CA2468048A1 - Process for purifying chemically synthesized rna - Google Patents

Abstract

Disclosed is a process for purifying chemically synthesized RNA having one or more chemical modifications, comprising: (a) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(b) eluting the RNA by passing a suitable buffer through the column to obtain an eluate; and (c) collecting the eluate from the column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.
The RNA to be purified may be produced by deprotecting one or more protecting groups.

Description

,0909-173D

PROCESS FOR PURIFYING CHEMTCALLY SYNTHESIZED RNA
This is a divisional application of Canadian Patent Application Ser. No. 2,183,992 filed February 23, 1995.
Field of Invention The parent application 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.
This divisional application relates to a process for (deprotecting and) purifying chemically synthesized RNA
having one or more chemical modifications.
However, it should be understood that the expression 'this invention" or the like contained in this specification encompasses the subject matters of both the parent and divisional applications.
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, e.g., ICAM-1, IL-5, relA, TNF-a, p210bcr-abi~ and respiratory syncytial virus genes. Such ribozymes can be used in a method for treatment of diseases caused by the expression of these genes in man and other animals, including other primates.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner.
Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, ,e909-173D

1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in traps (and thus can cleave other RNA molecules) under physiological conditions. Table 1 summarizes some of the characteristics of these ribozymes.
Ribozymes act by first binding to a target RNA.
Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA
target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. The advantage reflects the ability of the ribozyme to act enzymatically.
Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the ,n909-173D
2a 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 ratio 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 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.
Thus, in a first aspect, this invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave RNA species encoding ICAM-1, IL-5, relA, TNF-a, p210bor-abl, or RSV proteins. Particularly described are 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, p210bcr-abi or RSV proteins in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic uses.
Indicated here is that these ribozymes are able to inhibit expression of ICAM-1, IL-5, relA, TNF-a, p210bcr-ably 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, relA, TNF-a, p210bcr-abi~ 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 cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.
By "gene" is meant to refer to either the protein coding regions of the cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.
By "enzymatic RNA molecule" it is meant an RNA molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule.
This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to~ occur.
One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By "equivalent" RNA to a virus is meant to include those naturally occurring viral encoded RNA
molecules associated with viral caused diseases in various animals, including humans, cats, simians, and other primates. These viral or viral-encoded RNAs have similar structures and equivalent genes to each other.
By "complementarity" it is meant a nucleaic acid that can form hydrogen bonds) with other RNA sequence by either traditional Watson Crick or other non-traditional types (for examplke, Hoogsteen type) of base paired 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 Refroviruses , 8,183, .of hairpin motifs by Hampel and Tritz, 1989 Biochemistry. 28, 4929, EP 0360257 and Hampel et al., 1990, Nucleic Acids Res. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry, 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 ~, 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.
ci. U A 88, 8826-8830; Collins and Olive, 1993 ~iochemisfrv 32, 2795 2799 Guo and Collins, 1995 EMB ., 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.e., 1 CAM-1, IL-5, reLA, TNF-a, p210 bcr-abl or RSV proteins encoding mRNA such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required., Alternatively, the ribozymes can be expressed from vectors that are delivered to specific cells. By °vect.ors° is meant any nucleic acid andlor 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 (e.g., of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA
structrure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g. Scanion, K.J. et al., 1991, Proc.
Nail. Acad. Sci.. USA, 88, 10591-5; Kashani-Sabet, M., et a1.,1992, Antis~nse Res. Dev., 2, 3-15; Dropoulic, B., et al., 1992, . Virol, 66, 1432-41; Weerasinghe, M., et al., 191, Virol 65, 5531-4; Ojwang, J.O., et al., 1992, Proc. Natl. Acad. Sci.. USA. 89 10802-6; Chen C.J., et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver, H., et al., 1990 Science, 247, 1222-1225). Those skilled in the art would realize that any ribozyme can be J
expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from tl.e primary transcript by a second ribozyme (Draper et al.; PCT W093123569, and Sullivan et al., PCT W094102595; Ohkawa, J., et al., 1992, Nucleic Acids Svmo.
~ 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 . Biol hem 269, 25856 ).
By "inhibit" is meant that the activity or level of ICAM-l,Rel A, IL-5, TNF-a, p210bcr-abl or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to~ the level of 1CAM-1, IL-5, Rel A, TNF-a, p2lObcr-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, 1L-5, Rel A, TNF-a, p210bcr-abl or RSV proteins will relieve to some extent the symptoms of the disease or condition.
Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.
The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 2,3,6-9, 11, 13, 15-23, 27, 28, 31, 33, 34, 36 and 37.
Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such _ ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains a~ 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.

s 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, andlor 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 the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequence listed in the above identified Tables may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. , In another aspect of the invention, ribozymes that cleave target 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 ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol I), RNA polymerase II (pol Il), or RNA polymerase III (pol III). Transcripts from pol ll 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. ~Nafl.
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 (e.g. Kashani-Sabet et ai., 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. Nafl.
Acad. Sci. U.S.A., 90 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors ,0909-173D

(such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).
As stated above, the subject matter of this divisional application is a process for (deprotecting and) purifying chemically synthesized RNA having one or more chemical modifications.
In a first aspect, purification only is involved.
The process according to a major embodiment of the first aspect, comprises:
(a) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(b) eluting the RNA by passing a suitable buffer through the column to obtain an eluate: and (c) collecting the eluate from the column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.
The process may further comprise:
(d) recovering the RNA from the desalted eluate under conditions which allow for purification of the RNA.
In a second aspect, both deprotection and purification are involved.
In this aspect, the process according to a first major embodiment, comprises:
(a) loading the RNA onto reverse phase high-performance liquid chromatography (HPLC) column, wherein the RNA comprises a 5'-protecting group;

,n909-173D
7a (b) eluting the RNA by passing a suitable buffer through the reverse phase column;
(c) removing the 5'-protecting group from the RNA
to obtain unprotected RNA;
(d) loading the unprotected RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(e) eluting the unprotected RNA by passing a suitable buffer through the anion exchange column to obtain an eluate; and (f) collecting the eluate from the anion exchange column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.
In this aspect, the process according to a second major embodiment, comprises:
(a) contacting the RNA with an alkylamine under conditions suitable for removing any exocyclic amine protecting groups or phosphate ester protecting groups;
(b) contacting the RNA with triethylamine-hydrogen fluoride under conditions suitable to remove any alkylsilyl protecting groups from the RNA;
(c) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(d) eluting the RNA by passing a suitable buffer through the column to obtain an eluate; and (e) collecting the eluate from the column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.

7b The process may further comprise:
(f) recovering the RNA from the desalted eluate under conditions which allow for purification of the RNA.
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 ribozyme domain known in the art. Stem II can be >- 2 base-pair long.
Figure 2(a) is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2(b) is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2(c) is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2(d) is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.
Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with at least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is ? 1 base). Helix 1, 4 or 5 may ,6909-173D
~C
also be extended by 2 or more base pairs (e. g., 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate.
Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is z 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 self-cleaving VS RNA ribozyme domain.
Figure 6 is a diagrammatic representation of the genetic map of RSV
strain A2.
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 65 °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°C for 15 min. The sample is allowed to cool for 10 min before adding TEA~3HF reagent, to the same poi, 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
isUCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG.
Figure 17 is a schematic representation of synthesis of 2'-N-phtalimido-nucleoside phosphoramidite.
Figure 18 is a diagrammatic representation of a prior art method for the solid-phase synthesis of RNA using silyl ethers, and the method of this invention using SEM as a 2'-protecting group.
Figure 19 is a diagrammatic representation of the synthesis of 2'-SEM-protected nucleosides and phosphoramidites useful for the synthesis of RNA. B is any nucleotide base as exemplified in the Figure, P is purine and I is inosine. Standard abbreviations are used throughout this application, well known to those in the art.
Figure 20 is a diagrammatic representation of a prior art method for deprotection of RNA using TBDMS protection of the 2'-hydroxyl group.
' Figure 21 is a diagrammatic representation of the deprotection of RNA
having SEM protection of the 2'-hydroxyl group.

' 10 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 Nindlll-linearized plasmid. (23) HH Cassette, transcript containing the hammerhead traps-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 su ra).
The traps ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the traps-ribozyme contains 2 non-ribozyme nucleotides (UC at positions 50 and 51) at its 3' end. The 3' processing ribozyme is comprised of nucleotides 44 through 96. Roman numerals I, II and III, indicate the three helices that contribute to the structure of the 3' cis-acting hammerhead ribozyme (Hertel et al., 1992 Nucleic Acids Res 20, 3252).
Substitution of G70 and A71 to U and G respectively, inactivates the hammerhead ribozyme (Ruffner et al., 1990 Biochemistry 29, 10695) and generates the HH(mutant) construct. (24) HP Cassette, transcript containing the hammerhead traps-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 EMEMBO. JJ
12, 2567). The traps-ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the traps-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 G 52 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 8~ Bruening, 1993 Nucleic Acids Res. 21, 1991; Altschuler et al., 1992 su ra). (25) HDV
Cassette, transcript containing the traps-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 traps-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, Il, III 8~ IV, indicate the location of four helices within the 3' cis-acting HDV ribozyme (Perrota &
Been, 1991 Nature 350, 434). The ~HDV 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 efifect of 3' flanking sequences on RNA self-processing in vitro. H, Plasmid templates linearized with Hindlll restriction enzyme. Transcripts from H templates contain four non-ribozyme nucleotides at the 3' end. N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from N templates contain 220 non-ribozyme nucleotides at the 3' end. R, Plasmid templates linearized with Rcai restriction enzyme. Transcripts from R templates contain 450 non-ribozyme nucleotides at the 3' end.
Fig. 28 shows the effect of 3' flanking sequences on the trans-cleavage reaction catalyzed by a hammerhead ribozyme. A 622 nt internally-labeled RNA (<10 nM) was incubated with ribozyme (1000 nM) under single turn-over conditions (Herschlag and Cech, 1990 Biochemistry 29, 10159). HH+2, HH+37, and HH+52 are traps-acting ribozymes produced by transcription from the HH, ~HDV, 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. !n vifro 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 primer-exiension 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 stem-loop structures at the 5' and the 3' end following self-processing. 30, 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, 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-a Sequence of the primary IRNAimet and D3-5 transcripts.
The A and B box are internal promoter regions necessary for pol lil transcription. Arrows indicate the sites of endogenous tRNA processing.
The D3-5 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 d 3-5 RNA resistant to endogenous tRNA
processing.
Figure 34. Schematic representation of RNA structural motifs inserted into the o3-5 RNA, e3-51HH1- a hammerhead (HHI) ribozyme was cloned at the 3' region of D3-5 RNA; S3- a stable stem-loop structure was incorporated at the 3' end of the D3-5/HH1 chimera; S5- stable, stem-loop structures were incorporated at the 5' and the 3' ends of 03-5/HH1 ribozyme chimera; S35- sequence at the 3' end of the D3-5/HH1 ribozyme chimera was altered to enable duplex formation between the 5' end .and a complementary 3' region of the same RNA; S35PIus- in addition to structural alterations of S35, sequences were altered to facilitate additional duplex formatiow within the non-ribozyme sequence of the e3-5/HHI
chimera.
Figures 35 and 36. Northern analysis to quantitate ribozyme expression in T cell lines transduced with D3-5 vectors. 35) D3-5/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 8 Sacchi, 1987 Analytical Biochemistry 162, 156-159), and transduced with various constructs described iri Fig. 34. Northern analysis was carried out using standard protocols (Curt. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley 8 Sons, NY). Nomenclature is same as in Figure 34. This assay measures the level of expression from the type 2 pol 111 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 D3-5 constructs described in Figs. 35 and 36 In a standard ribozyme cleavage reaction, 5 pg total RNA and trace amounts of 5' terminus-labeled ribozyme target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCl2. RNAs were renatured by cooling the reaction mixture to 37°C for 10-15 min.
Cleavage reaction was initiated by mixing the labeled substrate RNA and total cellular RNA at 37°C. The reaction was allowed to proceed for - 18h, following which the samples were resolved on a 20 % urea-polyacrylamide gel. Bands were visualized by autoradiography.
Figures 38 and 39. Ribozyme expression and activity levels in S35-transduced clonal CEM cell lines. 38) Northern analysis of S35-transduced clonal CEM cell lines. Standard curve was generated by spiking known concentrations of in vitro transcribed S~5 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 6418 and then grown in the absence of 6418 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 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 S35 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 and S35 plus containing HHI ribozyme.
Figures 44, 45, 46 and 47 are the nucleotide base sequences of S35, 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 5T
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-iRNA chimera.

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

5 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 10 RNA polymerase III promoters. A. Diagram of an AAV-based 'vector containing minimal AAV sequences comprising the inverted terminal repeats (ITR) at each end of the vector genome, an optional selectable marker (Neo) driven by an exogenous promoter (Pro), a ribozyme transcription unit, and sufficient additional sequences (stuffer) to maintain a 15 vector length suitable for efficient packaging. B. Diagram of ribozyme expressing adenovirus vectors containing deletions of one or more wild type adenoviorus coding regions (cross-hatched boxes marked as E1, pIX, E3, and E4), and insertion of the ribozyme transcription unit at any or several of those regions of deletions.
Fig. 58 is a graph showing the effect of arm length variation on the activity of ligated hammerhead (HH) ribozymes. Nomenclature 5/5, 6/6, 7/7, 8/8 and so on refers to the number of base-pairs being formed between the ribozyme and the target. For example, 5/8 means that the HH ribozyme forms 5 by on the 5' side and 8 by on the 3' side of the cleavage site for a total of 13 bp. -DG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and th.e 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 .base-paired stem II (HH-H3 and HH-H4).

Figs. 63 and 64 show RNA cleavage activity of HH-I and its variants (see Fig.62). 63) cleavage of matched substrate RNA (15 nt). 64), cleavage of long substrate RNA (613 nt).
Figs. 65a-b is a schematic representation of a method of this invention to synthesize a full length hairpin ribozyme. No splint strand is required for ligation but rather the two fragments hybridize together at helix 4 prior to ligation. The only prerequisite is that the 3' fragment is phosphorylated at its 5' end and that the 3' end of the 5' fragment have a hydroxyl group. The hairpin ribozyme is targeted against site J. H1 and H2 are intermolecular helices formed between the ribozyme and the substrate. H3 and H4 are intramolecular helices formed within the hairpin ribozyme motif. Arrow indicates the cleavage site.
Fig. 66 shows RNA cleavage activity of ligated hairpin ribozymes targeted against site J.
Figs. 67a-b is a diagrammatic representation of a Site K Hairpin Ribozyme (HP-K) showing the proposed secondary structure of the hairpin ribozyme ~substrate complex as described in the art (Berzal-Herranz et al., 1993 EMBO. J.12, 2567). The ribozyme has been assembled from two fragments (bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent intramolecular helix formation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme). Left panel (HP-K1 ) indicates 4 base-paired helix 2 and the right panel (HP-K2) indicates 6 base-paired helix 2.
Arrow indicates the site of RNA cleavage. All the ribozymes discussed herein were chemically synthesized by solid phase synthesis using RNA
phosphoramadite chemistry, unless otherwise indicated. Those skilled in the art will recognize that these ribozymes could also be made transcriptionally in vitro and in vivo.
Figure 68 is a graph showing RNA cleavage by hairpin ribozymes targeted to site K. A plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-K2 (6 by helix 2) cleaves a 422 target RNA to a greater extent than the HP-K1 (4 by helix 2).

To make internally-labeled substrate RNA for traps-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-32P]CTP (Chowrira ~
Burke, 1991 supra). The reaction mixture was created with' 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 p.l DEPC-treated water and stored at -20°C.
Unlabeled ribozyme (1 pM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris~HCl 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 ~I were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA).
Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary structure of the hairpin 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 by helix 2) cleaves a 2 KB target RNA
to a greater extent than the HP-L1 (4 by helix 2). To make internally-labeled substrate RNA for traps-ribozyme cleavage reactions, a 2 kB region (containing hairpin site L) was synthesized by PCR using primers.that place the Z7 RNA promoter upstream of the amplified sequence. The cleavage reactions were carried out as described above.

Figs. 71 a-b shows a Site M Hairpin Ribozyme (HP-M) with the proposed secondary structure of the hairpin ribozyme~substrate complex.
The ribozyme was assembled from two fragments as described above.
Figure 72 is a graph showing RNA cleavage by hairpin ribozymes targeted to site M. The ribozymes were tested at both 20°C and at 26°C.
To make internally-labeled substrate RNA for traps-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 26°C 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 5.
Helix 4 is replaced by a nucleotide loop wherein q is z 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 ef 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 5.
Helix 4 is replaced by non-nucleotide linker molecule "L" (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 "s" at the indicated location, wherein s is >_ 1 base. Hairpin ribozymes containing spacer region', can be synthesized as one fragment or can be assembled from multiple fragments. Nomenclature is same as described under figure 3.
Figs. 75a-a shows the structures of the 5'-C-alkyl-modified nucleotides. Rt 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 5'-G
alkyl-D-allose nucleosides and their phosphoramidites.
Figure 77 is a diagrammatic representation of the synthesis of 5'-C-alkyl-~-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.
Figure 80 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al. Nucleic Acids Res. 1992, 20, 3252) showing specific substitutions.
Figs. 81 a-j shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention. R groups are alkyl groups, Z is a protecting group.
Figure 82 is a diagrammatic representation of the synthesis of 2'-G
allyl uridine and cytidine.
Figure 83 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene uridine.
Figure 84 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene cytidine.
Figure 85 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene adenosine.
Figure 86 is a diagrammatic representation of the synthesis of 2'-C-carboxymethylidine uridine, 2'-C-methoxycarboxymethylidine uridine and .derivatized amidites thereof. X is CH3 or alkyl as discussed above, or another substituent.
Figure 87 is a diagrammatic representation of a synthesis of nucleoside 5'-deoxy-5'-difluoromethyiphosphonates.

