCCAGGCC CVGPAGCCCGAAAGGCCGAA

AGGUUC

AZCUGA CUAGM-"-

AGCC

UAGGCAAU CUA GCCGAAAGGC~cGAA ACUAC&x3 CAtUCCCGA CaGAVGAGCCAAAGaGC;

AGCCA~CC,

ACCAUJCCC CUAUuAGCtrzAAAGGCCGAA AlAGCAG GUACAGGG C-G CCCGAAAG~ccGAA ACUCAAUA UCGUUUGU CUGAflGAGCCGAAAGGCCCA;L AUCCUcc ACCOCCAG CVGAUrGAGGCCGCAAAGCCCGA;L

AC,-A;AGG

ACI=CGUAC-CcAUUGGCCGA

AGGACCALG

UAGGUA L"UGUGAGCCGAAAGCCGAA

AIJGGACG

CCUGAGGC CIJQ GAGGCCGAAAGGCcGA ACAAGUAXJ UtkGGC-=CU ACAGGCCGAAAGGCcGAA AGGCU~.Cz, ACAL1CAAC VAUAGGCCGXUAAGGCC=A

AGAGOUG

ACCUCCAG CGUGGCCGAAAGGcC=a

AGGUCAWG

CAGGACCC LUGuflGAGGCCGAAAGGcA

AGUJCGGA)

GALYCAUG CUCUGAGCCGAAAGGCA ACAoCC;CU AGAGGCAG CtJGP GCGAAAGCCGA AAACAGGC AC-AUCAAC CUDGAflGAGGCCGAAAGcCCAA

AGAGUUG

AAGUUA CAGGCCGAAGccA

AIUUCUCAA

UAAL COU AGGCCGAA GCCA AACUGUCIL AUJUAA CO %A=GCCrGAAfGCCGA At2ACAUCA.

G-AAfltMU CUGAUGACGCCC)SAGGCCG; AADAkC=.J MJAMMUA CU-tA GAACCCGALA AAUk=~ AACAAAGG CUO ~jGwcaAAAGGCCGAA. AGGAAflGU CUCUGAAU CUG =GCCGAAAGGCCGA AALVA~LaX AAUUAkAV CUAG GG,-AAAGGCCGAA

AUACADCA

GAAUL'GW COGUGAGGCCGAAAGCGcG AAIUhCAflC UCUGAAUU CGUGACCCGAAAGCCGA

AUAAAC

VACUCAUT CGAGAC CCZGAAAJGCCG

IAU=

GAGGACCA CUA GCCGAXI.GCCCGAA

AUACCACA

AGCAGGGG CVGAIMAGCCGAAA AA~CCG~1hA TJGACUCGU CrAUAGCCGAAAGGCCGA

AAAGAAAXT

GUGGUUGG CUGAX1GAGGCCGAAAGCcGAA AcAfluric TJCA.AA CUGAUGAGccGAAAG~CGfl

AACUGUCA

ACL7CAAMA COGAGAGGCCGAIGCCA fAUCUGU M=CCAAU CUGAUGAGGCCGAA GCG; AAVAACUr GLMhCUCAA, CO rG~ccGAAAtGC

AAAUAACU

GGGUACUC CUGAUMGAGGCCGAAG-CA

AUAIA

CAAUTAAAU CGUACCGAXAGGCCCA

ACUGUCAG

UGACCUCG CUGAUGAGGCCGAA GCCGAA AGACAUUC CtIGGCAUG CUJGAUGA rCGAGCC GAA AAGAGUCTJ GCCGGG CUGAX3GAGC-CCGAAGCCGA

AAGUACCC

GA.CCM7UG CUGAtYGAGG-CCGA AGCCGAA AGXZGCCC CAGtJGGCU CUG~.A CGAAAGGA

ACACAA

CAUCCAGU CLTGAUGGCCGAAAGGAA

AGUCUCCA,

CCCAGGCC CUGAUGAGGCCGAA GGCGJ AGGUOCUC AAGGUAGG CUGAI3GAGGCCGAAAGCCGAA

AIGUAUG

231 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 UGUGGCCL7 CUGAVGA GCGAAGGC-'AA

AGGUCC-A

AGUrUC~UM CU fGAUGCCcGAAACCGAA jAsCaUG' ACt~hCUGA C.U =AAGGCGAA AGCUG=u GCGACCAG CUGAUAGA AAGGC~CGAA

AMIGAG

UUCAGtUGU CUGACGGGAAAGGCCGAA

AAMGGAU

GCA.CCGUG CUGAUGAGGCCGAAG--CA AM"Gj'C AGCACCGU CUGAUG G=GCCGA GG.aA

A.IGUGAU

AACUUtGUA CtJGIJGA=GkC3GCGAA

AUCCLUGA

UAhCA=U CUGAUGAGC-GA-GCC r.AA ACCOG--Cc ACCCGM.U COGAUGAG-CGAc GC- ~G

A=U~LU-CO

ACUCAA~k CUGAUGAGGCCGA AGGCCA. AUAcrjW CAUUOGGAG CUGAUGAGGCGAAGCGAA. AacCAG Gm.Cuz= UaruvAGGCGAAGCM

LUC

ACCCGOAu cuGAuGAc.G-caA cr~cGAA ALUCMtUCC CCUGUGGA CUGAUGAGGCCAAAC,.C-AlA

AAGCCCAA

GCCUGGGG Mu AIAGGUcA=^ UGAGCAcC UAGCaAGM

ACAGGCCC

GAGAGGUC CCGAUGAGGCCGAA

GGCL

UGUGGGAG CUC-flGCGAAAGGAA

AGGCAGG

UUCUGUG CW UGAG-CCAGMG,-GA

AUGGA=G

CUTUCCAGG CUGAG GCCaAG~CG

AAAAG

AAGAGGAAa UAGCWAGCCAAGCAGUrjC UAAt~hGAG CUGXAGrcc GAA AGGAAG UCGIrAAA AUAc CAA

AAAUCAGC

CGCAAGAG CtJGAGAGGCAC---,'ct- AA G-;.ArCAG ACUCGL1GA CGruGC;AGCGA;(A;T TGCACt7C.U CUGUAGCGAGGCA

AAAGAAAT-T

COUGtJGC CUGAUGAGGCCGXAGCCGAA AC'-CaA CGUCCAMCA c UGAG-GaAGCA

AGUU

GAGGACCA CUAGGCGAAGCA AtUGACkA UGPkAGCALU CUGAUGAGGCCGAGGCGL AGAAATJUGc AACE3UGaA CUGAflGAGGCCGAAGCG;

;CACCUA

AGUUCUGU COUGAGCCAGGCGA

AAGCMA

GAACUCOG CUGAUCACcGAGCA;

=AXAA

tJAGUCtJCC CUGAUGAGGCCAAG CGAA ACCCCAGG; AACUGUCAL CUGGGCGAGGC

AAMCCUA

UCGUUUGU CUA;UGAGCCGCCGAAQ AVCCt7CCG GGGGGAAG CUGAUMAGGCCG AACCGA;, ACOMtJECA CGAGGCAG CUGAUGAZCC

AAGGCC

WGGAG CGADGAGGCCG CCGAA

AACAGC

AGAGGCAG CUGAUGA G CAA,

AAACAC'

AACAAAGG CUGAUGAGGCCGAA GCCA AGGAAUGUr UGUGGOAG CUGAUGAGGCCG AGCCGA AGGCAGGcG UUGGAAC

CE

7 GAUGAGCCGAAAGCCA

AAGM=AG

GGCGGWAA CUGAUGAGGCCGA.GC-CCGAA AGGU-uMjA, GGGACA CUGAUGAGGCCGAAAGGCCG.A A~C-GCGAc ACAUUGGG CUGAUCGCG AGGCGAA ACAAGGtJ GACAUUGG CUAGCC

AACAAG

UAGGUGGG CGAGAGGCCGAAAGCCCAA AGG ,uC,,u S S

S

SS

.55555 212 0 0 000.* 9*o '06.

0000 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 MLCGGAAUG CUCGAOrACGCCGAAAGCCGtAA

ALGA=

AAGGCAGG CGUuCGCAAACGccCAA AlGfLtU AAAGGLAG CVGCVQAAGcc

AAUGJGTJG

AATAGGM AG AC~rcGCCCA

AAAUGGAC

ACAUUGGMGAUAGCCGAcccA AC~azCGU GACALIOW CUA CG AAAccCG;L

AACAAA,

UGAGGGGU CUM AGCCGGCCC AAflGCUGU GGmM=CWArACCt3 GccAG~cCr

AGCACCGAL

AAAGUC CGA GACCGU ,CMU~ AGCUGCCU CtUGACACA~ CUGAI GAGGCCGAAGCW AAUrCU=rJ CCAGGGCA CUAGCCGAG~rA

AGUGCAGG

GAGAGGUC COGAUGAGC-CCGAAC-,CGAA

ACGAG=~

GGCUGUM GAUAGCCGAAGCCCA

AGGAGGCA

CUCAA= CGUGGCCGAAAGGCCGA

AGGAAGAG

AGCAGGGG CGUAGCCGAAGGCCGA AAtUhGAGA GCGACCAG CGUAGCCGAAGGCCC- ACCAGGAGa GAGGPACCA COAG cG cc AJAGcCA ACAACGGC CUGAGA GAAGGCCGA ACCAGrAc CCrJGGOG C GAUGAGCCGAAC--

ACUCCCAC

UCCCACGG; CUGAUGAGCCG AGCML A CAUCCAGU CGUAGCCGAAAG~CCC

AGUCUCCA

AACUG=GU GCC~aGGAAc

AACUU

AGCAGCAC CMAGG CCGUGCC

ACUGAA

GGCUClGA CUAU GCCGAAGC

AAGUU~

GUGAAUM UGAUAC-CCGXGCCA

AUCUGUGA

UGGAUGGA CUGAUGAGGCCGA UZCCGA

ACCUGAGC

AAUGUAU ~CcrWG GGCGXGGC

AGGUGGGG

ACAGGCAG COGAUGAGCCGAAAGGC

AAACAGGC

AGCACCCU CUCQ ACCurGu GCUUGCWCGUGCAAG cG~CCG;

ACC==UC

AGCUAG CGGAGCCGAACcr AcCr-cuu AGOCCUCUT CUGAGGCCGACc-

AGGCCUQA

CCtUGGGGG CMUGAGGCAGG .A A= c UAGGUGG CUGAUGAGOCCGAAG CG; AGGUGGrJC ACCOUCCU Cc c~ AGGLJAG CACCMUC CUGWXGAGGCCGAACCCGAM

AAGGUAG

ACCCGITUUCGt~Gc

AUCUUC

CAAACCCG COPX UGAGCGAGCCc A At3CDU CatJGCACG CMGAAGWCMAWZC

AXJCCACCC

GGUUuuaL~ CGUAGCCGAACCGA

ACAGGGAC

CCTCGA CUGAUGAGCCGAAG CGAA. AGUUCGUC GGAASAU CUGCGAGGCA AAAGUCCcG AGGCGUAGGCCGAAAGGCCGAA

AGCAAA

GCAGGGGtJUAJAGCAAG

GAAAAA

UUGACCAU CUGAUGAGGCCCAACGCGAA

AUUJUCACG

GUUTCUGUG CUGAGCCAAGCC

AGAGA

AGUUCUGUT CUGAAGCCGAACGC

AAGCAUGA

AGGGUCAG CUGAUGAGGCCG AGCCA

AUGGGAGC

GGAAGAUC CUGAUGAGGCCGAAAGCCr.A AAAGt7CCG 213 2821 ACCAG CUAGGCGAAG~- ACGtCAGG 2822 GCGAGCUG;L CUAAG-GAAGC

AAGUUA

2823 UGGGAGCU CUGPIDGAGGCCrGAAAGCCAA AAAAGUrG 2829 GGAMCCU CUCA=ArCGAAAGCG,AA AGCACCGA 2837 GGGGGAAG CUGAUGAGCC~uAAGGCCGAA AcCCEGMG 2840 UGCGC-UG CUGAUGAGGCCGAAAGcccGAA AGc.C,7.GC 2847 AC-%GG-tC-acz C GGcGC .G cG AGCGGUAA 2853 Ct-MGUCGG CU3GAflGAGC=cCCG GcA A GAAAUCG-A 2860 UUCGG CCAUAGGCAAG~CCCAA A=cA(aG 2872 UGAGCACC CCGAUGAGCCtrAAGcrG ACAGCz- 2877 GGUGC-'CW CUCA~'v,-GACCG; AG C-crr_ 2899 AAAGUCCG Ct3GALGAGG-CCAGA GGCG AG,-UGCCU 2900 AGAGAAGG CUCVMGAGGCXC GccG XWCA=C 2904 AAGAGGAA CUGAflGAGCCGAAACGCCC-AA

ACAGUC

2905 AGA.GAAGG CUGAUGAGGCCCAAAGGccGA .AGUCAGC 2906 UU~AAA CUAGG-tAA~CA

ACAUCAAC

.*2907 CGCAAGAG CUG XGAGGCCGAAAGGccAA AAGAC= too: 2908 AIAULMALXA CDGAUGAG~CcGAAAGCM AMCAMjc 0.:.02909 AAQAGGAA CUGAUGAGGCCGMtAGc-CGA, AGCAGrjC *2910 GTA~ADC- CGLGCGCCGAAGGcCGaA

AAGGAAG-'

2911 GGGU-aAAML CUA~XCt.A~,CA

AGAAGG-AA

0:0*2912 UCAUTA C~zUAGCAAst= AAL~ 2913 CUGGGAC CCACAG-GAc~c;

AAMCACA

2914 ULCUGAAUtJ CGAGCc-GAA.CGcCCA

NLALA

*2915 CUCUGAAUT C GQGAAG~cG cc-AA GGCG AAMAj 2916 CUt7CGCAA CUGAUCAG~CCGAAA-C r-V ACGGA= *2917 GUj"JUCGC CUG UGAGCCGAAGGcCGAA AGAAGGAAG 292.8 UGCuC=GU CUGAUGAGC-CCGAAGGCCGAA AAAGAAAU *2919 CAGUGGCET CUGAUACG-CcGA GaZCAA ACAAAA 2931 GGCAG=G CUGAfLGAGGCCGAAGGcCGAA

ACCA

2933 GGUGCCUG AUGAGCCCCCAGccCGAA

AGXCUCCA

2941 GCCUGGGG CUUGACCCAAGcG~GCCGA

AAGUACUG

.*2951 GUCAGAflG CUACaGCAA~cA IGCAfUG 2952 GAAGATJCG L"UGAzGccc ;aGCCCA AAcGU 2955 CCAUGUCA6 CUGArGrLGcGAAGcCC.A

AGGAAGC;L

2956 AUUGADUC CUGAOQArAGCCGAA CGAA AAGGAAAG.

2961 CAGUGGCU CUC-AU ACGCCGAAACGCCA aCACAA 2962 CUGGCA&C CJG AGCCGAAAGGcCGAJA

AAIJACAC

2965 ACUUUT~ CUGAUGAdGCCGAAAGCCG;L

AUAA

2966 AGCUUGMA CUGAUGAGCCAAAGGcCGA

ACUUCCA

2969 tJAAAACUtJ Ctrz JGAGGCCGA ~jzcCGAA ADUGALUUC 2975 AZCUUGA Ct3GAtmAGGCCGAG~,-CCA

ASCULTCA

2976~ CAGGUGAG CtJGUGAGG.CCGAA GGCCCAA ACCAUAUJA 2977 UCAGCUUG CtTGAUGAGGAGCCC aGAC 214 Table 11: Humaynn IL-5 HE Target Sequence at.

Position EM Tar'get Sequence Ut.

Position NH ToxGret Sequence a.

a.

a a 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 AUGCACU U UGCACU U GCACUU C ACUUCU U CUUUcU Uj AGAACGU U G-AACGUU U AACGOUU C GGAUJGCU U GAUGCUU C UCUGCAUJ U CUGCAUJU U UEUGAGU U UUGAGUU U GUU7 A GCtChGCt C LVGCUCUT U GCUGCCU A MCGUGU A AUGCCAU C CAGAAAU U AGAAAU C AGUGCA.U U GAGA.CC U CACOCCJ U ACUGCU UJ CUGCUUU C GCU UUUM A uurca~cu c tU.CUCAU.C UCGAACUT C UGCUGAUJ A UGAGACU C UGAGGAD U GAGGAUU C UtJCCUG U UCCt3GUU C UUCCEIGtJ A

UCUUUGC

CUU~xrCc

UUUGCCA

UGCCAAA

GCCAAAG

UCAGAGC

CACA~CC

AGAGCCA

CUGCAU

UGCAUUU

UGPAGUU

GAGUUUG

uGcaAGc

GCCAGCU

GCU=

TJUGGAGC

GGAGCUG

cGuGL1AU

UGCCWUC

CCCACAG

CCCACAA

CCAMG

GGUGAAA

GGCACUG

UCUACUC

CUACOCA

TMCUMA

CUCAUCG

AUCGAAC

GAACUCU

UGCOGAU

GCCAAUG

UGAGGAU

CCUGUUC

CUGUUCC

CCUGUAC

CUGUACA

CAAAA

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 AAGAAAtJ

GAACU

UAAUTJ

AAUCUUU

AGGGAAU

GGAGAGU

AGCGGG

AAAGLCU'

AGACEUt GACUauuc

AA.AAACUI

AACUUGUC

UUGUCCU t UJGUCCUU 2

CCUCAAU

AAGAAAUJ AA.UACAU t: GGAGAGU A AACCAAU U ACCAAUJU C AATJUCCU A CLVGCU A CAAGAGU Uj AAGAGUJU U AGAGUUU C AGUUUCEJ U UUGGtJGU A AGUJGGAU A GGAUAAU A AGAAAGtT U UGAGACU A AACUGGU U ACUGGUTJE U GGUOUGU U CAAAGAU U AAAGATJU Uj AAG-AUUUj U AGGACAU u GGACAUJU u GACALTJU U ::UtUCAGG U UJCA~GA J CAGGGAA AGwAU k. GMCCAC

:AAACL'GU

k CLYGJGGA, k. UUCAAAA 7 CAAAAAC

:AAAAACU

I GUCCUUA

'CULGLAUA

IAAUAAAG

LAIMAAC-A

LA; LA AAU LCAtUGAC r GACGGCC

AACCAATJ

CCUAGAC

COU.GACU

GACUACC

CCUC-A

UCUUGG~u C7UGGUG

UUGUG

GOGIA

AUCGAAC-A

AUA~vAUA

G-AAAGU

GACA

AACUGGU

UGUUGCA

GVUGCAG

GCAGCCA

IUUGGAGG

UGGAGZGA

GWAGGAG

L7JACUGC

UACGCA

ACUGCAG

UGUACAU A AAAAUtCA LVLAAAAU C ACCAACU 215 *9*e 9* 9 9.

S S 9 500 531 538 539 542 543 544 545 549 551 554 555 556 560 561 573 577 579 580 581 588 597 598 611 616' 617 619 620 625 627 629 630 631 636 638 644 647 653 655 656 657 658 661 672 676 678 S81 682 ACAV=UU A CUGCAGU AAAGAGU C AGG=U CAGG=~t U AAflU=U AGGCCUU A PAUClUC CCOUAA U UEJCAAfLA CULULWU U UCAALIAU UUAAUUU U CAA tUAtUUM C AAMlUnA 013UCAAU A UUUUiA UCAALVM A AUUtMLAC AUAhAU U MLCUUC T-IULADU U AACUUC;L AMLWWU A Acuw-AG UUU AACU U CAGWGGG UMACUU C ACAGGA GGXAAGW A AA~kUUU AGUAAAU A UUUCAW UAAkt U flCAGGCA AAA~kTJU U CNG= AAM=lU C AGGcAUA CAGGCAU A CUGACA UGAXCACU U UGCCAGA GMACACOU U GccAGAA AAAGCAU A AAAUC= AtMhAAA U CU~kAAA MLZAAAUU C UuaAAALT AAAfUCU U AAAAMILU AAflUCUU A AAAUADA UtkAAAU A UAUUCA AAAAIMV A UUUA AAIEh.W. U UCAGAiM AM==fl U CAGA.MC tMkMUU C AGAMWC.

UUCAGALT A UCAGAAMJ CAGAUAU c AGAJCA UCAGAAUJ c AvuWAAG GAAUCAU U GAAGc~u UUGAAGU A UUUUCC GAAGtLhDU uuccC AAGM!tUU U UCCUCCA AGUUU u ccucCAG GMTfUCJ C CUCCAGG UUUUCCU C CAGGcA GCAAAAT-T u GA3akmkC AAU=GU A UACOU=r UUGAUAU A CLUUUM AMU U UU=O tiAM~CUU U UUUCUUA 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 MCUUUU U UCUUAUU CUUU=U C U~tIUUA UUUUUCU uT AUfLVA~C uOuurCUE' A UUaACU TUUAU U UAACUUA u CAW.U U AACUUM C;UtMuu A ACUUAAC UOU)AACU U AACAMXU UGaACUE A ACWuUCU UMCAr-U U CUGUAAM MAO=IJ C UGIMhAAA.

AXUUCUGU A AAAUruC AAAAflGU C UGOUAAC UGUCUGU U AACUA UCi A ACtUMAU GUUAACU U AAXkGuA UaAACUrj A AUaMU;= ACUUahW A GMW=~ MUAGU A UULIUA ATmGUADL U U0ALGAAA UAGtM=l U AUGAAAU AGMTfU= A UGAAATJG AAAUGGU U AAGAAUU MUGGUU= A AGAAUW UAAGAAU U- UGGUAAA AAGAAUU U GGEMAAU ALUUrrU A AAfaLUGtI GGt~hAAU U AGUUU GUAAMfl A GUUMU AAflUAGU A UUUAUU UMZ=UA U MULTUAA UkGUAU U AUuUAAU AGM=~t A tUUUAAUG MULUAU U UAAUGU AUUL~hVU UJ AAUCUUA UUUADUU A AUGUUAU UUAADU U AIUGU UAAIUU A tJGUUGUG GUUGU U GUGUUCU GUUGUGU U CUALAUAA UUW=rj C UAAA GUGUUCU A AIJkaAAC UCLIA A MAACA CAAku A GACAACu 216 Table 12: Human IL-5 HE Ribozyme Sequences

C.

C

be..

C.

Cbb*

C.

be..

ebb.

C C be..

CC

C

C

bC

C

nt.

Position 12 13 36 37 38 56 57 63 64 69 70 74 78 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 192 200 201 206 207 212 216 222 245 GCAAAGA CUGAMAG-CrC-AAAGGCCG-.A

AGUGC-;U

GGCAAAG CMU WC"AAAGGCC--

AAGUGCA.

TJGC-AA CUAGGCc--AoG-C-. AAAGtGC TUUUGGCA CUGAVGACGCC,-kAGGcC-A AGAAAGTj CUUUGGC CUGAflGAGGCC C-AA AAGAAAG GCOCT3GA C~a ~vG~GCCG ,AAGCC. LC=3UcU GGCUCt3G CUGAGAGGCCGAG C-.AA -AAC-rUCC 0 =J-r CUAGGAAAC,-CG

AAACGUU

AAUGCA CUGAUGAGGCAGGCCGAA

AGCAUC

AAAUGCA COGAUGAGGCCGUAGCWG.A AAGCAtJC AAACtJCA C GAGAG~rAACC-A

ALC-CAG-A

CAAACUC CUG-AUGAGGCCGAAG~c,-.A

AAL-GCAG

GC~kGCA CGJAGCIcA= -A ACUCAAA AGCEMWGC CUGAGAGCCGGG-CQ,- AAcCCA CAAGAGC CUAUGGCC-AAGG-,- AGC A~ GCOCCAA CU~NGAG GAAACc

AGCLVJAGC

CA==UC G-WCCG-AAAC,-rA

AGAGCUTA

AtkCACG CUGAUGAC-GC=-kA-GCC Ar;GGC GAUWGCA CUGAUGAGGCC AA ACACGU2.

CUGrJGGG CM AUGAGOCC ~azCCC-A

ACGCU

UtJGEMWt Ct AUGAGG.A-CGACL AUUUCtJG LCUUGUGG CUCAtUGAGGCCC-G-,C-,AA

AAUMU

EIUUCACC COGALGAGGCCGAAAGGCUG

AJGCACTJ

CAGt3GCC CUGAM3AGGCCGAAGcCCA ACGX=Cc GAGCAGA CMMM G=CGCkXCGAA AGCAGrJG UGPIGUAG CUGU CC CGUA AAGCAGU AUGAGUA CUGAt7GGCCGAA CCA

AAAGCAG

CGAUGAG CUGGCAA--CA

AGAAAGC

GUUCG-AU CUGAUGAGGCCGAAGCG- AGt3AGAA AGAGUUC CGUGAGGAc;C~CC-A

AUGAGUA

AUCAtGCA CUGAUGAGGCCG LCG-AA

AGUTJECGA

CALUUGC CUGArGGcGCGGGCCL

ATCAGCA

AUCCUCA CUAMGJC-XGCCGrAA

AGCC

GAACAGG Ct3UAGc.AG-cA

AU=CCA

GGAACAG CUAG -CWAGCA AAlCCUC GtMCAGG CUGIGA,-GACCQ C,-AA ACAGGAA, UGTJACAG CUAGGC-AA~-CAA

AACAGGA

ULTUtCAUG CUGAUGAGGCCG AAAGGCCGA

ACAGGAA

UGALt3UU CUGAUGAGGCCGAAAGCCA

AUGEJACA

AGUUGG;U CUGAUGAGGCCG AAAGCCGAA AUUrUrjL CCEYGAAA CUAJAGGCC-AG-CC-A

AUUUCUUE

H Ribozym. Sequence 217 e.G.

C.

C C 9C en.

C C. C a C

C

C

CO

C C

CC..

e.C CC.

C.

247 248 249 257 273 291.