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 5 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 io site N. Positions of 2'-hydroxyl group substitution is 15 indicated.
Figure 95 shows RNA cleavage activity of ribozymes containing a 2'-hydroxyl group modification of the present invention. All RNA, represents hammerhead ribozyme (HHN) with no 2'-hydroxyl group modifications. U7-ala, 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 ribozyrnes 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 provided to target the R-loop complex to cells wing 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 i 5 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 /CAM-1, iL-5, rei A, TNF-a, p210bcr-abl, 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 /CAM-1, Il- .5, rel A, TNF-a ,p210bcr-abl, or RSV mRNA in these systems may prevent or alleviate disease symptoms or conditions.
1 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 welt as by Draper et' aL, PCT/US94113129.
Rather than repeat the guidance provided in those documents here, below are provided specific examples of such 30. 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 ~i optimized and delivered as described therein.. .White specific examples to animal and human RNA ate 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 iri 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 (Jae,ger et al., 1989 Proc. Natl.
Acid. 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 W093123569.
Briefly, DNA oligonucleotides representing potential hamriierhead or hairpin ribozyme cleavage sites are synthesized. A polymerise chain reaction is used to generate a substrate for T7 RNA polymerise transcription from cDNA clones. Labeled RNA
transcripts are synthesized in vitro from DNA templates. The oligonucleotides and the labeled trascripts are annealed, RNaseH is added and the mixtures are incubated for the designated times at 37°C.
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 hai~pi~ 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 at., 1990 Nucleic Acids Res., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, phosphoramidites at the 3'-end. The average stepwise coupling yeilds are >98%. Inactive ribozymes are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 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, Mefhods 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 ace purified by gel electrophoresis using heneral methods or are purified by 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 the advantage of being generally immunologically inert, whereas significant neutralizing anti-IgG 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. ImmunoG 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 activily of T cells, NK cells, monocytes or granulocytes.
Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell interactions (Simmons et al., 1988 Nature (London) 331, 624-627).
ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J. lmmunol. 137, 245-254). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, ~-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 ef. al. supra; Dustin et al., supra; and Rothlein et al., 1988 J.
lmmunol. 141, 1665-1669). Induction occurs via increased transcription of ICAM-1 mRNA (Simmons et aG, supra). Elevated expression is detectable after 4 hours and peaks after 16 - 24 hours of induction.
ICAM-1 induction is critical for a number of inflammatory and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd,1988 Proc. Natl. Acad. Sci. USA
85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331).
Thus, ICAM-1 expression may be required for the extravasation of immune cells to sites of inflammation. Antibodies to ICAM-1 also block T cell killing, mixed lymphocyte reactions, and T cell-mediated B cell differentiation, suggesting that ICAM-1 is required for these cognate cell interactions (Boyd et aL, supra). The importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J.
lmmunoG 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 motifis we have designed several ribozymes directed against /CAM-1 mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance. These ribozymes cleave 1CAM-1 target sequences in vitro.
5 The sequence of human, rat and mouse /CAM-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 add human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility.
The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4. - 8 and 10. Those in the art will recognize that 15 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 rnay be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
20 The ribozymes will be tested for function in vivo by exogenous delivery to human umbilical vein endothelial cells (HUVEC). Ribozymes will be delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjectivn, or by expression from DNA or RNA
vectors described above. Cytokine-induced /CAM-1 expression will be 25 monitored by ELISA, by indirect immunofluoresence, andlor by FACS
analysis. /CAM-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 /CAM-1 protein and mRNA by more than 90°/a will be identified.
As disclosed by Sullivan et al.,. PCT W094102595, ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme wilt be monitored by its ability to block ex vivo induction of 1CAM-1 mRNA and protein. The effect of the anti-/CAM-7 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.
Use ICAM-1 plays a central role in immune cell recognition and function.
Ribozyme inhibition of ICAM-1 expression can reduce transplant rejection and alleviate symptoms in patients with rheumatoid arthritis, asthma or other acute and chronic inflammatory disorders. We have engineered several ribozymes that cleave ICAM-1 mRNA. Ribozymes that efficiently inhibit ICAM-1 expression in cells can be readily found and their activity measured with regard to their ability to block transplant rejection and arthritis symptoms in animal models. These anti-ICAM-1 ribozymes represent a novel therapeutic for the treatment of immunological or inflammatory disorders.
The therapeutic utility of reduction of activity of ICAM-1 function is evident in the following disease targets. The noted references indicate the role of ICAM-1 and the therapeutic potential of ribozymes described herein.
Thus, these targets can be therapeutically treated with agents that reduce ICAM-1 expression or function. These diseases and the studies that support a critical role for ICAM-1 in their pathology are listed below. This list is not meant to be complete and those in the art will recognize further conditions and diseases that can be effectively treated using ribozymes of the present invention.
~ Transplant rejection ICAM-1 is expressed on venules and capillaries of human cardiac biopsies with histological evidence of graft rejection (Briscoe et al., 1991 Transplantation 51, 537-539).
Antibody to ICAM-1 blocks renal (Cosimi et al., 1990J.~ Immunol. 144, 4604-4612) and cardiac (Flavin et al., 1991 Transplanf. Proc. 23, 533-534) graft rejection in primates.

A Phase I clinical trial of a monoclonal anti-ICAM-1 antibody showed significant reduction in rejection and a significant increase in graft function in human kidney transplant patients (Haug, et al., 1993Transplantation 55, 766-72).
~ Rheumatoid arthritis ICAM-1 overexpression is seen on synovial fibroblasts, endothelial cells, macrophages, and some lymphocytes (Chin et al., 1990 Arthritis Rheum 33, 1776-86; Koch et al., 1991 Lab Invest 64, 313-20).
Soluble ICAM-1 levels correlate with disease severity (Mason et al., 1993 Arthritis Rheum 36, 519-27).
Anti-ICAM antibody inhibits collagen-induced arthritis in mice (Kakimoto et al., 1992 Cell lmmunol 142, 326-37).
Anti-ICAM antibody inhibits adjuvant-induced arthritis in rats (ligo et al., lmmunol 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 Physiol262, 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 Dermafol 125, 211-6; Griffiths 1989 J Am Acad Dermafol 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., 1993) lmmunol 150, 2148-59 ).
~ Kawasaki disease Surface ICAM-1 expression correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989Lancef 2, 1298-302).
Soluble ICAM levels are elevated in Kawasaki disease patients; particularly high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992Arthritis Rheum 35, 672-7; Tsuji, 1992 Arerugi 41, 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
Immunol37, 377-80).
Example 2: IL-5 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, e.g_, by inhibiting the synthesis of LL-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-xB. 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-1 R and TNF-aR on keratinocytes, epithelial and endothelial cells in airways. Recent data suggest that certain neuropeptides may play a role in asthmatic symptoms. These peptides include substance P, neurokinin A and calcitonin-gene-related peptides.
These target genes may have more general roles in inflammatory diseases, but are currently assumed to have a role only in asthma.

Ribozymes of this invention block to some extent IL-5 expression and can be used to treat disease or diagnose such disease. Ribozyrries 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.
Res~ir. Dis. 144, 931-938; Larsen et al., 1992 J. Clin. Invest. 89, 747-752;
Mauser et al., 1993 supra). Ribozyrne cleavage of IL-5 mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.
The sequence of human and mouse IL-5 mRNA were screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 11, 13, and 14, 15. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. (In Table 12, lower case letters indicate positions that are 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 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form.
Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 15 and 16 (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 12; 14. - 16 may be formed of ribonuclectides 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 IL-5 expression levels. Ribozymes will be delivered to cells by incorporation 5 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.
10 Ribozymes that block the induction of 1L-5 activity and/or IL-5 mRNA by more than 90°!° will be identified.
Use Interleukin 5 (IL-5), a cytokine produced by CD4+ T helper cells and mast cells, was originally termed B cell growth factor I) (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 Immunoloav 71, 258-65) and in vitro survival of eosinophils (Lopez et al., 20 1988 J. Exp. Med: 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 I. Ex~. Med. 172, 1525-8). The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma:
Several studies have shown a direct correlation between the number 25 of activated T cells and the number of eosinophils from asthmatic patients vs. normal patients (Oehling et al., 1992 J. Investig. Allergol. Clin.
Immunol.
2, 295-9). Patients with either allergic asthma or intrinsic asthma were treated with corticosteroids. The bronchoalveolar lavage was monitored for eosinophils, activated T helper cells and recovery of pulmonary function 30 over a 28 to 30 day period. The number of eosinophils and activated T
helper cells decreased progressively with subsequent improvement in pulmonary function compared to intrinsic asthma patients with .no corticosteroid treatment.
Bronchoalveolar lavage cells were screened for production of cytokines using in situ hybridization for mRNA. In situ hybridization signals were detected for 1L-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 . Aller Clin. Immunol. 92, 313-24). Another study showed that upregulation of IL-5 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 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 interferon-gamma.
Bronchial biopsies from 15 patients were analyzed 24 hours after 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 IL-5 and GM-CSF mRNA significantly increased.
In another patient study, the eosinophil phenotype was the same for asthmatic patients and normal individuals. However, eosinophils from asthmatic patients had greater leukotriene C4 producing capacity and migration capacity. There were elevated levels of IL-3, IL-5 and GM-CSF in the circulation of asthmatics but not in normal individuals (Bruijnzeel et al., 1992 Schweiz. Med. Wochenschr. 122, 298-301 ).
Efficacy of antibody to IL-5 was assessed in a guinea pig asthma model. The animals were challenged with ovalbumin and assayed for eosinophilia and the responsiveness to the bronchioconstriction substance P. A 30 mg/kg dose of antibody administered i.p. blocked ovalbumin-induced 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 Vim. Rev, Re~pir. Did 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 (L-5 in eosinophil development and function makes IL-5 a good candidate for target selection. The antibody studies neutralized IL-5 in the circulation thus preventing eosinophilia. Inhibition of the production of lL-5 will achieve the same goal.
A st h m a - a prominent feature of asthma is the infiltration of eosinophils and deposition of toxic eosinophil proteins (e.g. major basic protein, eosinophil-derived neurotoxin) in the lung. A number of T-cell-derived factors like IL-5 are responsible for the activation and maintainance of eosinophils (Kay, 1991 . Allergy Clin. Immun. 87, 893). Inhibition of IL-5 expression in the lungs can decrease the activation of eosinophils and will help alleviate the symptoms of asthma.
Atopy - is characterized by the developement of type I hypersensitive reactions associated with exposure to certain environmental antigens. One of the common clinical manifestations of atopy is eosinophilia (accumulation of abnormally high levels of eosinophils in.~the blood).
Antibodies against IL-5 have been shown to lower the levels of eosinophils in ri~ice (Cook et al., 1993 in Immunopharmacol. Eosinoohils 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 supra). 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 IL-5.
Pulmonary infiltration eosinophilia- is characterised by accumulation of high levels of eosinophils in pulmonary parenchyma (Gleich, 1990 ~. Allergy Clin. Immunol. 85, 422).

L-Tryptophan-associated eosinophilia-myalgia syndrome (EMS)- The EMS disease is closely linked to the consumption of L-tryptophan, 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 IL-5 and transforming growth factor play a significant role in the development of EMS (Varga et al., 1993 supra) by activating eosinophils and other inflammatory cells.
Thus, ribo~ymes 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 IL-5 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 su ra) and can be used to optimize activity.
Example 3: NF-xB
Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-xB. One subunit of NF-x8, the relA gene product (termed ReIA or p65) is implicated specifically in the induction of inflammatory responses. Ribozyme therapy, due to its exquisite specificity, is particularly well-suited to target intracellular factors that contribute to disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A or TNF-a may represent novel therapeutics for the treatment of inflammatory and autoimmune disorders.
The nuclear DNA-binding activity, NF-xB, was first identified as a factor that binds and activates the immunoglobulin x light chain enhancer in B cells. NF-xB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-xB 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-xB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-xB (encoded by the nf-xB2 or nf-xB1 genes, respectively) are generated from the precursors NF-xB1 (p105) or NF-xB2 (p100). The p65 subunit of NF-xB (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 (N.D. Perkins, et al., 1992 Proc. Natl Acad. S
I~SA- 89, 1529-1533). For instance, the heterodimer of NF-xB1 and Rel A
(p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NF-xB2/ReIA heterodimefs (p49/p65) actually inhibit transcription (H.B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)).
Conversely, heterodimers of NF-xB2/ReIA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-xB1/ReIA (p50/p65) heterodimers have little effect (J. Liu, N.D. Perkins, R.M. Schmid, G.J.
Nabel, Virol. 1992 66, 3883-3887). Similarly, blocking re! A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-xB1 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 sssigned to NF-xB in transcriptional activation (M.J. 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 "knock-outs" 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-xB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-xB binding sites, and over expression of the natural inhibitor MAD-3 (an IxB family member). These agents have been used to show that NF-xB is required for induction of a number of molecules involved in inflammation, as described below.
~NF-xB 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, 5 1993 Mol. Cell. Biol. 13, 6137-46).
~NF-xB 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-xB 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-xB and 15 inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-xB. The glucocorticoid receptor and p65 both act at NF-xB
binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol.
Chem. 269, 6185-6192). Glucocorticoid receptor inhibits NF-xB-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 (Id.).
Ribozymes of this invention block to some extent NF-xB expression 25 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.
30 The sequence of human and mouse relA mRNA can be screened for accessible sites using acomputer 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 3s 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.
By engineering ribozyme motifs we have designed several ribozymes directed against rel A mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave relA target sequences in vitro is evaluated.
The ribozymes will be tested for function in vivo by analyzing cytokine-induced VCAM-1, /CAM-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, /CAM-1, IL-6 and IL-8 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS
analysis. Rel A mRNA levels will ~ be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.
Activity of NF-x8 will be monitored by gel-retardation assays. Ribozymes that block the induction of NF-xB activity and/or rel A mRNA by more than 50% 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 II_-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-relA ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

Use A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory. effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.
~Rheumatoid arthritis (RA).
Due to the chronic nature of RA, a gene therapy approach is logical.
Delivery of a ribo~yme 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 months would be expected (B.J. 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-xB 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-xB is required for the expression of the oncogene c-myb (F.A. La Rosa, J.W. Pierce, G.E.
Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-xB 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.

NF-xB 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-xB in the transplanted endothelium may be sufficient to prevent transplant-associated vasculitis and may significantly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme expression vectors. Recipients would receive the treatment prior to transplant. Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1, VCAM-1, and/or B7 an B7-2) can induce antigen-specific anergy. Tolerance to the donor's histocompatibility antigens could result; potentially, any donor could be used for any transplantation procedure.
~Asthma.
Granulocyte macrophage colony stimulating factor (GM-CSF) is thought to play a major role in recruitment of eosinophils and other inflammatory cells during the late phase reaction to asthmatic trauma.
Again, blocking the local induction of GM-CSF and other inflammatory mediators is likely to reduce the persistent inflammation observed in chronic asthmatics. Aerosol delivery of ribozymes or adenovirus ribozyme expression vectors is a feasible treatment.
~Gene Therapy.
Immune responses limit the efficacy of many gene transfer 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 immune-compromised 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 rel A mRNA and thereby NF-xB 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-xB

function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for NF-KB function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity.
Example 4: TNF-a Ribozymes that cleave the specific cites in TNF-a mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.
Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated leukocytes, that is a potent mediator of inflammatory reactions. Injection of TNF-a into experimental animals can simulate the symptoms of systemic and local inflammatory diseases such as septic shock or rheumatoid arthritis.
TNF-a was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, 1985 cien a 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., ~ 985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-a have been cloned and found to be related to TNF-f3 (Shakhov et al., 1990 ,J. Exo. Med. 171, 35-47). Both TNF-a and TNF-f3 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 cienc 248, 1019-1023). TNF-a secretion has been detected from monocytes/macrophages, CD4+ and CD8+ T-cells, B-cells, lymphokine activated killer cells, neutrophiis, 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 io 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. Trod. Med. HVQ. 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 5 cleavage sites (Sioud et al., 1992 J. Mol. Biol. 223; 831; Sioud WO
94/10301; Kisich and co-workers, 1990 abstract (FA B 4, A1860; 1991 slide presentation (J. 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, DavisJ 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 15 of septic shock and rheumatoid arthritis. Ribozyme cleavage of TNF-a mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.
The sequence of human and mouse TNF-a mRNA can be screened for accessible sites using a computer folding algorithm. Hammerhead or 20 hairpin ribozyme cleavage sites were identified. , These sites are shown in Tables 23, 25, and 27 - 28. . (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafiter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the 25 ribozyme prior to testing in humans. The nucleotide base position is noted 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 30 study are shown in Tables 24, 26 - 28. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Tables 24 and 26 (5'-GGCCGAAAGGCC-35 3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in 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 non-nucleotides. 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, I-473.; Nabel et al., 1990 Science, 249, 1285-1288) and both vectors lead to transient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, e.g., through the use of an injection catheter, scent 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 ace 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-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 i ml of sterile fluid thioglycollate broth (Difco, Detroit, MI.) was injected i.p. into 6 week old female C57b1/6NCR mice 3 days before peritoneal favage. Mice were maintained as specific pathogen free in autoclaved cages in a laminar flow hood and given sterilized water to minimize "spontaneousN activation of macrophage:.. The resulting peritoneal exudate cells (PEC) were obtained by lavage using Hanks balanced sail solution (HESS) and were plated at 2.5X105/well in 96 well plates (Costar, Cambridge, MA.) with Eagles minimal essential medium (EMEM) containing 10% heat inactivated fetal bovine serum. After adhering for 2 hours the wells were washed to remove non-adherent cells. The resulting cultures were 97% macrophages as determined by morphology and staining for non-specific esterase.
Transfection of ribozymes into macrophages:
The ribozymes were diluted to 2X final concentration, mixed with an equal volume of llnM lipofectamine (Life Technologies, Gaithersburg, MD.), and vortexed. 100 ml of lipid:ribozyme complex was then added directly to the cells, followed immediately by 10 mi fetal bovine serum.
Three hours after ribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride , (LPS) was added to each well to stimulate TNF
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. Ouantitation of TNF-a was done by a specific ELISA. ELISA plates were coated with rabbit anti-mouse TNF-a serum at 1:1000 dilution (Genzyme) followed by blocking with milk proteins and incubation with TNF-a containing supernatants. TNF-a was then detected using a murine TNF-a specific hamster monoclonal antibody (Genzyme). The ELISA was .developed with goat anti-hamster IgG coupled to alkaline phosphatase.
Assessment of reagent toxicity:
Following ribozyme/lipid treatment of macrophages and harvesting of supernatants viability of the cells was assessed by incubation of the cells with 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-Biphenyl 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 3D 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. 5 (Supp. A), 133-143].

44 ' 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 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-inflammatory cytokines which are not normally detected in the serum, such as TNF-a, interleukin-1 ~i (IL-1 f3), ~-interferon (IFN-Y), interleukin-6 (IL-6), and interleukin-8 (iL-8). Other non-cytokine mediators such as leukotriene b4, prostaglandin E2, C3a and C3d also reach high levels (de Boer et al., 1992 Immunopharmacoloav 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 suflra). In animal models, injection of TNF-a has been shown to induce shock-Pike symptoms similar to those induced by LPS injection (Beutler et al:, 1985 Science 229, 869-871); in contrast, injection of tL-1 C3, IL-6, or IL-8 does not induce shock, Injection of TNF-a also causes an elevation of IL-1 f3, IL-6, IL-8, PgE2, acute phase proteins, and TxA2 in the serum of experimental animals (de Boer et al., 1992 su ra). In animal-models the lethal effects of LPS can be blocked by pre-administration 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 toss 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-1 a and IL-1 t3, iL-fi, GM-CSF, and TGF-13 (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 pro-inflammatory cytokines detected in vivo. Addition of antisera against TNF-a 5 to these cultures has been shown to reduce IL-ia!(3 production by these cells to undetectable levels (Abney et al., 1991 a r ). Thus, TNF-a may directly induce the production of other cytokines in the RA joint. Addition of the anti-inflammatory cytokine, TGF-f3, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical 10 specimens clearly demonstrate the production of TNF-a, IL-ia/f~, and IL-6 from macrophages near the cartilage/pannus junction when the pannus in invading and overgrowing the cartilage (Chu et al., 1992 Br. ,~
Rheumatofoav 31, 653-661). GM-CSF was shown to be produced mainly by vascular endothelium in these samples. Both TNF-a and TGF-f3 have 15 been shown to be fibroblast growth factors, and may contribute to the accumulation of scar tissue in the RA joint. TNF-a has also been shown to increase osteociast 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 ll, lL-1a!(3, II-fi, 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 25 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. ci. USA 89, 9784-9788). In addition; a study of RA
patients who have received i.v, infusions of anti-TNF-a monoclonal 30 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 35 keratinocyte hyperproliferation and immune cell infiltrate (Kupper, 1990 ,j~, Clin. Invest. 86, 1783-1789). It is a fairly common condition, affecting 1.5-2.0% 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 T-lymphocytes are activated CD4+ cells of the TH-1 phenotype, although some CD8+ and CD4-/CD8- are also present. B lymphocytes are typically not found in abundance in psoriatic plaques.
Numerous hypotheses have been offered as to the proximal cause of psoriasis including auto-antibodies and auto-reactive T-cells, overproduction of growth factors, and genetic predisposition. Although there is evidence to support the involvement of each of these factors in psoriasis, they are neither mutually exclusive nor are any of them necessary and sufficient for the pathogenesis of psoriasis (Reeves, 1991 ~emin. Dermatol. 10, 217).
The role of cytokines in the pathogenesis of psoriasis has been investigated. Among those cytokines found to be abnormally expressed were TGF-a , IL-1 a, IL-1 f3, IL-1 ra, ll-6, IL-8, IFN-y, and TNF-a . In addition to abnormal cytokine production, elevated expression of ICAM-1, SLAM-1, and VCAM has been observed (Reeves, 1991 su ra . This cytokine profile is similar to that of normal wound healing, with the notable exception that cytokine levels subside upon heating. 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 (Oxhoim et al., 1991 APMI 99, 58-64).
Nickoloff et al., 1993 (J Dermatol Sci. 6, 127-33) have proposed the following model for the initiation and maintenance of the psoriatic plaque:
Tissue damage induces the wound healing response in the skin.
Keratinocytes secrete IL-1 a, IL-1 t3, 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 keratinocyte-expressed ICAM-1 and MHC class Il. IFN-y secreted by the T-cells synergizes with the TNF-a from dermal dendrocytes to increase keratinocyte proliferation and the levels of TGF-a, IL-8, and IL-6 production.
1FN-y also feeds back to the dermal dendrocyte, maintaining the activated phenotype and the inflammatory cycle.
Elevated serum titres of IL-6 increases synthesis of acute phase proteins including complement factors by the liver, and antibody production by plasma cells. Increased complement and antibody levels increases the probability of autoimmune reactions.
Maintenance of the psoriatic plaque requires continued expression of all of these processes, but attractive points of therapeutic intervention are TNF-a expression by the dermal dendrocyte to maintain activated endothelium and keratinocytes, and 1FN-'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), FSORCON
(diflorasone diac~etate) 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 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 TNF-a and TNF-fi levels, hypergammaglobulinemia, and lymphoma/leukemia (Rosenberg & Fauci, 1990 Immun. Todav 11, 176; Weiss 1993 cienc 260, 1273). Many patients experience a unique tumor, Kaposi's sarcoma and/or unusual opportunistic infections (e.g. Pneumocystis carinii, cytomegalovirus, herpesviruses, hepatitis viruses, papilloma viruses, and tuberculosis). The immunological dysfunction of individuals with AIDS
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 rnonocytes 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 . Vir I, 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 TNF-a compared to those not given zidovudine (Cremoni et al., 1993 AID 7, 128). This correlation lends support to the hypothesis that reduced viral 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.
Chronic elevation of TNF-a has been shown to shown to result in cachexia (Tracey et al., 1992 Am. J. Trop. Med. Hvo. 47, 2-7), increased autoimrnune disease (Jacob, 1992 supra), lethargy, and immune suppression in animal models (Aderka et al., 1992 Isr. J. Med. Sci. 28, 126-130). The cachexia associated with AIDS may be associated with chronically elevated TNF-a frequently observed in AIDS patients.
Similarly, TNF-a can stimulate the proliferation of spindle cells isolated from Kaposi's sarcoma lesions of AIDS patients (Barillari et al., 1992 ~
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.

50 ' ' 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 months would be expected (B.J. 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 traps-epidermal diffusion .
Organ culture systems for biopsy specimens of psoriatic and normal skin are described in current literature (Nickoloff et al., 1993 a ra).
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 vectors in terminally differentiated cells is longer in neonatal or immune-compromised 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 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-a I
Chronic myelogenous leukemia exhibits a characteristic disease 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 (i.e., 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.c~,~
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).
he 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 10-25% of all cases of acute lymphoblastic leukemia [(ALL); Fourth International Workshop on Chromosomes in Leukemia 1982, Cancer Genet. Cytogenet. 11, 316J. In virtually all Ph-positive CMLs and approximately 50% of the Ph-positive ALLs, the leukemic cells express bcr-abI fusion mRNAs in which axon 2 (b2-a2 junction) or axon 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;
Shtiveiman et al., 1987, 8100 69, 971). In the remaining: cases of Ph-positive 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 ai., 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; Weisterkamp et al., 1990 Nature 344, 251). The importance of the bcr abl fusion protein (p210bcr-abl) in the evolution and maintenance of the leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p2'lObcr-abl expression. These inhibitory molecules have been shown to inhibit the in vitro proliferation of leukemic cells in bone marrow from CML patierits. Szczylik et al., 1991 ci n a 253, 562).
Reddy, U.S. Patent 5,246,921 describes use of ribozymes as therapeutic agents for leukemias, such as chronic myelogenous leukemia (CML) by targeting the specific junction region of bcr-abl fusion transcripts. It indicates causing cleavage by a ribozyme at or near the breakpoint of such a hybrid chromosome, specifically it includes cleavage at the sequence GUX, where X is A, U or G. The one example presented is to cleave the sequence 5' AGC AG
AGUU (cleavage site) CAA AAGCCCU-3'.
Scanlon WO 91/18625, WO 91/18624, and WO 91/18913 and Snyder et al., W093/03141 and W094113793 describe a ribozyme effective to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to ex3emal 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 iLn viv administration to reduce the tumor burden, or ex v- ivo treatment to 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 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 et al., 1992 supra) is an in vitro transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments. This limits the utility of this approach. 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 p210bcr-abl expression and can be used to treat disease or diagnose such disease.
Ribozymes will be delivered to cells in culture and to tissues in animal models of CML. Ribozyme cleavage of bcrlabl mRNA in these systems may prevent or alleviate disease symptoms or conditions.
The sequence of human bcrlabl mRNA can be screened for accessible sites using a computer folding algorithm. Regions of the mRNA
that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Table 29 (All sequences are 5' to 3' in the tables). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.
The sequences of the chemically synthesized ribozymes most useful in this study are shown in Table 30. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table 30 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 30 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the 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-abi 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 compfexing with cationic lipids, by microinjection, .or by expression from DNA vectors. Expression of bcr-abl is monitored by EL1SA, by indirect immunofluoresence, and/or by FACS analysis. Levels of bcr-abI 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-abl) protein and mRNA by more than 20% are identified.
5 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 10 under the genus Pneumovirus (for a review see Mclntosh and Chanock, 1990 in Virology ed. B.N. Fields, pp. 1045, Raven Press Ltd. NY). The infectious virus particle is composed of a nucleocapsid enclosed within an envelope. The nucleocapsid is composed of a linear negative single-stranded 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 nrn in diameter. The complete life cycle of RSV takes place in the cytoplasm of infected cells and the nucleocapsid never reaches the nuclear 20 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)]
25 found in the inner membrane, three proteins localized in the nucleocapsid (N, P and L), one protein that is present on the surface of the infected cell (SH), and two nonstructural proteins [NS1 (1C) and NS2 (1B)] found only in the infected cell. The mRNAs for the 10 RSV proteins have similar 5' and 3' ends. UV-inactivation studies suggest that a single promoter is used 30 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 1 C, 1 B, 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 1 C and 1 B mRNAs are 35 much more abundant than the L mRNA. Synthesis of viral message begins immediately after RSV infection of cells and reaches a maximum at 14 hours post-infection (Mclntosh and Chanock, supra).
There are two antigenic subgroups of RSV; A and B, which can circulate simultaneously in the community in varying proportions in different years (Mclntosh and Chanock, supra). Subgroup A usually predominates.
Within the two subgroups there are numerous strains. By the limited sequence analysis available it seems that homology at the nucleotide level is more complete within than between subgroups, although sequence divergence has been noted within subgroups as well. Antigenic determinates result primarily from both surface glycoproteins, F and G. For F, at least half of the neutralization epitopes have been stably maintained over a period of 30 years. For G however, A and B subgroups may be related antigenically by as little as a few percent. On the nucleotide level, however, the majority of the divergence in the coding region of G is found in the sequence for the extracellular domain (Johnson et al., 1987, Proc.
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 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., NY.]. 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 naed 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 polymerise 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 NS7 (7C), NS2 (7B) and N viral genes.
These genes are known in the art (for a review see Mclntosh and Chanock, 1990 supra ).
Ribozymes that cleave the specified sites in RSV mRNAs represent a novel therapeutic approach to respiratory disorders. Applicant indicates that ribozymes are able to inhibit the activity of RSV and that the catalytic activity of the ribozymes is required for Their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in RSV mRNAs encoding 1C, 1 B 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 (P, M, SH, G, F, 22K and L) and the genomic RNA may be readily designed and are within the invention.
In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 31, 33, 35, 37 and 38.
Examples of such ribozymes are shown in Tables 32, 34, 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
Ribozymes of this invention block to some extent RSV production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of respiratory disorders. Ribozyme cleavage of RSV encoded mRNAs or the genomic RNA in these systems may alleviate disease symptoms.

While all ten RSV encoded proteins (1 C, 1 B, 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, 1 B, 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.
The sequence of human RSV mRNAs encoding 1 C, 1 B 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 >98%. Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 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 Enzymoi. 180, 51 ). All ribozymes are modified extensively to enhance stability by modification with nuclease . resistant so 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 hammerhead ribozymes listed in Tables 32 and 34(5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 37 and 38 (5'-CACGUUGUG-3') can be altered (substitution, deletion, andlor 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 anima! 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 1 C, 1 B and N protein encoding mRNAs by more than 90% will be identified.

Oatimizin Rq ibozyme Activity Ribozyme activity can be optimized as described by Draper et al., PCT
W093I23569. 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 e.g., Eck~tein et aG, International Publication No.
WO 92/07065; Perrault ef aL, 1990 Na_ furs 344, 565; Pieken et al., 1991 cienc 253, 314; Usman and Cedergren, 1992 Trends in Biochem Sci 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA
molecules, 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, 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 iontephoresis, 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 stmt.
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., su ra, 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 polymerise I (pol I), RNA poiymerase II
(pol ll), or RNA polymerise 111 (pol III). Transcripts from pol II or pol Ill promoters ,will be expressed at high levels in all cells; the levels of a given .
pol If promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.~ present nearby.
Prokaryotic RNA polymerise promoters are also used, providing that the prokaryotic RNA polymerise enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 proc. Natl. Acid. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev , 2, 3-15; Ojwang et al., 1992 Proc. Natl.
Acid. Sci. U S A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res_., 20, 4581-9; Yu et al., 1993 Proc: Natl. Acid. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992 EM8 . 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl, Acad.
Sci. U. S. A., 90, 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA
vectors (such as retroviral, or alpha virus vectors).
In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves target RNA is inserted into a plasmid DNA vector, a retrovirus DNA viral vector, an adenovirus DNA viral vector or an adeno-associated virus vector or alpha virus vector. These and other vectors have been used to transfer genes to live animals (for a review see Friedman, 1989 cienc 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, DNAlvehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, e.g., 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. 8y using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e~a., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes andlor 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-t, relA, TNF-a, p210, bcr-abl or RSV related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and. one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA
whose protein product is implicated in the development of the phenotype (i.e., 1CAM-1, rel A, TNF~, p210bcr-abl or RSV) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will ' decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
ii. Chemical Synthesis Of Ribozyrmes_, 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.
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 far 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 (l.e., 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 NH3lEtOH
(ethanolic ammonia) for 20 h at 65 °C. 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 fi5 accomplished by a two-step chromatographic procedure in which the molecule is first purified on a reverse phase column with either the trity) 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 oh an anion exchange column, using alkali metal perchlorate salt gradients to elute the fully purified RNA molecule as the appropriate metal salts, e.g. Na+, Li+ etc. A
final de-salting step on a small reverse-phase cartridge completes the purification procedure. Applicant has found that such a procedure not only fails to adversely affect activity of a ribozyme, but may improve its activity to cleave target RNA molecules.
Applicant has also determined that significant (see Tables 39-41) improvements in the yield of desired full length product (FLP) can be obtained by:
1. Using 5-S-alkyltetrazole at a delivered or effective concentration of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. (By delivered is meant that the 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 wilt recognize the meaning of these terms from the examples provided herein.) The time for this step is shortened from 10-15 rn, 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 groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S,, NOp or N(CH3)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 fib 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =0, =S, N02, halogen, N(CH3)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 groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated n electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazoiyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', 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, propy! or butyl) or NH40Hlalkylamine (AMA, with the same preferred alkyl groups as noted for MA) ~ 65 °C for 10-15 m to remove the exocyclic s~
amino protecting groups (vs 4-20 h C~ 55-65 °C using NH4OH/EtOH or NH3/EtOH, vide supra). Other alkylamines, e.g. ethylamine, .propyiamine, .
butylamine etc. may also be used.
4. Using anhydrous triethylamine~hydrogen fluoride (aHF~TEA) Q 65 °C fot 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.
5. The use of anion-exctlange 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 phosphorarnidites; 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 (e.g., with substituted 2'-groups), and allow efficient synthesis of large amounts of such RNA. Such RNA may also have enzymatic .activity and be purified without loss of that activity. While specific examples .are given herein, 3hose in the art will recognize that equivalent chemical reactions can be pertormed with the alternative chemicals noted above, which can be optimized and selected by routine experimentation.
fn another aspect, the invention features an improved method for the purification or analysis of RNA or en2ymatic RNA molecules (e.g. 28-70 nucleotides in length) by passing said RNA or enzymatic RNA molecule over an ~HPLC, e.g., 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 10).
Draper et al., PCT W093/23569, disclosed reverse phase HPLC purification. The purification of.long RNA
molecules may be accomplished using anion exchange chromatography, particularty in conjunction with alkali perchlorate salts. This system may be used to purify very long RNA~molecules. 1n particular, it is advantageous to 68 ' 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 saif of the RNA molecule during the purification.
In the case of the 2-step purifiication 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 5'-trityl-off method. Either molecule may be recovered using this reverse-phase 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 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 HPIC 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 ~, preferably 300 ~, and a particle size of at least 2 pm, preferably 5 Vim.
Activation The synthesis of RNA molecules may be accomplished chemically or enzymatically. In the case of chemical. synthesis the use of letrazole as an activator of RNA phosphorarnidites 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 5 min gives equivalent or bEtter results. The following exemplifies the procedure.
Example 7: Synthesis of RNA and Ribozymes Using 5-S-Alkyltetrazoles as Activating Aoent 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 5'-end, and phosphoramidites at the 3'-end. The major difference used was the activating agent, 5-S-ethyl or -methyftetrazole ~ 0.25 M
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 2'-O-rnethylated RNA. A 6.5-fold excess (162.5 ~L of 0.1 M = 32.5 pmol) of phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 ~L of 0.25 M = 100 umol) 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 anhydridell0% 2,6-lutidine in THF;
oxidation solution was 16.9 mM i2, 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 3.0 the solid obtained from Applied Biosystems. ~ .
All large scale syntheses were conducted on a modified (eight amidite port capacity) 3902 (ABI) synthesizer using a 25 umol scale protocol with a 5-15 min coupling step for alkylsilyf protected RNA and 7.5 m coupling step for 2'-Qmethylated RNA. A six-fold excess (1.5 mL of 0.1 M : 150 pmol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole ~{4.5 mL of 7~
0.25 M = 1125 ~mol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 3902, determined by colorimetric quantitation of the trityl fractions, was 95.0-96.7%.
Oligonucleotide synthesis reagents for the 3902: Detritylation solution was 2% DCA in methylene chloride; capping was performed with 1fi% 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°I° water in THF.
Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25-0.5 M in acelonitrile) was made up from the solid obtained from Applied 8iosystems.
Dearotection The first step of the deprotection of RNA molecules may be accomplished by removal of the exocyclic amino protecting groups with either NHQOH/EtOH:3/1 (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845-7854} or NH3/EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341 ) for -20 h ~ 55-65 °C. Applicant has determined that the use of methylamine or NH40H/methylamine for 10-15 min C~? 55-65 °C gives equivalent or better results. The following exemplifies the procedure.
Examt~le 8: RNA and Ribozvme Deorotection of Exocyclic Amino Protecting Groups Using Methylamine (MAl or NHAOH/Methylamine ~,."AMAI
The polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or NH4OH/methylamine (AMA) C~? 55-65 °C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oiigoribonucleotide was transferred from the synthesis column to a 4 mL glass screw top vial. NH40H 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 55 or 65 °C for 5-15 min. After cooling to -20 °C, the supernatant was removed from the polymer support. The support was washed with 1.0 mL
of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The same procedure was followed for the aqueous methylamine reagent.
Table 40 is a summary of the results obtained using the improvements outlined in this application for base deprotection.