305 307 308 316 319 322 323 326 334 338 380 388 389 392 397 409 410 411 413 419 437 440 447 454 462 463 466 479 480 481 497 498 499 500 531 538 539 542 543 544 545 549 551 UCCCUGA CM 3

AUGAGGCCGAAAG-GCCGAA

UUCCCUG

CUGACLCCAAAG-CCA

AUUCCC LTAGGAAGCCG

AA

GJGG=CC

GUAGCAAGCA

ACAGrJUU ~C~rArACG

AAGGCCA

UCCACAG CrArAcGAAGCGc UUUUGAA

CUAGGCAAACCCA

GUUUUUG

CJGUGACCGAAAGGCCA

AGUUUU CUGcAGcc UCAG COGALUAGGCCGAAAGGCCCGAA CUUATJUU

CGUGCCGAAGCCA

UCDUUU CWA~tAG-CGWCCGAA ATJUUCtJU CGAGCAGGCCA.A GUCAAUG COAGGGAAGCCGAcA GGCCGUC CUGArAGGCC~kAGCCGAA~ AUUGGUU

CVAAGCAGGA.A

GWCUAGG

CGGAGCGGCCGAA

AGucaAG CUAG GAA c GGLVLDC CtX3AUGAGCC

A

UWGG CUGAfl AGGCCGA ACCAAGA CUGAWGAGC iJ r CACCAAG CUGAVGAGGCCGAA GC

A

AM=CAA CMUGAGGCCGAA G-

A

urmcmcc CUGAUGAGGCCG

AAGC=U

UGUUCAIJ CUGUGAGGCCGLUG-

A

UUUCOAU CUGAX GIGCGA

A

AACUUUC CUG AGCC A G At ~.ukUC aMJAUGAGGCCGXUGCCAC ACCAGU3U CUGAGAGCCGAGC~

A

UGCA COGAI t<r-

AC

3"MCAACUGAG CCGL aCGc

AP

UkaGCUGC CUG MGGAAGGCCG~:~AC CCUCCAA CMUfGAGCCMUAGGCGAA

A

UCCUCCA CUAMGCC kGCAA CUCCUCC CGGGGCCGAAAGGCCQC

A

GCAGMLZL CUG-AUGAGGCCAA CGAA

A

UGCAGUA

COAGGCCGAXGCCGA

L;GC~r Kh CCUACCZJ;GGAA ACUGCAG GAVGCGAGCrA AAGGCCLT CUAGGGCAAGCrA GAAAAUJU CUAUAGCCGA~AACCr.AG UGAAAAU CUGAU

GGCCGAAAGCCAAC

UAUUGAA

CUAMWCGAAGCAU

ATJAUUG CUGA GGCCGAAAGGCCGMAAt UIUUG CUGAt GAGCCGAAAGCCGAA

A

UMM~tU cUGzAnrGGAAAG

CGAAAA

UAAAUJTA CUGAUGAGGCCGAAGGCCGA A~t UtAAU CTGAIGAGrCAGGCCcC Atm

AGAUUUC

AAGAUUU

AAAGAUU

AUUCCCtJ

ACUC

ACCCCCU

AGUCUrU AtJAGUCU

AAUAGUC

AGUUUU

A.CAAGUU

AGGACAA

NhAGGACA kWUAAGG

LCUC

AUUGGU

C7GAAUU

GTJCUAG

,CUCUU

AACUCTJ

3AAACU

:ACCAA

XCCACt7 MtAhCC _ItUUcu

"JCUCA

LCAGrJ

AACC

CUUUT

ATJCUU

Gi7ccu MumJC

M=IGC

;cu 7AAGG

XTAAG

XTEA

AuUA

CAAA

LXIGA

218 554 GAA~a CUGAGAGGCCGUG(C-CGA&

AU~UAL

555 UGAAW CUGA~r.AGrCCGAAAGr,-CCGAA AAaA 556 CUAG CUGAGCGG ,;CrA

AAAXXW

560 CCCUCUG MAGCXAGCA

AGULAA

561 UCCCUCU CUCAGAGCC-LCAA3CCA AjGTJ 573 AAAM= U ArCCCGA(;cG AjCUUUCC 577 CCUGAAA COGAUGGCCGAAGCCG A~UtLMC 579 UGCCUGA COAGGCGAGCCA AAUrjA 580 AUGCCCG CUGAlUGAGGCCGAAAGGCCG.A AAtLtrjr 58i LtAUGCCU CUMAXUGAGGCCGAAGGA AXVX=r 588 GUGUCAG CUGAflGAGGCCGAAGCCA 597 UCrUGGC. CUGAGAGGAAGC AG C 598 UUCUGGC CUGUGAGGCCGAAACGAA GUrUC 611 AGAUrU CUGAtGAGCCGAA-

AUCU

616 0 UUAAG CVGAUAGGCCGAAG=Cr.A

AUUUIU

617 AUOUUVLA CUGaUCAGccGAA rGCC AAtUUUUA 619 ADUiOU CUG~r-AGGCCGAACC-C AAGXjrj 620 iUAU= CGGAIJGAGGCCGLzr ,C AAGW 625 UGAAATI;L COAGGCGAMCA

AUUUUA

627 UCGAA;LCUGAUGAGGCCGAAAGGCCGAA

AWU

C.629 JUCUGACGUAGCAAGCLAAkTJ 630 A7JUCrUG CUGAVGGGCCGAAAC-G AALITJUu 631 CRAUX7CU CUGAUAGCGAGCCGA

AAAUAUA

S..636 ADJUCt3GA CUOGAGCGAAAGGCA AVCUcGx 638 UGAU=C C1GXfAUGGCCQ GG-CCGA AVU=~u 644 CUt3CAAU COGALGA GGAAAGGCC AUUCr3GA Ce S647 AM=~C~C CtGAXJGAGGCCAAGOCGA;

AIU=C

eee*653 AGGAAAA CUAGGCGAACCGL

ACUUCAA

eq655 GGAGGA CUCAUGA=CGACG~CCC- AUU~c 656 UGGAGGA CUG~AGAGCCGAAGGCGA .AAflAE= B::657 COGAGG CLXGAUMCAGG &JeCCGA AAA~kCE 66 CUGAUGAGGCCGAAAGGCCGAA

AAA

612 ULUC cOGAtGAGCCGAA GAA AuGA 672 AGUAl AUtG :*e678 AAAAAGU C GAGAGGCCAAA~ccGAA AucAAurJ e.681 AAAAAA CUGALVGGCC AACGAA

AGUAA

682 UA.AA

CUAGAGG-C-C-CCGAAAAGU

683 AMAGAA CUGAXGAGCCAAAGCCGAA AAG2Au 683 AALAGA Ct7GAGAGGCCAAOCA

AAAG~

684 AAUAAG CUAGGCGkGCG;

AAAAU,

686 UAAAUJAA CUAMGCGAAGCA

AAAAAMG

688 GUUAAAU rAMZCAAGCAAAAX 689 AGutahAA CUGAUGA r-CGXGGCCGAA

AAGAAAA.

691 UAAGUU C~UGAGAGCCGAAAGrCCGAA

AUMAA

692 ULMAGUt CUG GAocc GAAGCCA

AAUAAGA

693 GUEUhAGt7 CUAr.,-GAGCGL

MAAA

697 GAAUTGUU CUGAIJA~GG A BJGCGAA

AGUUAA

698 AQ;AXGU CUGU- GAAGCCGAA

AAGUUA

219 703 UUtUhCAG CMGA=~GCCGAAAGGCCGAA

AOGULIA

704 UUU~CAC CU--rAGCGAA,-)CA AAUrGUMi 708 CA.X CUGUA7,-CAGCGA

ACAGAA

715 GUAAC CUCA GAC-CCGAAGccGM ACAUu= 71.9 ULVLu CUAM3C,AAGGcc CcA AAGACA 720 AUtCAU CUGAUGGCAAjcc CGA AACAGAC 724 UACCAUU CUGAUGAGGCCGAGCAA

AGUA

725 AuACmuU CUGAUGAGGC-CGAAGCCCA

AAGUUA

728 GAAAXAC CUAGAGCCGAA GGCAA AUAkAGr 731 UCAMLZA CCGAUGAGGCCGAAGCCA

ACTJWJMJ

733 UUUCAUA CUGGAWaGGC--C-IA)GG-C-

AM~C=A

734 ALU=A CUMW-XGGCCGAA .GC.A AakA 73-5 CAMUuCA CjACNC-XAGCA AAAtMhC 745 AMUt cuG-AUAoGrCCGAA ~crA AccAuuu 746 AAAUUC CUGAVGAGGCCGAAG AACcxJr 752 UUUACCA CucGtUGAGGCCGcc G-.CG; ADUCUUA 753 AUGA CUGAUGAGGCCcc AMC AArjr=r 660*757 ACM COAL GCGAAG-rA

ACO

*7761 AAA37cU ctAGAGGCC~kGCCJ

ADAC

MAUAC CU~ArCGAA=rA AAtUUMC ~.765 AAAM.AA CUGAUGA=GAcc GCu;L ACLMAUU 0* 767 LZAAIM CUGAUAGGCcGAA cA AUACA 9:0768 AtUAAAU CW-NU AGGCCGAGG=GA AAkU 771 AACAMtM CUG VACrAGGC~CC AtMAU 0.*772 UAACAUU CMUA=AAGcrA AAmLAu 773 AMACAU CCAGCC

AAAUAA

*:.778 ACAACAU CWUXGCAAGCGL

ACAUCAA

779 CACAA CW-VXGAGGCCAAAGCCuAA AAcAULM AUAACAC CDWXfGAGGCCGAAAGGCCGA ACALXh.AC **.788 VGawmG CUWGA GAGGCGL AcACAAC *'*789 Ou M=lG CtjGAUGACGAAGG CA AACCA 791 WUCAtU C~zUACCGAGLCA

AGAACAC

794 UUOG1OUU CUGADCAGGCCGXGCrA AUUAG~hAA 00*805 AGUUGUC UAGC--

AUUC

k 0 220 Table 13: Mouse IL.,5 B ibozyme Target Sequence nt.

Position 8 1i 12 36 36 37 43 58 59 59 66 82 91 112 113 141 141 i58 167 196 197 197 202 202 206 212 212 218 218 218 232 241 241 241 241 243 243 244 HEl Target Sequenice CGCLC~t7 c CUUUGCu UCt3UcCtJ U tJGC-,AA, COEUCCty U GCugAAG GAAgacU U CAGAGuC GaAgAcU u cAgAGjc AAgaJUU C AGAGuCA.

OcaGaGtJ c AflGAgaA GGAtJGC U CDJGCAcU GAUGCrT C UGCAcUU gAUcUU c uGcAcU CUGCAcU U GAZUgt~u UgAcucU c aGctUt1 GcUgtJGU c uggGCCA LUgGAAU U CCCAuagA gGAgAIJ C CCAugAG GAGACCU U GaCACaG GAgACcU U GaCAcAg g'UCcgwCU C AcCGAgC cCGAgCUj C tUGditGAc UGAGGcU U CCtJGUC GAGGcUU C CUGUC gAGG~uU c CtUGuCcc t3UCCUGU c CCUacuC TJUCC~UG c CcUkcuc tJGUCccty a cuCaUAA tUhCUCAU a aAAaUca UacuCAU A AAAADCA UaaAaaU c aCcAGCU UAAAAAU C ACCAgCu uAAAAAU c acCAgCU uaUGCA'U U GGaGAAA gAC-AAAU C UULYCAGG gAgAaAU c TJUUCAGG gagAAAU c UUr3CAGG gAgAaAU c UULJCAGg gaAkucU U TUCAGgGg G-AAAUCU U tJCAGGGg AAAUCUU U CAGGGgc AAUJCUUtJ C 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 nt.

Position RN Target Sequence AGGGgcUJ A GaC-AuAC UagACAU a (=C-a-kgA GaAG-AaU C AAACUGUI GaAGAaU c M;,aCugU GAAgaAtJ c 8AAc

T

gU uGGGGGU A CLUGGA AAAugc A UUcCAAAL AAAugCU a uUJCCaaA ALUGCuAU u CCaAaAc ZAUgCkuA U CCAAAC u6C'=fUU C cAAAACc AACcUGU C alltAAA ctUGUCaU U AAUAAAG UGUCaUUT A AUAAAGA.

CaMtAh7u A AAGAAA1J -AAGAAAU A CArUGAC AAUACAU U GALCcGCC ;AAUaCatj u G-ACcgCC AggCAgU U CCUgG;Lu ggCAgUU C CU9GAuU C'JgGAuU A CCtJGCAA CAAGAGU U cCtxrt3Gou AAGAGrJU c CULTGUG7j -AGUUcCU U G--TnUgA LUcaCA;LU u t3AAgtUA CzAA~TU U .7A9UUaA kAA~UUUr A AgtlTaAa AcAA~juu a aGU'jVAAa AAAUUgU c AAcAgAu GCUGuUtj c CaUuUAU UUUCCAU UJ UauatUrj UUauAuiU u allgUccU ttuarUu u AugUcCU uaUAUULJ a ugUCCuG UAhUAuUUj a tUgUCcUg UUIAtJGt c cUGUaGu UTtUU c cUGUagU AAAGuUI u uaaCCuu AAgUGuUi u aACcUtUU 221 0 620 793 816 818 825 825 839 840 863 864 864 913 917 957 960 960 962 975 987 990 1000 1027 1034 1037 1039 1039 1041 1051 1148 1213 1213 1214 1215 1234 1236 1275 1276 1280 1298 1.310 1310 1310 1350 1358 1370 1.375 1377 1383 1405 caAGgCU Ct3GagtUu GALgutty AuCcucU UCcucuEJ AAgUAUU

AAGU;LUL

gAaCUCU, Ucuuggu tJUagcAU GCAuccU I GcaUcCtyI AtcCuuU c aGaLUGpW UGAuACU UGACuCY c CgggGCrJ T JCCUGcU C UgcUJCcU A cuccu c UGuGCAU a. UACUCcc cUCCcuC

=CCCCUCA

i cGUMGCA GtJUGCAhu I cCAGCCu CAGGCug caggCug rGGucCaG CAGAuGG

CUUUCUC

TJCUCCUA

UCUCcUa UCcUaGC AgAtIAgA CuuAAUG Akugacu ug~CuGA CUaUcuA UUAACtJ UAACUUc TUAAcu u uuuGrJAu 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 14.91 1491 1491 1491 1494 1502 1502 1507 1509 1509 1510 1510 1510 1510 1512 1515 cCAgUUrJ A CtCCAGg ccAgIuU a cucCAw gUUtyaCU C CACGaAA AT-gCtUtj U atluUaAU allgcuuU U JAUUUAAu aTgcuU u UAUUAAU L'gcUvU a UuUaATJU ugcUur=r a umtmauu UUUUAUu U AAuUcug UU~UE U AA~iucUga UUAILotj A AUucUgU UUUaAuL3 c UGuaA~a AUUCUGUj A kgAJGtUU ugt3UcaU a UUTAUUUA tUgTJUcAU A uUAUUUA UucAt=U u AUUEung UCADLUU A UUM=xjA AU=~JU U UAUGAug AuGgATJtj c aGtMAgU AUCaGU A AgUUTAaU aGuAAGLT U AAUADUU aGmkAgu u AaUaUU GUTAAgUU A allAUUUA agUt3hAU a TUuAUA AgUt~aU A UtUAUa tUtAAtlaU u uAUtACA, UUAAuAU u t3AUUaCA UUAaUJAU U tUAUUacA UAAX~auu u ;xumAcA UAaUAUu U AUuAcAc TUAaUJAT3U U APJUacAc AAMJUUU a uuaCAcg AAtLIUUU a UuAccg AaMU~IU A UUAC-ACG AaUUUr A T-r,-acAcG AUuUAUEJ a CAcgUAU cACGT~at A tUaauAtu cA.CgUAU a UJAAuaUEJ AtJAUJAaU a UtYcUaaU AUMUAt-T U Ct~aAuAM allaayaU U ctAAtThA UAAuATJ c UaAuAAa UAAUAUU c UaauAAM UAAuAU c UaatAAA UaatyaUrj C UATAAA allaUUCU A AUrAAAgc.

TJtJCL7ALt A AAgC:AgA CcUAUcU A A~ttcAa tlUcAAuty U AAuAcCC uGAcOUU u cuuamuG GCUgGaY u UUGGAaa gctJGGAU U uUgGAAA cugGAUty U UGGAaaA ugGAUUU U GGAaaAtG gGGACAU GACA.UcU UgGGCCtj gGGCCLTLT UgAA=U gcAAAGtJ GCAAAgU GcaAAgU

AAAGCAU.

AAAUGGU1 UgUuaUtI UUtCAGgU UCCUtJGC "CcUUGCAG U AcUUcUC A CUtICUCC a AGAaGCJA aAuAcCA a aAtyAcca a AAUAcA A AAAUggU UT ggGAugU C AGgEJAUc k UCAGggU UCAGggU C ACtJGgAG CCCCAgU U LACUCCA TIable 14: Human IL-5 Hairpin Ribozyrne Sequences nt.

Position 86 151 172 203 Hairpin Ribozyme Sequence Substrate UACNU PA3AA GUC A ACAGAAAC mi~naWXMiu~s LWGiXU OCC UzACGLU GAL-A PA1AA GJ3CCA ACCAA ACI OCC LU

L-M

UaW-ALG PA3AA GGAU AOAGAAWAh6XnamxrUG GWLJYXU QU OCUGW~A 223 fee* U I0 00 a

S.

a S a a S 0 a 0 a a S a a. *t a a S.

CS

a a SOS C a a a.

C

Sap a C S a SO 4e a ala 0** Table 16 :Mouse 11,5 Hairpin Ribozyine Sequences nit.

POsfltion 83 147 150 154 168 199 274 381 454 499 548 701 710 91-9 1030 1170 1205 1402 1421 CCN3ACAC ACIAA OUGlAGf AGAA WAGUAJ3 AGAA CCCXCAC AG1A AALCAGG

AGAA

CACCAE

AGAA

GUULG

AGMA

UAAArLWA PLGAA GAGGA AGA Hairpin Ribozyme Sequence GAACACArAAN2CCCXna

MMCII

G~L~r- ACGAMACAU

CU

GACAQ CWWNCCCG3~MXA~M GM CAGGOXXAX~aa-UMCX( GMU3AC ACCGA -AAAC-aXna- ~AUL)g GCW. COLGMCWAU GAAAUUs~,~*-

GAEWZA

OrCCA GG3GA fl- Substrate ACtUUA OCU GULMU WGACACA GCU GUCOOCL7 GCtU GCU GACAOAc oaGtUC GIXZ GCAAGC DZ-AACU GME CfLUOG CUGA GJU CCAEXX=A GCAACA oAU GCcAAAA AEUCW GAD UCAMAC AEJxtu GAU CCUCCLIO WCUM GCC IUC3AL UGUCCA GAU GD ozwxOU oCu C~tLAKX LUA GAC UZnXAU W3raDGA 0CU GCNLXM UVOZOA GUU UAL1CA MMAACA GAU GLJG~rXU AGL)AAA )A3AA UW3AUX)G ?AA ALGCACA AGAA CAAAALO NAA AAL-CRIAC AGAA 225 Table 17 Mouse re/ A nt. Position HH Target sequence 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 AAUGGCU a caCaGgA aG~tJct a cGUgG Cct~caU u GC~gACa GAUCt3GU U tUCCC AUCUGurJ U CCCC;C t~uCCCCU C AUCULuC CCCUcC UmuCcu CUCAU=E TU u.CCcuCA T-CAUCO u CCcuCAG CAGGCuU C UGGgCCuI GGgCCut3 A 3GUJGaAG UGGAGAU C ATcGMaC AGAUC=t C GAaCAGC AUGCGaU UJ CCGC~ku UGCGaTJU C CGCLt7A Ut7CCGCLT A uAAaUGC GGGCGCEJ C aGCGGGC GCAGuAU u CCuGGCG CACAGAU A CCACA CCACCAU C AAGCA= UCAAGAU C AA~rGCU AAUGGCU A CACAGGA T-uCGaAtJ C UcCCG CGaAUJCU C CCt2GGUC CCCM=G C ACCAAGG GGcCCCu' C Ct7Ccuga UCCaCCU C ACCGGCC CCGGCCU C AuCCaCA AuGAaCU u GtugGGgA AGaUCaU C GaAzAGC GAUGGCIJ a CUAUGAG AUGGucu c uccGgaG GGCUaCU A UGAGGCU CUGAcCU C UGC-CCaG GCaGuAU C C~uAGcU Cr-gCACGU a UCCAuAg CALLAGCU U CCAGAAC AkuAGCUU C CAGAACC UI2GG9gAU C CAGUGtJG GCUCCU U UUCUCAA GCUCCUUT U UCuCAAG Ct7CCUUU U CUCAAGC UJCCOUUU C uCAAGCU UGGCCAU U- GtJGUUCC 467 469 473 481 501 502 508 509 5127 514 534 556 561 562 585 598 61.3 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 1167 -1168 1169 2.182 1183

CCAGGO

AaGCcAT tuUUgAGm

AACCCC

UuAc--tC'

UUCCA

UCGCCUj Ct7CUGCLT

U-CUGCU

aAgCC,'bu

GGCCCCU

CCCCUCGU

CGCCU

CCUTJCCtJ I3CCUgCU AUCCgAU CCgAUuu CgAUuru 1 UGgCcAU1

CCGAGCU

UCAAGALU

CGgAACEJ

GCUGCCUC

AUGAGALT C GAGAUCU C2 AGAI3CEJE C UJUCUCCE) c AaGACAU

U

GAGGUGU A GGUGUJAU

U

GUGUAUu

U

'UG1UUUU

C

CGAGGCU

C

GAUGAGU

U

AUGAGEWt U UGA'-,N JUU U AUGcUGU

U

U-GcUGUtI a J CUgutJCg

AGCCAGC

3C AGauCAg 7C CAGACCA U UCACGUU U CACGUC U C=MUhAG C CUAUAGA A tACAgG3A A GAgGAGC A uGACuUG C UGC-UUCC U CCAGGUG C CAGIGA u AGCCAGC C CUCCUGa C CUcuaCaC c UCaCAUC C CUCAgCC- C AgCCaug Li CC.%L=C u Ut3UGAuA U GAUAACC .1 GIjGuuCC

AAGALUCU

UGCCGAG

LGGgAGC

GGUGGGG

UUCUUgC CU9CUG uU9CtGU Cau3GcG

GAGU

UUtJCACG

UJCACGGG

CACGGGA

ACGGGAC

CErJUCU

UUCCCCC

UCCCCCA

CCCCCAU

aCCaUCa CCaI3CaG 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

AUUGUGU

uUU cCAGGCU UUCGaG;U CGaGUCU Cg~CGC~ CGcGAC

CUGUUCG

UCCA=G

cADGcAG CGGCC= C uGALICGC GcGAGCLT C AGtXGAGC AT3GGAgU U CCAGM.C tJGGAqUU C CAG~kCu UtUCCAGU A CuUGCC-A GCCucAU~ c CkAGA AGAuGAU C GCCACCG CagUacUu gCCaGkLc ACCGGAU U GAaGAA GAgACcU u cAA~agu AGGACcU A UGAGACC GAGA=CT U CAAGAGu AGACCEJU C AAGAGuA AGAGuAU C AXJGAAGA GAAGAGU C CUUUCAa GAGUCCU U UCAauGG AGUCCDU Uj CAauGGA UCCU7 C AauGGAC CCGG-ctY C CAaCcCGr UaCACCU u GAucCA& GgCGuAU U GCtUGGC UGUGCCU a CCaM aaGCCrUU C CCGaAGu CGaAaCU C AaCUtju CUC~aCU U CVGUcc OCAaCUU C UGUCCCC CUtGU C CCCAAGC CAGCCCU A cCcuLC GCCatIAU a gCcu;C CAUCCCU c agCacc;L AcaCCUY c CCagCALU UCCaUcU c CagCuUc UM~hCUt u AgCgcc cCagCAU c CCUcAGC GCACCAU C AACtU ATJCAACU u UGAXJGAG GAAGACtU CUCCDCC AAGACtJU C UCCUCCA IACUt C CUCCWTU ULYCCCU C CAUUGCG CCUCCAU U GCGGACA.

1184 1187 1188 1198 1209 1215 1229 1237 1250 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 GGcccci- UUACCai GC-gAGut

CAGCCL-L

cuGGCCE GGUCCCt CCCAgcU CcA~CIC CCCaGCU

CCAUGGU

gtJGGgct3 AUgAGuU CUCCUGtY cCCCAGU

CAGLUCU

gGGuCCu

CUUUUCU

ACGCUGrJ CUMcAGU TJGcAGO

GGGGCT

CCtJUGCLT GgaGUGTJ gaGTIG=u

CUG-GCAUT

CUUCgau

GACAACU

UCaGAGEJ t CaGAGU L aGA.GUtx

C

gGuGCAU c AUGGAGU A tJGAaGCU A AaGCUMU A TJAUAACU

C

CUCUCCU A CCCAGCU c UJCCUGCU u CCGGCU u cU~aCCEJ c CUCLgCEJ U ucugcuy c CUCgcuuj u U C CUcCUGa 3c CUcaGCc 3C aGGGCAG Iu AGuCuGa 7a caCCraUc U aGCaCCG u CCucAGc C CUGCCcC C CAGgCuC C CUGCCcc c ccuuCcu C AGCUgCG u ucccccA U CgAGTJCu U CtJAaCCC A aCCCCgG C CCCAGuC C AaGCUa C gGAaGC^C U UGAU)GCU U GAt~cUG U GCUUGGC UT GGCAACA LT CACAGAC C ACAGACC :UGU9GAC IGggAAcu aGAGUUU

UCAGCAG

CAGCAGC

AGCAGCU

CCUGUGu CCCUGjAa

LMACUTCG

ACtTCGCC GCCUgGU GaGAggG

CUGCCCC

C99rUaGG

CCCAAUG

ugccCAG cCAGGuG

CAGGUGA

CGGAGgU 227 1704 ATJGW=C U CUoC!.-Gc 1705 UGGACUU c TUC--GcuC 1707 GACUUCU c uGCUCu 1721 UUUGAGU C AGAUCAG '1726 GUCAGAU C ACuccu 1731 AUCAGCU C CMAGGu 1734 AGCt3CCU A AG~u~Cu 1754 CaGugCT c CCaAGAG 228

U~

4 4 *4 *4 .4*

S

*5 Table 18a Human re/ A nt. Position 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 HH Target Sequences HH Target Sequence AAUGGCU C GtJCCGU GGCCUCGU C UGUAGMJ CGUCUGU A GWGCACG GAACUG TJ CCCCCUC AACUGUUT C CCCCUCA UCCCCC C AIJCUUCC C'CuCA C UUCCCGG CUCAVCU U CCCGGCA UCA.ucur C CCG A CAGGCC C UGGCCCC GGCCCU A tUGrGAG UGGAGAU C AUMAGC AGA.VCA.U U GAGCAGC AlGC= UJ CCGCMThC UGCGCUJU C CGCOkCA TJUCCGCU A CAAGUGC GGGCGCY C CGCGGGC GCAGCA.U C CCAGGCG CACAGAU A CCACCAA cCAcCAU C AAG=MC UCAAGAU C AAflGGC AATJGGC A CACAGGA UGCGCG= C UCCCDGG CGCAUCT C CCEUGGUC CCCUGG C ACCAAGG GGSACCCU C CUCACCG CCUCJC ACCGGCC CCGGCCU C ACCCCCA ACGAGCTJ U GUAGGAA AGCUDGU A GGAAAGG GAUGGCU U CUAVGAG AUGCUU C UALMAGG GGCUUCU A UGAGGCU CUGAGCU C TJGCCCGG GCTJGCAU C CACAGUU CCACAGU U UCCAGAA CACAGtJU U CCAGAAC ACAGUUE7 C CAGAACC t3GGGAAU C CAGUGUG GGCUCCU U UrJCGCAA GCUCCEJU U UCGCAAG CUJCCtJUU U CGCAAGC UCCJUUIJ C GCAAGCU UGGCCAU U GUGUUCC AUUGUGU UJ CCGGACC 467 469 473 481 501 502 508 509 51.2 514 534 556 561 562 585 598 613 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 1167 1168 1169 1182 1183 1184 UAUCAGt ArCGCAL ACCCCtIJt

UCCAAGU

CCAAGuU AGUC~r

UUCCMLU

GGGGA.CU

UGCGGCUj CUCUGG-tY

UCVGCUU

G-AC=CU

GGCCCCU

CG-CCGu"

CUGUCCU

TUGUCCU

CCUCC-

UCCtJCAU

AV'CCCAXJ

CCCAUCU

CC.!LICt-jt1

UGACAAU

CCG.AGCtJ UCAAGAtJ

CGAAACUC

GCUGCCUC

AUGAGAU C GAGAUCU Z: AGAUCUU C UcrxuCCU A: ;A-GACAuj U GAGGUGU A GGUGEIAM U GUJGCATU U TJGUATUUU C CCUAZGCt C GAUGAGU

U

AUGAGUU U t7GAGJE.U C AUGGU~TU U UGGUGU U GGUGUTUU

C

7A CAGtJCA C AGUCAGC C AGCG-CAU C CAGACCA U CCAAGUTJu C CAAG=UC *U CCLULVtAG C CUAUAGA A TAGAAGA A G-AACAGC- A CGACL-tIG C UGCtJUCC U CCAGGUG, C CAGGUGA C AGGCAGG C COCCUGC C CUUCCEUC U CCUCMtJC C CLUCAUCC C AUTCCCAU C CCATCtU C UG-r-ACA U7 UGACAAU j C-LAUC

GUGCCCC

AAGAUCU

TiGCCGAG

TGGCAGC

*GGUGGGG

UUCCtUC

CCEACUG

CEUhCUG;U

CUGUGUG

GAGG;UGU,-

TJOUCACG

UCACGGG

CAkCGGG-A

ACGGGAC

CrUUUCG

UCCCACC

CCCCA

CCACCAU

UCCcj

CCUJUCUG

CUUXtJGG nt. Position HH Target Sequence

S.