The second step of the deprotection of RNA molecules may be accomplished by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman et al. J. Am. Chem. Soc: 1987, 709, 7845-7854). Applicant has determined that the use of anhydrous TEA~HF in N-methylpyrrolidine (NMP) for 0.5-1.5 h C~3 55-65 °C gives equivalent or better results. The following exemplifies this procedure.
Example 9; RNA and Ribozyme Deorotection of 2'-Hydroxyl Alkvlsilvl Protectina Groups Using, Anhydrous TEA~HF
To remove the alkylsilyi protecting groups, the ammonia-deprotected oligoribonucleotide was resuspended in 250 pL of 1.4 M anhydrous HF
solution (1.5 mL N-methylpyrrolidine, 750 uL 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 Gaiagen 500~ anion exchange cartridge (Oiagen Inc.) prewashed with 10 mL of 50 mM TEAB.
After washing the cartridge with 10 mL of 50 mM TEAB, the RNA was eluted with 10 mL of 2 M TEAR and dried down to a white powder.
Table 41 is a summary of the results obtained using the improvements outlined in this application for alkylsilyl deprotection.
Example 10: HPLC Purification. Anion Exchan qe 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 NaCl04). A gradient from 180-210 mM NaCl04 at a rate of 0.85 mMlvoid volume for a Pharmacia Mono Q~ anion-exchange column or 100-150 mM NaCl04 at a rate of 1.7 mMlvoid volume for a Dionex NucIeoPac~ anion-exchange column was used to elute the RNA.
Fractions were analyzed by~ a HP-1090 HPLC with a Dionex NucIeoPac~
column. Fractions containing full length product at >_80% by peak area were pooled.
For a trityi-ofif 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 HiLoa~d 26!10 Q-Sepharose~ Fast Flow column. The column was thoroughly washed with 10 mM sodium perchlorate bufifer. The oligonucleotide was eluted from the column with 300 rnM 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 H20 to lower the salt concentration and applied to a Pharmacia Mono D~ 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 NucIeoPac~ column. Fractions containing over 85% full length material were pooled. The pool was applied to a Pharmacia RPC~ column for desalting.
For a trityl-on large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26!10 Q-Sepharose~ Fast flow column. The column was thoroughly washed with 20 mM NH~C03H/10%
CH3CN buffer. The oligonucleotide was eluted from the column with ~1.5 M
NH~C03H/10% acetonitrile. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material present in the synthesis. The oligonucleotide was then applied to a Pharmacia Resource RPC column. A gradient from 20-55°!° B (20 mM NH4C03H125%
CH3CN, buffer A = 2D mM NH4C03H/10% CH3CN) 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 H20, dried down and resuspended in H20 again. This material was analyzed on a HP 1090-HPLC with a Dionex NucIeoPac~ coturnn. The material was purified by anion exchange chromatography as in the trityl-off scheme (vide supra).
Example 11 Ribozyme Activity Assav_ Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-end labeled ribozymes (,36-mers) were both heated to 95 °C, quenched on ice and equilibrated at 37 °C, eparately. RiboZyme stock solutions were t uM, 200 nM, 40 nM or 8 nM and the final substrate RNA
concentrations were - 1 nM. Total reaction volumes were 50 ~L. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCl2. Reactions were initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 pL 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 15%
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 (e.g., 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 depratection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol (Fig. 12). According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 min and the reaction is allowed to coot for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA~3HF reagent.
The reaction is quenched with 16 mM TEAB solution.
Referring to Fia. 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 F_ ig. 14, hammerhead ribozymes targeted io site B (from Fi . 13 are tested far 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 l2a:lmproved protocol for the synthesis of hosphorothioate containina RNA and ribozymes using 5-S-Alkyltetrazoles as Activating A_4ent .
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-3-one 1,1-dioxide (Beaucage reagent; Vu and Hirschbein, 1991 supra).
TETD requires long sulfurization times (600 seconds for DNA and 3600 seconds for RNA). It has recently been shown that for sulfurization of DNA
oligonucleotides, ~ Beaucage reagent is more efficient than TETD
(Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med. Chem. 4, 1519).
Beaucage reagent has also been used to synthesize phosphorothioate oligonucleotides containing 2'-deoxy-2'-fluoro modifications wherein the wait time is 10 min (Kawasaki et al., 1992 J. Med Chem).
The method of synthesis used follows the procedure for RNA
synthesis as described herein and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The sulfurization step for RNA described in the literature is a 8 second delivery and 10 ruin wait steps (Beaucage and lyer, 1991 Tetrahedron 49, fi123). These conditions produced about 95% sulfurization as measured by HPLC analysis (Morvan et al., 1990 Tetrahedron Letter 31, 7149). This 5% contaminating oxidation could arise from the presence of oxygen dissolved in solvents andlor 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, 5-S-ethyitetrazole 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 wish a 0.05 M solution of 3H-1,2-benzodithiote-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 . (ABL) synthesizer using a modified 2.5 pmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 pl of 0.1 M = 32.5 pmol) of phosphoramidite and a 40-fold excess of S--ethyl tetrazole (400 ~L of 0.25 M = 100 ~mol) 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 trity) fractions, was 97.5-99%. Other oligonucleotide 5 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-futidine iri 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 10 bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems. Sulfurizing reagent was obtained from Glen Research.
Average sulfurization efficiency (ASE) is determined using the formula: ASE = (PSJTotal)1~n-1 15 where, PS = integrated 31 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 20 data suggest that 5 second wait time and 300 second delivery time is t'he condition under which ASE is maximum.
Using the above conditions a 36 mer hammerhead ribozyme is synthesized which is targeted to site C. The ribozyme is synthesized to contain phosphorothioate linkages at four positions towards the 5' end.
25 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.
Examofe 13' Protocol for the synthesis of 2'-N-phtalimido nucleosidg ~hosphoramidite .
30 The 2'-amino group of a 2'-deoxy-2'-amino nucleoside is normally protected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supra,; Pieken et al., 1991 Science 253,. 314). This protecting group is not stable in CH3CN solution or even in dry form during 76 ' 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 i7.
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 (15), dimethoxytrytilation (16) and phosphitylation led to phosphoramidite 17.
Since overall yield of this multi-step procedure was low (20%) 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 equivalents of Nefkens reagent in DMF overnight with subsequent treatment with Et3N (1 hour) only 10-15°!° of N and 5'(3')-bis-phtatoyl derivatives were formed with the major component being N-Pht-derivative 15. The N,0-bis by-products could be selectively and quantitively converted to N-Pht derivative 15 by treatment of crude reaction mixture with cat. KCN/MeOH.
A convenient "one-.pot° procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCIlEt3N 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 mls 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 N-carbethaxyphthalimide (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 CHC13) and 57 pl of-TEA (0.1 eq.) was added to effect closure of the phthalimide ring. After 1 hour an additional 855 ~I (1.5 eq.) of TEA was added followed by the addition of 1.53 grams (1.1 eq.) of DMT-C!

(Lancaster SynthesisC3~, 98°!°}. The reaction mixture was left to stir overnight and quenched with ETOH after TLC showed greater than 90°!°
desired product. Dmf was removed under vacuum and the mixture was washed with sodium bicarbonate solution (5°!° aq., 500 mls) and extracted with ethyl acetate (2x 200 mls). A 25mm x 300mm flash column (75 grams Merck flash silica) was used for purification. Compound eluted at 80 to 85°l° ethyl acetate in hexanes (yield: 80% , purity:
>95°!° by ~ H N M R).
Phosphoramidites were then prepared using standard protocols described above.
With phosphoramidite 17 in hand applicant synthesized several ribozymes with 2'-deoxy-2'-amino modifications. Analysis of the synthesis demonstrated coupling efficiency in 97-98% range. RNA cleavage activity of ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/or U7 positions (see Figure 1), 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 base-composition analysis. The coupling efficiency of phosphoramidite 17 was not effected over prolonged storage {1-2 months) at low temperatures.
Protecting 2' Position with a SEM Group There follows a method using the 2'-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base deprotection. At the same time, this group should also be readily removed when desired. To that end the t-butyldimethylsily) group has been efficacious (Usman,N.;
Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 709, 7845-7854 and Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.
1990, 18, 5433-5441). However, long exposure times to tetra-n-butylammonium 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, ?8 78, 5433-5441 and Stawinski,J.; Stromberg,R.; Thelin,M.; Westman,E.
Nucleic Acids.Res. 1988, 76, 9285-9298).
The (trimethylsilyl)ethoxymethyl ether (SEM) seems a suitable substitute. This protecting group is stable to base and alt 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 BF3~OEt2 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 various positions by methods well known in the art, e.g., as described by Eckstein et aL, International Publication No. WO 92!07065, Perrault et al., Nature 1990, 344, 565-568, Pieken et al., Science 1991, 253, 314-317, Usman,N.; Cedergren,R.J. Trends in Biochem. Sci. 1992, 17, 334-339, Usman et al., PCT W093/15187, and Sproat,B. European Patent Application 92710298.4 .
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 (BF3~OEt2) under SEM removing conditions, e.g., in acetonitrile.
Referring to Fic_ur~ e~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 (R) at the 2'-position in prior art methods can be a silyl ether, as shown in the Figure. 1n the method of_ the present invention, an SEM group is used in place of the silyl ether.
Otherwise RNA synthesis can be performed by standard methodology.
Referring to Figure 19, there is shown the synthesis of 2'-O-SEM
protected nucleosides and phosphoramadites. Briefly, a 5'-protected nucleoside (1 ) is protected at the 2'- or 3'-position by contacting with a derivative of SEM under appropriate conditions. Specifically, those conditions include contacting the nucleoside with dibutyltin oxide and SEM
chloride. The 2 regioisomers are separated by chromatography and the 2'-protected moiety is converted into a phosphoramidite by standard procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.
Referring to Fioure 20, a prior art method for deprotection of RNA using silyl ethers is shown. This contrasts with the method shown in Figure 21 in 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 Examofe 14: Synthesis of 2'-O-((trimethylsilyl}ethoxymethyll-5'-O- Di-methoxytrityl Uridine (21 Referring to Figure 19, 5'-D-dimethoxytrityl uridine 1 (1.0 g, 1.83 mmol) in CH3CN (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°C) at which time (trimethylsilyl)ethoxymethyl chloride (SEM-CI} (487 pL, 2.75 mmol) was added. The reaction mixture was stirred overnight and then filtered and evaporated. Flash chromatography (30%
hexanes in ethyl acetate) yielded 347 mg (28.0%} of 2'-hydroxyl protected nucleoside 2 and 314 mg {25.3%) of 3'-hydroxyl protected nucleoside 3.
Exam le 15: nthesis of 2'- trimeth Isil I ethox m h ridin 4 Nucleoside 2 was detritylated following standard methods, as shown in Fi r 1 Example 16: Synthesis of 2'-D-((trimethylsilyllethoxymethyll-5' 3'-O-Acetyl Uridi Nucleoside 4 was acetylated following standard methods, as shown in Fi ure 1 .
5 Example 17: Synthesis of 5'.3'-D-Acet~rl Uridinel6l Referring to Figure 19. the fully protected uridine 5 (32 mg, 0.07 mmol) was dissolved in CH3CN (700 pL) and BFg~OEtp (17.5 ~tL, 0.14 mmol) was added. The reaction was stirred 15 m and MeOH was added to quench the reaction. Flash chromatography (5°l° MeOH in CH2C12) gave 10 20 mg (88°I°) of SEM deprotected nucleoside 6.
Example 18: Synthesis of 2'-D-((trimethylsily~etho~methy~-3'-Q
Succinyl-5'-O- Dimethox~rtrityl l~ridine~21 Nucleoside 3 was succinylated and coupled to the support following standard procedures, as shown in Figure 19.
15 Example 19: Synthesis of 2'-O-((trimethylsilyl ethoxymethyl -5~Di-methoxytrityl Uridine 3'-(2-Cyanoethyl N N-diisopropylphosphoramiditel i~1 Nucleoside 3 was phosphitylated following standard methods, as shown in Fi ur 1 20 Example 20: Synthesis of RNA Using 2'-O-SEM Protection Referring to Figure 18, the method of synthesis used follows the general procedure for RNA synthesis as described in Usman,N.;
Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 709, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.
25 ~ 1990, 78, 5433-5441. The phosphoramidite 8 was coupled following standard RNA methods to. provide a 10-mer of uridylic acid. Syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 pmol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 pL of 0.1 M = 32.5 pmol) of phosphoramidite and a 80-fold excess of tetrazole 30 (400 pL of 0.5 M = 200 ~moi) 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: Detrityiation 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 15.9 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 NH3/EtOH at 65 °C. 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 CH3CN (1 mL). BF3~OEt2 (2.5 ~L, 30 umol) was added to the solution and aliquots were removed at ten time points. The results indicate that after 30 min deprotection is complete, as shown in Figure 22.
111. Vectors Expressing Ribozymes There follows a method fior expression of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a decreased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful for scaling up production of a ribozyme, which may be either modified or 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, trans-acting or desired ribozyme instead of processing only one end, or only one ribozyme. This allows smaller vectors to be derived with multiple trans-acting 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 t7raper et al., PCT WO 93!23509.
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 isolation techniques currently used to purify chemically synthesized oligonucleotides. Thus, the method allows synthesis of ribozymes in high yield at low cost for analytical, diagnostic, or therapeutic applications.
The method i5 also useful for synthesis of ribozymes in vifro for ribozyme structural studies, enzymatic studies, target RNA accessibility studies, transcription inhibition studies and nuclease protection studies, much is described by Draper et al., PCT WO 93/23509.
The method can also be used to produce ribozymes in situ either to increase the intracellular concentration of a desired therapeutic ribozyme, or to produce a concatarneric transcript for subsequent in vitro isolation of unit ,length ribozyme. The desired ~ibozyme can be used to inhibit gene expression in molecular genetic analyses or in infectious cell systems, and to test the efificacy 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 double-stranded, partially double-stranded or single-stranded RNA, formed by sife-directed homologous recombination into viral or viroid RNA genomes.
Such vectors..need not be circular. Transcriptionally linked to the promoter region is a first ribozyme-encoding region, and nucleotide sequences encoding a ribozyme cleavage sequence which is placed on either side of a region encoding a therapeutic or otherwise desired second ribozyme.

Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA expression system. The desired second ribozyme may be any desired type of ribozyme, such as a hammerhead, hairpin , hepatitis delta virus (HDV) or other catalytic center, and can include group 1 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-recognition sequence to aid in vector construction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector.
When the promoter of such a vector is activated an RNA transcript is produced which includes the first and second ribozyme sequences. The first ribozyme sequence is able to act, under appropriate conditions, to cause cleavage at the cleavage sites to release the second ribozyme sequences. These second ribozyme sequences can then act at their target RNA sites, or can be isolated for later use or analysis.
Thus, in one aspect the invention features a vector which includes a first nucleic acid sequence (encoding a first ribozyme having intramolecular cleaving activity), and a second nucleic acid sequence (encoding a second ribozyme having intermolecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA
transcript encoded by the vector. The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have . intermolecular cleaving activity.
As discussed above, the first ribozyme can be chosen to be any self-cleaving 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 prefierred embodiments, the vector includes a plurality of the nucleic .
acid sequences encoding the second ribozyme, each flanked by nucleic acid sequences recognized by the first ribozyme. Most preferably, such a plurality includes at least six to nine or even between 60 - 100 nucleic acid sequences. In other preferred embodiments, the vector includes a promoter which regulates expression of the nucleic acid encoding the ribozymes from the vector; and the vector' is chosen from a plasmid, cosmid, phagmid, virus, viroid or phage. In a most preferred embodiment, the plurality of nucleic acid sequences are identical and are arranged in sequential order such that each has an identical end nearest to the promoter. If desired, a poly(A) sequence adjacent to the sequence encoding the first or second ribozyme may be provided to increase stability of the RNA produced by the vector; and a restriction endonuclease site adjacent to the nucleic acid encoding the first ribozyme is provided to atfow insertion of nucleic acid encoding the second ribozyme during construction of the vector.
In a second aspect, the invention features a method for formation of a ribozyme expression vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a single-stranded 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 polymerise 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 ' ~ 85 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. A11 three ribozyme motifs self-process 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 su r ;
and Altschuler et al., 1992 .G_ene 122, 85. The present invention enables the use of cis-cleaving ribozymes to efficiently truncate RNA molecules at specific sites in vivo by ensuring lack of secondary structure which prevents processing.
fsoiation of Therapeutic RiboZ~rme The preferred method' of isolating therapeutic ribozyme is by a chromatographic technique. The HPLC purification methods and reverse HPLC purification methods described by Draper et al., PCT WO 93/23509.
can be used. Alternatively, the attachment of complementary oligonucleotides to cellulose or other chromatography columns allows isolation of the therapeutic second ribozyme, for example, by hybridization to the region between the flanking arms and the enzymatic~RNA. This hybridization will select against.the short flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybridization can be accomplished in the presence of a chaotropic agent to prevent nuclease degradation. The oligonucleotides on the matrix can be modified to minimize .nuclease activity, for example, by provision of 2'-O-methyl RNA oligonucleotides.
Such modifications of the oligonucleotide attached to the column matrix will allow the multiple use of the column with minimal oligo degradation. Many such modifications a.re known in the art; but a chemically stable non-reducible modification is preferred. For example, phosphorothioate modifications can also be used. w 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 Teirahymena can be used in an alternative vector of this invention. If desired, the full-length as 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 ribozyme-encoding 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 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, to allow a timed expression of the therapeutic second ribozyme, as well as an appropriate shut off of cell or gene function. Thus, the vector wilt include a promoter which appropriately expresses enzymatically active RNA only in the presence of an RNA or another molecule which indicates the presence of an undesired organism or state. Such enzymatically active RNA wiH 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: Desi4n of self-processing cassettes In a preferred embodiment, applicant compared the in vitro and in vivo cis-cleaving activity of threedifferent ribozyme motifs-the hammerhead, the hairpin and the hepatitis delta virus ribozyme-in order to assess their potential to process the ends of transcripts in vivo. To make a direct comparison among the three, however, it is important to design the ribozyme-containing transcripts to' be as similar as possible. To this end, all the ribozyme cassettes contained the same trans-acting hammerhead ribozyme followed immediately by one of the three cis-acting ribozymes (Fioure 23-25). For simplicity, applicant refers to each cassette by an abbreviation that indicates the downstream cis-cleaving ribozyme only.
Thus HH refers to the cis-cleaving cassette containing a hammerhead ribozyme, while HP and HDV refer to the cassettes containing hairpin and hepatitis delta virus cis-cleaving ribozymes, respectively. The general design of the ribozyme cassettes, as well as specific differences among the cassettes, are outlined below.
A sequence predicted to form a stable stem-loop structure is included at the 5' end of all the transcripts. The hairpin stem contains the T7 RNA
polymerise initiation sequence (Milligan & Uhlenbeck, 1989 Methods Enzymol. 180, 51) and its complement, separated be a stable tetra-loop (Antao et al., 1991 Nucleic Acids Res. 19, 5901). By incorporating the T7 initiation sequence into a stem-loop structure, applicant hoped to avoid nonproductive base pairing interactions with either the traps-acting ribozyme or with the cis-acting ribozyme. The presence of a hairpin at the end of a transcript may also contribute to the stability of the transcript in vivo. These are non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using a variety of promoters, initiator sequences and stem-loop structure combinations generally known in the art.
The traps-acting ribozyme used in this study is targeted to a site B
(5'~~~CUGGAGU_C~GACCUUC~-~3'). The 5' binding arm of the ribozyme, 5'-GAAGGUC-3', and the core of the ribozyme, 5'-CUGAUGAGGCCGAAAGGCCGAA-3', remain constant in all cases. In addition, all transcripts also contain a single nucleotide between the 5' 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 Packing 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 traps- and cis-acting ribozyme is; however, designed so that there is minimal extraneous sequence left at the 3' end of the trans-cleaving ribozyme once cis-cleavage occurs. The only differences between the constructs lie in the 3' binding arm of the riboZyrne, where either 6 or 7 nucleotides, 5'-ACUCCA(+!-G)-3', complementary to the target sequence are present and where, after processing, two to five extra nucleotides remain.
The cis-cleaving hammerhead ribozyme used in the HH cassette is based on the design of Grosshans and Cech, 1991 su ra. As shown in Figure 23, the 3' binding arm of the traps-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., 1990 supra) which remain on the 3' end of the traps-acting ribozyme following self-processing.
The hairpin ribozyme portion of the HP self-processing construct is based on the minima! wild-type sequence (Hampel & Tritz, 1989 su ra). A
tetra-loop at the end of helix 1 (3' side of the cleavage site) serves to link the two portions and thus allows a minimal five nucleotides to remain at the end of the released traps-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;
Fioure 4). This slight destabilization of helix 2 was intended to improve self-processing, activity by promoting product release and preventing the reverse reaction (Berzal-Herranz et al., 1992 Genes & Dev. 6, 129;
Chowrira et al., 1993 Biochemistry 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing interactions in helix 2 (U~7C and A52G substitution; Figure 24). Another modification to discourage the reverse ligation reaction of the hairpin ribozyme was to shorten helix 1 (Fioure 24) by one base pair relative to the wild-type sequence (Chowrira & 8urke, 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 (F_ is 25) was designed to generate the traps-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 8~ Been, 1992 su ra) but includes the modifications of Been et al., 1992 (~iochemistw 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 (Fi ur , 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 Fiq_ 261, The single-strand portions of annealed oligonucleotides were converted to doubie-strands using Sequenase~ (U.S. Biochemicals). Insert DNA was ligated into EcoR7lHind111-digested pucl8 and transformed into E. coli strain DHSa 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.
Larger scale preparations of plasmid DNA for use as in vitro transcription templates and in transactions were prepared using the protocol and columns from OIAGEN Inc. (Studio City, CA) except that an additional ethanol precipitation was included as the final step.
Example 22. RNA Processing in vitr,Q
Transcription reactions containing linear plasmid templates were carried out essentially as described (Milligan & Uhlenbeck, 1989 Su r ;
Chowrira 8 Burke, 1991 a ra). In order to prepare 5' end-labeled transcripts, standard transcription reactions were carried out in the presence of 10-20 pCi [7-32P)GTP, 200 pM each NTP and 0.5 to 1 ~g of linearized plasmid template. The concentration of MgCl2 was maintained at 10 mM above the total nucleotide concentration.
To compare the ability of the different ribozyme cassettes to self-process in vitro, each construct was transcribed and allowed to undergo self-processing under identical conditions at 37°C. For these comparisons, equal amounts of linecrized DNA templates bearing the various ribozyme cassettes were transcribed in the presence of ~[Y-32p~GTP to generate 5' end-labeled transcripts. In this manner only the full-length, unprocessed transcripts and the released trans-ribozymes are visualized by autoradiography. In all reactions, Mg2+ was included at 10 mM above the nucleotide concentration so that. cleavage by all the ribozyme cassettes 9~ .
would be supported. Transcription templates were linearized at several positions by digestion with different restriction enzymes so that self-processing in the presence of increasing lengths of downstream sequence could be compared (see Fig. 26). The resulting transcripts have either 4-5 non-ribozyme nucleotides at the 3' end (Hind111-digested template), 220 nucleotides (Ndel digested templates) or 454 nucleotides of downstream sequence (Real digested template). .
As shown in Figure 27, all four ribozyme cassettes are capable of self-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 (Fi . 2 ). 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 traps-ribozyme (Fia~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(GG) is more efficient than the HP(GU) ribozyme, both in the presence and in the absence of extra downstream sequence. In addition, the aclivity 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 (sub-optimal) HP(GU) casserie.