445 4 229

S

C

C. 835 845 849 872 883 885 905 906 919 936 937 942 9 5 3 962 965 973 986 996 1005 1006 1015 1028 1031 1032 1 033 1058 1 064 1072 2.082 1083 1092 1097 1098 1102 11i25 1127 1131 1132 1133 2.137 1140 1153 1158 1680 1681 1683 1686 1.690 1.704

UUGU

GCCCCLT

GCcAccU

CGGACCC

CCM=CG

CGCAGAC

t3GCG= C t3CO~LTGC CGtJGUCEJ C CAX3GCAG GCGGCCU U CCGACCG CGGCCULT C CGACCGG G-GGAC-U C AGtGAGC AUGGAAU U CCAG~kC UGGAAUUL C CAG~ccc UUCCAGT A CCDGCCA GCCAGAU A CAGACC.A AGACGAU C GUCACCG CGAUCGU C ACCGGAU ACCGGAU U GAGaGAA GAAACGU A AAAGGAC AGGACAU A UGAGACC GAGACCLT U cAAGAGC AGACCUt7 C AAGAGCA AGAGCALu c AuGAAGA GAAGAGT C CtUUUCAG GAGtJCCU U UCAGCGG AGUCCE3U U CAGCGGA GUCUt C AGCGGAC CCGGCCtU C CACCLTCG UJCCACC C GACGCAU G-ACGCAU U GCUGUGC UGUGCCtI U CCCGCALG GUGCCrJU C CCGCAGC CGCAGCrJ c AG-cuUCEJ CUCAGCU U CE3GUCC UJCAGCUU C tUGCCc CUJ C CCCAAGC CAGCCCU A UCCCUUU GCCCUAU C CCUUtMC UAUCCCU u UiACGtCA AUCCCUU u AcGucAu UCCCDUU A CGUCAIJC UUtkCWt C AUCCCEJG ACGUCAV C CtArC GCACCAU c AAcLYAD AUCAACU A U.GAflGAG GAAGACU U CuCCjC 1187 1188 1198 1209 1215 1229 1237 1250 1268 1279 1281 1286 1309 1315 1318 1331 1334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 1664 1665 GUUtCCu U CtIGGGCA UUUCCU C tUGGGCAG GGC.k M-U C .AGCCAGG CAGGCCU C GGCCUG UCGGCCU U GGCCCCG GGCCCCu C CCCAAGU CCCAAGrJ C CUGCCCC CCAGGCU c CAGCCCC CCCtIGCEJ C CAGCCAU CCALIGG-U A UCAGCUc AUGGUu C AGCLTCrG AUCAGCTJ C UGGCCCA CC CCL7G C CCAGUCC UCCCAGU C CUAGCCC CAGt7CCU A GCCCCAG AGG,-CCE C CUCAGGC CCCUCCU C AGGCUGU ACGCrIGU C AGAGGCC CUGCAG U UGAUGAU OGCAGtIU U GAUGAUG GGGGCUt U GCUUGGC CCUUGC-U U GGCAACA GCUGUGU UJ CACAGAC CUGUMU C ACAGACC CtGGCAU C CGUCC CAUCCGU C GACAACU GACAACrJ C CGAGJEJT UCCGAGEJ U UCAGCAG CCGAGUU U CAGCAGC CGAGUUJU C AGCAGCU AGGGChtI A CC=JLGG AUGGAG!) A CCCUGAG UGAGGCU A LMACUCc; AGGCUIAU A ACUCCC UJAUAACU C GCCt]AGU CUCGCCU A GUGACAG CCCAGCEJ C CGCUCC UCCEJGCU C CACtJGGG CGGGGCtJ C CCCAAUG AUGGCCU C CUUUCAG GCCt3CCU U UCAGGAG CCUCCEUU U CAGGAGA CUCCUEJU C AGGAGAU

C.

AAGACUU

C

GACtUtCU c tJUCLJCCU C CCEYCCAU u AUGGACU U

UCCUCCA

CtYCCAUU

CAUUGCG

GCGGACA

CUCAGCC

230 1705 LrGGACUU C UCAGCCC 1707 G-ACUUCt3 C AGCCCUG 1721 GCUGAGU C AG~A 1726 GUCAGAU C AGCUCCU 1731 ADCAGCU C CUAAGGG 1734 AGC=CC A AGGGGGU 1754 CUGCCCCU C CCCAGAG 231 Table 19 Mouse rel A HH .Ribozyme Sequences nt. HH Ribozyme Sequence 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 UCCUGUG COGAUGAGC-CCGAAAGGCCGAA

AGCCATJU

CACCACQ CGUAGccGAAGCGAA AGCCr, UGUCCGC CUAGAGGCACGCCGA

AUGGAGG

GAGGGGA CUGAI GAGG-CGAt QCCrA

ACAGAUC

T3GAGGGG CUG A GAGCCAGCCG

AACAGAU

GAAAGAU CGU-c- GkAAaC-C,-rA

AGGGMA

AGGGAAA cGaX-AGGCGAA GGCCGAA

AUGAGGG

UGAGGGA CUGAUG GCCGAAAG-.CCG

AGAUGAG

CUGAGGG COAGCrcGAAGCCGA

AAGAUGA

AGGCCCA CUGAUGAGGCCAGCGA

AAGCCU;G

CUCCACA CUGAUGAGGCCGAACUCA

AAGGCCC

GOUCGAU COAGGCGUGCA AI2CUCC;L GCOUa CUGAlGA-CGGG,CGAA ADU~u AUAGCG-G CUGADGAGGCCG AGCGAA

AUCGCAU

T3AUAGCG CUGAflGAGGccGAAAGAA AAuflCAc GCAUUA CGUGAGCCGAAAGGCCA

AGCOGAA

GC-CCGCt7 CUGAIUGAGGCCGAA CCA;

AGCGCCC

CGCCAGG CUGAflGACCG GA

AUACUGC

UUGGG=GA AGCGACCCA

AWUGUG

UGAUCU CUGAG ArC GXUOCGA

AUGGUGG

AGCCAflU CUG AUGAGGCCGAAAGGCCGAA

AUCOUCA

tJCCUGUG CUG flGAGGCC GaNGCCGAA

AGCCATU

CCAGGGA CUGAI2GGCCGAAGCGAA ALWtCGAA GACCAGG CUGAUTGAGGCCGACA

AGAUUCG

CUU COAr CCGh.CCCG

ACCAGGG

UCAGGAG ta~um=GCCG"~G=AA

AGGGC

GGCC7%GU GAGCCGAACG

AGGUGGA

UGWGGAU CVUGAGAGCCAXXGCt .A AGGCCGG UCCCCAC COGAUGAGCCG AGCGAA

AGUJUCAU

GCUGUU CUGAUGAGGCCGAAGCGAA, AUGAUCtJ CUCAUAG CUGAUGAGCGCCGAAAGGCCAA

AGCCAUC

CTJCCGGA CUGAUGAGGCCGAAGCGAA

AGACCAU

AGCCUCA CUGAUGAGGCCGAAGCCA

AGUNGCC

CUGGGCA CUGAUGAGGCCGA AGCCA AGtJCAG AGCUAUG CUGAGAGCCkGGA

AXJCUTGC

CtWUGGA =AGGCGAAGCA

ACUGCGG

GUUCt3GG CUGAUGAGGCCGAAGCCA

ACUW=

GGEJUTJ CUGAt 3 GA=CGAAAGGCCr.AA AAGCau CACACUG CUArGCGAAGCA

AUCCCCA

CGAACAG CUGAt3GAGGCCGAAAGGCCGA

AGCCUGG

GCUGGCrJ CUGAUGAGGCCG AGGCCGAA AUGGCUrj CUGAUCT) CUGAUGAGGCCGAGCL

ACUCAA

UGGUCEJO CUr-AUGG=CGAAGCCQAA

AUJUCGCT

a.

*0 a a 232 0 a.

a.

a S a. a a.

a 501 502 508 509 512 514 534 556 561 562 585 598 613 616 617 620 623 628 630 631 638 661 667 687 700 715 717 718 721 751 759 761 762 763 792 795 796 797 798 829 834 835 845 849 872 883 885 905 906 AACGUGA CGAGAGCCGAAGGG

AGQGGUU

GAACGWG CUGAUGAGC-CGAAAGCCGALA AAGGG-u CUDMG COUAGCCCGAC

ACGUGA

OCUG CUAGGCGAGC

AACGUA

UCCUCUA COGUGA G-CGAAGCCA

AGGA

GCU7C CU VGAC cc

AUWGGAA

MAAGUCA CUGAUt GAAAGCGCGrAA AGtUCCCc GGAAGCA COGAUGAGGCCGAA GcAA AGGCGrk CACC= AGAGGLCGCA

GOA

UCACCtIGA ACC CA AAGCAGA GCt3GGcr3 CO V-C-CAGGCCGAAc

AUGGC=

UICGG CUGAM A C-C--IA=G

A=G-,

GUGAGAG CUAUGAGGCCGLUCGAA.

ACAGGW

GAUGOGA, CUGAGGCCGAAGr.CGAA

AGSA=A

GGCUGAG COGAUGAGGCCGA ACGC AA GGGAC CAUGGCL3 COA GAGCCMAAAG

AGGAG

GAGATUGG CUAGGCGAGC

AGCAC-

GUMAA CMUWGCCUAGCCCA AJCGrA~U GUMUfCA CUAGGGMUZ=GAA

AAAUCG

GGUtWJ CUGAUGAG~CCGACGA

AAAAUCG

GGXACAC CUGAUMIGGCCGUGCCA

AUGGCCA

AGAUCt3U CUAUGGCCG AGGCCG.AA AGCUCcG CT3CGGcA CGUAGCCGAGGCCGA

AT-TC=M

GCUCCCA CUACGGCCA AGUrJCCG CCCCACC CMAGAGGCC GrCGAA

AGGCAGC

GCAAGAA CUGAM AGGCCC 't.GCAA

AUCUCAU

CAGCAAG CUGAflGAGGCCGAA GCGA AGAUUc ACAGCAA CUGAUGAGGCC~aGGAA AArAuctj CGCAATJG CUGALTGAGGCCGAA 1 C AGGAA AAMC C GUAGCGU AUGUcU CGUGAAA CMUtGAGGC CAGCG; ACACCUc CCGUGA C==GAUGAGCC AtkCACC UCCCGUG a=UGfGAGGCCG AZCGL AAt7ACAC GUCCCGU CGUGAGGCGCCCA AAAc AGAAAAG UA UGAGGCC GGcccAA AGCCUCG UGAGAACUA =====WWCAA

AGGAGCC

CtJUGAGA CUGAt7GAGGCCGA AGGCcG

AAGGAGC

GCDUJGAG CG&UGAGGCCGGCAA

AAAGGAG

AGCUUGA CUGAUGAGGCCGAAGCGAA

AAAAG

GGAACAC CUGAUGAGGCACCC AA AUGGCCA AGUCCGG CUGAt3GAGGCCGAA C ACACAAty GAGUCCG CU AUGAGGCCGGCGA

MCACAA

GCGUACG CUGAUGA7GC GAA AGGAGrJC Gt3CGGCG a AtAGGCCGA GAccGAA

ACGGAGG

CGAACAG CUAt kGCCGAAGCGc

AWCCUG

GCATJGGA CUGAUGAGGCCGAA GlCCGAA

ACUCGAA

CUGCA.UG CUGAUJGAGCCG AG CCAA AGACtJcG CGAUCAG cuGuGAX cAA GGCC .GAA AGGCCGC GCGAUCA CUGAUGAGGOC GGCCCA

AAGGCCG

233 0 0 *0 0 0**0

C

91i9 936 937 942 953 962 965 973 986 996 100 1006 1015 1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 i098 1102 1125 1.127 1131 1.132 1133 1137 1140 1153 1158 1167 1168 1169 1182 1183 1184 1187 1188 1198 1209 1215 1229 1237 1250

GICUCA

4 CU =GrAUGAGGCCGAAA CCGAA

AGCEXG-

G~kCUGG MM =GC aCA AmxmCuG

AACUCCA

UGGCAAG CMX~-MAA-C ACtJGGAA T3CAUGrJG CMAMAWJCc

AUGAO-

CGGUGGC CWMGCUWo AtICAXJM GUCUG-GC CMU=CWGCA AMA=cu UCaCLTUC CMAAG=CGU

AUCCC-GU

ACUCOG CUGAM AMCCGIAW-, AGGUCrjC GGUC~cGGUC

C

AkCtcuuGCGIG M'c Gcu UMCAU~ cc.uAG-C,

AAGGU

UUAAAG CUGAnAGCCGAA AUCUCutjc CCAUUGA C D A G C c M A GGAC LyC UCCA=~U C U ~AGNri mCCwtu LIAfLCIIii 3ccC!%i3AG GCG AAG CGGGOU CUGC- AGCCGAAf^Q AGA UrGG D f

AGG=

G-CACAGC

AGGUGM

UUC GG A ~kG CC ACUrJCG CA

AGGCCA

AGAA= AGGu GGCAG

AGUXG

GGGGAC

~AGUUG

GACUGG CUGAUG

ACAAA

GAAGUGCUAX3AGGCG~CGMJ

AGGGCU

GUMAGGC C=Dht'WAkCCA

AM=,GC

UGGOCCU

GCMAG

GAAGCUG CGW G I GACccr=

AACGUM

GCUGAGG Wn~~

ADGUG

CAAA=O AfGC=u CUC=CA Ct c~ ADGrar GGGGGAA CUGAAGCG=AA

ACUCAU

GGGGA

CGU

AUGcS Ct7GAI AACr CUGAUGG CUGAUG CCQAAQC

AAU

OCAGGA CUG UGAC A~AGGG U GGCUAG AGGAU 2WCCu CUGCCC

AAGGGAC

UCAGAM u C G fl A C AUGtICC CGGCUG MGA GGGAAAGGACCGAA

AGGCCG

GCUGGG ~AGGGACc GGGGCAG CUGAGAGCC~kAGCCGAA AGCOGCr, GAGCCUG C UCAGC AGGaIGG 234 a.

a.

a. a a a. a a.

a a a.

a a 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 CUGAMGC=CGAAAGGCCGAA

AGCOGG

AGGAAG CtGAUGAG~CCC AG-- ACc-nrG CGCCCU CtTAfl.GG'CGAAA,,C.:L AGLcc UGGGGGA COGAVGAGGCCGAAGCG aAECCjj AGACUCG CMUGXGAGGCCMU MCGAA

ACAGGAG

GGGOMG CMJAGCAAG=

CGG

CCGGGU CoUGAGGfl AGAGGccc C AGACuJ GACUGG CU ====GGCCCCGAA

AGGACCC:

UCAWcJU CUGAUG GCCGAAGj=

AGAAGA

GG-CUOC LCUGAGGCCr,;GG= ACAGCcrjU AGCAtJCA C GAUGAGC'C :GXACG-xA AMt'CAG CAGCAUC COGAUMAGGCC01 =GA AACtCA GCCAAGC CUGtMAGGC, ,AGG- AGCc, UGUUGc cuGAI3GAGcc-AA G c CC GUCMGU CMUGAGG--CMCGCoc, ;AMacCC GG3CUU CUGAGAGGCCG AG,-CGAA AACACT-c GUCCACA CVAMGC,-WaAG--CGAA AflGCC AGOOCCC

CMGGCGA~,-GAAGA

AAACUCU CVUAGGCCAAMGGCCG AGrjrzIC CUGCUGA CUAGAGCCGkAGGACUCUGA GCUGCUG CUCUGAGG,-C,-aGGAACQ

~JILAG

AGCt7GCU CUGAUGA GCC AAA CUCUAA AA= AcA.CAGG CUGAIUGAGGCC% AGCCG

AUMCACC

UUCAGGG CUGAUGAGGXU.-CC-AA~

ACUCCAIJ

CGAGMM~ CUGAX3GAGCG="- CGM AGUI GGCGAGrJ CUAGAGGCCMAGCCCG;. AUAG=tT ACCAGGC CUGAlGAG.- CcAAGCAA, AGUta CCCDCUC Ct GAUGAGG-C,'A-CC AAG AGG.AGAG GGGGCAG U =GA4AAGGA-CCA

AGCUG,

CCUACCG CUGAXUGGCAGC CCG

AGCAGGA

CAVUGGGCUAGGCG

CAACCG

COCGGGCA C=U G- AGGUCCAG CACCL7GG CUGAI GAGGCCG ;LAGCCA AGCAJ

A

UCACCL7G GU cCAA AA~cAG ACCUCCG COGAUGAGGCCG~a.AC,-C

AAGCGAG

GGAGGAG CUGAUGAGMC CCGAA AGUCUc t3GGAGGA C=U GGCGAAXCCG

AAGUJCU

AAUGGAG CUGAI CC 'GM

AGAAGTJC

CGCAAUG CUAGGCGAC%-CA

AGGAGA

UGUCCGC cuGAuGAGrCGAAC CC-

AUGGG

AGCAGAG CtUGAC CcUGGCCGA AGTJCCAu GAGCAGA CUGAX GGAGCZ-AGGCCA

AAGUCCA

AAGAGC-A CUGAUGAGGCCCGA GCGAA

AGAAGUC

CUGAUCU CU&GAG I-C ACE7CAA) AGGAGCr CUtACCGAGGCCGQA

AUCUGAC

ACCUUAG C JAUGAGGCCGAAGCGA

AGCUGAU

AGCACCU CU ATTAGCGAG;CA AGGAGCrj CUCUG CLTGTJGGCCGA1rCCCGA

AGCACUG

235 Table Human re/ A nt. Position 0* 0 0* 19 22 26 93 94 100 103 105 106 129 138 148 151 180 181 186 204 217 239 262 268 276 301 303 310 323 326 335 349 352 375 376 378 391 409 416 417 418 433 467 469 473 481 501 HH Ribozymne Sequences HH Ribozyme Sequences TJACAC-AC CUGA GCCGXAC-GCCCA

AGICCAU

CXA COGAUAG

ACGAGCC

CGOGCAC CUAGGC~%Ac-c,, AczA GAGGGWG COG IGAGGCCGACGCC ACAM-jrC UC-*Z= G UAGCCAArCr AACAGoU GGAAGA arUAGCAAG-CM

AGGGGM.

CCGGAA aMUAW-W.,.C-L

AUGAW.-

UGC'GG CO GCCkCC-A

AGAUMG

a=GCG GAAGCAM CCA

AAGAU=

GGGGCCA CD 3AMAGG-CGC~r-CGA AGGCCUjG CE3CCACA CUGAUGACGaC AAGCCAA

AGGGGCC

GCOCAAU CUAGGCGAC-CA AUCUCcA GCOGCUC CO MWW4QAGGCC GAA AUGAU GtMC7G CO IAGAGG ACCCA

AGC

UGCAGCr. CO A GAGCGA-CCr-

AAGC-

GCACUUG CUAACA AGCGcA CCCC k COGAflXGGCCCAAGCC

AGCGCC

CGCCUG%7G W -*CLU-C

AUGCUG--

UUGGUGG CVar-X GAuocG UU=CUCUU c G~GCCGG^C

AUGGUGG

AGCCAUtI COUGAW4GCC ,3C-3A

ATUUG

UCCUGUGaMMC=UGAX--tC ACG=Tjr CCAGGA CUAUAGCCXU;-.CCr

AUGCGCA

G-ACCAG GAGGC cj AGAUGCG CCUUGGU CUGCCAGGG7 .CGGOGAG CIGUGCCGIAAccc .CQ AGG=tCC GG-CCGU CO l~ C~

AGGAG

UGGCG-=

AGGCG

OUCCLAC CUAU CAWCCGU~crA AGCUCC~u CMUUCCCC C ACAAGCrJ CtYCAUTAG CG GGC AGCCAxyC CCUCAUA CO UGGGCCAG-CCA

AAGCCAXJ

AGCCtJCA CL

UAGC~LGC~AGAAGCC

CCGGGC-A CUAUGAC<C GGCGAA. AGCUcA AACUGU CO AUGAUGCC GAAGCCGL

AUGCG

UUCUGGA CUAJ GC CGAA~ ACOGu,- G;UUCUGrG COGAUG GGCCGAACJ

AACUGTJG

GGUU=U C OAGGGCGAG-Ca AAACJu CACACOG COGAUGACC-CGAA,-. At7UCC UGACtJA GA A

AGCCUGC

GCtJGACU CGGC.. CGA

ATJAGCTJ

AUGTCGCUT CUGAUGAGCCGAAGCCG

ACTJU.A.

UGGULTG CUGAUGArGc M AC;CGAA AtJGCC MCUtG COGAUGAGGCCGAAGCCC;A AGGGrJr 236 502 GAACUJtG C

AGGE

509 UCtMh.G CVAC:WCCAAACr

AACUTJGG

512 UCUc CUGGGGCCGXU-.CGAA

AGGAACEJ

514 GCUCEJUC CJiGAUG~GCCr C~kGGAA AUAGG17AA 534 CAGGU7CUG C XU G AGUCCCC 556 GGAAGCA CUAGAC

AGCC

561 CACCUGG CUGAtMAGcc C AGCAAG 562 L=ACUG CM-UAG-bGCGA A GCAGA 585 cCGCCU CUGAGCC =GGAA AUGGGC 598 GCAGGCO CUGAUGAGGCC-GAAGGCCGAA

AGGGGCC

613 GAGGAAG CUC-AWAGGCMW-G ACAGr.G 616 GArGAGG aUGAG-GC CCGAA ANGGACAG 61.7 GGAUGAG CUAMGCGXG-CA

AAGC

620 AUGGGAU CMUA-CGU-CCA

AGSAAW

623 AAGALGG CrGXGC~UGCA

AUAG

628 UGUCAAA CUGUGGCGAAGCC AtJGG=~J 630 AUUCAL CUGAX3GAGGL--AAGMW

AGATUGG

*09.631 GAUUGt7C CUGAUGAG-GAAAGCCC-

AA=G

638 GGGGCAC CUAGG-CG Zr-CA

VGC

661 AGAIJUU CtGAUG GCAAAG GCGk

AGUC

667 CUCGGCA cuGAflGAGGCCGAAGCCGAA AucuuGA 700 CCCCACC CUGAU kGCC-GCC

AGGCAGC

9.715 GCUhGGAA CEUGAUGAGGCAGGCC A AUCUCAU 717 CAGUAGG CUGAUGG;G AGAA

AGAIDCUC

718 ACAGUAG

CGUAG-GUG-GAAGLC

721 CACACAG CVGAUGAGGC-CGAAAGGCGAA

AGGAAGA

751 ACACCUC CE3GAUGAG--C AGCCGA AtJUCCU ;759 CGt2GAAA CUGUGA-GCCGAXIGGC AA cc ::*761 CGVA

ACACCC

762 U.CCCGUG CM MUGAGGCAGCcCGAA

AAUMCAC

763 GUCCCGU COGADGAGXGCA JGCCGA AAAMMh 792 CGAAAAG CUMAG~C-rj e

AGCCUC

"e:795 TJTGCGAA CtXAIGAGGCCG GCGA AGGAGCc *9796 CtJUGCGA CUGAUGIGAUAG GCA A AGGG 797 GCrJUGCG CUAGC--CX~-CA

AAAGGAG

798 AGCtJUGC CUGAUG GGCCGAAGCCA

AAAAGGA

829 GGAACAC C TGAI GAGCGAAGCGAA,

AUGCCA

834 GG%-UCCGG CUGAUGAG-GCCGAAGCCA

ACACAAU

835 GGGUCCG CUGAUGA GGCCGAGCA A A 845 GCGUAGG CUGAUGGGCCGAAWCGA

AGGGGUC

849 GUCUGCG CUGAUGAGG-CCGkUW.L

AGCGAG

872 CG-CACAG CUr-AUGA--CAGGCCG AA G AGCCUGC 883 GCAUGGA CUGAUGAGGC GAr CGAA

ACACGCA

885 CE3GCAIJ"G CUGAUrAGGCGAAAGGCCC-AA

AGACACG

905 CCGGUCGG CUGAtJGAr-GCACGAA AGGCC GCc 906 CCGGUCG CUCAUGAGGCCGAAAGGCCA

AAGGCCG

919 GCt3CACU CUC-AUGAGGACCGAC

AGCUC

237 0 0* 9360 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 1.131 1132 1133 1137 1140 1153 1158 1167 1168 1169 1182 1183 1184 1187 1188 1198 1209 1215 1229 1237 1250 1268 GtTACt3GG CUMUtGAGGCCGAAAGGCcG, GGMVhCU CUGADAGGCCGAAAG~Cc-MA

AAUUCCA

UGGCAGG Ct3GAUGAGZCCGAAAGGCC.-A~ ACtUGGA UCGUCTJG COGU AGGC ~CGAA

;AUCTJGC

CGGU3GAC CVUGA AGCC CGAA AUCGt7CU ATCCGGU CUGAUGAGGCCGAAA~GcaA

ACGALT~C

UJCUCCDC CUGAUGAGCGAAGCCGAA

AUCCC,-U

GUCCUUU CDGA~rzAGGccGAACCrrX

ACGUUUC

GGUCUCA CUGAU GGCCGAkAGGC- AMMCrJ GCUCUUG CUGAUGAGG-CGA UCGCAA AGGtICUC UGCUJ=CUCAGC,-GA G-CA AACGUUi UCDUCAU CUG-AUGAG--GGGcC -CW.A ALMCUL-i CUGAAAG CUGMAGG--CGAAG,-=AA ACtTC-.UC CCGCUGA CUGAX3GAGGC-C GAA CCG-AA AGGACUC UCCGCUG COAGG-CAAGCA

AAGSACU

GUCCGCU CtMAtGAGCCCAGCC GA A;L,-GW-C CGAGGUG CUGAIJGAGGCGAAGGCGAA

AGGCCGG

AUGCGUJC CUG.AUGAGGccGA cCCAA

AGGUG

GCACAGC 'CUGAUGAGGCCZaCGAA~

JAUGC

CtJGCGGG CU~TAGCA GL AG~CACA CE2GCGG- CUGAUG GGCCGAA G-C,-AA

AAGGCALC

AGAAGCt7 CrJGAUGAGGCCGAAACGr-CA

AGCUGCG

GGGACAG CUGAUGAGGCCG -AC^,GAA

AGCG

GGG M-CA CUGAUGAGGCC-GG C-AA AAG%-CGA GCUU=G CUGA GCCGAAAGCCA

AC-AZAAG

AAAGGGA CUGAUGAG-G .C-,AA

AGGGCLG

GUAAA.GG CUC-AUGACGcAAGCCG At2GGGC: UGACGUAarU GCGAGGCA AGGGAuJA AtJGACGVJ CUGAUGAGGCCGAAAGGCCGA AAGGGArj GAUGACG CUGAUIGAAGGCCGAA~ AAAGGC2A CAGGGAU CMGAUGAGGCCGAAGCCGAA

ACGAA

GCUCAGG Ct GAfGAGGAAAGC A AUGACGU CAUAGUU CUGAUGAGGCCGAAAGCGAA

AUGGUGC

CUCAUCA CUGAUGAGC-CCG AGCCCGAA AG'auO~ GGUGGGA CUGAUGAGGCAGCGQ,;L

ACUCATJC

tJGGt7GGG arAUAGG,-CGAAAGGCCGA;

AACUCAJ

AUGGLUGG CUGAGAGoAGCCG;I.

XACUJA

AGAAGGA CUGAUGAGG-GAGCCU CAA ACACCAU CAGAAGG Ct3GAtGAGGCCC-AAC-CGA AACACC-2 CCAAA CUGAUGAGGCCGA ~r-CGAA

AAACACC

t3GCCCAG CUGAUGAGGCCG AAGC=GAA

AGGAAAC

CUGCCC-A CGAUGAGGCCGAUGCGAA

AAGGAA

CCU-GC CUGAUGAGGCCMAGGGAA

AT-CVGCC

CAAGGCC CUGAUGAGGCc CGA).CA

AGGCCUG

CGGGGCC CUGATJGAGGCCGAAAGCCGk

AGCCGAL

ACUUGG CUGAUGAGGC-CGAArCGAA

AGGGG&-C

GGGGCAG CUGAUGAGGCCGAA.GCCC-A.A

ACUUGG

GGGGCYG aUGAGr.GGC C-AA ArCrCUC-G AUGGCUG CUGAUGAGGCCGAAGCCG,,

ACAG

238 0 flee C C

C.

6*@C C C

CO

C

*CCC

C

C

b.C.

C C C. 1279 1281 1286 1309 1315 1318 1.331 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 '754 GAGCUGA CUGAUGAGGCCGAXLCGGCCMAA

ACCAUGG

CAGAG-CU COACCCCGZaAG- C~rA ALMCAU UGGGCCA CUGAG CrW.XGCCGA

.AGCUGAU

GGACUGG CMUGGGCCGAAAGGCCA

ACAGG=

GGGCtMG CGUA CAA C, C CoGGGG CO AUGGGCCGAAAGGCCCAA

AGGACUG

GCCUGAG COGAGAGCCGAAAGCCG

AGGCC

ACAGCCU Uv-3A,-CGAAAGCA aGAGC, GGCCOCUT CVAU CCGXUZGCC;L

ACAGCGU

AUCAUCA CGUAGC CG

ACU=GCG

CAUCAUC C.UGGCCGGGCM,

AACUGCA

GCrAG CUGVGA GCCMAAGGCGAAL

AGGCCCC

UGUUGCC CUGA GCCCXG,,-GCcU

AGCAAG

GUCOUG am -CA

ACACAGC

GGUCOGU CUG UAGCCG aAGCGAA

AACAC=

GUCGACG COAGAAGCCGAA-.CA

AUGCCAG

AGUUGUC COGAUGAGGCCGXUGCCA

ACGM

AAACUCG CrXAfG G c' u t 2 AGUWGUC CuGCOGA COA WCCGAAAGGCCGM

ACUCGGA,

GCOGCOG CAGGCCGAA

ACV

AGCtJGCU CGUAGCLGCA

AAACUCG

CCACAGG COGAUGAGGCCGAAAG--CCQAA

AUJGCCCU

COCAGGG CUGAXJGAGGCCG AA-CCGAA,

ACUCCAU

CGAGM CGGAGGGGcc-r-A

AGCCUCA

GGCGrU COG-AUGACGCCAGGJ- AUPGC=r ACOAGGC COGAI GAGGCC kArCGAA

AGUUAIJA

COGUCALC CUGAGCAcm cCCGAA AG~rccA GGAGCAG CMUGGCCAG~CCr.

AGCUGG

CCCAGUG CUAGAGGCcAAGAc

AGCAGGA

CADGGG CUWAW'A~CC GGA AGCCCCG CUGAAAG CUAU GCCGGCCGA AGGCCAL3 CtJCCUGA UAGGCMAG

GA=

UMCUCM GCMAGCCA

AAGGAGG

AUCU=~ COGAUGAGGCCGAGCA

AZ..G

GGAGGAG CCGAflGAGCC CC AGUCTuC UGGAGGA CUAGGCGUGCA

AAGUCOU

AAUGGAG CUGAT GAGGCCWGAGCCGAA

AGAAGUC

CGCAAUG CUGAUGAGGCCGAAAGCCGAA,

AGGAGAA,

UGUCCGC CWAGGCXAGCA

AUGGAGG

GCUGAG CUGAUGAGGCC AAGCL AMXucAT-T GGGCOGA C OGU~GGCMUZCrA

AAGUCCA

CAGGGCUJ CUGAUAGGCCGAACG A AGAAGUC CUGAtJCU COGAUGAGGCCGA- GGCGAA

ACUCXGC

AGGAGCL CMO GGGCCGAAAGCCG,'

AUCUGAC

CCCUCUAG CMUGAGGCCGkAG=CcAA

AGCUGATJ

ACCCCCU CUGAUGAGGCCa a'A.CCG AGrC CUCUGGG arWkGCAUG-GA

AGGGCA.G

C.

C C

CCC.

C

OCCCC*

C a.

@6 6 0@ a a 6* 6 a. a a 6 64 C a a 46 a 6 *Ca a aa a.

at 6 6 S a a S 6 SG6a 6 6 0 Table 21 Human re/A Hairpin Ribozyme/Targel Sequences nt. POSjtjon Hairpin Ribozyme sequence Substrate 156 362 413 606 652 695 853 900 955 1037 1045 1410 1453 1471 UGAGGOG AGAA GUUC

GCUGCUUG

GCCAUCCC

GrJUCUGGA

GAAGGACA

UUGAGCLJC

CCCACCGA

AGGCUGGG

GGUCGGAA

UGACGAUC

GLJCGGUGG

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCUC

GLJCC

GUG

GCAG

GUGU

GCUJG

GCGU

.GCCG

GUAU

GCUJG

GUGG

GCAG

GUGC

GUGA

ACCAGAGAAACACGUUGGGUACUUACCIGGO

ACCAAGAAACACAGLGUUACUACCUGU

ACCAGAGACACAGUUUUACUUACCUGUA

ACCAGAGACACrJUUGGACUACCUGGUA ACCAGAGAMACCGUGGUACAUUACCrGU

GAACLJ

GAGCA

GGACU

CCACA

CUGCC

ACACU

CAGCU

ACGCA*

CGGOG

AUACA

CAGCG

CCACC

G~LR

GCC

GC

GUU

GCC

GCC

GCC

GAC

GCC

GAC

GAC

CCCCCUCA

CAAGCAGC

GGGAUGGC

IJCCAGAAC

UIGUCCUCC

GAGCUJCAA

UCGGUGGG

CCCAGCCLJ

UUCCGACC

GAUCGUCA

CCACCGAC

CAUCAUCA AGAA ACAGCUGG

AGAA

GAUGCCAG AGAA GCACA GAC CC-AGCUGUU UCACA GAC

CUGGCAUC

Table 22 Mouse re/A Hairpin Ribozymefrarget Sequences nt. Position Hairpin Ribozyme sequence

V.*

Substrate 11 137 273 343 366 633 676 834 8a1 1100 1205 1361 1385 1431 1449 1802 2009 2124 2233 2354 GUUGCUUC AGAA GUIIC GAGAUUCG

AGAA

GCCAUCCC

AGAA

GCAGAG'AGAA

UUGAGCUC

AGAA

CCCACCGA

AGAA

AGGCUGGO AGAA GAUCAGAA AGAA AGGUGUAG

AGAA

GCAGAG

AGAA

GGGCUUCC

AGAA

CAGCAUCA AGAA ACUCCUOG

AGAA

GAUGCCAG AGAA AAGIJCGGG AGAA UGGCUCCA

AGAA

UGGUGUICG

AGAA

AUUCUGAA AGAA

GUUC

GUCC

GCCJ

GUGU

GCUC

GCGU

GCCG

GUGC

GCGU

GCAG

GUGC

GUGA

GCUIG

GUCC

GCAC

ACCAGAGAAAC!ACACGCUUGUGGUACAUUIUGGUA

ACCAGAGAAACACUUGUGGUAQJTACCGUA

ACCAG-AGAAACACACGUGUAAUUACCJGGUA

ACCAGAGAAI~cACACGUJ~GUGUAAJACCUGGUA

ACC-AGAGAAACACACGUUGGUACAUUACCLJGGUA

ACCAGAGAAACACACGUIGGUACAUUA~.JGUA

ACCAGAGAAACACACGUUGLIUACAUACCJGGUA

ACCAGAGAAACACGUCGUUACAUUAIJCCUGGUA

ACCAGAGAAMCACACGUUTGUGOIAAUACCUGGUA

ACCAGAGAAACACACGUUGGGUTAATJACCUGGUA

ACCAGAGAAACACACGUUGUACAUUACCUGGUA

ACCAGAGAAACACACGUGU~GG1AAUUACCGGUA

ACCAGAGAMCACACUUGUGGUIJAUUACGGUA

ACAAAA~AGUGGUCUACGU

ACAAAAAAGUUGAAuC~GU GAACA GCC GAACA GUUl GGCU

C

AGGCU

ACCL

AGCC

CGCC

CCGCG

CCC

GACC

ACGXCA

CCA

GAC

CC

0CC

GAC

GCC

0CC

GTJC

GUC

GUU

GAAGCAAC

CGAAUC

GGGAUGGC

CUCUGCCC

GAGCUCAA

UCx3GUGGG

CCCAGCCU

UU)CUGAUC

CUACACCU

CUCUOCCC

GGAAGCCC

UGAUGCEc3 UCACA GAC CAGCU Gcc GGACA GAC GUGCu 0CC UGGCC GCC AGACA

GCC

CCAGGAGU

CCCGACUU

UOGAGCCA

CGACACCA

UUCAGAAU

UUUACUGA

UCAGUAAA AGAA GrJCU 241 Table 23: Human TNF-a H Ribozyme Target Sequence nt.

Pea ti± n HE Target Seque.nce Pont.

Poi -i on 28 29 31 33 34 37 39 44 58 65 67 69 106 136 165 177 180 181 184 190 192 193 195 198 199 205 226 228 229 243 244 253 273 286 288 290 292 295 302

GGCAGGU

C=AGGrju

AGGU=C

GUUJCU=t oc JICE7

CACGGCU

CCAC=C

ACCCUCEJ

GCAUGAU

AGGCGCU

CAGGG-CU

CGGUGCI

UGCUUGUt

GC-UUGUYC

U TJUCCU C UCAGCC C AGCCUCU U GCUCUU C CuUuLCU C UUCU U UCUCCLUu C LUCCUGAU c CCACGC-U C ACGCUCU

U

CGCUCt7U C CUGCACU Ui tJGCACUU U GAGUGAU c GAAGAG3

C

GGGACCU C GACCt7CU C ccUCUCU

C

U CU=C C UCUUCCU C

T

UUCCMICU

U CCTJCUCA C COCUCAC C UCACAUA C ACXACu A CUGACCC C CACCCUC C ucuicccc C UCccCETG CCCCO7GGA C CGGGACG

CAGGCC.G

IGOUCCLTC

CCtICAGC

CUCAGCC

AGCCETCU

UUCUICCU

CUCCUuc

UCCUUCC

CUUCCOG

CCUGAUC

CUGAUCG

GtJGGCAG

UCUGCC

UGCCUGC

UGGAGUJG

GGAGLMA

CCCCAGG

DtCUCUAA

UCUAAUC

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 ER Tarxgt Sequence GUCULAtj C AUCUCU AGALUCAU C UCUCGCA

AUCA

1 ICU U CUCGAAC UCAUCMM C UMC3AACC AUCUUCU C GAACCCC AGCCUGU A GCCCAUGM CCCAUG U GUAGCAA AUGU=G A GCAAACC AAACCCtJ C AAGCUVGA GGCAGTJ C C-AGUGGC AVGCCCU C CUGGCCA GAGAGAxj A ACCAGCU GUGCCAU C AGAGGGC GGCCrJGU A CCUTCAUC UGUACCU C AUCUACtJ ACCUCAU C CrCAUCU A Ct3CCCG AUCt&CU C CCAGGC CCCAGrGU C CUCUUCA.

AGGLUCCU C UUCAAGG GUCCUCY U CAAGGGC UCCUCUU C AAGGGCC IGCCCCU C CACCCALU AUGUGCU C CUCACCC UGCUCCU C ACCCACA ACACCAIJ C AGCCGCA GCCGC:AU C GCCGUCU UCGCCGU C tTCCUACC GCCGUCU C CUACCAG GUCUCCrj A CCAGACC CCAAGGU C AACCt3CC UCAACCU C CUCJCtJG ACCUCCU C UJCUC-CCA CrUCCUCM C UGCCAUC CUGCCAU C AAGAGCC CCCUG7GU A UGAGCCC AGCCCAU C UAUCUGG CCCAUCU A UCUGGGA UcUCUCU CrJCE]AAU AC-CCCtJ A AUCAGCC C AGCC=tJC C OGCCc 242 0

S.

S *5

S

*SS.

S S S. S.

S.

S

*5*S

*SSSSS

S

671 682 684 685 709 721 725 735 737 739 744 745 753 763 765 768 769 775 778 801 808 809 820 833 837 838 839 841 842 849 852 853 863 869 871 872 878 890 898 899 904 917 918 924 925 926 945 946 959 CAIXVCE C UGGGAG GM=GG C UUCCAGC GGGGUCU U CCAGCUGM GGGUOU C CAGCUGG ACCGACU C AGCGCUG CUGAGAUJ C AAX.CGGC GPAUCAAU C GGCCCGA CCCGACU A T.CUCGAC CGACtU.t C UCGAC= ACLEhAUCU C GacuuUG CDCGACU U- UGCCGAG UCGACUU U GCCGAGtJ GCCGAGUJ C UGGGCAG GGCAGG;U C UACUOUG CAGGUCU A CUUUGGG GUCtJACU U t1GGGAUC UCtkCMu U GGG&UCA UUGGW~ C AOUCCC GGtAU U GCCCaGU CGAACADU C CAACCtU CCAA.CC U CCCAAAC CAACCUU c ccAAACGr AACGCCU c =CCC CCCCAAU C Ccr3U=.

AAUCCCU U umX3Lcc AUCCcu U AVUACCC UCCCUJUU A UUACCCC CCUU U ACCCCCu CUUtM=E A CCCCCUc ACCCCCE3 C CUUCAGA CCCUCCU U CAGACAC CCUCCUU C AGACA~CC ACACCCu C AACCLTCU UCAACCT c UOCUGGC, AA.CCUCU U CUGGCUC ACCUCUU C UGGCUCA UCUGC-J C AAAAAGA AGAGAAU U GGGGGCU G-GGGGCU U AGGGUCG GGGGCUU A GGGUCGG UUAGGGrJ C GGA.ACC CCAAGCU TU AGAACU CAAGCUU A GAAcurU tW.GAACU UJ UAAGCAA AGAACU uU AAGCAAc GAACDUUU A AGCAACA CA~CCACU U CGAAACC ACCACUJL C GAAACCU CtJGGGA U CAGGAAU 960 1001 1007 1008 1021 1029 1040 1046 1047 1051 1060 1067 1085 1086 1090 1091 1111 1124 119 1135 1151 1152 1158 11l59 1162 1164 1166 1174 1175 1176 1183 1184 1187 1208 1224 1228 1230 1232 1233 1234 1238 1239 1245 1251 1252 1254 1255 1256 1258

UGGGALU

AACCPACU

AAGAAUU

GGC-Gcu CAGAACrJ

GGGGCCU

TJACAGCU

ACAGCLU

CUUUGAU

CJGACA.U

4 GG).U GGAGCCU1

GAGCCUU

CUOUGGU

UUUG.-UUC

CAGGACUt

AAGACCUC

CUCACC z.

MkC-GAAAU UJ UGGACCU TU GC-AC~JU

A

UAGGCCU TU AGGCCMu C CCUUCCtY c UU)TCCLTtY c CCUCUCtJ c CAGAUtW U AGA~Uur U GAUGUJUU c CCAGACU UL CAGACU c ACUUiCCU U CACCCLT C GCCAGCU C GCUCCCu C U-CCCUCUj Ak CCUCtJA u CUCtUTU u UCEJUU A UUUAUG.U Uj UUGUU UC UUGCACLT UC tUUGUC-ATU u; U'GtJG-ATU A UC-AUUAt3 U GAMUAUU r AUUALUt A UJAUUUTAt

UA

C AGGAAUG A AGAAJUrC U CAAACUG C AAACUJG C CAGAACrJ C ACUGGGG A CAGCUJI U UJGAJC-C ,U G-AUCCC-U C C,-t7G;LcA C GAATJC C tJGGAGAAC :3 tG rrJuCr J GG-UUCUG 3CUGGCCA t7GGCCAG 7GAGAAGA

ACCUAGA

GAAAUUG

GACACCA.

AGC-CCUU

GGCC

CCUCUCU

CUCUaCC UCrJCCAG

UCCAGAU

CAGAUGU

UCCAZAC

CAGACU

CAGACUU

CCUUGAG

CUUGAGA

GAGACAC

CCCAUGG

CCUCtIh.u

UAUUUAU

UUUAUGU

TJUGUU

.'OUGITUG

GtJUUGC

.,GCACUU

;CACUUG

JGAUL1A MLuAUU

T"ULCJ

'AUlUAUU .MUruAU

UALUUA

UUUAUU

243

S

a.

a efr..

a a a. a a. a a a. a 1261 1262 1263 i265 1266 1267 1269 1270 1272 1273 1274 1276 1277 1278 1280 1281 1282 1294 1296 1297 1298 1300 1301 1315 1317 '1334 1345 1350 1359 1360 1361 1362 1.386 1394 1401 1414 1422 1423 1425 1426 1427 1431 1 432 1436 1437 1438 AUUtUhUU A UUMtUU UtMW= Ti u umuw.

MWM=fl U AUUtM= ATUfU= A UUUM thLMUMUu U UCfl~hu AUUEahWr U AIJfU U UtUUU A UM3UUM.

tu~imu U Auurzuu AnU~aUU A U~aAUUU UUU U MU:uJOt MUDJM=l U ATUU AIJU=U A UfUU LMU~.u u UAUCUAC AUuwaU AUtUkCA UUMfUM A UUt~hCA ~ukutm U UkACQAu AUUCWU U ACAGAUG UUU A CAGAUA UMIAUU A u~uUm AAUMLT U ~fUADG., AUGMMuuU T AUUG UGM~U A tUUUGGA MUUMLU U UGGGAk AVUM.u Uy GGGACAC CCGGG,--J A JCcr.QG GGGCt=U c CUGGGG-- CCAPG A GGAGCLTG GC-GCCU U GGCUCAG CUUGGCU C AGAAUG GA.CAflGU U uOc=j ACAUGUU U CGGA CAUGUUU U CCGMA 1440 1441 1446 1448 1449 1451 1456 1457 1461 1464 1466 1479 1480 1494 1498 1501 1512 1517 i528 1533 1537 1540 1546 i549 1551 i552 1566 1572 1576 1577 L'GUUMU U AAAATUAT- GLVUUUrj A AAAXUhxjr U~AAAU A UUAUCUG AAAAUAU U -AUC0=AU -AATAUU A UC-C7GAjUu AA~U= C UGAUUAA, AUCLUGAU U AAGUUGEJ UCUC-AUU A AG-UtGUC AULJAAGUJ U GUCrJAAA, AAGr3UGU C MAACAA GUUGUCU A AACa.UG L GrCUGAU- U UC,-GUGAC GCGCAUEJ U GGGC CAACtJGU C ACUCATU UGU~Crj C AUL-C,-- CUCCAiI U GC=GAGW GAGGCCtI C UGMt7CCC CCUGCU C CCCA=G AGGGAGrJ U GLrG~CC,- G'3G=G C tGtATAC OJGUCTUGU A AUCGGCc C"UGtMAy C GGC-t3AC U"CGGCCU A CU~ITUCA GCCtACU A tUCA.LG CtJACUU U CAGUGGC LrAC~kUU C AGUGr-- GAGA;AAU A AAGGUL'M U;AAGU U GC-UUAGG GGD"CU U AGGAAAG~ GLJUG'CTJU A GGAAAQG.

a.

a a a

AUGOU

GAACAAU

AGGCtJGU

GGCOUU

CCCAUGU

CUGGCCU

LIUGCCU

GUGCCDJU

GCCUUCT

CUUM-t UEMhUGU t tUJGUU t C CGVGAAA A GGC=3 U CCCAUWr C CCAUtGUA A GCCCCCU C UGUGCCU U CUtUUGA C UTUtIGAU- U UUGAJM U tUGAWAMU J7 GAUUALI J AUGUUMu

UGUUU

7 juaAA I UUAAAA

ULMAAAU

244 Table 24: Hum~an TNF-a Hammerhead Ribozyme Sequences .nt.

Position RN Ribozym. Sequence 4 4 9 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

GGAAG

AGGA

GAG

UALGD

GGGUC2

GAGGGE

GGGGC

CAGGGG

VCCAGG

CGUCCC

UCUUGG

CCGCCU

GAGGAA

GCUGAG

GCGCUG;U

AGAGGCI

AGGAGA;

GAAGGAC

GGAAGG3

CAGGAAC

GAUCATC.

CGXt7CAC CrJGCCAC

G-GCPAGAA

CAGGCAG

GCAGGCA

CACUCCA

TUCA~cU

GGGGGCC

CCUGGGG

ULU.GAGA

GAULWA

CtGAUTA GGCUGAtJ

GAGGGCU

TJGGGCCA

.CUAtr'.GCAGGCCGAA

.CUGAAGAGCCGAA

CMACUGAGCGAAGCC

J~CUGAUGAGGCCGAAAGGCCGAA

SCUGAUGAGGCCGAAAGGCCGAA

SCUGAUGAGGC-CGAXAGGCCGAA

.G CUGAUGAGGCCGAAAGGCCGAA 0CM AUGAGCC XGGCGAA3

CMOAUGAGGCCWMCGAA

SCtJGAUGAGGCCGAA.AGGCGAA) SCDGAflAGOCCGAAAGCGAA C UGAUGAGGCGAAAGcc-,

A

SCrUGAGGCCGAA cG.A A :Ct XJAW-Cc GAAc SCUGAX3GAGGCCGAAAWA-C

A

CUGAUAGGCCGAAAG-GCCGAA

A

CUGAUGAGOCCGAAAGGCCCAA

A

CUGAUG-AGGCCGAAAGG-CCGAA CUG-AUGAGGCCGAAGGC

AC

CUGAUG.AGG.CCGAAAGGCCGAA

A

CUGAUGACGCCGAAAGGCCGAA

A

CUGAUGAGG;CCGAAAG.GCCGAA

A

COGAUGAGGCGAAAGGCCrAA C0GAUGAGGCC

,CCAU

CtJGALMAGGCCGAAAGG.CCGAA

A

CUGAUG-AG.-CCGAAAG-GCGAA

A

CUGAUGAGGCCGAALAGGCCWAA

Ct 3 GAtJGAGGCCGAAAGGCCCGAAAG CUGAUCGAGGCCGAAAGGCCG

AU!

CUGAUGAGG-CCGAAALGGCCGAA

G

ACCUGCCC

AACCUC

AGAACCU

AGAGAAC

AAGAGAA

AGGApAGA

AGAGGAA

AUOGAG

AGCCGtG

AGGGJG

P.GAGGGU

WAGGAG

WCAUGC

kCGCG=c

LGCCX

CACCG

JZAAG-CA

ACAAGC

GGAACA

GOUG

GAGGCU

A.GAGGC

3AAGAG 3GAGAA kGGAGA

JCP.GGA

;CGUGG

MAG=G

UGAGCG

,VGCAG

GUGCA

CACDC

UCUUic

GEJCCC

PkGUC kGAGG kGAGA MkGAG

;GCUG

4.

C C 94t 4*4**g 4 245 a..

a a a a. a a 321 324 326 327 329 352 361 364 374 391 421 449 468 480 484 487 489 492 499 502 504 505 525 538 541 553 562 568 570 573 586 592 595 597 604 657 667 669 671 682 684 685 709 721 725 735 737 739 744 AGAAGAU CUGAMGAGCCGAAA.-CCAA AUCtUGAC UCGAGALA CUGAtMAGGCCGAAAGCCG;LA

ADGAUC-I

GTUCGAG CUAMGCCGUGAAA

AGAXUGAI

GMUCGA C GUGGCG-c,AGGoCCC-L AAr.At C-GGGt7UC CUG UGGC-CGGY-cc

AGAAAU

CAtJGGGC CMGC CGr-

ACAGC-

UUG--T1AC Ca PArXG-CCGAXA=C-CCA6A ACAflGGG GGUUGCa a~LAGGCCG-AAAGCCOA

ACAACAU

UCAGCECtY COGAflGAGGCC AAC,- AGGGUrjrj UGCC= COG AGC.ICCCGAck

AG,-G=

AGCGUt CMAW-V"CCGAAGCC-A ADut3UC GCCCU.CU CUGAUGAGGCCMX-GCCA,3 AUGGrCAC GAUGAGG' CUGAfLG -Cr i.GAA ACAGGCC AGOAGAtI CGVAGGCC'- C~CcA AG~kr-c, GG=-AGM UA GGG ;G-CA AfGAGGU CUGGGAG C

AGAUGAGGC-A=--AAAA

GACCUGG CGGAOAGCCAAAC AA AGMU UGAAGAG CUGA CCGAAAGCC ACCr3G CCUUGAA, CUAAGCSAGC AGGACCrJ GCULG COGAUAGGCCGAAC-=A AGAflGAC GGCCCUU CUM GGCGAAAGGk

AAGAG

AUGGGU CUAuAGGCCcGXUGGAA

AGGCG'-;,

GGGOGAG CUX WrGGGCC;

AC=

UGt3GGU CcAUk GCCAAGC,- AGGAc UG-C-GCU CMUAG-CWAXA AT3GGOUr AGACGGC CUGALAGCCA

AIJGC

G.-JAG.A6 COG-AUGAGXC U~,CGAA ACGc-.

CLGGUAG ar~AGCACCC,.

AGAC

GGUCUGG C GAGCC4GCG

AGGAA

GGAGGOUU CUGAMG CAGCCGAA~ ACCUu= CAGAGAG CUAGAGCGAGGCUA

AGGULVA

UGGCAGA C-AGCCAGCC,-A AGcGG GAUGGCA CUGAUGAWGAXVCG-CCAA

AGAGAG

GGC-UCUty CUGAGAGGCCGAGCC-L AtJGGCAG GGGCUCA

CUGAI

3 GAGGCCGAAAGGCCGAA

ACCAGGG

CCAGAUA CLGA GAGGCCGAACCcr-t-

AUGCU

tTCCCAGA CUGALUGAGGCCWA.-

AGUG

CCECC;CA-A UGACCCAAAGAAc

AUAGAUG

GCUGGAA CUGAUGAGGCCGAAGCGCAA ACCCCrJC CAGLt3G CUGAM G AGGC CGAA AGAC=Cc CCAGCtjG CUGAUMIGGCCGAA GCGAA

AAGACCC

CAGCGCU CUAA-CGAAGCA

AGUCGC;U

GCCGAUU LMGAUGAGCGAG CC-A AtYCUCAG UJCGGGCC C=GUGAGC-CCAA CA~ AMUAC GUCGAGA6 CUCAGGC.AGG CCC-a,

AGUGGG

AAGtCGA COG-AOGAGCCCGAAAGGCCGAA

AM~G

CAAAGUC CUC-AUGAGGCCGX6ACGA6A

AGAXIAGTJ

CUCGM CA GAtGAGGCACGCCGA

AGTUCG

246 745 ACUCGGC COGAtr-AC-CCrAAACGCCQaA

A.,G=JGA

753 CUGCCCA CUOGAtGA=GCGAAA~kGG,-,,.A ACtUCGC.3 763 CAAAGUA CUGkMAGXGXU CG--CAA ACCUCC 765 CCCAAAG CUL GAGGCCGAC-GC.;

AGACCUG

768 GAX3CCA CUGAflGAGGC~CGA)CC.A AGUkA~C 769 t3GAUCCC CUGAUflAGGCCGAGC=.A

AAUG

775 GGGCAAU CUGaaX GAAVCCC 778 ACAGGGC CUAUGM AGGCGW

AZGUCC

801 AAGGUG CUGAVGAGGCCGAAAGGCC,-AA

AUGUUCG-

808 GUUUGGG CUGAUGAGGCCG AG-ZG AGGUUjGG 809 CGUOUGG COGAUGAGGCCG

AAC,-U,-G

820 GGCAGGG CUGAflGAGCC,-GAAG AGC~rr 833 AUAAG CUGAVGAGc3Ccz-- a-M.U 837 GGUAUA CUAGCCGAA-CC; ACC W~ 838 GGGMAU CUCQU3AGGctraa.GA AGUj 839 GGGUAA CM AX3GA=GC GGC-G^A.

AAAGWA~,

841 AGGGG3 LLOrA1rn1VCCGXC-,-IA A1 AAGG 842 GAGGGGG CUGAM~AC=GAAAGGGCC

AAMSUAAG~

849 UCOGAAG Ct GAGAGCCGAAGCGAA AC,.Wrj 852 GGUCUGCUGAtJGAGGCCGAAAGGCCGAA

AGGGG

:0853 GGJGUCU Ct3GAM =AGGCCGA CGA AAGGAGG 00863 AG-AGGUU CUGAUGA=GA CGCCA. ACGUT ***869 GCCAGAA amamc

GUJGA

0**871 GAGCCAG CVGUGAlGAAAC~.,-CG AGAJt--UU 000872 UGAGCCA CUCGAGGAAAGcc

AAGAGGTJ

878 UCUUOUU CUGAX GAAA CCGAA AGCCCAGA 89 G00 0A0AGCGAG-,CA

AMTC=C

.o:o898 CGACCCU CUG AGG-CCGAGGCCA

AGCCCC

**0899 CCGACCCCU aMMGGAAAG GjAA~~

AAGCCCC

904 GGGAUU'CC CVGAM~AGGCCAA GCCGAA

ACCCEA

917 AAGULTCU CMUGfGAGGCCGAAGCCGA AGCjrjG **.918 AAAGUJUC

CGVAGCAAC-AAGCU

000924 UGCUUA CUGAIX-AGGCCGAAGGCG AGM=rJ 925 GtUUuw CUGAt7GAGGCCGAACG-CA A ArUrC .00. 926 UGUUGCtJ CUGAGAGG-CCAAACCGAA

AA.AGUC

.00945 GGUUUCG Ct3GAIJGAGCGAAGCGAA

AGUGG.-UG

0: 0 946 AGG=tC CUGAGAGGCCAGCGAAAcZUC 959 A~TJCCUG CUGAGGCCAGGCC~GGa

AMCCCA

960 CAUUoCCL CUGA=CGAAGC7CGA

AAJJCCCA

1001 GAULTU CUGAt AGCA=CG;L

AGGU

1007 CAGUUJG CUGAGWX CCG;L ATJUCU~k 1008 CCAGUUL UGWAGCkZg AAUUCUr 1021 AGUCUG CUAGCCGAGIGk AG Cc 1029 CCCCAGty CUAGGCGAA.CG

AGZUC

1040 AAAGCUG CUIGAUGAGCCGAAGGCCCAA

AGGCCCC

1046 GGGAUCA CUGAUACGAGCCGAc AGC3GTJA 1047 AGGGAUC CUGAUGAGCCGAAAGGCCA ACGu 1051 UGUMCGG CUJGAUGAWGG AAGGCC-AA AJCA6AAG 1060 GADJUCCA CUGAIGGAGGCCAA~

AUCAG

a.

aa a 1067 1085 1086 1090 1091 111,3 1124 1129 1135 1151 1152 1158 1159 1162 1164 1166 1174 1175 1176 1183 1184 1187 1208 1224 1228 1230 1232 1233 1234 1238 1239 1245 1251 1252 1254 1255 1256 1258 1259 1261 1262 1263 1265 1266 1267 1269 1270 1272 1273 Gucc AGA~u

CAGX

CUETC

CAAM~

UUGUCG

AA=G

GAAGG

AGAGA

GAGAG

AUCTJG

GCUC

AGUCUC

AAGU=

CUCAA(

AUAGAG

At3AAAD

AAACATJ

CAAACAJ

GCAAAZ

AAGUGCa

CAAGUG(

t~UCA

AALTAAXT

UAAA;

AAUAW.