9t Example 23' Kinetics of self-orocessina reaction Hindlll-digested template (250 ng) was used in a standard transcription reaction mixture containing: 50 mM Tris~HCl pH 8.3; 1 mM
ATP, GTP and UTP; 50 ~M CTP; 40 pCi jcc-32P]CTP; 12 mM MgCl2; 10 mM
DTT. The transcription/self-processing reaction was initiated by the addition of T7 RNA polymerise (15 U/pl). Aliquots of 5 ~I 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, 8~
dyes) and freezing on dry ice. The samples were resolved on a 10°!°
pofyacrylamide sequencing gel and results were quantitated by Phosphorlmager*(Molecuiar Dynamics, Sunnyvale, CA). Ribozyme self=
cleavage 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-~) where t represents time and k represents the unimolecular rate constant for cleavage (Long ~ Uhlenbeck, 1994 Proc. Natl. Acid. Sci USA
91, 6977).
Linear templates were prepared by digesting the plasmids with Hindlll so that transcripts will contain only four to five vector-derived nucleotides at the 3' end (see Fioure 23-25). By comparison of the unimolecular rate constant (k) determined for each construct, it is clear that HH is the most efificient at self-processing (Table 44). The HH transcript self-processes 2-fold fester 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 traps-cleavage activity of the hairpin ribozyme (Hampel et al:, 1990 su ra; Chowrira 8~
Burke, 1991 su ra). The rate of HH self-cleavage during transcription measured here (1.2 min-1) is similar to the rate measured by Long and 3'D Uhlenbeck 1994 s_u_pra using a HH that has a different stem I and stem II1.
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 testedafter isolation from a denaturing gel (Been et al., 1992 su ra . This decrease likely reflects the.difference in protocol as well as the presence of 5' flanking sequence in the HDV construct used here.
*Trade-mark 92 ' Example 24: Effect of downstream seauences on tram-cleavage in viirg Transcripts containing the traps ribozyme with or without 3' flanking sequences were assayed for their ability to cleave their target in traps. To this end, transcripts from three templates were resolved on a preparative gel and bands corresponding both to processed traps-acting ribozymes from the HH transcription reaction, and to full-length HH{mutant) and ~HDV
transcripts were isolated. In all three transcripts the traps-acting ribozyme portion is identical-with the exception of sequences at their 3' ends. The HH traps-acting ribozyme contains only an additional UC at its 3' end, 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 withi the three ribozyme transcripts in a standard reaction buffer.
To make internally-labeled substrate RNA for traps-ribozyme 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-32P]CTP (Chowrira 8 Burke, 1991 suflra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel,, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 pi DEPC-treated water and stored at -20°C.
Unlabeled ribozyme (1pM) and internally labeled 622 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris~HCl 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 ul were taken at regular time intervals, quenched by adding an equal volume of 2X forinamide 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 traps-acting ribozyme cleaves the target RNA approximately 10-fold faster than the oHDV transcript and greater than 20-fold faster than the HH(mutant) transcript (Fi ure 2 ). The additional nucleotides at the end of HH(mutant) form 7 base-pairs with the 3' target-binding arm of the traps-acting ribozyme (Fi ure 3). This interaction must be disrupted (at a cost of 6 kcallmole) to make the traps-acting ribozyme available for binding the target sequence.. In contrast, the additional nucleotides at the end of ~HDV were not designed to form any strong, alternative base-pairing with the traps-ribozyme. Nevertheless, the aHDV sequences are predicted to form multiple structures involving the 3' target-binding arm of the traps ribozyme that have stabilities ranging from 1-2 kcallmole. Thus, the observed reductions in activity for the ~HDV 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.
Exam,~le 25: RNA self-processing in vivo Since three of the constructs (HH, HDV and HP(GC)) self-process efficiently in solution, the affect of the mammalian cellular milieu on ribozyme self-processing was next explored by applicant. A transient expression system was employed to investigate ribozyme activity in vivo. A
mouse cell line (OST7-1) that constitutively expresses T7 RNA polymerase in the cytoplasm was chosen for this study (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA 87, 6743). In these cells plasmids containing a ribozyme cassette downstream of the T7 promoter will be transcribed efficiently in the cytoplasm (Elroy-Stein & Moss, 1990 supra).
Monolayers of a mouse L9 fibroblast cell line (OST7-1; Elroy-Stein and Moss, 1990 supra) were growr5 in 6-well plates with - 5x105 cells/virell.
Cells were transfected with circular plasmids (5 pglwell) using the calcium phosphate-DNA precipitation method (Maniatis et al., 1982 su ra). Cells were lysed (4 hours post-transfection) by the addition of standard lysis buffer (200 ullwell) containing 4M guanadinium isothiocyanate, ,25 mM
sodium citrate (pH 7.0), 0.5% sarkosyl (Chomczynski and Sacchi, 1987 Anal. Biochem. 162, 156), and 50 mM EDTA pH 8Ø The lysate was extracted once with water-saturated phenol followed by one extraction -with chloroform:isoamyl alcohol (25:1 ). Total cellular RNA was precipitated with an equal volume of isopropanol. The RNA pellet was resuspended in 0.2 sa 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 Irg/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90°C
for 2 min. in the absence of Mg2+, and then snap-cooling on ice for at least 15 min. This protocol allows for efficient annealing of the primer to its complement~a~ry RNA sequence. The primer was extended using Superscript II reverse transcriptase (8 U/~I; BRL) in a buffer containing 50 mM Tris~HCl pH 8.3; 10 mM DTT; 75 mM KCI; 1 mM MgCl2; i mM each dNTP. The extension reaction was carried out at 42°C for 10 min. The reaction was terminated by adding an equal volume of 2x formamide gel loading buffer and freezing on crushed dry ice. The samples were resolved on a 10°~o polyacrylamide sequencing gel. The primer sequences are as follows: HH primer, 5'-CTCCAGTTTCGAGCTTT-3 ; HDV primer, 5'-AAGTAGCCCAGGTCGGACC-3'; HP primer, 5'-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 self-processing in OST7-1 cells appears to be strikingly similar to the extent of self-processing in vitro {Fi ure 2 "In Vitro +MgCl2" vs. "Cellular").
Consistent with the in vitro self-processing results, the HP(GU) cassette self-processed to approximately 50% in OST7-1 cells. As expected, transfection with, plasmids containing the HH(mutant) cassette yielded a primer-extension product corresponding to the full-length RNA
with no detectable cleavage products {Figure 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 for 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 TY~- ~.~.

metal ions such as Mg2+ and Ca2+ 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 5 regions of the processing ribozyme. Binding of these primers . should prevent the 3' cis-acting ribozymes from folding into the conformation essential for catalytic activity.
Two experiments were carried out to further eliminate the possibility that self-processing is occurring either during RNA preparations or during 10 the primer extension analysis. The first experiment involves primer extension analysis on full-length precursor RNAs that were added to non-transfected 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 15 of low Mg2+ (5 mM) and high NTP concentration (total 12 mM) in an attempt to eliminate the free Mg2+ required for the self-processing reaction (Michel et al. 1992 Genes 8 Dev. 6, 1373). The full-length precursor RNAs were gel-purified, and a known amount was added to fysates of non-transfected OST7-1 cells. RNA was purified from these lysates and 20 incubated for 1 hr in DEPC-treated water at 37o C prior to the standard primer extension analysis (Figure 29, in vitro "-MgCf2" control). The predominant RNA detected in all cases corresponds to the primer extension product of full-length precursor RNAs. lf, instead, the purified RNA containing the full-length precursor is incubated in 10 mM MgCl2 prior 25 to.the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (Figure 29, in vitro "+MgCl2"
control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-processing, even though the RNA in question is inherently able to 30 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 internally-labeled precursor RNAs were carried through the RNA purification and 35 primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-processing. 8y this analysis, the ss 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 ~ST7-1 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.
In addition, those in the art will recognize that Applicant provides guidance through the above examples as to how to best design vectors of this invention so that secondary structure of the mRNA allows efficient cleavage by releasing ribozymes. Thus, the specific constructs are not limiting in this invention. Such constructs can be readily tested as described above for such secondary structure, either by computer folding algorithms or empirically. Such constructs will then allow at least 80°l0 completion of release of ribozymes, which can be readily determined as described above or by methods known in the art. That is, any such secondary structure in the RNA does not reduce release of the ribozymes by more than 20°t°.
IV. Ribozvmes Expressed by RNA Polymerase lil 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 ill gene unit, a number of other pol Ill promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell29, 3-5), 5S RNA {Nielsen et al., 7 993, Nucleic Acids Res. 21, 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 Cell 22, 405-413), U6 snRNA (Gupta and. Reddy, 1990 s7 Nucleic Acids Res. 19, 2073-2075), vault RNA (Kickoefer et al., 1993 J.
Biol. Chem. 268, 7868-7873), telomerase RNA (Romero and Blackburn, 1991 Cell67, 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 5' 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 iri decoy, therapeutic editing and antisense protocols as well as for ribozyme formation, in addition, the molecules can be used as agonist or antagonist RNAs (affinity RNAs).
Generally, applicant believes that the intramolecular base-paired interaction betwean 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 II1 promoter system (e.g,s, a type 2 system) used to synthesize a chimeric RNA
molecule which includes tRNA sequences and a desired RNA (e~a., a tRNA-based molecule).
The following exemplifies this invention with a type 2 pol I11 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 111 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 "3' 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 z 0 nucleotides from the 3' terminus. For example, in the S35 construct described in the present invention (Fi . 4 the 3' region is one nucleotide from the 3' terminus. 1n 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 techniques generally known in the art. Generally, it is preferred to have the 3' region within 100 bases of the 3' terminus.
By "tRNA molecule" is meant a type 2 pol Ill driven RNA molecule that is generally derived from any recognized tRNA gene. Those in the art will recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol III transcription. , By "chimeric tRNA molecule" is meant a RNA molecule that includes a pol III promoter (type 2) region. A chimeric tRNA molecule, for example, might contain an intramolecular base-paired structure between the 3' region and complementary 5' terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol III promoter boxes. Thus, such a foreign RNA may be provided at the 3' end of the B box, or may be provided in between the A and the B box, with the B box moved ~to an appropriate location either within the foreign RNA or another location such that it is effective to provide pol III transcription. In one example, the. RNA molecule may include a hammerhead ribozyme with the B box of a type 2 pol 111 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 ~99 recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.
By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev.
Biochem. 60, 631-652). By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J.American. Med. Assoc. 260, 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 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 H!V 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 RNAn 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. .

boo ' 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 af., 1992 Proc. Natl. Acad. Sci. USA 89, 8864-8868).
In other aspects, the invention includes vectors encoding ~ RNA
molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this RNA.
Thus, the invention features a transcribed non-naturally occuring RNA
molecule which includes a desired therapeutic RNA portion and an intramolecular stem formed by base-pairing interactions between a 3' region and complementary nucleotides at the 5' terminus in the RNA. The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs.
In preferred embodiments, the 5' terminus of the chimeric tRNA
includes a portion of the precursor molecule of the primary tRNA molecule, of which >_ 8 nucleotides are involved in base-pairing interaction with the 3' region; the chimeric tRNA contains A and B boxes; natural sequences 3' of the B box are deleted, which prevents endogenous RNA processing; the desired RNA molecule is at the 3' end of the B box; the desired RNA
molecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA; the molecule has an intramoiecular stem resulting from a base-paired interaction between the 5' 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 111 based promoter system, e.g., a type 2 pol 111 promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol Ill promoter separated by between 0 and 300 bases; DNA
vector encoding the RNA molecule of claim 51.

In other related aspects, the invention features an RNA or DNA vector encoding the above RNA rnolecule, with the portions of the vector encoding the RNA functioning as a RNA pol lil 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 a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerise 11 (pot II) and RNA polymerise III (pol III) based expression systems have been used to produce therapeutic RNAs in cells (Sarver & Rossi, 1993 AlOS Res. &
Human Retroviruses 9, 483-487; Yu et af., 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.
Pol II produces messenger RNAs located exclusively in the cytoplasm, whereas pol III produces functional RNAs found in both the nucleus and the cytoplasm. Pol II promoters tend to be more tissue restricted, whereas pol III genes encode tRNAs and other functional RNAs necessary for basic "housekeeping" functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types. Finally, pol 111 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.

~~2 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 03-5 (Fi . ; Adeniyi-Jones et al., 1984 supra), has been adapted to express antiviral RNAs (Sullenger et al., 1990 MoL CeII. Biol. 10, 6512-6523). This element was inserted into the DC retroviral vector (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523) to accomplish stable 'gene transfer, and used to express antisense RNAs against moloney murine leukemia virus and anti-HIV decoy RNAs (Sullenger et al., 1990 Mol. Cell.
Biol. 10, 6512-6523; Sullenger et al., 1990 Gell63, 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 ~3-5 vector system (These constructs are termed u~3-5/HHI"; Fia. 34). On average, ribo2ymes were found to accumulate to less than 100 copies per cell in the bulk T cell populations. In an attempt to improve expression levels of the ~3-5 chimera, the applicant made a series of modified D3-5 gene units containing enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (F, ia. 34). One of these modified gene units, termed S35, nave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original e3-5/HH1 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 d3-5/HHl version of this vector.
The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decay, 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 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.
d3-5 Vectors The use of a truncated human tRNAimet gene, termed D3-5 (Fia~33;
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 111 promoters, the antisense and decoy RNA sequences were expressed as chimeras contGining 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 03-5 vector combination has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cloned ribozyme-encoding sequences (termed as °d3-5lHHI") 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 {Fi4~35,). To try and improve accumulation of the ~ibozyme, applicant incorporated various RNA structural elements (Fi . 4 into one of the ribozyme chimeras (a3-5/HHI).
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 1 oa 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 (Fi ure 4). 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: S35 in which the 3' end was altered to hybridize to the 5' leader and acceptor stem of the tRNAimet domain, and S35Pfus which was identical to S35 but included more extensive structure formation within the non-ribozyme portion of the e3-5 chimeras {Fi ur 4). These stem-loop structures are 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_,_sup~a~_and__.CEM. .(tiara 8 Fischinger, 1988 supra) cell lines were established (Curr. Protocols Mol.
Bio!. 1992, ed. Ausubel et al., Wiley 8 Sons, NY). The RNA sequences and structure of S35 and S35 Plus are provided in Figures 40-47.
Referring to Fioure 4 , there is provided a general structure for a chimeric RNA molecule of this invention. Each N independently represents none or a number of bases which may or may not be base paired. The A
and B boxes are optional and can be any known A or B box, or a consensus sequence as exemplified in the figure. The desired nucleic acid to be expressed can be any location in the molecule, but preferably is on those places shown adjacent to or between the A and B boxes (designated by arrows). Fi ur 4 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 2fi: Cloning of o3-5-Ribozyme Chimera Oligonucleotides encoding the S35 insert that overlap by at least 15 nucleotides were designed (5' GATCCACTCTGCTGTTCTGTTI'TTGA 3' and 5' CGCGTCAAAAACAGAACAGCAGAGTG 3'). The oligonucleotides {10 pM each) were denatured by boiling for.5 min in a buffer containing 40 mM Tris.HCl, pH8Ø The oligonucleotides were allowed to anneal by snap cooling on ice for 10-15 min.' The annealed oligonucleotide mixture was converted into a double-stranded molecule using Sequenase~ enzyme (US Biochemicals) in a buffer containing 40 mM Tris.HCl, pH7.5, 20 mM MgCl2, 50 mM NaCI, 0.5 mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 37°C for 30 min. The reaction was stopped by heating to 70°C for 15 min.
The double stranded DNA was digested with appropriate restriction endonucleases (BamHl and Mlul) to generate ends that were suitable for cloning into the D3-5 vector.
The double-stranded insert DNA was ligated to the o3-5 vector DNA
by incubating at room temperature (about 20°C) for 60 min in a buffer containing 66 mM Tris.HCl, pH 7.6, 6.6 mM MgCl2, 10 mM DTT, 0.066 ItM
ATP and 0.1 U/~I T4 DNA Ligase (US Biochemicals).
Competent E. colt 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 far 1 min. The reaction mixture was diluted with LB media and the cells were allowed to recover for 60 min at 37°C. The cells were plated on LB agar plates and incubated at 37°C for - 18 h.
Plasmid DNA was isolated from an overnight culture of recombinant clones using standard protocols (Ausubel et al., Curr. Protocols Mol.
Biology 1990, Wiley & Sons, NY).
The identity of the clones were determined by sequencing the plasmid DNA using the Sequenase~ DNA sequencing kit (US Biochemicals}.
The resulting recombinant D3-5 vector contains the S35 sequence.
The HHI encoding DNA was cloned into this ~3-5-S35. containing vector using Sacll and BamHl restriction sites.
Example 27: Northern analysis RNA from the transduced MT2 cells were extracted and the presence of e3-Slribozyme chimeric transcripts were assayed by Northern analysis (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiiey & Sons, NY).
Northern analysis of RNA extracted from MT2 transductants showed that D3-5/ribozyme chimeras of appropriate sizes were expressed (Fig. 35.3f,).
In addition, these results demonstrated the relative differences in accumulation among the different constructs (Figure 35.36). The pattern of expression seen from the D3-5/HHl ribozyme chimera was similar to 12 other ribczymes cloned into the ~3-5 vector (not shown), In MT-2 cell line, 03-5/HHI ribozyme chimeras accumulated, on average, to less than 100 copies per cell.
Addition of a stem-loop onto the 3' end of D3-5/HHI did not lead to increased D3-5 levels (S3 in F, i-.q 35,36). The S5 construct containing both 5' and 3' stem-loop structures also did not lead to increased ribozyme 4evels (Fig; 35.36).
Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original D3-5/HHI vector transcripts (Fia. 35,36}. This may be due to increased stability of the S35 transcript.
Example 28: Cleavage activity To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T
cells were incubated with a labeled substrate containing the HH1 cleavage site (Fi ure 7). Ribozyme activity in all but the S35 constructs, was too low to detect. However, ribozyme activity was detectable in S35-transduced T cell RNA. Comparison of the activity observed in the S35-transduced MT2 RNA with that seen with MT2 RNA in which varying amounts of in vitro transcribed S5 ribozyme chimeras, indicated that between 1-3 nM of S35 ribozyme was present in S35-transduced MT2 RNA. This level of activity corresponds to an intracellular concentration of 5,000-15,000 ribozyme molecules per cell.
Example 29: Clonal variation Variation in the ribozyme expression levels among cells making up the bulk population was determined by generating several clonal cell lines from the bulk S35 transduced CEM line (Curr. Protocols Mol. 8iol. 1992, ed. Ausubel et al., Wiley & Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (Figure 38 and 39).
All the individual clones were found to express active ribozyme. The ribozyme activity detected from each clone correlated well with the relative amounts of ribozyme observed by Northern analysis. Steady state ribozyme levels among the clones ranged from approximately 1,000 molecules per cell in clone G to 11,000 molecules per cell in clone H (Fig=