AAATA

UAAAUJAA

AALMhAAt1

AAAUAA

ALVAAXJA

AAAU

UAAUAAA

AAUAAA

AAAUTAAU

UAAAUAA

AAUAAAU

AAAU~AA

AtU.AAUA

AAUAAAU

247 CCA CUGtUGflG ccAAGG--CGAA AUtUCCAG C CUGAWGAGGCGAAAGGCCG-AA

AGGCUC

CcCUGADGAGGCCQAAAGGCCAA

ACCACUC

AG CUu~3G CAACCA AcCAA :..CUGAfl G---CGAAGGCCGAA

AGUCCUG

GCUGAW-AGGCCAAGCCGAA AG~rJCU ltCUGATWGGGC-AAG=c,.A

AGGUGAG

CWU AGGCCGA 'GCCGAA

AUOUCUA

:CU CUGAUXAGGCCGAA,--GAJL AGM=~c~ ~c CUamwAGGCCGAAAC-CGAA AAGGuCC GGLr-VTfCCC kaaCGAA AGCCrA A G WGGC-GAA~_-C-

AAGGCCY

CUG CA AGGAAGG ~CUGAW.AGGCCGAAAC-CCG;L AGAGc ~CU~GAMGGCCA C A AACG -G CUGAUGAGGCCGAAAGGCCGAA A~CW=c CWG I-IGG CGAAGCGAA

AACUC

GCUGAUGAGGCCGAAAGG-CGAA AAtICU LCUGAUGAGGCCGXU '-CGAA AAGt7CtG C' C AGGCC

G

~CW-rIG -CA CGC-A

AGGGCUG

GCUG-tJAOGCCGXAG-CCGALA

AGCUGGC

ACUGWGGAAGCCGJL

AGGGA}GC

ACUGAWAWAAGCJ CGAA

AGAGG

ACUGVAC--C~CGAOCGAA At3AAGG ~COGAUGAGGCCGAAAGG.CC,-AA AAtJAG SCUGAW.GAGGCCGAAAGGCCGAA6

AAAUAGA

SCUGAUGAGGCCGAAAGG.CCGAA

AGCAA

CUGAUGAGGCCGAAAGGCCGA

AGOCA

7CUGAfGAGGCCGArCCG AUCaAA CtrAMGCCGGG~rA

AUAUA

CUGAGAC G GA~

AAATTCA

CUGAUGA=GCaAAAGGCCGAA

AAAXJAAUJ

CUGALGc-AAGGCC- AUAAAum Ct3GAMAGr-CGX"GGCCA AAtUXA aUAGGCCGAAAGGCCGAA

AAAA

CUGAUGAGGCCGLr~CGA6A

A;LAAMU

CUGAUGAGGCCGAAAGGCCGAA

AIYAAATUA

a=MAC-GGAAGCCGAAC AAtUAAU

CUGAM

3 G GUGAAGGCCG

AAAIJAAA

CtJGGAAGCCGXCCC-

AUAAAUA

CUGAUAGG-CCAAAG-.CCGAA AAt7AAAU CUA-GCGAAGCCA

ATJAAUA

CUGAUGAGGCCGACAGGCC-AA

AALUWA,

a.

a a a. aaaa a 248

S

*5 S

S

S

1274 1276 1277 1278 1280 1281 1282 1294 1296 1297 1298 1300 1301 1315 1317 1334 1345 1350 1359 1360 1361 1362 1386 1393 1394 1401 1414 1422 1423 1425 1426 1427 1431 1432 1436 1437 1438 1439 1440 1441 1446 1448 1449 1451 1456 1457 1461 1464 1466 AAADAAA COGAUGAGGCCGAAAGCCC-A

AAAMWAU

GiUATA~ CUCNUc CCGAAAGGCG ALMAAk UUAAA7 CUGAflGAGGCCGAAAGCCGA

AAXUU

CrJGLEMAA UGGAGCCGAAAGGccGA;

AAMMA

AtUCUGaA CUGAflGAGGCCAAACGCC:A

AIJAA

C-AUTCUGU CUGAGAGcC CGACCA AAlarj UCAIUCU Ct3GAUAGGAAAGGCGA AAATkAA AAAtJAAA CUAGAGGCCGAAGCCAA

ACAUCA

CCAAAUIA CUAGCr-CAA=G; ArUfrju CCCAAAUG UAGCGACCA

AAUXAC;LU

UJCCCAAA, CUGALGA GCCGAA GCGA

AAADACA

t7CUCCCA CW-T-C AACGCG-AA

AUAAAT-TA

G=CJCCC CUGvXGAGCGCCGAA GGCGA AAMJL2LU CCCAGGA6 CUCGAtrAGGC,-CGA ACCGA;L

ACCCCGG

CCCCCAG CUGAGAGCc ~CAA CAGCt7CC CG UAGGCGACG,-C-, ACAUMG7 CW.AGCC CU CAGCGAAGCMA AGGCAGc CAJUCU CUAUAGCCGAAGGCC

AGCCA

CACGGAA CtJGAflGAG=CCG.CCC. Ac=WC UCACGGA CVU C GAAG(C~CC,

AACAUGU

UJUCACGG CUGAUAGCCAAC.-CA

AAACAVG

UUCACG aJUGAGGC'W-,CGA AGGCCGAA

AAAACATJ

AACAGCC Ct7GAUGAGGCCGAMG AUTX.tJC tAm aUGAAGGCCGAA '=CCGA A~CAGcc AGcGGGC CUGAMGAGXCGGCC

ACAIGGG

AGGCACA CUGAUGAGGCCGAA C-CCGAA

AGGCCAG

TJC-;AAAG CUGUGAGGCCGAAGGCCC-A

AGGCACA

AUCAAAA CUGAflGAG--GCAC-GCCG.A-

AAGGCAC

UAAUCAA CUGAflGAGCCGAACGAA

AGAAGGC

AUAMICA CUGAfLVA GCG-C r GA AAGAAGG CAL]AAUC CUAUAGGCCGAA ,-CCGAA

AAAGAAG

AAAACALU CUGAXAGGCCGA-.C,.

AUAA

AAAAACA c~.~.~cGAcGcA AAuCA UUUAAAA CLTGAUGG-GA AAGGCCGAA

ACAMJAU

UUUEMhAA CUArA-CCAACC

AACMAU~~

AUUU7LA COAGAGGCCC GC-'cc-A;L.

AAACAU

UAOUUUA CUGAUGAGGCCGAA rCGAA

AAAACA~U

AtULUU C UiA~AGG CGAAG'C-;

AAAAAC

AALM7WUU CUGAUGAGGAGCCG;.

AAAAAC

CAGAflAA Ct3GAUCACGCCGAAG'CrAA AUUUtMA AX3CAGU CrJGAXAGGCCAAGCGA A3AUULTU AAUCAGA COAUGGCGGCC;L

AA-LT~

ULIAAUCA, CUGAUGA=GAAACCGAM AtUhAT3AU ACAACUT CuGAUGAGGCCGAWCGAcc ;TaUAU GACAACtJ CUGAUCAGGCCGAGCCCG -AAtICAGA UE3UAGAC CUGUAGGCCAGCCC-C ACUtIAAU UUGUEM UA GCCCAGCCGA

ACAACUT

CAUUGUU CUGAUGACGC-AAt.GCCC-

AGAC

249 0 1479 1480 1494 14.98 1501 151.2 1517 1528 1533 1537 1540 1546 1549 i551 1552 1566 1572 1576 1577 GUCACCA CUGAM AGCGAAAGCWGA AtJCAGCA GGUCACC CUAUAGCGA c-CCGAA AAtUCAGC AAVGAGT CGUGGCCGAAAGCCA

ACAG=U

C-XGCAAU COUAG GGCGAA

AGUGACA

CCUCAGC CUGz~lAGGAAAG;GCG

AMMAGUG

GGGAGCA CUGA GAGC-CGAAAGCGrAA

AGGC

CCCUGGG CUMGA AGCC GAACC--M AG~kAG CAGaCAC CUGXUGAGGCCAAGCCA

ACUC=C

GAfltawA CMAMAGGCCGAAGGCA

ACACAAC

GG;CCGAU CUAG.GCIl A ACAGACA GLGC C CVAGXC AUUkrcAt T3GAAG COAAGCGAA

AGGCCGC-.

CACOGAA arUGGCCGAAAGGCC AGtkWC GCCACUG CUGAGAAGCA AL1AGh CGCCACT C~UGA CGAAC AA IGU CAACCUU CUGAtUGAGCCGA GCC AUUUCUc CCEMh.AGC CMUGWGGCCGAGGCCGAA,

ACCUUUA

CUUUCCOGAM-AGGCAAG=r.A AGcMACC UCOC CUGAUGtX A AGGCCA A MGCAAC 4.

S.

S S. S

S

S S

S

250 Table 25: Mouse TNF-a HETarget Sequences at Position

V

a a 0* a a a.

a.

a p 66 101 101 102 102 106 i16 137 139 177 207 228 228 236 236 249 249 H Target Sequence UgCAAAU a GcucCcA GGCAGGU U. CUgUcCC GCGgU u CuGriccC G=AGO= C UgUcCCU gCAGg'tJ c ugUcCC GUUCUgU c CCUUUCA UgUcCCtJ u UCAcA gUCcCDU U CaCOCA gUCCCuU u CACUCAc U~CC~uU C ACucACU UuUCACrJ C ActJGgcc GCCaCALU C UCCcUCC CaCAUCU C CCUCCAg GCAUGAU C CGcGACG AG3CaCU C CCCcAaA GGGGCuU C CAGAACU GGGCuU c CA.GaacU CAGaaCtJ C CAGG=G CAGaACU c c.AGgcGg Gr-ugCCU a UgUCt~cA GGUGCCEJ a UGucUCa UCAGCCU C tUUCaU UCAgCCU C UUC~cau AGCCUCU U CtJCafUC AgCCUICU U CUcaUUC GCUCtUU C TUCaUUCC gCCUCUU C UcauUc r.;UCUF. C alUCCG UUCUCAU U CCUGcUu UCUCat3U C CUGCUuG TUGCU u GUGGCAG CCACGcu C UUCt3GuC ACGCUCUT U CUGUCEUa CGCDCUU C UGuCUaC CLtuCUgU c uAcUGaa UCUG~cU a cUgAAcU Ct3GaACU U cGGgGUG tTGaACUt7 c GGgGUGA, uGaaCJU c GG~guGa gGGLIGaU c GgUCc 324 347 364 366 366 369 376 390 396 401 404 406 406 407 409 409 409 432 444 501 5.60 560 564 567 569 572 572 572 579 580 580 582 582 584 585 608 615 61.5 618 Pa it on EE Target Sequence GgGUGA C G-GUCCCC GAQAagU u CCCAaaU CCUCCCU C UCAUCAG TUCCU=CU C MXCAGuu U~cCCUCU C auCAGuU CUUCAU C AGuuCUa CAGuUCU a UG-GCCCA AgACCCU C ACaCLUcA UcacA.cU C AGAUCAI cCcAGAU c A3CuUCUT AGAUCA C DUCUaA AUCAnCrJ U CUCaAAa ATcAUcU U cUcaAAA.

UCAt3CUU C UCaAAau AUCUUcu C aAAauuC AuCuuCUT c AaAADJUC allcUUcU c AAAauUc AGCCDGU A GCcCAcG 261 261 263 263 264 264 266 269 270 276 297 299 300 304 306 314 315 315 324 ACGUcGU AcGCCCU gGgUUGU GGgUUGU

TJGUACCU

ACCtUU CUUgUCrJ 9UCtmcu GUCEaCU GUCUac-U CCCAGGU1 CCAC~gUt CCaGGuu AGGUOCrJ AGGUuCU GUuCuCUI UUCUCUtJ CCCGaCU aCgUGcU

C

AcGUGCEJ C UG-CUCCU

C

A. GCAAACC C CUGGrc a CCUUguC k CCLUguc a1 gUCOACU

UACUCCC

LCUCCCAG

CCAGGUu *CCAGguu CCA9GUu

CUCUUCA

UCUUcAa tlCuUcaa UUCaagg

UUCAAGG

CAAGGGa AAGGGac CgugCUC Ct3CAccc

CUCACCC

ACCCACA

251 0 9* 0 5

S

S

*0Se

OS

a 630 630 638 643 645 647 663 669 669 672 674 681 681 68i 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 ACACCgU C AGCCGau ACACCgU C AgCCgaU agcCgAU u uGCt~aUc allUUGcU a uC~c.AuA TUGCuU C tJCa~CC GCuaUCtJ C allACCAG agAAaGU C AACCUCCtUCAAcCCU C CUCUtrG UcAAccu c cuetuctG, ACCU~Cu c UjCt"GCCg CECUkCU C UGCCgUC CUGCC9U C AagaGcC Ct7GCCgU C AAGAGCC- CUGcCgU C aaGAqcC CCCUGGU Az UGAGCCC CccUGGUj a UgaGCCc AGC-cA= a UAcCVG.- CCCAUaU A CGGGA GAgGAGLt C UUCCAGc GAGGaGtj C UUCCAGC- GGaGrJCU U CCAGCU GaGUCUUt C CAGCUGG ACCaACU C AGCG=U CUGAGgU C AAUCuGC- GgUCAAu C UGCCCaA CCCaAgU A cuUaGALC AgtUkcuU a GACuUUG uUaGACTJ U UGCgGAZ UAGACUU UJ Gr-GAGU Gf-gGAGU C cGGGCAG GGCAGU C UCUUUG CAGGUCU A CUUWGGa CAGgucU a CLTUUgGA cagGuCt) a CUUUgGA GVUCCU U U~agqUC UCEUhCUL U GGagUCA UUGagU C AUUGCuC GagUCAu UJ GCuUGE AUCCaUU c uctMCc AuucuCU a CCcaGCC CCcCaCU c UgaCCCC CCCCacU c UgACCCC CCCCAkCU c uGAccCC GACCcu Ui uacuUG ACCCCuty u acUCuGA CL7UUAcU c ug'aCCcc GACCcCU u uauuguc gAcCCCtj U UATJguC CCtUUAU U guCuaCt) 940 943 972 972 973 984 984 985 997 1010 1017 01 1019 1073 1096 1106 1107 11I08 1115 1 113 1164 ill80 1203 1210 121 1214 1218 1218 :21.8 1218 1219 1219 1226 1226 1227 1227 1228 1238 1262 1283 1283 1285 1287 1287 1288 1289 1293 1293 1294 GUcCUC c CUCAGaG t3M.CUt C AGaG3CCC UC~aaCUT u AgAAAcg UcUaaCU u AG-AaAgG Ct~aACuU A GAAAggG AG9GgAU U auGGcuc AGGGgaY U allgcUc GGGrGauU a UGcUCa UCAG-CAgU c CkAC-cu CuguGcy c jkAG=~r CAGAgCU U Uc-NaC.AA AG-AgCE-U U CAaCAAC G-Agctjuu c AarmACu U9gG~CCt c ucAUgCAL AAC~gAcU C -AAAugGC; aUCrGGCU U- uccGAAU UGGCtU u ccGAAflu GGgCuUU c CGaaUrC CcG-AAuTJ C ACDGGaG CGAAngU C CALUCCU gagU;GgU c AgGUUGC UJcUgucu c agaAUG; aaGAL.CU c AGGCCU cCGCCU U C-_UacCU AGGCCUU C CUacCt~u CCtJUCCrJ a cC-JuCAG CCUACCU u C&GACCu CCUaCCj u rCAccu CCIACcU u C-kgACCU CCUacCtjU CAkG;ccrj Cue.CCUu C AGACcuu CUaAcCUt) c agAcUt CagACCrj U uCCAgAC CAG-AcCU U UCCAGAC aVCLUj U CCAgACu AG;LccUJU U CCAGACU GAccuUU c CAGcACc gALCU c cCUCAGG CAGCCt'U C CUCACaG CCC%-CcU C uauLUAu CCCCCCU C tUUUAU CCCCUCTJ A tUUaU CCUCUAU U UauAuTU CC'JCUAU U tjAUaUUU CUCEMIJ U ALUaUUEJG UCTJ-AcLr) A U~arUUGC ULMUaU U UC-CacUr u-UUaUaU u UGCAcUu UUAtarUE U C-CACUUa 252 9.

*sa.

a.

a a 1300 1303 1304 1306 1307 1307 1308 1310 1310 1310 3.311 1311 3.311 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

UUGCACUT

Cacuuau acUuA~jU UuAU uAUMtUU UaUUaUTJ

AUMUUU

UauUuALT

AUCUU

AAUU= A uuuauuu UUUU U MUfUAT-T uailtum U UAUGU uUAUtW. u UaUruk~ MMUUt U AUDU= tUDRUkU U AUUU AUaW=UO A UUrBAD= AUUUAUU U .AfUX=~t UU~Uku A UUMUA ATUA=U A UUUAUruU AUUMDu Uj AUCgCu UUMUU A UU~gCuu tUUtUA.u UUgcwjAu AUUUAUJU U- gCuuAtjG auUUGCU U AuGAAuG uUUGCU A uGAAuiGu tMAU A UOUAIjrOU AAU Uj UMTG AUGrMnU U AIUWGa TUzUr A UDUG~aA UAUULW. u UGcaAGG UAUWh, U UGGaA~G AUUtM.U U GGaAGgC GGGgU C CUGGaGG gCt~guCU T- cAGACAg GCEJGUCU U cagaCAG GACAUGU u uU~cuGG, ACAUGUU ucuUGGA CAUGOUU U CuGVCAA AtJGUUJLE c uGuGAAA AtJGUUUTJE c UgugAaA gaGC~UG c CCCAccU CUGGCCU C UcUaCCU ggCCLICE C UaCCUUG U alluAUlu u AuUuAU A UUM.UM~ U AULTAVUU U AuuAUU A ULVhUUA U AUUUAraU U AUUtMUL LY AUUMUU hi UUULtIU 1462 1470 1472 1473 1474 1478 1479 1479 1484 1498 1511 1514 1516 1529 1529 1530 1530 1563 1563 1568 1589 1592 1617 1623 1633 25 accuuGu cuccUCU CcUCU

UUUGCUUT

TJUUGCUU

UUUGUrJ AAAuauU AcccAaU cAaUUGTJi allUGUcuJ CgcugAU cGCUGAU T gCUGAUuTt GCUGAULt UgTaAcCUy c ugaaCCLT C CUCUGCU C UGaCUGU A CUGtUhAU u GAGAAAU A UAAAGaU c UUAaaaU a AgGgaCtj a U GCCUCCTJ C vuJut3ct U- ULJGCUUrA U UJGct3LA U GcUUAUJG U AUGUUUa I. UG.UuuAa k. UGUUUaa I aaaAcAA 3 AUCt~aAc I GtJCUUAA UuAAuAA LAAuAAc-,

LUGGUGAC

gGUgacC

GGUGACC

UGcUCCC UGCrUCCC CCCAcGG ATJUGcCC GcCCUAC GCTJUTAaa aaAAaCC gCCagGA 253 Table 26: Mouse T.NF- Hammerhead Ribozymne Sequences nt.mouse MI Pbazyme. sequienc UCCGC CUGILrkCGCC,-%A=CAA AGt3CCcuI 00 t2GGGAGC C=UA CkAXCI AUUuc.

101 GGGA CUA.~,C-%A3-CA ACCUrc C2 GGGACA CUGAVCG GC, -rA G'-C GA A C -_G 102 AGISGACA Ct2UGAANGG-CG- -CGAA

AACC=G

2.06 UGAAACG CUAG=C- =A ACAGAAC iiO ~UGAGUGA COG AGC,CGAA CG; AGGGAM~ 2.1GUGAGMJ CUGAUG,GAGCC GA AAGGGAC 112 G~GU G COGAVGAGGCCGA Gr-,.JL AAGGGI.C 12AGAU CUAVAGCCGAAAG~Cc AA-GGa 9*99 2U6 GGCCAGU CMUat2G G ACC AGUC~, 2.37 G-AGGCGA CUG UGAC-GAACCC~GA

AUGG

COGAG IU~it GAACZ~GCGAG AGAtJGO **177 CGCCG CUC-AUWZ-CG CGA AU~CAC 207 UUUGGGG CUGAXMAGG-CC

U.AGGCC

228 AGUOCUG CUGA! AG-CG CCC.A AGCCC 228 AGtUG TG!UGc= GG AAGC=c *:.236 CCGCCt3G CUGAUGGC-XAA GGC GA AGU 236 CICXccU

CUGAOG-AGGCC~GAAGM

24 UGAGACA C AUGAGGCCC-G .G AGC 249 GAGACA CUCACCCGAAC GCCGA,

AGGCACC

29i -UAA CUGAUGAG~C-CGAAAGGCcGA 29. *MAA CMUGGG-rAAGCCA

AGGCUGA,

9. 2 6 1 A GAAU G A 263 GAAUGAG Ct2GAUGMGAAGG CG AG-AGGCU 263 GAAUGAG CUGAt2G GCCeAC-

AGAGGCU

264 GGAAUGA CUG;LUGAG-,CGAGGCCG

AAC

264 CGGAU

CUGAGAGGCCQAGCCALAGAA

269 AGCA G CU AUGA-G--GAAA GCC AA GAGGC 297 GACAGAACUGALUAG--CCGAAAGGCCGAA

CGTG

299 400v& CAi.AUCUA,,-CI--CCGA

A

269 GAA~CAAGG .GCoCAX

AIJAGA=

T~CAGc. COGAUGAG--CCGAXGGCGAA

ACAGAG

276 CGUMCAC Ct GAUGAGCC aACrCGAA

AGAGG

314 CACCCCG COGIAVGAGCCC- AACCCA A~JUCAG UCACCCC CUC.AOG-AG~C-caAAGAAC

AAM-UCA

254 315 ucAccc CUAUAGGCCGAAAGGCCGAA

AAGUMA

324 GGGGACC CUGArUaGGCCGAAAGWCC-GAA A33CACCC 324 GGGGACC CUGAUGAGcGAAAGGCGAA, AU~Ccc 347 AVUVGGG CUCA.GAGGCCGAAAGG-CcGAA ACotp= 364 C~UGA CUUG~AG-cAAAGCCccAA

AGGGAGG

366 AAU CUAGG-CAA r, ACCG 369 AACt7 CUGV~vGGCCGAAAGGCM-AA

AGGG

376 UGGGC=CA. GGCC =G ACAAc 390 UGAGUGU C~aUAGCGAAG-_A AG---Cr 401 AGAG CUGAUGAGCCGAAAGGcc,-- .?ifCGGAG 404 UAGAAA CUGzADGCC-AAG~ccGAA,

ADC-AWU

406 UUUUGAG CUGat~rAGGCCG AGccA ACAG;aU 406 UUUGAO CUGAflGAGGCCGAAGcc-jG

AGAIGAU

407 AfUCUUGAL CUACAG-CGAGCrA

AAGAUGA

409 CAAfUU CUGLAUGCGCCC C-ZA ArMGCAU 409 GAAU= CUGAflGAGGccrGAAAGGC,-AA

AGAAGAU

409 GAAD= CU CUGA~CGCAA

AGAACGAU

432 MOUG=G COMIDGAGGCC,-AAAGGCCGAA

ACAG=C

*444 GGUUC CLTuGAC-AG,-CCGA GGC AC% ***501 UGCCAG CGUGCGCAACGA AGG=c 560 CGACAACGG CUGAUGCG,-MAAGC,-CGA

ACA.ACCC

560 GACAAGG CUuDA--C~-LG. A ACAACCC 564 AGLMAC CUAGCG CIaG--_C AG IGGAGM CUGAGCGAACGCGAA

ACA

S.*569 COGGGAG CUMVJGAGGG WAA AGACAAG 72 AACCUGG CUG-M G~cAAGGCCV CC-C-AA AGtAGAC 572 AACC=~ CUGAW-IGGC.-AAAGGCCGA; AG~kMC 572 AACCUGG COGAnAGCCGe.AGtGA

AGAA

3 79 t3GAAGAG CGUAGCAAGCA

CV

580UUGAGACUGAflGAGGCCGAAAGOCCGAA AACC=j 580 UUGAAGA CUGAGAGCCGAACC-=,A

AACCUGG

=82 CUUGAA CCGAUGGc GCCGcrAA AGAAccEJ *582 CCUUGAA CUG-NflAGCCAAAWCCCA

AGAA=CT

584 UCCCUUG L GMAGCCrGG CCCM AGAL3AAC *585 GT~UC UGAGCC-,-,jGC-A

AAGAGA'A

608 GAGCACG CUArAGCGArCrA

AGUC=G

615 GGG--UGAG CUArcCrAjCGA

ACC=

615 GCrGAG CE. GAAGCA

AGCA=G

618 UGUGGGU COGAUGAGGCCG kGG~rA

AGGAGCA

630 AUCGGCEJ CAUGAGG-Cc GGr-CG

ACGG=~

630 AUCGCCU CuGUGAG~A GGrc cGA AcGGmu 638 C GA CUGA MG. GAAGCG

AUCWGCU

643 UAUGA.GA CUGAIJGAGGCGAAAGGCCGAA

AGCAAU'

645 GG-3UUGA CtGAUGCGAGGCCGAA

AUAGCAA

647 CUGUAU CUG;LtAGrCCAAGGCCG,

AGALTAGC

255 663 C-GACG-UU CUU-GCAA-.-rA ACUuMcrj 66.9 CAGAGAG CWAG=CAAGCC., AGM07M 66.9 CAGAGAG CU~c=GAACG

AGU;

672 CGGCAGA CUMGU3GGCCGAGC.I

W=

674 GACGGCA CWAAGWGCGAAAGGcca

AGAGGAG

681 GCCU CUMW----Gcc GCGAA

C~

681 GGC uC CM3AVGAGGCC=C-J r Av- GCAG 6581 GG=u~ CMUGG--CcAA~AGG-=a

ACG=CA

734 GGC-=CA CODGAGGCAGCW

ACCGGG

734 GCEJCA CMUGXGAGGCCGXUGGJ A ACCAGC-G 744 CCAGGA CGMWa=~.CMAAGG- 746 t7CCCAG C~GUZAGCCGAAGC-LA

AUALIGG

759 GCt3GGAA CUAAG

CC--

759 COGGAA CUGAAGGCCGXUGcC.-A

AC~C~C-,

761 CAGCTJG CUC-GGACCGXA

AC--

762 CCAGCU CMAMflAGGCGAA,-CGAA

AAGCVIC-

786 CAGCGc-aAUAGCGk AGOUi 798 GCAGA1UU =GMGCGAGCG;

ACCOCA

802 UtUGGck rAGCGAGGCA 81 *LA

AD~_

9*~eCUGAVWGC CGCC U :816 CAAAGUC COAGG-C,.AGC. AAaxu 821 CtuccrcA CtUGAGCUGC CA AGCM **822 ACt7CCGC CUGAUGlCGAAAGA AAGa A 9830 CUCCCcc CT3GAXMAG---GCC iJC-GACU 840 CAAAGUA Acu4--r L 0.0842 UCCAAAG CMXAIXMAGCCGAAfW4CcA AC-NCCr 842 UCCAAAG CUGAWlAGcGCCGAA~- AGACCEUG **842 t7CCAAAG CUGtAUGGC C-G-CGAA GAL t *85GACUCCA. COGAVGAGGCCGAZGCt A1MW LrxhAcU CUGAMlAoC-CGAA-,CA A ~r- 852 GAGCAAU CJrArAf-CC~UGCr

ACCA

855 .ACAGAGC CUAAWGX=CA

AUAC

887 GGGUAGA CGMGCLMCA AG .0 90 .CI.C CMUAGAGXCcGAAAGCt..t... 00905 GGG-GUCA CUWAct~AGUG= 905

GGGGUCA

0*.914 CAGAGUA Ct3G4ACAGCCGAAGGCGAAL

AGUGGGC

*999915 UCAGAGU tI;.W *AGCGAAGC-A AALGGG:;U 919 GGGGt3CA CUGAUGAG CXA CC;A

AGW

928 GACAAtIA CMUAG-GUGLC

AGG=GC

928 GACAAUA arLVUG AXCC CCWA, AGG~c 932 AGUAGAC CGUAC

XAG

940 AULUA

CGUGAGG.

943 GGG-t-U G CGAA AGGAGyah 972 CCUUU=t UAG~tt~E,~~~m 972 CCUUUCu CUGuGahGC., vjm 973 CCCUULUC CrUGGCGAAAGCCG cl AGUU&GhQ 984 GAGCCAU CrJGAUGAGr.CCGAAAGG.CCGA AUCCC,_j 256 984 GA'GCC;LU CUGAUGAGGC-CGAAAGCCaA

AUCCCU

985 LrAGCCAL CUG =AGGCCGAAAGCCG

AA=C

997 AGAIGUUG CDGAAGG

ACVCUGA

1010 AAGCUCU COU AGGCCG QGCQ

A~C~CA.C

1017 UUGOUGA CUG UAGCGAGGCA AC-UrG 1018 GuUjGlUG CUC-V-C---GAAAc GCC A AACI~j 1019 AGOUGUU CUGAtflAGGCCM'AAG GA AAAGctj 1073 UGCAMGA CUGAUMAGGCCGAIG CG; AG-c-., 1096 CCCAUUO' CUGAMAGG--CA1 GG AGUCCU i106 ATJECG-GA CO;GGCGkZZ-a

AC-Z

1107 AADO=G CUG GADGCGA

AAGCCCA

1108 GAAUUCG CUAGA~GGCCG C-A AAAGC-C 111s CUCAU CUG M~-GXU=CA

AAU--

1133 AGGAAUG CMWXGCMAG. ACAU=c 1164 GCAACC L~ COAMGc CuGc-a;; ACCxvcC 1.180 ULDUCU CMUWGGCCGXUG-.A ArNAJA A 1203 AAGGCC CtXADGAGcGCCGAAGCC

AGWCU

1210 AGGTMG CUGAUAGGCCM~;GG; AGGCj r G 1211 AAGUG CUkG-CCAGCGIL-

GCE

1214 COU= AGG DMGCAAXAA AGGAWc ***1218 AGGUCM UMGGCGXrCGXAG~, 1218AGG CUGATJGAGGCCGAAAGGCCC-U

AGGGAGG

1218 AGGUCCG CUGAGGCCAXAG- C ACGGGA= 1218 AGGOCUG CTXMAGGOAGGCCCAA AGa;G i 218 AGGUCU CUGAGG UGGC

AAG-M

1219 AAGGU C GAGC

AAGGG

O,1226 GUCUGGA CMUMJGGGCCMUGGCCG~AA

AGGUCUG

1226 GUCLIGGA, CUGAUMGAGCCGU=ZAA AGGUCE7G 1227 .AGUCUGG CGUAGC AGUr 122 AGCG aG *122 AGUUGGCLMA=XA~C-CQ CGAA AA= 1228 GAGUCUG CUGAUGAGGCCGAAGC-=

AAAGGUC

1238 CCUCAGG CMUGXGAGCC AA AAGAGIJC 1262 CUGUMG~ CUGAUGAGOCCMUAGGCMAA

AAGGCU

*1283 AXIAAAUA CUGALJGAGGCCMUGCA

AGGG

1283 ALMAA~ UA~G~ck Q~AA

AGGGGG

1285 AUWAA CG GCC GG

AGAGG

1287 AAAMMh CUGA17CA.CCGAAGGCr.A AMC.,A= 1287 AAAMflA CUGAGcc

AMGAGG

**1288 CAAAXIT. CUGAUG GGCCG XXGCCGAA AAt~kWZ 1289 GCAAAL.h.. _:AAGCMA CMGA AMAIA 1293 AAGUGCA CMMGCGAGCC;

AMUA

1293 AGUGMA CUMAMAGc GGCCMA Ar_ AAUI.AA 1294 LAULGmcC UMUGGCAG CGQAA AAMUAA 1300 AAADW CUGAAG-UACM;A

AGUGCAA

1304 ALMAUA CUGAncAGCCMUAWCGA

AMLA=

1306 UAUA

CUGAUWMG-C

1307 AAMAGAA 1307 AAA7LAU CUG At3AGGCCGAA-C

AA~

257 1308 UAAATIAA CrOGAUGAGC--CGAAAGGCG AA AUAAU 1310 AAMAAU Ct 2 GIMaGGC=AAA,-CCGAA AMALtM 1310 AAMLUAUx CUGA--AGGCC ZC-GM AX7AM~~ 23ii AAAAA CWUGAG-GAA G,-GA ATAArU 1311 AAALMAA CMMM&U AAGGC CGAA AAAA 1311 AAAUAAA

CMAA)C-CA

Z-123 AUAAA CUXGA=GC AAG,-GA AAZkTJ 1 2 1 X.ZX A G G C A A U A A DA A 13 ALMAAU CtMXaGGWCMG ,CGAA AMWAAh 2.313 ADAAAUA CUGAi;aG-ctA6 134AADAAU C't3WAtGC 'AAC-CGA

AAUAAXIA

1314 AAMA CUGAUGG~- MatGC-CA 1 ~i5 tMAUAAA.DAGGC AGCcAXUM 1 1'7 TJ CMr-IGGCCGAAAGGC ALt3AAMt 1318 AAALTAAkU CMUGA C,,-CGAAC-,-G

AAU~AAD

13i.9 U~AALAA CM CVr-C.AAGCGA

AAAMLAA

1326 AAAUA COGAUGAnG tCGA1AGCM- ;AAUmA '1328 G-CAAALA CMGAGG MA-CrAA AMUjAUA 1329 AG-CAAAU CUG=AX WCMUG.C-AA AAUkAAUz 1330 AAGCAACG AGCG-GWA~m~ 1332 AU=C CE2GAUAG-- "GACG-=

AMAAA,

133 CUGA~GAG3CCC'AAAGGCCGAA AA u i337 CAUUCAU UA AGC

P.

1338CMAU =C-SAAW-C- AGOAAU 38ACUUCDMUXG.CGAGGcA

AA~C-A

0:01346 AAATkhA 0*01348 cc~AACUALr -=GAG--XA

AICDUU

1349 UCCAAI3 CUGAUMAG:-CCQ ACG'GCA *2350 UcAAC G c CG AAflAC 1352 CAAcA CtGAUGAGC-CGAA GCCGAA AtAAAUAA 1352 CCUU=C=XG'-

CA

CCU CCA GAA-GC -CGAA AAAJ 1 35 3 GcUC UAGGc

AAAA

1369 GCCUcc CUAGGCAACCA A~kMU CUGUGCCGA -GCCAA,

AGACAGC

1398 CUGO=CUG MG--GC-CGAA6 AGAG 1412, CACAGA;LA rcC--AAAG,.A ACAZ3GUc 01,2413 UCAcAGA AACAU C 1414 UUCACAG CVGGA~rAGr.CCG.CC~r 9 aA AA WX.G s* 1415 UUUCACA6 CUGA AGGC,-CGAAGGCCAA~ AAAC 141.5 UUtJCACA CUAMGCGAG.CA

AAAACU

1438 AGUG CGXA GAcc~kGGCG

ACAGCUC

14 51 AGGUAGrA CUG rAGCGX 14A53 CAAGGM UGLc a,-CAA

AGAGCCG

145AACAAGG CGAGGCA

AGAGAGG

1462 AGGAGGC

ACAGG

1470 AGA UAM cc

AGGAGGC

2.472 UAAGCA6

AGAGAG

1473 AUAAGCA CUGAUGAGGccC cAGC, AAGAGG 1474 CAZAAC Ct3GAUGAGcc CA AAGAGG 1478 UACAU CGtA CGAGC~CGc

AAGAGG

258 1479 ULU~aAcaA CUGAUGAGCcGAGCCpaA

AAGCAA

1479 0 U M~A~ CUGAIGAGGCCGk;GM AaArCa A 1484 UUU CUGAUAC-AA GGccG CGAA AMMMA 1498 GO M=A CUGATJGAGGCCG 3GCCrGM

AAU

1511 UMAGAC CUGAflGAGCcrQ GACGr- AUUG=~ 1514 UUAUlAA C~UGAG XAAG G -CGAA ACAAWu 1516 c~uCGGAVaGGCc

AGACAAU

i529 GUCA CUAGGCGAGL

AUCAGCG

1539 GGUCCC CCJGAD GCAAACG CGA AUCAGC~ 1530 GGUCACC CVGAGXfl AAGG AA AAUCAGC 1563 GGGAGC;L CUAUG--CUGCC CG-A AGMUCA, 1563 GGAGC;A CUAG=GAAGC

AGMUCA

1568 C03UGG CCAAG CC AGCAGA i589 GGGCAAU CUGAU GGCCGAA -LL- ACAGUCA 1592 GUGGGC CUGAGA=CGcc GCCGCA tC 1617 cGnu UA c C AucAtG 1623 U AGC COAGGCMAGCA

AUUUA

*1633 U CADGAGCCGAACCCCA

AUUU

*0 .0 0@

S

0*e

S

S 55S

S

S

S

0e

S

S

SS*

S* S

S

5@

SO

S 555 5. 5* 55 5** .5 5 5 55 5 5*5*S S S S S S 5 0 5*Se S S S

S

Table 27: ilumnian 'I'NI-a Hairpin ItiI)ozytflC Sequences 'ft.

Position Hlairpin Ribozyme Sequence 185 201 230 234 254 296 317 387 404 453 518 554 565 576 687 704 726 730 824 1042 1168 1178 1202 1.220 1284 1340 1390

C

A

U

AGCCGUGG ACMA GIAUGU

ACAAAAAAGUGGUCUACGU

GAGGULGG AGAA GUGGU ACCAGAAAAcACAUJGUGUACAUJACCr.UA GGAGAAGA ACAA GACGAA ACCAGAGAAACACWYCJTJYGTJAGUACuuceqJGuA CUGCCACG ACMA GCAAGG ACCAGAGAAACACACQJurJEJQGTAAUUACC.JGGUA GIICCACCA ACMA GMGAG

ACCAGAGAAACACA(YGITJGJ(G!ACAUACCGUA

CAAAGIJGC ACMA GCCACA ACCAAGAACACACCTJ1JJCAJAACCUGGUA CCUCUCGG AGAM GCACc

ACCAACACACACGUYJUUACAUUACCLJGUA

GGCCAGAG ACMA CAUUAG CC AAAh~~uUGU~UACGU AGAAGAUG ACMA GACUGC ACG

CACACGUUJIGJGUAAUUACCOGGUA

GCCACIJGC AGAA GCCCCIJ

GAUCUACGU

AUUGGCCC ACMA GUUCAG

ACAAAAAAGUGGUCUACGU

GCACCACC ACMA GUUIAU ACCAGAGAAACACACY3YJ1Jr1TJUACJWIJACC.JGGUA GGUGGAGG ACMA GCCUUG

ACCAGAGAAACACACGUUGUAJJACCUGGUA

~GCGAUGC ACMA GAUGGU

ACCAGACMAACACACGUJTJ(GUGGTAIJ.JACCUGGUA

.JGGUACGA ACMA CGAUG

ACCAGAGAAACACACUJJVJGGUAAUACCUGGUA

-rGACCUUG ACMA OGUAGO ACCAGAGAAACACACGU1JGUGGUAJ.JACCUdGGUA XULCUCC ACMA GGAAGA ACCACAAAAcAcAcr3uuYGOuAcAuuACQCU~A IGCGCUGA ACMA GUCACC

ACCAGAAAACACAGYEJTJGGACAUUACCJ(GGUA

AUAGUCG ACMA CAUUGA ACCAGAGAMACACAC~urJT(JGGuA.ACCJGGUA JCGAGAUA ACMA GGCCCA ACAAAACc 1GU~~AUACGU GGAUUGG ACMA GGGAG ACAAACAC YJTJTJUJ(JACAUUACCQJGGUA4 GGAUCMA ACMA GUAGGC ACCAGACAMACA CCUUGUI.UACAJUACC.U3GUA4 UGGAAAC ACMA GGAGAG ACCAGAGMAACAC UUGUGGUAAJeGGu CAAGGAA ACMA GGAAAC ACCAGAACACACGUUpj UACAUUACCrGUA tJGGGGAG ACMA CGGCUJC ACC-rAACAC uUiGuGGuACAUUACCQJG (JAGAGOG ACMA CGCUCC ACCAGAACTGGUAC ACctUGO (JACAVUC AGAM GUAAAU ACCGAAccUGGUACA UUA CCGGU

A

3AGCCMA ACMA CCUCCU ACAAAAACCU

UGACUAC(GA

~CAUGGG ACMA GCCUAU ACCAGAGWACACACGUU GUACAUACCJGOU

A

Substrate ACAIJACU CAC CCACGGClJ ACCCACcJ C CCACCCUC IJUCCUCA CCC UCULJCLCC CCUUCCU CAU CGUG3GCAG CUCUIJCU CC UGCUOCAC UCUGCCU Cl GCACUUUG GUGAUCG GCC CCCAGAGG CUAAUCA GCC CUICUGGCC CGUCA GAU CAUCUL)CU AGGGCA GCU CCAGUGGC CUGAACC CCC GGGCCMAU AUMOCCA GCU GGIJGGU)GC CAAGGCU CCC CCUCCACC ACCATJCA CCC GCPUCCC CAUCCCC GUC LICCUACCA CCUACCA GAC CAAGGLICA UCUUCCA CI GGAGAAGG GGUIGACC GAC UCAGCGCU IJCAAUCG CCC OGACUAUC UCGGCCC GAC UAUCUCGA CUCCCCU CCC CCAAIJCCC GCCUACA Cl UUGAUCCC -UCUCCA GAU GUUUCCAG UuUCCA CAC UUCCUUGA ;ACCCA CCC CUCCCAI ;CACCCA Cl CCCUCUAU ~UUUACA CAul GAAuGuAu ,GGACU CCC UUGGCUICA UAGGCIJ CUU CCCAUGuA 145 ACU *GA GAA* ACGAACAGULGUCAUACGA *UAC G* UAA*. 145 ACAAA AGAA GAUMU. ACCAGAGAAACAcC U OGUAJUACCLGGUA AAU~CU GAul UAG~uJGAJ 1513 CCCrJGGG AGAA GAGGCC ACCAGAGAAACACACGUIJEJGUACAUUNCCJGGUA GGCCUICU GCU CCCCAGGG 1541 GAAUAGUA AGAA GAUUAC ACCAGAGAAACCACGTWJ(TJGUACAUUACCJGGUA GUAAUCG GCC UACUAUUIC 0 p 0* p p p. *pp p.

p p p pp p p p p p Table 28: Mouse TNF-a Hairpin Ribozyme Sequences nt.

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 Hairpin flibozyme Sequence Substrate GnMAAG AGAA GAACCU GUXuLIOMrA ~LGAMAGA AM GAGACA ACCN3N3M

IULW

CUXMACA AGAA WGAAW ACXCCUON

LUCA

G1ZZNCA AGMA GAAGAG CU M G~UC an z CtUUUGGG AGAA GALCAC GJM LC OAG GXCCAUAG AGAA GALP GACUUCM GL CWpC GUGUGAG AGAA GG3CCA L1CCNM AC AL~flA AGAAGAlN) AGAA GGKU ACGA CAWA& GNU CNLUjX)C GCCACUC AAA GtU L 3A OC

GAU

AACtCAUr ?A3AA GOACC G3LtCC GC G IIGL UACAACCC NAM GLUGOC OCGCGUGa )GrA QG UM AGAA GC t CACOCU GOCCrG

CUA

AGCA2Mn AGAA GOO=~a CWUN3CC GACyr~ L XIIC GCIAA AGAA OU ALN3GAa GOil GMXx3C QLUAC AGAA GCMA cujo Go Lmuu GtUCuG AGAA GGGGCU ACCOC W OANIA CCUCUC AGMA OGAAGA UX)AO aW

W

AGUALUG AAA GALJA ~CA WUCAUC GO an

LLC

ACIALIG AGAA GGLA CMCAAACm GO

AT

GJAAO3J AGMA GGUG CAU GAC COttrXuA ALMAAGMl AGAA GA A UaM LM GAC OCtmXWj AGGACACA AGAA 09909C OCOCCAGU N3AAEo ICLM AGNAA GAGO2A LUx)C GU LGM CtUWMAQ fAM GA09 ACMCA ACCUUCw AWOGAAGA AGAA OGAAAG ACGGACCLUG)--- tXAGCI~CC uAGA1G323 PAAA GXoflJ ACACAAA C IGGnjA OXc C=tXI a 1-4 Ln K On 0.

Ch N) L'i tj Ln ul

III

I

I'

V C

I..

9**e ~9 9 9 9 9- 9 9 999* 9..

9999 9 ~9 9 I'll' 263 Table 29: Hum~an bcr/abl EH Target Sequence Sequence =f NO.

b3-&2 HETaxget Sequence GkCAG CWU C%0,=Z

S

S

95 9 *Q S 9 LMAGAGtL= AAAAa= LMAAAG= au c~m CAAAG= L=t AO33Cnj 264 Table 30: Human bcr-abi l Ribozyme Sequences Seque~nce IDNo.

26 27 28 HE Pibozyme sequence GGCUCCU CGAUGAGGCCGAAAGCCG-A

AUUGAGG

ACUGGCCGCtG CUGAVGAGGCC',GAAAC-L-,c:kA ACG:,---U=Cu UACUGGCGCVJ CUATAG--,AG-r-:AALC=U GAAGGCVDDU=U GAGv-CAAGCCA AACtUCUGCMuM ACtGGCCGCi GAUGGGCCGAAAGGCU AGGGCUUrUUG UACUGGCCGCU cUAAG-rGAGCc=A

AACGG-UU=

a.

265 Table 31: RSV (1B) EH Target Sequence nt.

Position *a i0 14 18 i9 54 57 77 94 97 101 110 113 118 122 134 137 148 149 150 152 154 157 161 165 176 188 208 209 210 214 215 221 226 239 241 242 251 261 265 267 274 HE Tazget Seque~nce G~CCAAAU A AAUCAAU AALMh.AA C AAVUUCAG MAUCAAU U CA.GCCAA AUMMAU C AG-cctMc CAUGAU A AMACACC UGAflAAU A CACCA t1GAUA.U C ACAGACA AGACCGU U GOCACtU CCGDLIGU C ACUUGAG UUCACLT U GAGACCA AGNACCALT A .ALMACAU CCAMLAZ A AO.A AMM=A C ACUAACC CAUCACU A ACCAGAG GAGACAU C AUAACAC ACAUCAU A ACACACA CACAAAU U UAMU=c ACAAAVU U AM~UM CAAATLU A UA~kUU AAUUUAU A tU.COUGA UCUktW A CUOGADA AtMCU U GAU.AAAx ACUUGAU A AAUCAM3 GAMAAU C AUGAAUG AAUG=A A GUGAGAA GAAAACU U GAUGAAA GCCACAU U U~AAUM CCACAVU U ACAX3UCC CACADUL A CAOC uuaA= U CCUGGUC UUACAUJU C CUGGUCA UCCU=G C AACCAhUG GCAACU A VGAAAIJG UGAAACU A ULTACACA AXA~t== U ACACAAA AACM=j A CACAAAG ACAAAGU A GGAAGCA AAGCAME A AAIA ACUAAAU A UAAAAAA M~AAX.MU A AAAAAUA AAAAAAXJ A MACUGAA 276 283 2.95 303 304 305 309 317 319 320 323 327 337 338 340 341 350 356 357 363 372 375 380 383 385 391 396 398 402 406 410 411 412 421 423 4.24 432 434 446 448 454 nt Positioni EMTa.xget Sequence AAA.AkTjA A CUGAAUA ACUGAAU A CAACACAC ACAAAAU A UGGCACIJ UG=CACU U UC-CUATJ GGCXct= U ,CM~r, Gc*aCUjU C CC,*'AUGC UUUCCCU A UGCCAA UGOCCAAU A TJUCAMCA CCAAXMrU U CA7UC;AUt CAAUAhUU C AUCXAADC UAU=~ C AAUCAM~ CAUCAAU C AUGAI7G GAUGG,-U U CUUAGAA At3GGU C ULVZGAU GGGUUXCu U AGAAUGC C-OCCU= A GAAI3GCA AAUGCAU U GGCAUUA UL==CA U AAGCCUA UGC-CAUjU A AGCCUjAc UAAGCCU A CAAAC-CA-; AAAGCAU A CUCCCAU GCALmCU C CCAUX;,U CUCCCAU A AUAUAcA CCAM.AU A UkAA= AUAXMU A CAAGUAU UACAAGU A UGAUCU;C GUAUGAU C UCAAUCC AUGAIICu C AAUCCAU UCUCAAU C CAI3AAAt3 AAUCCAU A AAUUUTCA CAUAAAU U UCAACAC AUAAATJU U CAACACA tJAAAUUEJ C AACACAA ACACAAU A UUCACAC ACAAUAY U CACACAA CAAUXUU C ACACAAU ACACAAU C LV~AACA ACAAUCU A AAACAAc AAC~aAU C UXCAtJ CA.ACUr A UGCAU-AA UAUCCAU A ACTkUAC m 266 458 CAIDM=C A UCU=c 460 UAACMU A CUCA 463 CM~U C A~kG= 467 ACCCAU A GUCM'GA 470 CCA~kG C CAGAinGG 489 tGAAAAU U .AUA 490 GAAAUU A MaahAU 492 AAUW A GUAAUU 495 UAGU A AVUMQA 267 Table 32: RSY-(1B) EHRibozyme Sequence HE RiLbazyme sequaence Posi tion AUUGXTU CUGAUGAGCAAGCA ArUMCC 14 CUGAAtUU CUG-VXrAGGCC AAGCC

A~UULULJU

18 UUGG-t7G CcXAGC rr

A=G.AUU

19 UWrGCU CUGAUGACGCCQCGAA~;-cAA AAfLrmoA 54 GGOGUG~ COXfGA cC Ci *ACrU 57 0 G=U CMGAGGaGGC AA Aflt.tJCA 77 UUCDG CUGA GCCQ Q AUC~UM~ 94 AAGtJGAC CUAMGC~UGCA ACGj-j 97 CUCAAGU UMMG=AAGCA

ACA=G

101 ~CUC CMM

AGMAC;L

110 AUGUUMM COAGGCCAG-CA AxUGMMc 118 GMCJ CJA UGG~aAGOCA

AU-

122 CDCUGGEI CMUAC

AGMAMJ

*134 Gt3G~TMU CUAGGGG.GO A AX3GUC 37UGUG=G COGAVGTAhCcr.-1 e AUGA=~r 148 GtUfLAfA

CUGAGCCQGC;AAUUV

149 AGtUXMu CUAMG=AAGCA AAIUU=r 150 AAaM CDM~AG GCCG WC-AA AAAIuu= *.a152 UCA.AGUACOAGCAGCGA

AUAAAUU

a154 UUAGCGUAGCAX4-GAA

MMAAA

***157 1IUUUAUC COGALXAGOCC XGCCA AGtWMj 161 CAflGA~UU CMA~XnAGCcQUGG t CGA

AUAAU

176 UCfUCAu CUGAUAGGCCGQ G

AUCCUC

188 UUUCAUC CUG~AAGC CGAAAMCCGA

AGUUU=

209 GGAAfl= CWUGAWGccr AA G .210 AGGAAtUG CU~lA~~AAWU= 214 GACAG CVAUGGGCCMQ GCG AfGIAA 215 UGACCAG I-UAWAG C,-CA

AAUGUAA

221 CAX~hGUU aUGAACG hWCMA~CAc 226 CAfUUCA CUG~rWGJ

AGLMG.AC

239 t3GU aMUAGCX~~A AGUUrUCA 241 UUrJGUGU Ct30 XG AA=C~C=QA AtUtJU 242 CUUUGUG alDACGAAGCA AAUkGU 251 VGCMUC CIGAUGAGGCCGAAAGCCr.AA

ACUUMUG

261 UtCAUAU CUIGA CAXGcCG AGOG= C~ 265 UUUUUUA CtX11WGAGGCCGAAAGGC=A AMU=Ar 267 MVUU CVArG3CAAG=n AtUhWturj 274 UUCAGTJA CUArACCGACGC

AUUUUUU

276 tIOUCAG CtUG GCCGA9--GA

AMUAU

0 C C C. C C C C. C 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 4z-1 412 421 423 424 432 434 446 448 454 458 460 463 467 470 489 490 492 495 268 UGUG CUGATAGGCCGAaGCCMA

AUCAG

AG=GCA CMUGAGGCGXGG=GaA WtUOUu AUMA OGAUUCMAGCGAV CA=G C~~AG=AAGC;

AAMM

GA~MGMGAMGCCG&aGCcZ'; WGaAA AUGGCAA axukC=XGCXA

ADGGMA~

UAwm cuvcc o ADEx= GAfUuGAtU aMUGGCAGcc C CA A&UAM'r CAUGAUrJ CUAA=M6C--A

MM

cc,.UCA CMAMGCAGGCCGAA

AUMA

UUCMAG CT.AMWG1GAX;GryG E..cA

CXAUC

AUUCrIAA CUAMG="GCM AAcC= GAUCM CUAAGcMUGCA AGAA=c

UGCAIUUCMAAG-G&GCGA-Z

~aG=CUGW cAGCCMAG=X

AUGCCAA

GrkGGCL UGAGGXAGC;

AMC

AMGAG ~COGMGGCLa-G; AMlu AUM~a lGGAMWC=CrA AGAG-r, UMMUCcGAAaCMZC=A

AGA

AtCUUGLCGW aA C

MU

AGC= CMUAGCMA~-

AUMW

GQXtWUGA CMUGCCGWCC

AUM

ATMGAW CMGC=cr

AUGA~

AUGGU UAUA 4 XUGGGAAGU U A M A A U UA M C A A W A U M G GE; cc,%GAcGAA~CCG

AUU

UGUUG CUGADMGArCCGAA~ AAflUUT UGUGUU AAAfUM

OUGUGUG

AUGUUM ~a AAU GIUUCG UC c~ AUAxG= AUGCXUA

F=A=="CGM~AU

uaAUGCA

AGA=MU

UGGAGGA CMWWo,

AGUUAM

UACZG CCMccM GACMM GGCCGAA)

AGM

UCGAC U At CCAU=CU AaMVACW4= UGA..U AUUUUcM AUOA=CUAVGGGrGAAC-CG AAc AAMAMC =U~AGGCC AMAUjj UUGAA"t CU ==MWC

ACUA

CC

CC

C

CC..

CCC...

269 Table 33: RSV (IC) *Htarget Sequence ut Peasiti±on Target SequenIce Posilion Tax-Gret Sequence ft...

ft.

ft ft ft.

ft...

ft...

ft...

ft ft ft. ft ft...

ft ft ft ft ft ft. ft ft ftft.

ft ft ft...

ft ft.

ft. ft ft ft ft.

*ftftftftft 16 2.7 21 31 32 36 37 38.

42 46 50 51 67 68 71 76 81 87 88 92 93 100 10.1 104 105 120 125 128 129 135 143 145 151 155 156 i59 163 164 GGCAAAU; A AGAA=U TIAAGAAU U UGAML;.G AAGAATJU U GaAkAWu AUUUG;LU A AGUACCIL GAUXhAGU A CCxamO tACCACU U AAAfUt= ACCALt. A AAUUt7A CUALAU U UAACtTCC UWUU U AACUCC tMhLU= A ACUCrL TuuaACUJ C CCUu CCUUGGU U AGAGMG CUUGG--UU A GAGAUGG- CA~C-AAU U CAUQGAG AGCAAUrI C AUUGAG- AA.UUCA~U U GAGUmy= AUGAG A tJGAXUAA GWUX3GAU A AAAGUtM LMhAAAGU U AGAvtax AAAAGOEJ A GAUMLtCA GOAA U ACAAAALT UMGAWU A CAAAAU ACAAAAJJ U UGUOUGA CAAAAXJU U GUUUlAC AAUOUG U VGACAAU AUUUGUU U GACAAUG ALIGAAGEJ A GCA.UU= GUACAU u GUMAAA GCAt3UGU U AAAAATM, CWUTGUU A AkaA, MAAAAAU A ACADCCU ACA.UGCU A UACUGAU AUGCthU A CUGAM UkCt3GAUt A AAUUAAU GAUAAAU U AAUAC-AU AXYAAALIU A ADACAUIE AAUUAAUT A CAUUUAA AAUACAJU U tjAACrUM AUACAUU U AACtAAC 16m i69 175 2.76 '181 1.92 i96 201 206 216 221 222 231 2 32 234 235 241 247 249 250 256 259 262 265 267 270 273 278 283 284 285 300 303 316 317 319 321 338 339 346 UACAUU A ACW.Ac.