~$,). 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 i4 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 t~3-5 vector. Therefore, the S35 gene unit should be much more effective in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.
Example 3Q: Stability -Finally, the bulk S35-transduced line, resistant to 6418, was propogated for a period of 3 months (in the absence of 6418) to determine if ribozyme expression was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme expression after 3 months. This difference probably arose from cells with varying amount of ribozyrne 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 S35 motif (Fioure ).~ A desired RNA (e.g. ribozyme) can be inserted into the indicated region of TRZ tRNA chimera. This construct might provide additional stability to the c+esired RNA. TRZ-A and TRZ-B are non-limiting examples of the TRZ-tRNA chimera.
Referring to Fig. 53-54, a hammerhead ribozyme targeted to site 1 (HHITRZ-A; Fig. 53) and a hairpin ribozyme, (HPITRZ-A; Fig. 54), also targeted to site f, is cloned individually into the indicated region of TRZ
tRNA chimera. The resulting ribozyme trancripts retain full RNA cleavage activity (see for example Fia~55). Applicant has shown that efficient expression of these TRZ tRNA chimera can be achieved in mammalian cells. .
Besides ribezymes, desired RNAs like antisense, therapeutic editing , RNAs, decoys, can be readily inserted into the indicated region of TRZ-tRNA chimera to achieve therapeutic levels of RNA expression in mammalian cells.
Sequences fisted in Figures 40-47 and 50 - 54 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily generated using techniques known in the art, are within the scope of the present invention.
Example 32: Ribo_zyme expression in T cell lines Ribozyme expression in T cell lines stably-transduced with either a retroviral-based or an Adeno-associated virus (AAV)-based ribozyme expression vector (Figure 56). The human T cell lines MT2 and CEM were transduced with either retroviral or AAV vectors encoding a neomycin slelctable marker and a ribozyme (S35/HHI) expressed from pol III meti tRNA-driven promoter. Cells stably-transduced with the vectors were selectivelyt expanded medium containing the neomycin antibiotic derivative, 6418 (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 meti 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 W0 93104573, and Suflivan WO 94!04609, and 93!11253 describe methods for use of vectors decribed herein. ~ In particular these vectors are useful for administration of antisense and decoy RNA
molecules.
Examale 33: Ligated Ribozymes are catalyticallyr active The ability of ribozymes generated by ligation methods, described in Draper et al., PCT WO 93123569, to cleave target RNA was tested on either matched substrate RNA (Fi . 5 or long (622 nt) RNA (Fi~~59,, f~0 and 61 ~, Matched substrate RNAs were chemically synthesized using solid-phase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res.
18, 5433-544'1). Substrate RNA was 5' end-labeled using [~y~-32P) ATP and polynuc~eotide kinase (Curr. Protocols Mol. BQI. 1992, ed. Ausubel et al., Wiley 8~ Sons, NY). Ribozyme reactions were carried out under ribozyme excess conditions (kcat~KM~ Herschlag and Cech, 1990 Biochemistry 29, 10159-10171). Briefly, ribozyme and substrate RNA were denatured and renatured separately by heating to 90°C and snap cooling on ice for 10 min in a bufter containing 50 mM Tris. HCI pH 7.5 and 10 mM MgCl2.
Cleavage reaction vuas initiated by mixing the ribozyme with the substrate at 37°C. Aliquots of 5 pf~were taken at regular intervals of time and the reaction was stopped by mixing wish equal volume of formamide gel loading bufifer (Curr. Protocols Mol. Biol. 1992, ed. Ausube) et al.; Wifey 8~
- Sons, NY). The samples were resolved on 20 % polyacrylamide-urea gel.
Refering to F. ig_58, -DG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA
(Turner and Sugimoto, 1988 a ra). , RPI A is a HH ribozyme with 6/6 binding arms. This ribozyme was synthesized chemically either as a one piece ribozyme or was synthesized in two fragments followed by ligation to generate a one piece ribozyme. The kcat/KM values for the two ribozymes were comparable.
A template containing T7 RNA pofyrnerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone, The target RNA (containing HH ribozyme cleavage sites B, C and D) was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [a-32p) CTP as one of the tour ribonucleotide triphosphates. The transcription mixture was treated with DNase-1, fiollowing transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. RNA was precipitated with fsopropanol and the pellet was washed two times with 70°l°
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 su ra . 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.HCl, pH 7.5 and 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 37°C. Aliquots of 5 lrl were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved an a sequencing gel.
Examale 34' Hammerhead ribozymes with >_ 2 base-paired stem I1 a_re catal,~tically a~tivg 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 by stem ll) can be shortened without decreasing the catalytic efficiency of the HH ribozyme. The length of stem ll was systematically shortened by one base-pair at a time, HH ribozymes with three and twa base-paired stem II were chemically synthesized using solid-phase RNA phosphoramidite chemistry (Scaringe et al., 1990 su ra .
Matched and long substrate RNAs were synthesized and ribozyme assays were carried out as described in example 33. Referring to fi4ures , ~2, 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 Z 2 base-paired stem II region are catalytically active.
Examflle 35: Synthesis of catal fcally active hairpin ribozvmes RNA molecules were chemically synthesized. having the nucleotide base sequence shown in Fia~65 for both the 5' and 3' fragments. The 3' fragments are phosphorylated and ligated to the 5' fragment essentially as described in example 37. As is evident from the Fiau-~- re 65, the 3' and 5' fragments can hybridize together. at helix 4 and are covalently (inked via GAAA sequence. When this structure hybridizes to a substrate, a ribozyme~substrate complex structure is formed. White helix 4 is shown as 3 base pairs it may be formed with only 1 or 2 base pairs.
40 nM mixtures of ligated ribozymes were incubated with 1-5 nM 5' end-labeled matched substrates (chemically synthesized by solid-phase synthesis using RNA phosphoramidite chemistry) for different times in 50 mM Tris/HCl pH 7.5, 10 mM MgCl2 and shown to cleave the substrate efficiently (Fia~66).
The target and the ribozyme sequences shown in Fip~. 62 and 65 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using other sequences and techniques generally known in..the .art. ~ --V , Constructs of Hairpin Ribozymes There follows an improved traps-cleaving hairpin ribozyme in which a new helix (i.e., a sequence able to form a double-stranded region with another single-stranded nucleic acid) is provided in the ribozyme to base~
pair 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 i ur In addition, at least two extra bases may be provided in helix 2 and a portion of the substrate corresponding to helix 2 may be either directly linked io the 5' portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atlEast one base. By traps-cleaving is meant that the ribozyme is able to act in traps to cleave another RNA molecule which is not covalently finked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecufar cleavage reaction.
By "base-pair" is meant a nucleic acid that can form hydrogen bonds) with other RNA sequence by either traditional Watson-Crick or other non-traditional 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. Trans-ligation reactions catalyzed by the regular hairpin ribozyme (4 by helix 2) is very inefficient (Komatsu ef 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 5) the rate of ligation (in vitro and in vivo) can be enhanced several fold.
Results of experiments suggest that the length of H2 can be 6 by 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 by H2 have been designed against five different target RNAs and all five ribczymes efificiently cleaved their cognate target RNA, Additionally, two of these ribozymes were able to successfully inhibit gene expression (e.g., TNF-a) in mammalian cells. Results of these experiments are shown below.
HP ribozymes with 7 and 8 by 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 by H2.
Example 3~: 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 Modificat~n Oliaonucleotides with 5'-C-alkyl Group The introduction of an alkyl group at the 5'-position of a nucleoside or nucleotide sugar introduces an additional center of chirality into the sugar moiety. Referring to Fia~75, the general structures of 5'-C-alkylnucieotides 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 ~-talose (Ri = CH3 in 2 and 3 in Figure 75). Useful specific D-allose and L-talose ' 113 nucleotide derivatives are shown in Figure 76. 29-32 and Figure 77, 58-61 respectively.
This invention relates to the use of 5'-C-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As the term is used in this application, 5'-C-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 5'-C-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 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid o.r 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 5'-C-alkylnucleotides, 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. 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 Fi ure 7 , where each Ry 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 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 groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino, or SH. The term also iricludes 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 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO~, halogen, N(CH3)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 alkynyi 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 groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated n electron system and includes carbocyclic aryl,. heterocyclie aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazofyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
In other aspects, also related to those discussed above, the invention features ofigonucleotides having one or more 5'-C-alkylnucleotides; e.g.
enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, the invention features 5'-C alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.
The 5'-C-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.
In another aspect, the invention features a method for conversion of a protected allo sugar to a protected talo sugar. In the method, the protected allo sugar is contacted with triphenyl phosphine, diethylazodicarboxylate, and p-nitrobenzoic acid under inversion causing conditions to provide the protected talo sugar. While one example of such conditions is provided below, those in the art will recognize other such conditions. Applicant has found that such conversion allows for ready synthesis of all types of nucleotide bases as exemplified in the figures. .
While this invention is applicable to all oligonucleotides, applicant has found that the modified molecules of this invention are particulary useful for enzymatic RNA molecules. Thus, below is provided examples of such yes -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 Fi ur 1, the preferred sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 5'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Substitutions of any nucleotide with any of the modified nucleotides shown in Fi ur 7 are possible.
' The following are non-limiting examples showing the synthesis of nucleic acids using 5'-C-alkyl-substituted phosphoramidites and the syntheses of the arnidites.
Example 37: Synthesis of Hammerhead Ribozymes Containing 5'-GAlkvl-nucleotides & Other Modified Nucleotides The method of synthesis would follow the procedure for normal RNA
synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.;
Cedergren,R.J, J. Am. Chem. Soc.1987, 709, 7845-7854 and in Scaringe,S,A.; FrankIyn,C.; Usman,N. Nucleic Acids Res. 1990, 78, 5433-5441 and makes use of common nucleic acid protecting ahd coupling groups, such as dimethoxytrityi at the 5'-end, and phosphoramidites at the 3'-end (compounds 26-29 and 56-59). These 5'-C-alkyl substituted 2,5 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.
Examale 38: Methyl-2.3-O-Isooropylidine-6-Deoxy-J3-D-allofuranoside (4_1_ A suspension of ~-rhamnose (100 g, 0.55 mol), CuS04 (120 g) and conc. H2S0~ (4.0 mL) in 1.0 L of dry acetone was mixed for 24 h at RT, then filtered. Conc. NH40H (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 °C. 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 ice-water (0.5 L) and, after mixing for 0.5 h, was extracted with chloroform (0.75 L). The organic layer was washed with H20 (2 x 500 mL), 10% H2S04 (2 x 300 mL), water (2 x 300 mL), sat. NaHC03 (2 x 300 mL), brine (2 x 300 mL), dried over MgS04 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 °C, neutralized with dry C02 and evaporated to dryness. The residue was suspended in chloroform (750 mL), filtered , concentrated to 100 mL and purified by flash chromatography in CHC13 to yield 45 g (37%) of compound 4.
Example 39: Methvl-2.3-O-Isooropvlidine-5-D-t-Butyldiphenylsilvl-6-Deoxv~D-Allofuranoside f5~
To solution of methylfurancside 4 (12.5 g 62.2 mmol) and AgN03 (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 CHC13 (200 mL), filtered and evaporated to dryness (below 40 °C using a high vacuum oil pump). The residue was dissolved in CH2C12 (300 mL) washed with sat. NaHC03 (2 x 50 mL), brine (2 x 50 mL), dried over MgS04 and evaporated to dryness.
The residue was purified by flash chromatography in CH2C12 to yield 20.0 g (75%) of compound 5.
Example 40: Methvl-5-O-t-Butvldiphenylsilyl-6-Deoxy-Q-D-Allofuranoside Methylfuranoside 5 (13.5 g, 30.6 mmol) was dissolved in CF3COOH:dioxane:H20 l 2:1:1 (v/vlv, 200 mL) and stirred at 24 °C
for 45 m. The reaction mixture was cooled to -10 °C, neutralized with cone.
NH40H (140 mL) and extracted with CH2C12 (500 mL). The organic layer was separated, washed with sat. NaHC03 (2 x 75 mL), brine (2 x 75 mL), dried over MgS04 and evaporated to dryness. The product 6 was purified by flash chromatography using a 0-10% MeOH gradient i~ CH2C12. Yield 9.0 g (76%).

Example 41: Methyl-2.3-di-O-Benzoyl-5-D-f-Butyldiphenylsilyl-6-Deoxy_(3-p-Allofuranoside (7).
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 CH2C12 (300 mL), washed with sat. NaHC03 (2 x 75 mL), brine (2 x 75 mL) dried over MgS04 and evaporated to dryness. The product was purified by flash chromatography in CH2C12 to yield 9.5 g (89%) of compound T.
Example 42: 1-O-Acetyl-2,3-di-D-benzoyl-5-O-t-Bu~ldiphenylsi~l-6-Deoxy-J' -D-Allofuranose (8).
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 cooled 0 °C. 98% H~S04 (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.
NaHC03 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 MgS04, evaporated to dryness and coevaporated with toluene (2 x 50 mL). The product was purified by flash chromatography using a gradient of 0-5%
MeOH in CH2C12. Yield: 4.0 g (82% as a mixture of oc and p isomers).
Example 43: 1-(2',3'-di-O-Benzovl-5'-O-i-Butyldiphenylsilyl-6'-Deoxy-Q-D-Allofuranosyl}uracif (91.
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 CH3CN (100 mL), followed by CF,S03SiMe3 (2.8 g, 12.6 mmol). The reaction mixture was kept at 24 °C for 16 h, concentrated to 1/3 of its original volume, diluted with 100 mL
of CH2C12 and extracted with sat. NaHC03 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO~, and evaporated to dryness. The product 9 was purified by flash chromatography using a gradient of 0-5% MeOH in CH2C12. Yield:
5.7 g (80%).

Example 44: IVg-Benzoyl-1-(2' 3'-Di-O-Benzoyl-5'-O-t-But,~rldiphenylsilyl-6'-Deoxy-D-D-Allofuranosyl)Cytosine (101 IV4-benzoylcytosirie (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 CH3CN (100 mL), followed by CF3S03SiMe3 (4.76 g, 21.4 mmol). The reaction mixture was boiled under reflux for 5 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH2C12 (100 mL) and extracted with sat. NaHC03 (2 x 50 mL), brine (2 x 50 mL) dried over MgS04 and evaporated to dryness.
Purification by flash chromatography using a gradient of 0-5% MeOH in CH2C12 yielded 1.8 g (55°l°) of compound 10.
Example 45: IVY-Benzoyl-9-(2'.3'-di-O-Benzoyl-5'-O-t-Butyldii~henylsilyl-6'-Deoxy-~~D-Allofuranosyl)adenine X111.
!V6-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 CH3CN (100. mL) followed by CF3SO;,SiMe3 (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 CH2C12 (100 mL) and extracted with sat. NaHC03 (2 x 50 mL), brine (2 x 50 mL). dried over MgS04 and evaporated to dryness. The . product 11 was purified by flash chromatography using a gradient of 0-5%
MeOH in CHZCIp. Yield: 2.7 g (60%).
Example 46: I~-Isobutvrvl-9-l2'.3'-di-O-Benzoyl-5'-O-f-Butyldiphenvlsilvl-6'-Deoxy-f3-o-Allofuranosyl)duanine,~l2~
!Vz-Isobutyrylguanine (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 'f 20 solution of of acetates 8 (3.4 g; 5.3 mmol) in dry CH3CN {100 mL) followed by CFJSOaSiMe3 (6.22 g, 28.0 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1I3 of its original volume, diluted with CH2C12 {100 mL) and extracted with sat. NaHC03 (2 x 50 mL), brine (2 x 50 mL) dried over MgS04 and evaporated to dryness. The product 12 was purified by flash chromatography using a gradient of 0-2%
MeOH in CH2C12. Yield: 2.1 g (54%).
Example 47: IVY-Benzovl-9-(2'.3'-di-O-benzoyl-6'-Deoxy~~i-D-Allofurano-~r1)adenine (151.
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 CH2C12 to yield 1.0 g (85%) of compound 15.
Example 48: IVY-B_enzoyl-9-(2'.3'-di-D-BenzoVl-5'-O-Dimethoxvtri~ tvl-fi'-Deox -f3-D-Allofuranosyll-adenine~191.
Nucleoside 15 (0.55 g, 0.92 mmol) was dissolved in dry CH2C12 (50 mL). AgNOa (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 stirred for 2h, diluted with CH2C12 (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 CH2C12 yielded 0.8 g (97°l°) of compound 19.
Example 49: IVY-Benzovl-9-!-5'-O-Dimethoxytrityl-6'-Deoxy_ -D-Allo-furanosylladenine (23L
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 Dovi~ex 50 (Pyre form), filtered and the resin was washed with MeOH {2 x 50 mL). The filtrate was then evaporated to dryness. Purification by flash chroillatography using a gradient of 0-10% MeOH in CH2C12 yielded 1.1 g (80°l°) of 23.