UUM;hCUj A AGU UAC~Cj U UGr,--,A AACG,-cr j UGGcUAAG UM-G~C,- A AGCAG C-AGMUa A CAU~cA GAIUhCAT. A CAAJC-AA ALIrAAU C AAAILTMc AUCAAAU UJ GXu.Crj; AUGGCAU U GUGU=U AUUMJGUt U UGUCAU UUGUGJr U GtGCUG UGCAtjGJ U AUMAc GCALU' A U~LCXAG ALUUEAZ U ACAAGUTA UGuLmur Ak CAAGLV4 UaCAAGtJ A GUGAAU UAGurGAU AL UtJC~CCC G--GAt7AU U UGCCCU-rA LUAUU U GCCC--;AA UUGCC.CU A ALrAA.

CCCMIJ -A A~thALAU MUAU A AIT=~lG MAMAU A t2UGUAGIJ AUXAMxU U GtUGMA AAUUICCrj A GUAAAU UUGr.McrJ A AAAUCCA GUAAU C CAAU~tc ATUCCAAU Uj UCAC;AC UCCAAUrj U- cAcxkm CCAAUrJrj C ACAACA LCCAGTJ A CZACAAA.

CAGMThCr A CAAAAUG UG C-rj U AUAUA GGAGGM~ A UT.MakU AGGUUAU AL UAUGGGA~ GLMUj A UGGGAAA AUGGAAU U AACA.CAU Z:G-AATU AL ACACAUU AACACAUZ U GCt3Cc

U

a a a S a.

U

271 Table 34: RSV (10) HRE Ribozyme Sequence

S

0000 0* *ee m.t.

16 17 2i 31 32 36 37 38 42 46 so 51 67 68 72.

81 87 88 92 93 100 101 104 105 120 .125 128 1.29 135 143 145 1=1~ 155 156 i59 1563 2.54 1 65 H Ribcoym. Sequence AAAUUCLT CUGAGCGCACCCIAA

ADUC-C

cuUaUCA ccGc-Ac;-=A~Cm,

AUUCUU

ACUUAUC CMtUMCZ AAvUCMU CUGXW-CC-UGC.a

;LC%

UM.AGUGG CMAMGCCM6GG- ACtEJAL'C ULM=AU Cr-A~k.-CAG-

AGUGGM

GGAGUm MMI~.CGA AflthA.

GGGAGUU CMUA CCGAGCCAA AAfUUAAL AGGGAGU C=U-CCCG~UaM

AAAU=U

AccAAGG CUAGGC- AGO~kA U0rMA=C=U c cCCGAAG

AGGGAGU

CAMCU CMA-GCtYAOC-A

AC=

CC~UX7J CMUGAG-WAGGC .A AAcCAG ACOcAA CCAMG-CGAGCA AAUUWci CAM=~' CW-VrGAI. G ~CMW AMLAM UUCA CUAGGCCXAG-M ACUCAAv akAcuUU CCAGCMGCGAaL

AUM

GUAAULJ CUG GGCCG~zC--, ACUUAu U~~CUGAAAAUCX=CQA

AAUU

AM"tUUcU CUGAMAr--CC AG-CG AucaAAC AAUUU'UG CUIMG-GAAGA

ACA

UCAAACA, CUGGflGAG--CMAA CCX

AUUU=~

GUCAANC COA GCrGAAAGGCC;L AAUUUUr.

AtUUGUCA ~CGAU~--CAACCG ACXAAfU CADUG U AGX GAGGCA

AAMAAU

ACAATJUGC COAGGCGU4CGAACM UUUtkAC CUACV"'WAAGCA ATX3GMc~ U~flUUrU CUAGCCGAACCC ACAAzJGC LUaUUwUGAVGA-GAAGCC

AACAATJG

AG-CAUM UGAWG GAACC AMMUuM AMUCA COAI=CAkr-G; ACC~UMr UWLJCAG CUGAtXrAGGCCGAAGCCA AMGc= AUMLhAUU CGUAGCAACCA

ACAG

AUMUCUMGcAWC AtDUUA13C AAt2G=A CUGAflGAGCc~uMcc AAUxUAcyi TUAAAIM CUAGGCGUGCAX AU n umJAUU CUG=AG--C ACCAA AUGMzW& CULtIA= CUGAt GAG=CGAA .CGAA

AA~UG.U

CGUULAGU CrMGAt~AGcC GCGCCGL AAurVGM 272 169 AAAG=G CUGAUGAG~CCGXUAGGCCGAA AG~kAA 175 U~CAG=CGUA--CAA-,CA

C-=

176 COCAGC CUGAUAGGCCGA~r-CCAA

AACCU

181 ACCGC=r CTGAVGAGGCc ACCGAA

AGCCAAA

192 UtUUAUG CtOGJWGAGGCCGAGCrA U 196 LMMUUG CUGAaAGCGAAG=LccG., AUGOMtC 201

UCAA

2 fl= COGUG CCCAGC~-CGA

AUG

206 GCCAX3UC CUAGAG

AUU~UG

216 CAAACAC CUAGGCGAAOCA AflG-CAU 221 AUCACA COGAUGA=GGr~ac ,-CGAA AcCAAUr 222 CAGA CUGVGCrhf-i-CCGA

AACACAA

231 UUGCmAuL CUAMC--GAAOC- ACAX3GC;, 232 CMUUA COAGGCGAr---A

AAAU-

234 UACM=G CUGAGAGCCGcQGGCGA AtkAC;U 235 CtUhCUUG CtUGAGAGGCCGAAGGA

AAIMACA.

241 AMUXAC cu~GAGccAAGc-G AcuuG.am 247 GGGCAAA CUG~rGGAAA G CGA AUCALtm.

249 UAGGGCA COUGGG~CCGAAGC

AUWCA

250 U~GGGC CUGAtXGAGCCGc GCC AMvA :.256 UtAMn~ CUCAM-AGGCCM VZCCGAA

AGGA

259 AXUMLY CEX AGCCGckGGCGA AVrMC,- 262 ACAAMlU CUGGflGAGGCCGXU-.CGALA AUMU=rJ 265 AcaAA CUGAXGA4CCGAAAGCCGAA

AUAM

267 tutkcramc CUGAMAGGCGaGGCAA AtUXIhU 270 AUt3~kC CcGwDGGCCGXGCC

ACAAUAU

273 UGGAJUUr CUGAtJGAGCCM AG

ACMICA

*278 GAAAUEI C UGAGC-AGGCC G AA AtUUUAC 283 GULMUGA CUG~AGCCG GCGAA AUUGGAty *284 CGUGC

AAUUGGA

285 ULUGUUGU c CGAG!- AAGGCCG AA~G AAAIUMG 300 UUUGUAG CUGAtGAGGCCGUAGC~CcA

ACUGGCAL

316 CAXUhMu CtLrAUMW4GXUGCCGA ACCU7~~ CCA 317 CCAX3At CGc~~GAcc AACCUCC^ 319 VCCCAUA CMXAM-GGC~CGAAG C; AaLA=c ~321 Uuu C~ C Uit GG;A"I--A 338 AUGUrJMUGG-CGAG

ALMMCC

339 AUG= CGAUGAGCG AA=CAA AAMXUCA 346 VGAGAGC CUGAGA G-CCA-WCGAA, AUGU~vrj 350 AGGUUGA CUGAt7GAnCGXUGOCC

AGCAAUG

352 umG=U Ct3AUG AGGCCGA

AGAGCAA

358 AGACCAU CUGAt GQ AAGGCA

AGUG

364 CC AGML COUGAAGCCGAAG

ACCAUU=

366 CAUCM~G CUG-AUGAGGC AACC

AGACCAU

369 UGUCpAnC CUGAMG=CGcc -CGAA~ AGMkGA~C 379 AXUUUCAC CUGAUGAGcCCGAAJ C- AUtzUCA 387 AGAAU=t CUGAIGA-CCU GCGL

AUUCAC

388 GAGAAJ CUGAI

.CGCCGAACCCAA=C

392 UUUC-GAG COUAGrCCGAAGCGAA AUUUAAu- 273 393 395 405 412 413 427 428 430 436 440 441 443 449 450 453 458 459 463 465 466 469 473 477 478 480 483 484 487 489 494 502.

507 511 519 520 523 524 UUGGA CUMGIAGGCCCGka.Cr,-CGAA

AAVUUA

UUUOUUG CMJ-GCGAACCA

AGAM=U

AWCACE7 VGUaGCMGC~CGM

AMGUU

AVUGUUG CGW4Ck=C;Aflc~c=U CUGUU Gc A) AAUCACt7 0 MU~A CUGAtXGAaCCCGAAAGC

AUC

ADUCADA CUAG= cAAU UCAUUCA CMVAGCGGCCr-A

A~W

GAMLWU CUAMGCOUCGMC-

A~

UCk~r- CUGAUMWZCCGAA C-CGA AtUW=l UCCAAGU CGMGCMAGCA

UC-

AUCCAAG

UAGGWAOCAAA=

CAAUCGM GCAaa'M AGtIA;Dj AAGAUCA ~CVDaCCCXUGCG

AUCCAA

UMAUC COGAUMAG AAGCCA

AAU=;A

GG MMAflLAGGXAAACXA AWGMW C OMAGMAGG~rA

AG=

MWMAU CMUWCMMGCM AAGWflC AUUMWG GG-cGAA.

AUMLAGA

UALD= CCAMGGCGAAGGCGAAAlGMIUU M~UUW UG M--GAAGC- AUUUMnG tUrAUM CUMVAGCCMUAGGCGAA AAAfUUUA aUAAU CtJVGAICGAAGC~r.A AMtJUU GtAUU CUVAGCGGCCGAA

AXUCAA

GUAUc CUGA--mvwc AGua AtUUGAG~

AGUA

AUUAA CMAW=GAA~

ALGUGC

UMRAC COGAUMGCUMCA

AAUGM

UMMAU CVGAGAW4akAGGCCGAA

ACMAM

UOAMUW CM~AGCGAGCA

AACUAU

*woo a.

0 274 Table 35: RSV HE Target Sequence nt.

Position 4 V V V *V*V

V.

VVV

V.

VV

32 37 60 65 66 70 73 82 89 108 Ili 113 117 120 123 126 127 146 150 154 155 166 167 159 170 173 186 i89 192 196 197 205 206 209 213 E Target Sequence GGCAAAU A CAAAGAXI GAflGGCU C ULMZCAA UGGCUC= V AGrCAAAG GGC-UCU A GCAAAGU GCAAAGU C AAGMCA GUCAAGU U GAAUMGA CNAUU A CACUCAA ALACACU C AACAAAG CAAAGAU C AACt3UCU AZCAACL U CUG=CAU UCAACE7U C UGICAnJC CDUC=u C AUXCAGC CUGUM.U C CAGCAAA AGCAAAUJ A CACCA.C ACACCAU C CAACGGA AGGAGAU A GLUWUGA AGAM=hG A UUGAIMC AU U GAMCC amhuGY A CaCC-.UA UGAmkCU C CtLA t MCUCC A AUMUfG;L TJCCUAAU U AUGA=G CC MA~U A UGAV=U AACACAU C AAMAW~ CAUCMAU A AGOUM AAMAG U ALXGUGGC AMLAAu A UGUG=C GGCAUGtj U AuUAAI7c GCIAflUU A UMLVJCA AUGUMLU U AAIJCACA DGUMAMr A JACACAG tUhltMAU C ACAGAAG AGAUGCtJ A AUMA -MCAU7 C AUAAAU UAUCAUI A AAUCAC CAMLAAAU U CAC3GG AMAA=U C ACUC-GGU ACUGGGU U AAXUhGGU Ct7GGGUE A At7AGGLTA G,.tIUAAU A GGthAUGU AAUAGGE A UGUUAIjA position 217 218 220 229 231 235 236 254 260 263 277 279 284 299 305 315 318 326 327 346 347 355 356 361 370 371 383 384 389 395 401 406 408 415 418 431 449 453 460 472 474 HE Target sequence GGUTGU U aUn-G GUAU.GUW AL ML-c-,A AUGUtmW A L-rCG-.tJ GC-AUVu CtG tm G AU MC U a a;c L'CUAGG-U a AGCGkAA Ct3AGGU A G-GAAG ACACCAU A AAAAIjAC UAAAAAU A CUC.rAGAG AAAMCrJ C AC-AGAtx G-CGG A UCAUGt~.

GC-GAUAU C AflGUAA AUC=WG A AAAGMA Atr-GGAGU A GAUGUAA tJAGAUGU A ACAACA AAcCAU C G-,rAA ACALUCGU C A.GACAI AAGACU TJ UAArGGAA AGAC.AU A ALM.C.ALA ALUGAAAU U UGAAGUG LrGAAAUU U GAAGU GAAGUGU U AAC.A= AAUU A ACPIUo= UUAACAU U GC-C.AZGC GCAAGC-U U AACAACU CAAkG=t A ACAACL-G CUGAAAU U CAAA UGAAIU C AAAUCAA TUCAAUAU C AACA=U UCAACAU U GAGAGA ULGAGAU A GAAUCtIA AUAGAAU C tLh.GAAA AGAALTCU A GAAAUC AGA'AAAU C CUACAAA AAAUC=t A CAA AAAM3Gct A AAAj2A.AA GAGAGGU A GCEXCCAG GGtJAGCU C CAGAAUA CCA6AAU A CAGGCAU CAUGACU C UJCCUGAU UGACUCEJ C CUGAUUrG V V

V

V

VO

V V

V

4., 0 0 S:4 -480 491 494 496 497 501 503 518 522 526 527 544 549 551 552 563 564 573 576 581 584 603 604 613 614 617 629 640 641 643 652 653 663 670 671 672 674 680 681 682 683 686 687 690 691 692 UCCUGAU Uj GUGGGAU CG-fGAU A Ar~MUX UG~tA A UtMIUA AIUA4U U AI3GMtM UAAUU A TG1MG AfltUU A UIGCAGC UMafG=~ A GCA.G=A GCAGCAU U AG~A CAGcAJUu A GMJ=~ C3JAU= A AU 4

ACM~

tMGkA A ACMAAXY AAU A AADUGC ACL;AU U AGCAGCX CUAAADU A G-ACAG GACAGA C uGLuU AUCUGGU C UMWAGC LMGUCU U ACAGCCG UC-GUC= A CAGCMU CCGOMAU U AGGAGAG C=MkDU A GGAGAGC GAGAGCU A AUAWGTJ AGCaAAU A AUGUCCU AUAAGU C LMAAAA AUGUCCU A AAAAALir, GAAA;L u AcAAAGG AAACGOU A CAAAGGC 275 696 698 706 708 709 711 726 731 740 741 742 743 751 754 755 756 766 787 788 800 802 803 811 815 816 822 824 825 829 830 840 866 869 875 876 87 883 895 913 914 916 921 923 925 943 946 947 949 950 UUUUCG-a A UAGCACA, UUG~TmU A GCACAU GCACAAU C UUCUACC ACAAUCU U CUACCA CAAcU= C MVACCA At7CUCTJ A CCAGAGG- UGGCAGU A GAGUrJiah GMWGAGU U GAAGG AAGGGAU U UUUGCAG AGGGAW3 U UUGCACG,- GGGAM-Uu U UGCAGGA GGAflDUU u GCAC,3AU GCAGGA U Gt7UCThA GGAUUG U UJAUGAAU GA.UU U AUGAAUG AtJUGMJU A UGAAUJGC AAUGCCU A UGGUGCA GUGAUTGU U ACGG=Jc UGATU= A CGU=GG GGGGAGU c uaGCAA 1 GGAGUCU U AGCAAAA~ GAGUCU A GCAAAAU GCAAAAU C AGUUAA AAUCkGU U AAAAAtjA AT3CAGUU A AAAXflAU !ThAAAAIJ A UUUG AAAAUAU U AtLGUUAG AAATJAUU A t3GUUAGG AutaUAr U AGGACAU UM.JUGW A GGACAL-G ACAUGCU A GtUGUGCA AACAAGU U GVUGAGG AAGUrJGU U GAGGUuu UUGAGG UJ MkhUGAAUJ UGAG=U U AMAAX3A GAGGUUU A UGAAL7AU LUUAAU A UGCCCAA CAAAAAU UJ GGGUTG,-U GCAGGAU UJ CUTACCA CAGGAriX C TUACCAIIA GGAUUCE A CCAtU.tJ CUACCAU A LT;LUGAA ACCAMJU A UUGAACA CAUAtJAT U GAACAAC AAAG-CAU C AUUA~h.UU GCAUCAU U AL7UtWCE CAIJCATjU A UUX=CtL UCAUEAU UJ AUCUDUG CAtJUATJU A UCEJUUGA AAAZ=C V AAGGCU A GCUUACU A AGGACAU A AACAGCU

U

ACAGCU C AGCUUCU A GAAGtU U

ACWLCCC

CLCCCA

CCCAAGG

GCCAACA.

UAUGAAG

UGAAGtX3G

UGAAA

SS

S S 4 5 AAAACAU c iccCA=U CCCO=C Ui UAMAU CCCACU U AUAGAUG CCACUUU A UA=~E ACUUUWr A GAJUGUUU MkGADCU U- UOUGUC AGAUGOU U UUGUMC AUGUUU U GUUCAU UUUUWXU U CATUUT= uuuucUu C AUUWUG UGUUCAU U TUGCGUX GUUCAUUrJ U UG=rJA UUCAUUU U GGLMhUAG 276 .952 UCtOh.U C UUUGA=C 954 AUC= U UACUCA 955 u UCEu U cAcucA;x 960 UUUGACU C AAtRMU= 964 ACUCAPAD U tTCCU=~ 965 CUCAAflU U CCUCACU 966 UCAflU C CUCAC= 969 AlXuUcc C ACCUCtIC 973 CCUCACU U CUCCW 974 Ct3CA=U C UCCAGr 976 CACM=C c cAGurA 983 CCAGUGU A GMU3EG 986 GUGThGU A UL~kGGCA 988 GT~G= U AGGMWA 989 MGU=fU A GGCAAG 1007 CUGCC A GGCAMA 1013 tUhGGMU A AUCGG Gos1024 GGAAU A CGAGGU 1032 CAG A CACA *.*1044 GAZGGAAD C A :to* uu 1052 AAAJU A MU~r S* *1054 GAflCUNU A UAUGMA 1072 AAGGAU A UGUM i085 AACAACET C AAAAA 1103 GUGAG;U U AAMC S:1104 UGU A ACkA :108 AUXAACAT A CAUG 1115 ACAGUGU A C~aWcu U118 GGUACLTA GACUlA 0**!=113 CUAGACU U GACakCm to .1.39, AAu A GAGGcE= Soto ,1146 AZAGGU A UCAA; 1148 AGGCku c AAAcAuc U155 CAAACAU C AGCVL17A Sos U~ 60 AUCAGM~ u A~flcCAA 1161 t3CAGUU A ACCM *a1164 GCUtAAU c cAAAGA 1173 AC-u A AGA~uu 1181 AGA~UG A GAGCUUU 11.87 MkGACU U UGGU 1188 AGAGCUU U GAUOA 1193 UUUGAGU U AAMk U194 tUGAUL A AXkAAAA 277 Table 36: RSV H[H Ribozyme Sequence at.

Po sition RE Ribezyme Sequzence

S.

*5

S

S.

S S

S.

9 21 23 24 32 37 s0 66 73 82 89 108 111 117 120 123 126 127 146 150 154 i55 166 167 169 170 173 186 i89 192 196 197 205 206 209 213 AUULA CUGAUGAGGCCG-AAAGGCcG-AA AU,-tJGC FUuu -tmU CUGAGAG=GcAAAGC--.A ACCK-rA CUUMC aMWAGCCGAAAW,-CCA

AGAGCC

UCAACUU CU AawccAcG-,; ALCrUW AtCAUUC CUGAMAGGCACCG--a

ACUL'GAC

UCGAGU CUGAUGAGGCCQAAAGCC,- AncAM-C C.UOUGUU =MAGAGAGCCGAAG -CA AG-wMMU AGAAGUt7 C~AG~C, AAGCA CMUGGCCGUAA-c-=

AGUUGAU

GAr3GACA CUADAGCCAGAA~

AAGUOGM

GCr3GGAU CMUMWGCCXUGGC;L

ACAAA

UUUG-=CUGAUGAGGCMUAG--CGA;

-AUGACAG

GAUGGUJG CA GGCCA=WA~

AUU=GU

UCCGUU C=G--CXG--CA UCAACAC CUGAVGAGGCCcGAXAG=CCGX2L

AUCDCCU

GaWJCAA =MUAG

ACUAJJC'

GAGUAUC CUGMAGCCGAAAC1 CG Aaca UrkGGAGa UAGCGU,--CA

AUCZAIA

t3AADUAG CUG AGGCCGXU G~ AG~gUCA UcAMTAU CUGA~tGCCQXXG~J=

AGA

ACAUCAU CUGAUGAGGAAAGCGAA

AUUAS

CACAUCkL CUGALGAGGC-C GArCGA; AiAiUAo ACUCJTU CUAGAX

AGU

CALVA= C UAGCCGUGC AfltGAUG GCCACAU CUGWAVGGrCC

ACW

UGCC&CA~ aMUM CGAGr.-,-C AZUMLTy GAUUAAUT CUGAtMXrGGC CG ACM=iC UGAUUAA aMUAGCAAGCG;

AACAUGC

t3GUGAUU CUGJ GAGGCCGAAACCGA AMlACAU CUGUGAU COGAVGAGG,-CXGL-.X AAM C LMUCOU CUAAGCAG

AVMW-TA

UtMUGU CUGAUGAGGC-CGAA CCA AGCAUCrJ AAULUAU CUGAI GCG~AAAGCCAA

AIUWCA

GUGxAAUU CTG~zG;CAACCCA AI~rU CCCAGTJG CUGAUGAGGCCAAAG-CUG ALtJUAUG ACCCAGT CUr-AUGAGCGAAGCGA-A

AAUUUAU

ACCUAUU CU CA ACCCAGU tUACCUAU CtJGATMAGrCGAAGGCGA)A

AACCCAG

ACA' *C Ct7GAMlAGGCCWAAG--CAA

AUULACC

tMhUMACA CUGAUrC,-GAcAAG,-CG

A.CCUATU

278 217 CGCAfLA cxtAccAAGGA

CAUACC

218 VCOCAM

AAMC

220

CAUCGCA

229 GACCACUGGWA GCC L t aQP~Q 231 CC~kACC COUGAGCMAGGC GQAj AGACAjUC 235 UCUU CUGAVGWrCGAGGCcC A AccaAGA 236 CtUL"cc

A.ACM

254 GWAWAUMUWAAGGCGAGcA AI7GG= 260 CUkU~ tvirrj 277 UcACM GW 279 UUUACyCUCIXC 284 UOU~C3M~~~~~ 299 UhCMM==UGcCA cucwxUcc~ 305 GUUGUA aMGAMAGGcCCGAAW-CJ AC CA 315 0U=GA COGWAGGCCrAAAGX,,

AU

326 CV mmGcw~cA 327 AUGU=u~ 346 cmuc' CvAWAtX A AGC AUU 347AC;L~C= CU~kUGAGGX LfCG~AJA AAflUUCA 355 CXADU= CMUMWGCXtAcAeWr AC1UC 356 aWCM=G tto361

GC"UU-C

0, t. 370 CUGWlnAGGCCMAGCG

AGU

30AGUO=am~cUuACUG ~371

CAGOUUAG=G

83 UAU=c=OQWM=~ccAMGWCCGAA

AGO

383 ul=C=XAGCA MM 389 CAUGUUCMMMGCMMAG AGCMUUM CUU AUUGcA 4 MGAUu Cr3A=zx A A AUUC, 395 ADUU= GA~a 418 ucuumAUCUcAA ***431 ~UUC

AGAU

449 UGGAC UM~GXWcAAArWt.CG, AI 453 UADUCDGm 460 AUG--= UGWG~

A=CC

42ALJLM~~a G cAA AUUCUGG 474 CAAtUCAG CGA AGUAG 480 AUCCCAC C 3 tAGrr?1 9 GAA UCA 491 ALAU AMGGCCAW-

AUCAGGA

494 UaCAtJAA CU~G~AfltAhjc 496 UtM=~CGX Cr~l~

AUMVA

497 CMMCA Awaw~J 501 GC0 AW Cc CGA AAI 503 AGCU= COAUAGCCGAAGCC AhCAUXU 51i UAMt~ CUGAUGAG AGGCCW AUGCurC 279 512 UMUXW CUGUAGCAAGC

AAGCUG

518 LUA

CUG

3 AUGAGGCCG~a.GGCCG.AA ACM)LflG 522 AflUUGU CUGAXGCCaAj AUt~kCM~ 522GCMLWAU COAA--GAGCCA AGUXJWr 526 utdcuccu CUGUGflAGOCGc CGAA AZUaAGtJ 527 CtOGL CUG A=CGAGGCG; AAtUrJA 544 AAGACCX. CtIGWtWGCCMVGG~r AUCUMtC 549 GCOGMA. Ct3AGAGCC~kAGGGAA ACCAAaU 551 CGGCUGU CUGAOGCGAAGG CCGAA

AMCAG

552 ACGcG C.A Pzmcc

A-C;

563 C; U;.CC CLXALGAGCMcAGGCG

AUCAC=

564 GCUCUCC CVAGA=GXGCCA AAI3CACG 573 AO~UtXW COGAUGWGCC-"VCCA AGCUCtJC 576 XGAM CUGAMAC---GXMGGCC AUtUhGCt 581 UUU=U~ CUGA~G G-ACCA

ACAULIAU

584 CAIUUUEJ' CUGzAVAGCXN-r

AGGC;L

603 CCUUUGU CDQAXJGAG-CCQ 'J ACGUMr 604 GCCUU= CDGAXXA(GC AAG-C AACMorx 613 GGGUAkGU CtxGA'xAI. cc AGCCLUu *614 UGGtGG COWWC~XXCA AAGCCou 617 CCUO=G CVMGMGC2C 7 e AGt~hAGC 629 UGOUGGC- CUXGAXAGXGACC CGA AUGUCCrj :9640 UtICAflAG CO GAJ3GGCC U CGAA. AGCUG=r 641 CUUCA COMU=W GCCGQ 'G"GAA AAGCoUG 643 CACOUC UMVAGM&== GAC 9652 UUOUUMc CV=WGMA GCA

ACACE)UC

653 Guouurjc WMG=AkGCA CCU 663 AAGL7GcCO7 uGc-c AUGUUrJrj 670 AUCW= CM, MGCAA-,c

GG

~671 CU CAT UAGC.AGGCA ATo 672 ~L3~A AAGtG 674 AAACAUC CMUAGGCWAAy AWAKA-r 680 GAAAA CM kGGGCCGAAAGGCcA AcAAcrj 681 UGAACAA CGXAGCrzkA AMCU 9 9682 AACALTcA AUGAM CUAMXXM-"G-ZC-AAAAACAUC 683 AAUGMAC G G c~

AAC

686 CAAAAxUG

AAACI

690

AUACCAA

691 UM=AUGAACA 692 ctIAc CU G

AADAAC~

696 UGVG=U CCGAA

AUAA

698 AL3UGLUGC CtGAUGAGGCC 2 .A~rCCC,

AXJACC

68 ~7 G C G c "C AA AULtJGtC 708CtJGZt COUVCGt AGc GCCMAA

AGAU=T

709 UCUGGUA CGAGCMArCCGA AAG;=Gr 711 CCUCUGG CUGA-GCCAAC ,-CCCA

AGA,

726 ucAACEc -CrAIGC GAAAGGCGM

ACUGCA

731 t7TCCCUTJC CtGAAGr-CW W, ACUCEuA 280 740 CUCIAM CUGAAGGCCuIGGC A AUCU 741 CCUGCAA CUGAUMGGCCG AC-Mh AAtMCc 742 VCCUGCX, COUGAGGCCGAAG WA AAAflCC- 743 AUCCUGC CUGAVMAGCCQ