Examrle 50: fV5-Benzoyl-9-(-5'-O-Dimethoxytrityl-2'-øt butvldimethvlsilyl-6'-Deoxv~3-~-Allofuranosyl)adenine (271, Nucleoside 23 (1.2 g, 1.8 mmol) was dissolved. in dry TNF (50 mL).
Pyridine (0.50 g, 8 mmol) and AgN03 (0.4 g, 2.3 mmol) were added. After the AgN03 dissolved (1.5 h), 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 CH2C12 (100 mL), filtered into sat.
NaHC03 (50 mL), extracted, the organic layer washed with brine (2 x 50 mL), dried over MgS04 and evaporated to dryness. The product 27 was purified by flash chromatography using a hexanes:EtOAc / 7:3 gradient.
Yield: 0.7 g (50%).
Example ~1: N~-Benzovl-9-(-5'-O-Dimethoxytrityl-2'-O-t-bu~ldimethylsil~rl-5'-Deoxy-B-D-Allofuranosvlladenine-3'-(2-Cyanoethyl N N dii~oprogvl-phosphoramidite) (311.
Standard phosphitylation of 27 according to Scaringe,S.A.;
FrankIyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield.
Example 52. Methy;-5-O-o-Nitrobenzoyl-2.3-D-Isooropylidin~-6-deoxy-Q-u-Tallofuranoside (51 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 lefit 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 CH2C12 (300 mL) washed with sat. NaHC03 (2 x 75 mL), brine (2 x 75 mL) dried over MgS04 and evaporated to dryness.
Purification by flash chromatography using a hexanes:EtOAe / 9:1 gradient yielded 4.1 g (78%) of compound 33. Subsequent debenzoylation (NaOMe/MeOH) and silylation (see preparation of 5) fed to l.-taiofuranoside 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 anti~ense 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 94J 02595.
The ribozymes and the target RNA containing site 0 were synthesized, deprotected and purified as described above. RNA cleavage assay was carried our at 37°C in the presence of 10 mM MgCl2 a s 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-O'1 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-01, 2, 3, 4 and 5 are also extremely resistant to degradation by human serum nucleases.
Ofiaonucleotides with 2'-Deoxy-2'-Alkylnucleotide This invention uses 2'-deoxy-2'-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, 2'-deoxy-2'-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 2'-deoxy-2'-aikylnucleotide components replacing, but not limited to, double slranded stems, single stranded "catalytic care" 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 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 ofigonucieotides (examples of which are shown in the Figures), and to methods for their synthesis.
Thus, in one aspect, the invention features 2'-deoxy-2'-alkylnucleotides, that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the 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 "-D-alkyl" group, where "alkyl" is defined as described above, where the O is adjacent the 2'-position of the sugar molecule.
In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2'-position. an alkyl group. In other related aspects, the invention features 2'-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.
The 2'-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall 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.
1n another aspect, the invention features hammerhead motifis having enzymatic activity having ribonucleotides at locations shown in Figure 80 at 5, 6, 8, 12, and 15.1, and having substituted ribonucleotides at other positions in the core and in the substrate binding arms ifi desired. (The term "core" refers to positions between bases 3 and 14 in Figure 80, and the binding arms correspond to the bases from the 3'-end to base 15.1, and from the 5'-end to base 2). Applicant has found that use of ribonucleotides at these five locations in the core provide a molecule having suffiicient enzymatic activity even when modified nucleotides are present at other sites in the motif. Other such combinations ofi useful ribonucleotides can be determined as described by Usman ef al. supra.
Figure 80 shows base numbering of a hammerhead motif in which the numbering ofi 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 5'- to 3'-direction of the catalytic core is CUGANGAG[base paired with)GGAAA. In this invention, the use of 2'-Galkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance ofi 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 a!. , EMBO J. 1592, 11, 1913-1919) and ease of synthesis, but is not limiting to this invention.
Ribczymes 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 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 (3 was calculated (Table 45). This f3 value indicated that all modified ribozymes tested had significant, >100 - >1700 fold, increases in overall stability and activity. These increases in fi indicate that the lifetime of these modified ribozymes in vivo are significantly increased which should lead to a more pronounced biological effect.
More general substitutions of the 2'-modified nucleotides from Figure 81 also increased the t1 i2 of the resulting modified ribozymes.
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.
The following are non-limiting examples showing the synthesis of nucleic acids using 2'-C-alkyl substituted phosphoramidites, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance.
Example 53: Synthesis of Hammerhead Ribozymes Containing 2'-Deoxv-2'-Alkvlnucieotides & Other 2'-Modified Nucleotides 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, 709, 785-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 78, 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 ef al. International Publication No. WO 92/07065; and 5 Kois et al.
Nucleosides & Nucleotides 1993, 72, 1093-1109. The .average stepwise coupling yields were -98%. The 2'-substituted phosphoramidites were incorporated into hammerhead ribozymes as shown. in Figure 80.
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 11 intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.
Example 54: Ribozvme Activity Assav Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-end labeled ribozymes (-36-mers) were both heated to 95 °C, quenched on ice and equilibrated at 37 °C, 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 MgCl2. Reactions were initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 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 15%
denaturing polyacryfamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).
Example 55: Stability Assay 500 pmol of gel-purified 5'-end-labeled ribozyrnes were precipitated in ethanol and pelleted by centrifugation. Each pellet was resuspended in 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 0.5X 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 20%
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.
Exam I 56: ' '-Q Tetraiso ro 1-disiloxane-1 3-di I - ' Ph nox hi -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 Chemistry, ed. Leroy Townsend, 1986 pp. 229-231) and dimethylamino-pyridine (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 (85%) of 7.
Example 57: 3'.5'-O-(Tetraisooropvl-disiloxane-1 3-d~~~-2'-C-Ally) -Uridine To a refluxing, under argon, solution of 3',5'-O-(tetraisopropyl-disiloxane-1,3-diyi)-2'-D-phencxythiocarbonyl-uridine, 7, (5 g, 8.03 mmol) and allyltributyltin (12.3 mL, 40.15 mmol) in dry toluene, benzoyl peroxide (0.5 g) was added portionwise during 1 h. The resulting mixture was allowed to reflux under argon for an additional 7-8 h. The reaction was then evaporated and the product 8 purified by flash chromatography on silica gel with EtOAc:hexanes / 1:3 as eluent. Yield 2.82 g (68.7%).
Example 58: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine X91 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 5% 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 (57%) of 9 as a white foam.

128 ' Example 59: 5'-O-Dirnethoxvtritvl-2'-GAIIyI-Uridine ~C~anoethyl N N-diiso~ropylphosphoramidite~ ~1 Ol 5'-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 g, 1.12 mmol) was dissolved in dry dichloromethane under dry argon. N,N-Diisopropylethyl-amine (0.39 rnL, 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 °C) 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 (90°1°), white foam.
Example 60: 3'.5'-D-fTetraisopropyl-disiloxane-1 3-diyl)-2'-C-All~l~_ Acet~~l-Cytidine (111 Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile. To the resulting suspension a solution of 3',5'-O-(tetraisopropyl-disiloxane-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. NH40H 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 30. 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 (3% MeOH in chloroform). Yield 2.3 g (90%) as a white foam.

Example 61: 5'-O-Dimethoxytrityl-2'-GA11y1-N'4-Acetyl-Cytidine This compound was obtained analogously to the uridine derivative 9 in 55% yield.
Exam,~le 62: 5'-O-Dimethox~rtrityl-2'-C-allyl-N4-Acetyl-Cytidine 3'-12-Cyan~oeth_yl N,N-diisoprooylohosphoramiditel 1121 2'-O-Dimethoxytrityl-2'-C-allyl-N4-acetyl cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichloromethane under argon. N,N-Diisopropylethyl-amine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.
2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.38 mL, 1.7 mmol) was added dropwise to a stirred reaction solution and stirring was continued for 2 h at room temperature. The resction 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 'C) and purified by flash chromatography on silica gel using chloroform:ethanol / 98:2 with 2%
triethylamine mixture as eluent. Yield: 0.91 g (85°l°), white foam.
Exam~~le 63: 2'-Deoxy-2'-Methylene lJridine 2'-Deoxy-2'-methylene-3',5'-D-(tetraisopropyldisiloxane-1,3-diyl)- v uridine 1 a (Hansske,F.; Madej,D.; Robins,M. J. Tetrahedron 1984, 40, 125 and Matsuda,A.; Takenuki,K.; Tanaka,S.; Sasaki,T.; Ueda,T. J. Med. Chem.
1991, 34, 812) (2.2 g, 4.55 mmol ) dissolved in THF (20 mL) was treated 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 CH2C12.
Example 64: 5'-O-DMT-2'-Deoxy-2'-Meth~rlene-Uridine (151 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 far .12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken. up in CH2Cl2 (100 mL) and washed with sat. NaHCOg, water and brine. The organic extracts' were dried over MgS04, concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as efuant to yield 15 (0.43 g, 0.79 mmol, 22%).

Example 65: 5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine 3'-(2~yanoethyl N, N-diisoprowlahosphoramidite,}~1~
1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-(i-D-ribofuranosyl) uracil (0.43 g, 0.8 mmol) dissolved in dry CH2C12 (15 ~mL) was placed in a round-bottom flask under Ar. Diisopropyiethylamine {0.28 mL, 1.6 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diiso-propylchlorophosphoramidite (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 °C). The product (0.3 g, 0.4 mmoi, 50%) was purified by flash column chromatography over silica gel using a 25-70% EtOAc gradient in hexanes, containing 1°I°
triethylamine, as eluant. Rf 0.42 (CH2C12: MeOH / 15:1 ) Example 66: 2'-Deoxv-2'-Difluoromethvlene-3'.5'-O-lTetrai°oprop,~ldi°ilox~
ane-1.3-d~rl)-Uridinj 2'-Keto-3',5'-D-(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 °C. A warm (60 °C) solution of sodium chlorodifluoroacetate in digiyme (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. CH2C12 and chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine (3.1 g, 5.9 mmol, 70°!°) eluted with 25°I°
hexanes in EtOAc.
Example 67: 2'-Deoxv-2'-Difluoromethylene-Uridine 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine (3.1 g, 5.9 mmol) dissolved in THF (20 mL) was treated with 1 M
TBAF in THF (10 mL} for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on silica gel column.
2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4:0 mmol, 68%) was eluted with 20% MeOH in CH2C12.
_Examole 68: 5'-O-DMT-2'-Deoxv-2'-Difluoromethvlene-Uridine i16) 2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (1.42 g, 4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuv and the residue taken up in CH2C12 (100 mL) and washed with sat. NaHC03, water and brine. The organic extracts were dried over MgS04, concentrated in vacuo and purified over a silica gel column using 40% EtOAc:hexanes as eluant to yield 5'-O-DMT-2'-deoxy-2'-difluoromethylene-uridine 16 (1.05 g, 1.8 mmol, 45°!°).
Example 69: 5'-O-DMT-2'-Deoxy-2'-Difluorometh~lene-Uridine 3'~j2-Cyanoethyl N.N-diisopropyl~hosphoramidite~~l8,~
1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-~-D-ribofurano-syl)-uracil (0.577 g, 1 mmol) dissolved in dry CH2C12 (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-diiso-propylchlorophosphoramidite (0.44 mL, 1.4 mmol). The reaction mixture was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). The product (0.404 g, 0.52 mmol, 52%) was purified by flash chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1 % triethylamine, as eluant. Rt 0.48 (CH2C12: MeOH ! 15:1).
Example 70: 2'-Deoxv-2'-Methvlene-3'.5'-O-(Tetraisoprooyldisiloxane-1 diy,-4-N-Acetxl-Cytidine 20 Triethylamine (4.8 mL, 34 mmol) was added to a solution of POC13 (0.65 mL, 6.8 mmol) and 1,2,4-triazole (2.1 g, 30.6 mmol) in acetonitriie (20 mL) at 0 °C. A solution of 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldi-siloxane-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 CH2C12 (2 x 100 mL) and~washed with 5% NaHC03 (1 x100 mL). The organic extracts were dried over Na~S04 concentrated in vacua, dissolved 30~ in dioxane (10 mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine, (2 x 20 mL). Acetic anhydride (3 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.
NaHC03 (5 mL). The mixture was concentrated in vacuo, dissolved in CH2C12 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The . 132 organic extracts were dried over Na2S04, concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes.
Example 71: 1-(2'-Deoxy-2'-Methyrlene-5'-O-Dimethox~~tritvl-8-D-ribo-furanosYl_)-4-N-Acetyl-Cytosine 21 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-N
acetyl-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 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on silica gel column. 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol, 80%) was eluted with 10% MeOH in CH2C12. 2'-Deoxy-2'-methylene-4-N
acetyl-cytidine (0.56 g, 1.99 mmol) wGS dissolved in pyridine (10 mL) and a solution of DMT-CI (0.81 g, 2.4 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12.h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH2C12 (100 mL) and washed with sat. NaHC03 (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgS04, concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes ! 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, 75%).
Examale 72: 1-(2'-Deoxv-2'-Methvlene-5'-O-Dimethoxytrityl- -D-ribo-furanosyll-4-N-Acetyl-Cytosine 3'-f2-Cyanoethyl-N N-diisopropylphosohor-amidite~~221 1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-(3-v-ribofuranosyl)-4-N-acetyl-cytosine 21 (0.88 g, 1.5 mmol) dissolved in dry .CH2C12 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.8 mL, 4.5 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After 10 m the mixture evaporated to ~a syrup in vacuo (40 °C).
The product 22 (0.82 g, 1.04 mmol, 69%) was purified by flash chromatography over silica gel using 50-70% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.38 (CH2C12:MeOH / 20:1).

Example 73' 2'-DeoxX-2'-Difluoromethylene-3' S'-O-(Tetraisopropvl di°iloxane-1,3-dyl~-4-N-Acetyl-Cytidine (241 Et3N (6.9 mL, 50 mmol) was added to a solution of POC13 (0.94 mL, mmol) and 1,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 mL) at 0 °C.
5 A solution of 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisilox-ane-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 CH2C12 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The 10 organic extracts were dried over Na~S04 concentrated in vacuo, dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (5 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.
NaHC03 (5mL). The mixture was concentrated in vacuo, dissolved in CH2C12 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The organic extracts were dried over Na2S04, concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol, 78%) was eluted with 20% EtOAc in hexanes.
Example 74' 1-f2'-Deexy-2'-Difluoromethylene-5'-O-Dimethoxytrityl~-D-ribofuranosyl)-4-N-Acetyl-Cytosine 2~
2'-Deoxy-2'-difluoromethylene-3',5'-D-(tetraisopropyldisiloxane-1,3-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 vacuo.
The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol, 72°l°) was eluted with 10% MeOH in CH2C12. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (1.03 g, 3.1 mmol) in pyridine (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 CH2C12 (100 mL) and washed with sat. NaHC03 (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgS04, concentrated in vacuo and purified over a silica ge! column using EtOAc:hexanes / 60:40 as eluant to yield 25 (1.2 g, 1.9 mmol, 68%).
Example 75: 1-(2'-Deoxy-2'-Difluoromethylene-5'-D-Dim~thoxvtritvl-Q-D
ribofuranosvll-4-N-Acetylcytesine 3'-(2-c anoethyl-N N-diisonrowlflhos phoramidite,~ j261 1-(2'-Deoxy-2'-difluoromethylene-5'-D-dimethoxytrityl-~i-D-ribofurano-syl)-4-N-acetyicytosine 25 (0.6 g, 0.97 mmol) dissolved in dry CH2C12 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.5 mL, 2.9 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL).
After 10 m the mixture was evaporated to a syrup in .vacuo (40 °C). 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. Rt 0.48 (CH2CI2:MeOH
l 20:1 ).
Example 76: 2'-Keto-3'.5'-O-(Tetraisoprooyldisiloxane-1 3-diyll-6-N-(4-f Butylbenzoyl~-Adenosine (,2~~
Acetic anhydride (4.6 mL) was added to a solution of 3',5'-O-(tetraiso-propyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (Brown,J.;
Christodolou, C.; Jones,S.; Modak,A.; Reese,C.; Sibanda,S.; Ubasawa A.
J. Chem .Soc. Perkin Tans. l 1989, 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 MgS04 and concentrated in vacuo. The residue was purified on a silica gel column to yield 2'-keto-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylben-zoyl)-adenosine 28 (4.8 g, 7.2 mmol, 78%).
Examnfe 77: 2'-Deoxy-2'-methvlene-3'.5'-O-(Tetraisooropvldisiloxane 1 3 _diyll-6-N-(4-t-Butylbenzovl)-Adenosine Under a pressure of argon, sec-butyllithiurn in hexanes (1.1.2 mL, 14.6 mmol) was added to a suspension of triphenylmethyiphosphonium iodide (7.07 g,17.5 mmol) in THF (25 mL) cooled at -78 °C. The homogeneous orange solution was allowed to warm to -30 °C and a solution of 2'-keto-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 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 CHZC12 (250 mL), water was added (20 mL), and the solution was neutralized with a cooled solution of 2% HCI.
The organic layer was washed with H20 (20 mL), 5% aqueous NaHC03 (20 mL), H20 to neutrality, and brine (10 mL). After drying (Na2S04), 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-(tetraiso-propyldisiloxane-1,3-diyl)-6-N-(4-i-butylbenzoyl)-adenosine 29 (3.86 g, 5.8 mmol, 79°I°).
Example 78: 2'-Deoxy-2'-Methyfene-6-N-f4-t-Butytbenzoy~-Adenosine 2'-Deoxy-2'-methylene-3',5'-O-(tetrai~opropyldisiloxane-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 ge! column. 2'-Deoxy-2'-methylene-6-N-(4-t butylbenzoyl)-adenosine (1.8 g, 4.3 mmof, 74°I°) was eluted with 10°l°
MeOH in CH2C12.
Example 79: 5'-O-DMT-2'-Deoxy-2'-Meth~~fene-6-N-(4-t-6utylbenzoyll-Adenosine (29) 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75 g, 1.77 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI
(0.66 g, 1.98 mmol) in pyridine (10 mL) was added dropwise over 15 m.
The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH~C12 (100 mL) and washed with sat. NaHC03, water and brine. The organic extracts were dried over MgS04, concentrated in vacuo and purified over a silica gel column using 50°!°
EtOAc:hexanes as an eluant to yield 29 ~{0.81 g, 1.1 mmol, 62%}.
Example 80: 5'-D-DMT-2'-Deoxv-2'-Methvlene-6-N j4-t-8utylbenzoyll-Adenosine 3'-(2-Cyanoethvl N,N diisopro ylohosphoramiditel 1317 1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-(3-D-ribofuranosyl)-6-N
(4-f-butylbenzoyl)-adenine 29 dissolved in dry CH2C12 (15 mL) was placed 136 ' in a round bottom flask under Ar. Diisopropylethylamine was added, followed by the dropwise addition ofi 2-cyanoethyl N, N-diisopropylchlorophosphoramidite. 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 °C). 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 mrnol, 68%). Rf 0.45 (CH2C12: MeOH / 20:1 ) Example 81: 2'-Deoxy-2'-Difluoromethylene-3'.5'-O-(Tetraisoprooyldi~ifox-ane-1,3-d~r1)-6-N (4-t-But,~lbenzoyl)-Adenosine 2'-Keto-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butyl-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 °C. A warm (60 °C} solution of sodium chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period ofi ~1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The residue was dissolved in GH2Cl2 and chromatographed over silica gel. 2'-Deoxy-2'-dilluoromethylene-3',5'-O-{tetraisopropyldisiloxane-1,3-diyl}-6-N
(4-i-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol, 64°I°) eluted with 15%
hexanes in EtOAc.
>=xample 82: 2'-Deoxv-2'-Difluoromethvlene-6-N-~4-t-Butxlbenzovl)-Adenosine 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-i-butylbenzoyl)-adenosine (4.1 g, 6.4 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'-difluoromethyl ene-6-N-(4-i-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in CH2C12.
Example 83: 5'-O-DMT-2'-Deoxv-2'-Difluoromethvlene-6-N (4-t-Bu~i-benzoyll-Adenosine j301 2'-Deoxy-2'-difluoromethylene-6-N-(4-f-butylben2oyl)-adenosine X2.3 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 CH2C12 (100 mL) and washed with sat. NaHC03, water and brine. The organic extracts were dried over MgS04, concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as efuant to yield 30 (2.6 g, 3.41 mmol, 69%).
Example 84: 5'-O-DMT-2'-Deoxy-2'-Difluoromethvlene-6-NS4-t-Butyt benzo~~IlAdenosine 3'-(2-C~ranoethyl N N-diisonrop~lphos~horamiditel 1-(2'-Deoxy-2'-diffuoromethylene-5'-O-dimethoxytrityl-j3-D-ribofurano-syl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3:4 mmol) dissolved in dry C H 2 C 12 (25 rnL) was placed in a round bottom flask under Ar.
Diisopropyfethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.06 mL, 4.76 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). 32 (2.3 g, 2.4 mmol, 70%} was purified by flash column chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.52 (CH2C12: MeOH /
15:1).
Example 85: 2'-Deoxy-2'-Methoxycarbonylmethylidine-,~-O-(Tetraiso-propyldisiloxane-1.3-diyl)-Uridine (331 Methyl(triphenylphosphoranylidine)acetate (5.4 g,. 16 mmol) was added to a solution of 2'-keto-3',5'-O-(tetraisopropyl disiloxane-1,3-diyl)-uridine 14 in CH2C12 under argon. The mixture was left to stir at RT for 30 h. CHZC12 {100 mL) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCI. The organic layer was washed with H20 (20 mL), 5% aq. NaHC03 (20 mL), H20 to neutrality, and brine (10 mL). After drying (Na~S04), the solvent was evaporated in vacuo to give crude product, that was chromatographed on a silica gel column.
Elution with light petraleurn ether:EtOAc / 7:3 afforded pure 2'-deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 33 {5.8 g, 10.8 mmol, 67.5%).