AAAAVCC

751 CAtUhAAC CtGAXGGAXGGCGA

AUCCUW

754 AUUCAUA CVGAtZA GCCGAAGC AZCAuCC 755 CWfUCAU COAMGCCAAGCA

AAC.WC

756 GC~nAU CU AAGAAAGCG

AACA

766 UGCA=CCA ~r-MGCT CCGA AGX1rLTUr 787 CCACCGt3 CUGAXGAW--GCCQ GC,-A ACAUCc 788 CCCACCG COGAIflAGCCCAAGA AAMCAr~ 800 UO.CCE]A COAGGCGAGCCA

AC=

802 LUCUGCU COUGAGGAGGccL

AGX-U

803 AcUUCGC CMUGA=GAAAGC A AAG&,-_,C 811 UUr.Thcu CUGAtr---GGaAAAGGccGA.L

AUUUWC

815 LMUUUUU COG AMGCc C ACUWaU 816 AMU=a COGAGGCCGAGG

AACUG.AD-

822 AACAMA CUGAVGaGGCc MUUr= 824 Cavw CUMrCGAX AAA AMflUM 825 CCM.ACA CUAUatxAGCC

AMU

829 AUGUCCU COGAUMAGI

ACAAA

830 CAGUC CUGATJGCMAAA rGCrA

AACAUAA

840 WICACAC CtX GAGCkAGcc GCCG AGC~A=,Tj *866 CCUMAAC CUGAIUGA CCAAGGrA Aax~j~u 869 .AAACCCC COUAGAAGCCG~AOC ACU=r 875 AlUCAM. CUIGGCGXG-CA

ACUA

876 MUD=~c COUGA GCCMZAGC AACoCU 877 AifUCAM CUGAUGAG~CCC--A CC

AAACCEUC

*883 UOGGGCA CUGATJGAGGCCGAA e-fCGA AflUCAn 895 ACCC A CUAGA~r-uc;-cAA AUt3000 *913 AMM~tAG C1Ui rCC CGAA At3CCUGC 914 U~AWUGG COUGAGGCGAAC,=A AAcc .921 uU M~~l CMAGAGGACCG AGC=

AUGGA

923 Juk;AA CtXAXMAG~CXAAGOCG

AUAU

925 GOUGUIC; Ct7GAUMGGUA~c G XC M XU= 943 UUMAAU CVGAUxG CGGac

AVC

:*946 AGAUhAU CUAGGCGAG=A

AUAG

947 AAGAUAA Ct3GAUMAGCc AGCC AAUGra= 94 CAAAUC-A .AGGCAACM

AMMMGA

950 UCAAAGA CUMAGGGCCGc AG XA AAXUh.AU 952 AGUCAAA CUGMtXAGGCGXG~rA

AML

954 UGAUCX CL7G-XUGAGCC AAGC AGAXMhA 955 UUGAGUC CMUAGCMUGCCA

AAGAUAA

960 GGAAAUU CUGAUGAJ CCGXU=CGAA AGt7CAAA 964 GM3AGGA CMUAGCAAGCA AUrA 965 AGUGAGG Cr7GA.GAGCCU CCr-AA AAflUGAG.

966 AAGUGAG CtTGUGAGGCCGAAGCCA AAAtIUGA 969 GAGAAGt3 CUGAA~rG .WCCGAA

AGGAAAU

281 973 AUGGA CU-~ CGA Acc UM 976 MCACUG CUGD-CGA Ac '-CGrA AGAAGG of983 CtUAtC COGAOG rCCGAAGCCG

ACACOW

986 UGCCUAA CUGAUGA=CGcAJA=CG

ACUACAC

988 AUUGCCtI CUAGG=.AA C AUAcu3AC 989 CA.UUGCC CUAGAC---GAAGCCA AAUCnM 1007 UMUGcC COGA GGCCGXAGCGA AGCCr-A 101.3 CI3CCCAU CUGALGAWGCCGXAGGCCGAA A=CCr 1024 ACCraCu =GUG't ~GC--X G.CCGAA

ACL;COC

1032 CUCGS3o CUAUAGCC.-AAAGXGAA~

ACC

1044 AGWUU C GAAGCC--A=GAA AUtCICIUC 1050 UC~kUATUA CGAGGCCAG-L,, ACr-A 1052 CAUCAUA =MGA=GXU CGAA AGAUCIU 1054 UGCADGA

CUMA=GAAG-AALGU

1072 UUCAGCA C=UtGA=GCGAAAGGCCG AUG-Cct 1085 UUUCUoU CrGAUGAG-GAAAGCGA AG~tUUG 1104 CUGU= CUGAoWCCGA c AnACCA U* UA10 CACOG CUGAr oc A~uUu *11.5 AGCCG CUGAtGAGCCGAAGGM

ACACGU

1118 UJCAAGUC aUGACG-XU C=U AGMCA 2.*.23 GCCGUC CVGAtJGAGGCCGXAAGG-CGAA AGtCUuA 0 1146 UG0ULGA CUGi&U~GCC CG--AA AGMCCUUr 1148 GAUGUD UGAGGGC CA AGCCrJU 1155 tmA Cu AuMAIJ CC cc AUG=~j 11605 tUMLGAU CUuXGAClCA-AGG CCC AGUGM 1161.UUGA

AAGCUGA.

1164 U~tUUW UG A CAASCA A,-G CUUGCUGAUGAGrCCGAAAGC;CCGAA tC a.*1173 ACAUCAU CUGAUGACi ccCGAA ALCtjUuU 1181 AAGCUC CUAGGCGANCG;

ACAU=

1187 LhACOCA CUaUGaL,3

AGCC

118Ut~ACUC CUGXVGAGGCCGAA;CGAA AAGCUr t3UtTuU CUGnGAG c

ACOCAAA.

o. 1194 LUUUU~ Mr3AUGAG CCG AACUCA Tal 37: *8 HP .ioyeSusrt Sequence Table37: ISV 113) HP Ribozyubtae Sequence Position HPRbzm euneSubstrate IClGUGAUC AGAA GUCUUU ACCAGAhACAc!A J'JUUACAUCCWTJU AMAGACU GAU GAUChrhG 91 CAAGUGAC AGAA GUCUCA AcCAGAG;AICACACGUUG~ 1 ACAiJUACWMU IGAGACC GUUl GUCACUU 472 CAGGCtJCC AGAA GGACUA ACAhAA-rX UAGUCCA~MA~r".T GAUl GGAGCCUG Tnl 38: RS (N *I Rioy4Sus t Sequence Tnble38: Hai(Nrpin RloyncS borate SequenceSusrt Poitio 476 AUCCCACA AGAM GGAGAG ACAAAAAAGUG=A-UAO U CUcuCCU GAU UGGGU 540 MCGACCAG AGAA GUCCCC ACCAGAGAAACCACJfJJ1JcUACAUACCJQGUA GGGGAcA GAU CUGccUCUc 554 CIJAAUCAC AGAA GUAAGA ACAAAAAAQUUG~-UACGU UCUUACA GCC GUGAUUAG 636 UUCFAAGA AGAA GUUGGC ACC-AGAGAACJAcA1GUUGUGJUCcu U AcuG A GCCAAcA GCU UcuAUGAA 996 CCUAGGCC AGAA GCAUUG ACCAGAGAAAr-CACYIGJI~uACIW4JACCYUA CAAuGCIJ GCU GGCCUAGG 1156 UUGGAUUA AGAA GAUGUJU ACAAAAAA rnv-l"LMOUM AACAUCA GCU UAAUCM Li) 284 Table 39: Large-Scale Synthesis Sequence a.

S.

S

S.

S

S

*SSS

S*

A9T A9T

(GGU)

3

GGT

(GGU)

3

GGT

CeT

C

9

T

U

9

T

UST

A (36-mer) A (36-mer) A (36-mer) A (36-mer) A (36-mer) Activator [Added/Final] (mini) T [0.5010.33] S [0.25/0.17] T (0.50/0.33] S [0.25/0.17] T (0.50/0.33] S (0.25/0.17] T [0.50/0.33] S [0.25/0.17] T (0.50/0.33] S [0.25/0.17] S (0.50/0.24] S (0.50/0.1 a] S (0.50/0.18] Amidite (Added/Final] (m 1 110.02] 1/0.02] 1/0. 02] 1/0.02] (0.1/0.02] 1/0.02] 1/0.02] [0.1/0.02] [0.1/0.02] (0.1/0.02] (0.1/0.03] 1/0.05] 1/0.051 15 m 15m 15m 15sm Ism 15/15m 15/15 m 15/15 m 15/15 M Time* Full Length Product 89 78 81 97 21 38 *Where two coupling times are indicated the first refers to RINA coupling and the second to 2 -O0-methyl coupling. S 5 -S-Ethyltetrazole, T tetrazole activator. A is 5' -ucu ccA IJCU GAU GAG GCC GAA AGG

CCG

AAA Auc ccu where lowerecase represents 2 1 -O-methyinucleotides.

285 Table 40: Base Deprotection Sequence iBu(GGU) 4 iprP(GGU) 4 9* a a a a. a a a.

a a a Deprotect Jon Reagent NH4OHIEtOH

MA

AMA

MA

AMA

NH4OHIEtOH

MA

AMA

MA

AMA

NH4OH/EtOH

MA

AMA

MA

AMA

NH4OI-vEtOH

MA

Time (min) 16 h 10 M 10 M 10Mn loin 4 h 10 i loin 10 i 4 h 10 M 10Mi 10 i 10 m 4 h 10

M

T C 55 65 65 55 55 65 65 65 55 Full Length Product 62.5 62.7 74.8 75.0 77.2 44.8 65.9 59.8 61.3 cqu 65 65 55 55 65 75.2 79.1 77..1 79.8 75.5 22.7 28.9 A (36-iner) 286 Table 41: 2'-O-Alkylsilyl Deprotection Sequence AgT

(GGU)

4

C

10

U

10 Deprotection Reagent

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF Time (m 1 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h T OC 20 65 20 65 20 65 20 65 Full Length Product 84.5 81.0 60.9 67.8 86.2 86.1 84.8 84.5 B (36-mer)

TBAF

1.4 M HF

TBAF

1.4 M HF 24 h 1.5 h 24 h 25.2 30.6 29.7 A (36-mer) B is UCU CCA UCU GAU GAG GCC GAA AGG CCG AAA AUC CCU 09 Soo 99 o 9 9 99 99 .9 9 9. 0 0*9. 90 9 9 9 9 Table 42 NMJ Data. for UC Dimers containin~g.

Phosphorothioate Linkage Synthesis It Type Delivery Eq. Wait ASE 3524 ribo 2 x 3s 10.4 2 x 100 95.9 3525 ribo 2 x 3s 10.4 2 x75 s 92.6 3530 ribo 2 x3 s 10.4 2 x75 s 92.1 3526 ribo I x 5 s 08.6 1lx 3003s 100.0 3578 ribo 1 x 5 s 08.6 1 x 250 s 100.0 3529 ribo Ilx 5s 08.6 I x 150s 73.7 9* 0 0* ~0 0@ 000 0 0 0*@ *0 *0 0 ~0* *0 *e 0 0 00 00 0 006 of 0.0 0 0 0 Tabl e 4 3: NMR Data fior l 5 -lner RNA containing Phosphorothioate Linkages Synthesis i 3581 3663 'lyrpe ribo ribo Delivery I x5 S 2 x4 s Eq.

08.6 13.8 Wait I x 250 s 2 x 300 s A SE 99.6 3582 3668 3682 2'-O-Me Ix 6S 2 x4 s I x5S 08.6 13.8 08.6 1 x 250 2 x300s I x 300 100.0 99.7 c 99.8 99.8 289 Table 44. Kinetics of Self-.Processing In Vitro F Self-Processing Constructs; k ,i HH 1.16:t 0.08 HDV 0.56 t 0.15 2EP(GC) 0.36 t 0.06 BPI(GUJ) 0.054 0.003 Irepresents the unimoecla rateconstant for ribozyme self-cleavage determined from a non-linear, least-squaries fit (K-aleidaGraph, Synergy Software, Reeding, P-A) to the equation: (Fracton tUncleaved Tr~anscr~ipt) (Iet *The equation describes the extent of ribozyme Prcssn in the presense of ongoing transcription (Long tUhlenbecc, 1994 Po.N t A a S SA 9 1, 6977) as a function of time Wt and the unimolecular rate constant for cleavage Each value of kc represents the average range) of values determined from two experiments.

290 Table Entry Modificationi t1/2 tu2 (m) Activity Stability tS/tA t A) (ts)x 1 U4& U7 1 0.1 1 2 U4 U7 2'-O-Me-U 4 260 650 3 U4 2=CH 2 .l 6.5 120 180 U7 2=CH 2 -i 8 280 350

U

4 U7 2'--H 2 U 9.5 120 130 6 U4 2'5CF2U 5 320 640 7 U7 2'CF2 550 8 U4 U7 =2'CF2-U 20 220 550 9 U4=2'-F-U 4 U72'p-U 8 400 500 11 U4 U7 2'-F-U 4 300 750 12 U4 2'-C-Allyl-U 3 >500 >1700 13 U72'-C-AllyiU 3 220 730 ovS 14 U4 U7 2 -C-A~yl-U 3 120 400 U4 2'-araF-U 5 >500 >1000 16 U7 2'-araF-U 4 350 875 17

U

4 U7 2'-araF-U 15 500 330 18 U4='-NH 2 -U 10 500 500 19 U7 2'-NH 2 -U 5 500 1000

U

4 U7 2'-NH2'U 2 300 1500 21 U4 dU 6 100 170 22 U4 U7 dU 4 240 600

Claims (91)

1. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, IL- mRNA, rel A mRNA, TNF-a mRNA sites shown in Table 23, 27, or 28, CML associated mRNA selected from those identified as SEQ. ID NOS 1-25, or RSV mRNA or RSV genomic RNA in a region selected from the group consisting of 1C, 1B and N.

2. The enzymatic nucleic acid molecule of claim 1, the binding arms of which contain sequences complementary to any one of the 10 sequences defined in any of those in Tables 2, 3, 6-9, 11, 13, 23, 27, 28, 31, 33, 34, 36, and 37.

3. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said nucleic acid molecule is in a hammerhead motif. S4. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said 15 RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA or RNaseP RNA motif.

5. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 12 and 100 bases complementary to said mRNA or genomic RNA.

6. The enzymatic nucleic acid molecule of claim 5 comprising between 14 and 24 bases complementary to said mRNA or genomic RNA.

7. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 5 and 23 bases complementary to said mRNA or genomic RNA.

8. The enzymatic nucleic acid molecule of claim 7 comprising between and 18 bases complementary to said mRNA or genomic RNA.

9. An enzymatic nucleic acid molecule consisting essentially of a sequence selected from the group of those shown in Tables 4-8, 12, 14-16,

19-22, 24, 26-28, 30, 32, 34 and 36-38. 10. A mammalian cell including an enzymatic nucleic acid molecule of claims 1 or 2. 292 I 11. The cell of claim 10, wherein said cell is a human cell. 12. An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of claims 1 or 2 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell. 13. A mammalian cell including an expression vector of claim 12. 14. The cell of claim 13, wherein said cell is a human cell. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, re/ A, TNF-c, or RSV by administering to a patient an enzymatic nucleic acid molecule of claim 1 or 2. S* 16. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, relA, TNF-c., or RSV by administering to a patient an expression vector of claim 12. 17. The method of claims 15 or 16, wherein said patient is a human. 15 18. The method of claim 17 wherein said condition is selected from the group consisting of atherosclerosis, myocardial infraction, stroke, restenosis, heart diseases, cancer, rheumatoid arthritis, asthma, reperfusion injury, inflammatory or autoimmune disorders, transplant rejection, myocardial ischemia, stroke, psoriasis, Kawasaki disease, HIV and AIDS, and septic shock. 19. A nucleoside selected from the group consisting of alkylnucleoside, 2 '-deoxy-2'-alkylnucleoside, nucleoside nucleoside methylphosphonate, nucleoside 3 '-deoxy-3'-dihalo- methylphosphonate, and 5',3'-dideoxy-5',3-bis(dihalo)- methylphosphonate. A nucleotide selected from the group consisting of alkylnucleotide, 2 '-deoxy-2'-alkylnucleotide, methylnucleotide, 5'-deoxy-5'-difluoro-methynucleotide, 3'-deoxy- 3'-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3-bis(dihalo)- methylphosphonate. 293

21. A nucleotide triphosphate comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, methylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy-3'-dihalo- methylnucleotide, and 5',3'-dideoxy-5' ,3'-bis(dihalo)-methylphosphonate.

22. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in a talo configuration.

23. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in an allo configuration.

24. An oligonucleotide comprising a nucleotide selected from the group consisting 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and dideoxy-5',3 '-bis(dihalo)-methylphosphonate.

25. An oligonucleotide comprising a moiety having the formula I: wherein B is a nucleotide base or hydrogen; R1, R2 and R3 independently is selected from the group consisting of hydrogen, an alkyl group containing between 2 and 10 carbon atoms inclusive, an amine, an amino acid, and a peptide containing between 2 S:and 5 amino acids inclusive; and the zigzag lines are independently hydrogen or a bond.

26. An oligonucleotide comprising a 3'-amido or peptido group. o" 27. An oligonucleotide comprising a 5'-amido or peptido group.

28. The oligonucleotide of claim 24, 25, 26, or 27 having enzymatic activity.

29. Method for producing an enzymatic nucleic acid molecule having activity to cleave an RNA or single-stranded DNA molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'-position. [n:\libc]00132:MER 294 Method for conversion of a protected allo sugar to a protected talo sugar, comprising the step of contacting said protected allo sugar with triphenyl phosphine, diethylazodicarboxylate, p-nitrobenzoic acid under inversion causing conditions to provide said protected talo sugar.

31. Method for the synthesis of a nucleoside 5' or a 3'-dihaio- methylphosphonate comprising the step of condensing a difluoromethylphosphonate-containing sugar with a pyrimidine or purine under conditions suitable for forming a nucleoside or 3'- difluoromethylphosphonate.

32. The oligonucleotide of claim 3, wherein the normal hammerhead U4 and/or U7 positions are substituted with 2'-NH-amino acid.

33. A method for the synthesis of RNA comprising the step of providing at a delivered 0.1-1.0 M concentration for the activation of a RNA amidite during a coupling step for less than or S: equal to 10 minutes.

34. A method for the synthesis of RNA comprising the step of providing 5-S-alkyltetrazole at 0.15-0.35 M effective, or final, concentration for the activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

35. A method for the deprotection of RNA comprising the step of providing alkylamine (MA) or NH40H/alkylamine (AMA) at between 0 C 70°C for 5 to 15 minutes to remove any exocyclic amino protecting groups from protected RNA; wherein said alkyi is selected from the group consisting of methyl, ethyl, propyl and butyl.

36. A method for the deprotection of RNA alkylsilyl protecting groups comprising, contacting said groups with anhydrous triethylarnineehydrogen fluoride (aHF*TEA) trimethylamine or disopropylethylamine at between 60 °C-70 "C for 0.25-24 h.

37. A method for the purification of an RNA molecule by passing said enzymatic RNA molecule over an HPLC column, wherein said HPCC column is an anion exchange chromatography column. 295 W

38. Method for one pot deprotection of RNA comprising, contacting a protected base with anhydrous methyl amine at between 60 °C for at least 5 min, cooling the resulting mixture and contacting said mixture with TEA-3HF reagents under conditions which remove a protecting group of the 2'-hydroxyl position.

39. Method for synthesizing RNA containing a phosphorothioate linkage comprising the step of contacting 6-10 equivalents of 3H-1,2- benzodithiole-3-one 1,1-dioxide (Beaucage reagent) with the growing RNA chain for 5 seconds with a reaction time of at least 10 300 seconds.

40. Method of synthesizing RNA containing a phosphorothioate linkage comprising the step of achieving coupling with 5-S-ethyltetrazole or 5-S-methyltetrazole prior to sulfurization.

41. Method of claims 38, 39 or 40 wherein said RNA is enzymatically 15 active. S42. Method for synthesizing 2 -deoxy-2'-amino-nucleoside phosphoramidite, comprising the step of protecting the 2'-amino group with a N-phtaloyl group.

43. The method of claim 42 wherein the said nucleoside lacks a base. 20

44. Method for synthesis of RNA comprising the step of: protecting the 2'-position of a nucleotide during said synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group. Method for covalently linking a SEM group to the 2'-position of a nucleotide, comprising the step of: contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions.

46. The method of claim 45, wherein said conditions comprise dibutyltin oxide and tetrabutylammonium fluoride and SEM-CI.

47. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, comprising the step of: contacting said molecule or oligonucleotide with boron trifluoride etherate (BF 3 "OEt 2 under SEM removing conditions. 296 W

48. The method of claim 57 wherein said (BF 3 -OEt 2 is provided in acetonitrile.

49. One or more vectors comprising a first nucleic acid sequence encoding a first ribozyme having intramolecular or intermolecular cleaving activity, said first ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif; S.and a second nucleic acid sequence encoding a second ribozyme having intermolecular cleaving activity, said Second ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif and said second nucleic acid being flanked by other nucleic acid sequences encoding RNA which is cleaved by said first ribozyme to release said second ribozyme from RNA encoded by said vector; wherein said first and second nucleic acid sequences may be on the same or separate nucleic acid molecules, and said vector encodes mRNA or comprises RNA which lacks secondary structure which *20 reduces release of said second ribozyme by more than

50. Cell comprising the vector of claim 49.

51. A transcribed non-naturally occurring RNA molecule, comprising a desired therapeutic RNA portion, wherein said molecule comprises an intramolecular stem formed by base-pairing interactions between a 3' region and 5' complementary nucleotides in said RNA, wherein said stem comprises at least 8 base pairs.

52. The RNA molecule of claim 51, wherein said molecule is transcribed by a RNA polymerase III based promoter system.

53. The RNA molecule of claim 51, wherein said molecule is transcribed by a type 2 pol III promoter system.

54. The RNA molecule of claim 51, wherein said molecule is a chimeric tRNA. 297 S The RNA molecule of claim 53, said RNA having A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases.

56. The RNA molecule of claim 53, wherein said desired RNA molecule is at the 3' end of said B box.

57. The RNA molecule of claim 53, wherein said desired RNA molecule is in between the said A and the B box.

58. The RNA molecule of claim 53, wherein said desired RNA molecule includes said B box.

59. The RNA molecule of claim 51, wherein said desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 12 bases of said 3' region.

61. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 15 bases of said 3' region.

62. DNA vector encoding the RNA molecule of claim 51

63. The vector of claim 62, wherein said vector is derived from an AAV or adeno virus.

64. RNA vector encoding the RNA molecule of claim 51. The vector of claim 64, wherein said vector is derived from an alpha virus or retro virus.

66. The vector of claim 62 wherein the portions of the vector encoding said RNA function as a RNA pol 111 promoter.

67. Cell comprising the vector of claim 62.

68. Cell comprising the vector of claim 53.

69. Cell comprising the RNA of claim 51. 298 O

70. Method to provide a desired RNA molecule in a cell, comprising introducing said molecule into said cell a RNA comprising a desired RNA molecule, having a 5' terminus able to base pair with at least 8 bases of a 3' region of said RNA molecule.

71. The method of claim 70, wherein said introducing comprises providing a vector encoding said RNA molecule.

72. Hammerhead ribozyme having 2 or 3 base pairs in stem II with an interconnecting loop of 4 or more bases between said base pairs.

73. Hairpin ribozyme lacking a substrate moiety, comprising at least six bases in helix 2 and able to base-pair with a separate substrate RNA, wherein the said ribozyme comprises one or more bases 3' *of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

74. The ribozyme of claim 73, wherein said ribozyme comprises six bases in helix 2.

75. The nrbozyme of claim 73, having the structure of Fig. 3, wherein each N and N' is independently any base and each dash may Srepresent a hydrogen bond, r is 1-20, q is 2-20, o is 0 20, n is 1 20 4, and m is 1

76. Method for increasing the activity of a hairpin ribozyme by providing one or more bases 3' of helix 3 able to base-pair with a substrate RNA to form a helix

77. Trans-cleaving Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

78. Trans-ligating Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

79. The ribozyme of claim 73 having the structure of Fig. 73. The ribozyme of claim 73 having the structure of Fig. 74.

81. A cell including the ribozyme of any of claims 73-80. I_ 299 W

82. An expression vector comprising nucleic acid encoding the ribozyme of any of claims 73-80, in a manner which allows expression of that ribozyme within a cell.

83. A cell including an expression vector of claim 82.

84. Method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule, comprising the steps of: contacting said nucleic acid moleculee in vivo with an oligonucleotide or peptide nucleic acid able to form a duplex or triplex molecule with said nucleic acid molecule, wherein formation S. of said duplex or triplex molecule directly, or after nucleic acid *repair in vivo, causes at least one base in said nucleic acid molecule to be chemically modified to functionally alter the nucleotide base sequence of said nucleic acid sequence. 15

85. The method of claim 84, wherein said oligonucleotide is of a length sufficient to activate dsRNA deaminase in .vvo to cause conversion of an adenine base to inosine in an RNA molecule.

86. The method of claim 84, wherein said oligonucleotide comprises an Senzymatic nucleic acid molecule which is active to chemically 20 modify a base.

87. The method claim 84, wherein said nucleic acid molecule is DNA or RNA.

88. The method of claim 84, wherein said oligonucleotide comprises a chemical mutagen.

89. The method of claim 88, wherein said mutagen is nitrous acid. The method of claim 84 wherein said oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methylation of cytosine to

91. The method of claim 84, wherein an endogenous mammalian editing system is co-opted to cause said chemical modification. 300 U

92. Method for introduction of enzymatic nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under conditions in which said enzymatic nucleic acid molecule is produced in said cell or tissue.

93. Method for introduction of a desired nucleic acid into a cell or tissue, 15 comprising the steps of; providing a complex of a first nucleic acid molecule encoding said desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired 20 structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under conditions in which said desired acid molecule is produced in said cell or tissue. 94 Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired 1 301 Sstructure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and wherein said second nucleic acid further comprises a localization factor; and contacting said complex with said cell or tissue under conditions in which said desired nucleic acid molecule is produced in said cell or tissue. 10 95. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule :o having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid •molecule at a location which promotes expression of RNA from said to first nucleic acid under said conditions.

96. Complex of a first nucleic acid molecule encoding a desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

97. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said 302 first nucleic acid under said conditions, and wherein said second nucleic acid further comprises a localization factor.

98. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, mRNA, rel A mRNA, TNF-a mRNA sites,

99. Method for producing an enzymatic nucleic acid molecule, substantially as hereinbefore described with reference to any one of the Examples.

100. Method for conversion of a protected allo sugar to a protected talo sugar, substantially as hereinbefore described with reference to any one of the Examples.

101. Method for the synthesis of a nucleoside 5' or a 3'-dihalo-methylphosphonate, substantially as hereinbefore described with reference to any one of the Examples.

102. A method for the synthesis of RNA, substantially as hereinbefore described with reference to any one of the Examples.

103. A method for the deprotection of RNA, substantially as hereinbefore described with reference to any one of the Examples. t. 15 104. A method for the purification of an RNA molecule, substantially as hereinbefore described with reference to any one of the Examples.

105. Method for synthesizing 2'-deoxy-2'-amino-nucleoside phosphoramidite, substantially as hereinbefore described with reference to any one of the Examples.

106. Method for covalently linking a SEM group to the 2'-position of a nucleotide, substantially as hereinbefore described with reference to any one of the Examples.

107. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, substantially as hereinbefore described with reference to any one of the Examples.

108. A transcribed non-naturally occurring RNA molecule, substantially as 25 hereinbefore described with reference to any one of the Examples.

109. Method to provide a desired RNA molecule in a cell, substantially as hereinbefore described with reference to any one of the Examples.

110. Hairpin ribozyme lacking a substrate moiety, substantially as hereinbefore described with reference to any one of the Examples.

111. Method for increasing theactivity of a hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples.

112. Trans-cleaving Hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples.

113. Trans-ligating Hairpin ribozyme, substantially as hereinbefore described with reference to any one of the Examples. [n:\libc]00132:MER 303

114. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule, substantially as hereinbefore described with reference to any one of the Examples. Dated 15 September, 1999 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *se @1 Seeo o O [n:\libc]00132:MER