Example 88: 2'-Deoxy-2'-Methoxvcarbonylmethylidine-Uridine (341 Et3N~3 HF {3 mL) was added to a solution of 2'-deoxy-2'-methoxy-carboxylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 33 (5 g, 9.3 mmol) dissolved in CH2C12 (20 mL) and Et3N {15 mL). The resulting mixture was evaporated in vacuo after 1 h and chromatographed on a silica gel column eluting 2'-deoxy-2'-methoxycarbonylmethylidine-uridine 34 (2.4 g, 8 mmol, 86%} with THF:CH2C12 / 4:1.
Example 87: 5'-O-DMT-2'-Deoxv-2'-Methoxycarbonylmethylidine-Uridine 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 CH2C12 (100 mL} and washed with sat. NaHC03, water and brine.
The organic extracts were dried over MaS04, concentrated in vacuo and purified over a silica gel column using 2-5% MeOH in CH2C12 as an eluant to yield 5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 35 (2.03 g, 3.46 mmol, 86%).
Example 88: 5'-O-DMT-2'-Deoxv-2'-Methoxycarbonvlmethylidine-Uridine 3'-l2-cvanoethvl-N.N-diisonropv Ir ohosphoramiditel (361 1-(2'-Deoxy-2'-2'-methoxycarbonylmethylidine-5'-O-dimethoxytrityl-(i-D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in dry CH2Cf2 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the drapwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.91 mL; 4.08 mmol).
The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 °C).
5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1.8 g, 2.3 mmol, 67%) was purified by flash column chromatography over silica gel using a 30-60°!° EtOAc gradient in hexanes, containing 1% triethyiamine, as eluant. Rf 0.44 (CH2C12:MeOH / 9.5:0.5).

Examele 89' 2'-Deoxv-2'-Carboxymethylidine-3' S'-O-(Tetraisoprooyldi-~iloxane-1.3-diyILUridine 37 2'-Deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldi siloxane-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 1 N HCI solution, extracted with EtOAc (2 x 1.00 mL), washed with brine, dried over MgS04 and concentrated in vacuo to yield the crude acid. 2'-Deoxy-2'-carboxymethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 37 (4.2 g, 7.8 mmol, 73%) was purified on a silica gel column using a gradient of 10-15% MeOH in CHZC12.
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' Dihalc~phosphonate This invention synthesis and uses 3' and/or 5' dihalophosphonate-, e.g., 3' or 5'-CF2-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, 5'- and/or 3'-dihalophosphonate nucleotide containing ribozymes, deoxyribozymes (see Usman et al., PCT/US94/11649, and ' chimeras, of nucleotides, are catalytic nucleic molecules that contain 5'-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-8z-D-ribofuranose 5-d-5+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-O-isopropylidene-t3-D-ribofuranose 5-deoxy-5-dihalomethylphosphonate is described (e.g., for the difluoro, in Figure 87). Condensation of this suitably derivatized sugar with silylated pyrimidines and purines affords novel nucleoside 5'-deoxy-5'-dihalomethylphosphonates, These intermediates may be incorporated into catalytic or antisense nucleic acids by either chemical {conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into suitably protected phosphoramidites 12a or solid supports 12b, e.g., Figure 88) or enzymatic means (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into their triphosphates, e.g., 14 Figure 89, for T7 transcription).
Thus, in one aspect the invention features 5' andlor 3'-dihalonucleotides and nucleic acids containing such 5' and/or 3'-dihalonucleotides. The general structure of such molecules is shown below.

II
(R~O)2pCXp R II
O B 2 O 8 (R30)zPCXz O B
i I
Rz R~ i Xz R~ CXz R~
_ I
(R30~zP - O (Rs0)zP = O
where R1 is H, 4H, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, er carbonate; each R2 is separately H, OH, or R; each R3 is separately a phosphate protecting group, e.g,, methyl, ethyl, cyanoethyl, p-nitrophenyl, 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 anti enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleoside 5'-deoxy-5'-dihalo and/or 3'-deoxy-3'-dihalophosphonates by condensing a dihalophosphonate-containing sugar with a pyrimidine or a purine under conditions suitable to form a nucleoside 5'-deoxy-5'-dihalophosphonate and/or a 3'-deoxy-3'-dihalophosphonate.
Phosphonic acids may exhibit important biological properties because of their similarity 1o phosphates (Engel, Chem. Rev. 1977, 77, 349-367). Blackburn and Kent (J. 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 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 & Nucleofides 1985, 4, 165-167;
Blackburn et al., Chem. Scr. 1986, 26, 21-24). 9-(5,5-Difluoro-5-phosphonopentyl)guanine (2) 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 ei al., Biochemistry 1993, 32, 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.

' NH2 O P x-F O F O
O' O' 0' OH OH

N H
I ., N.
N

(H0~20PCF2~

(ETO}2POCF2Li One common synthetic approach to a,a-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive substrate by diethyl (lithiodiffuoromethyl)phosphonate (3) (Obayashi et al., Tetrahedron Left. 1982, 23, 2323-2326). However, our attempts to synthesize nucleoside 5'-deoxy-5'-difluoro-methylphosphonates from 5'-deoxy-5'-iodonucleosides using 3 were unsuccessful, i.e. starting compounds were quantitatively recovered. The reaction of nucleoside 5'-aldehydes with 3, according to the procedure of Martin et al. (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842), led to a complex mixture of products. Recently, the synthesis of sugar a,a-difluoroalkylphosphonates from primary sugar triflates using 3 was described (Berkowitz et al., J. Org.
Chem. 1993, 58, 6174-6176). Unfortunately, our experience is that nucleoside 5'-triflates are too unstable to be used in these syntheses.
The following are non-limiting examples showing the synthesis of nucleoside 5'-deoxy-5'-difluoromethyl-phosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon 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.
Example 90: Synthesis of Nucleoside 5'-Deoxv-5'-difluorometh~rlpho~honates Referring to Fia. 87, we synthesized a suitable glycosylating agent from the known D-ribose a,a-difluoromethylphosphonate (4) (Martin ef al., Tetrahedron Lett. 1992, 33, 1839-1842) which served as a key intermediate for the synthesis of nucleoside 5'-difluoromethylphosphonates.
Methyl 2,3-O-isopropylidene-~-D-ribofuranose a,a-difiuoromethylphosphonaie (4) was synthesized from the 5-aldehyde according to the procedure of Martin ei al. (Tetrahedron Leit. 1992, 33, 1839-1842) (Figure 87). Removal of the isopropylidene group was accomplished under mild conditions (12-MeOH, reflux, 18 h (Szarek ei al., Tetrahedron Lett. 1986, 27, 3827) or Dowex 50 WX8 (H+), MeOH, RT
(about 20-25°C), 3 days) in 72% yield. The anomeric mixture thus obtained was benzoylated with benzoyl chloridelpyridine to afford the 2,3-di-O-benzoyl derivative, which was subjected to mild acetolysis conditions (Walczak et al., Synthesis, 1,993, 790-792) (Ac20, AcOH, H2S04, EtOAc, 0°C. The desired 1-O-acetyl-2,3-di-O-benzoyl-D-ribofuranose difluoromethylphosphonate (5) was obtained in quantitative yield as an anomeric mixture. These derivatives were used for selective glycosylation of silylated uracil and N4-acetylcytosine under Vorbruggen conditions (Vorbruggen, Nucleoside Analogs. Chemistry, Biology and Medical Applications, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F3CS020Si(CH3)3 as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1-. ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron Lett. 1987, 28, 3623-3626 and references cited therein) (SnCl4 as a catalyst, boiling acetonitrile) to yield p-nucleosides (62% 6a, 75% 6b).
Glycosylation of silylated N6-benzoyladenine under the same conditions yielded a mixture of N-9 isomer 6c and N-7 isomer 7 in 34% and 15%
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
TEAR gradient for elution and obtained as their sodium salts (82% 8a; 87%
8b; 82°l° 8c).
Selected analytical data: 31 P-NMR {31 P) and 1 H-NMR (1 H) were recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H3P04 and TMS, respectively. Solvent was CDC13 unless otherwise noted. 5: 1 H
8 8.07-7.28 (m, Bz), 6.66 (d, J 1,2 4.5, aH1 ), 6.42 {s, ~3H1 ), 5.74 (d, J2,3 4.9, pH2), 5.67 (dd, J3,2 4.9, J3,4 6.6, ~iH3), 5.63 (dd, J3,2 6.7, J3,4 3.6, aH3), 5.57 {dd, J2,1 4.5, J2,3 6.7, aH2), 4.91 {m, H4), 4.30 (m, CH2CH3), 2.64 (m, CH2CF2), 2.18 (s, ~iAc), 2.12 (s, aAc), 1.39 {m, CH2CH3). 31 P 8 7.82 (t, Jp,F 105.2), 7.67 {t, Jp,F 106.5). 6a: 1 H 8 9.11 {s, 1 H, NH), 8.01 (m, 11 H, Bz, H6), 5.94 (d, J1~,2~ 4.1, 1H, H1'), 5.83 (dd, J5,6 8.1, 1H, H5), 5.79 (dd, J2,,1 ~ 4.1, J2~,3~ 6.5, 1 H, H2'), 5.71 (dd, Jg~,2~ 6.5, J3~,4~ 6.4, 1 H, H3'), 4.79 (dd, J4~,3~ 6.4, J4~,F 11.6, 1 H, H4'), 4.31 (m, 4H, CH2CH3), 2.75 (tq, JH,F
19.6, 2H, CH2CF2), 1.40 (m, 6H, CH2CH3). 31P 8 ?.7? (t, Jp,F 104.0). 8c:
31 p (ys DSS) (D20) b 5.71 (t, Jp,F 87.9).
Compound 7 was deacylated with methanolic ammonia yielding the product that showed ~~max {H20) 271 nm and min 233 nm, confirming that the site of glycosylation was N-7. .
Example 9l:Svnthesis of Nucleic Acids Containing Modified Nucleotide ~ntaininp Cores The method of synthesis used follows the procedure for norrria! 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, y8, 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 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 Triphosohate The triphosphate derivatives of the above nucleotides can be formed as shown in F_ig. 89, according to known procedures. Nucleic Acid Chem., Leroy B. Townsend, John Wiley & Sons, New York 1991, pp. 337-340;
Nucleotide Analogs, Karl Heinz Scheit; John Wiley 8 Sons New York 1980, pp. 211-218.
Equivalent synthetic schemes for 3' dihalophosphonates are shown in Figures 90 and 91 using art recognized nomenclature. The conditions can be optimized by standard procedures.
The nucleoside dihalophosphonates described herein are 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.
Olic~onucfeotides with Amido or Peptido Modification This invention replaces 2'-hydroxyl group of a ribonucl.eotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and 5' 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 1 below:

O
B

~~ R2 O N"
H R~ R3 p",. p..p I
O.
F,C~RMULA I
The base (B) 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 addition, either R1 or R2 is H or an alkyl, alkene or alkyne group containing between 2 and 10 carbon atoms, or hydrogen, an amine (primary, secondary or tertiary, egj, R3NR4 where each R3 and R4 independently is hydrogen or an alkyl, alkene or alkyne having between 2 and 10 carbon atoms, or is a residue of an amino acid, i.e., an amide), an alkyl group, or 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 R1, R2 and R3 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 farmed 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.

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 1, above.
In other aspects, the oligonucleotide may include a 3' or 5' nucleotide having a 3' or 5' located amino acid or aminoacyl group. Iri all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard modifications can be made. For example; an "O" may be replaced with an S, the sugar may lack a base (i.e., abasic) and the phosphate moiety may be modified to include other substitutions (see Sproat, supra).
Example 93: General procedure for the preparation of 2'-aminoacwl-2'-deoxv-2'-aminonucleoside coniuQates.
Referring to Fig~92, to the solution of 2'-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or D-) amino acid (1 mmol) in methanol [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDD) [or 1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to 50 'C from 3-48 hours. Solvents are removed under reduced pressure and the residual syrup is chromatographed on the column of silica-gel using 1-10 % methanol in dichloromethane. Fractions containing the product are concentrated yielding a white foam with yields ranging from 85 to 95 %.
Structures are confirmed by ~ 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 procedurQS (Oligonucleotide Synthesis: A Practical Approach, ~ 148 M.J. Gait ed.; IRL Press, Oxford, 1984). incorporation of these phosphoramidites into RNA was performed using standard protocols (Usman et aL, 1987 supra).
A general deprotection protocol for oligonucleotides of the present invention is described in F-_ia. 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 (e.g., adenosine, cytidine, guanosine) and/or abasic moieties.
Example 94: RNA cleavacte by hammerhead ribozymes containing 2'-aminoacyl modifications.
Hammerhead ribozymes targeted to site N (see Fig. 94) are synthesized using solid-phase synthesis, as described above. U4 and U7 positions are modified, individually or in combination, with either 2'-NH-alanine or 2'-NH-lysine.
RNA cleavaoe 2~say m vitro: Substrate RNA is 5' end-labeled using (.~-32p) ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace amount (< 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37°C for 10 min in a buffer containing 50 mM
Tris-HC1 and 10 mM MgCl2. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 ~tt 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 F_ia~95, hammerhead ribozymes containing 2'-NH-alanine or 2'-NH-lysine modifications at U4 and U7 positions cleave the target RNA efficiently.

' 149 Sequences listed in Figure 94 and the modifications described . in Fi ure ~ are meant to be non-limiting examples. Those skilled in the art wilt 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: Aminoac~rlation of 3'-ends of RNA
!. Referring to F- ig. 96. 3'-OH group of the nucleotide is converted to 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.

11. Preparation of aminoacyl-derivatized solid support A~ S..ynthesis of O-Dimethox rityl f0-DMT,) amino acid, Referring to Fig. 97. to a solution of L- (or D-) serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4,4'-dimethoxytrityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25°C) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was partitioned between 5% aq.
NaHC03 and dichloromethane, organic layer was washed with brine, dried (NaZSO~) 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 °l°
yield).
B~ Preparation of the solid suonort and its derivatization with amino acids Referring to Fig. 97, the modified solid support (has an OH group instead of the standard NH2 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 NH-monomethoxytrityl (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 and the support. This support is suitable for the construction of RNAlDNA
chain using suitably protected nucleoside phosphoramidites.
Example 96: Aminoac~rlation of 5'-ends of RNA
1. Referring to Fia. 98, 5'-amino-containing sugar moiety was synthesized as described (Mag and Engels, 1989 Nucleic Acids f?es. 17, 5973). Aminoacylation of the 5'-end of the monomer was achieved as described above and RNA phosphoramidite of the 5'-aminoacylated monomer was prepared as described by Usrraan ef al., 1987 supra. The phosphoramidite was then incorporated at the 5'-end of the ofigonucleotide using standard solid-phase synthesis protocols described above.
II. Referring to Fia. 99, aminoacyl groups) is attached to the phosphate group at the 5'-end of the RNA using standard procedures described above.
V11. Reversing Genetic Mutations Modification of existing nucleic acid sequences can be achieved by homologous recombination. In this process a transfected sequence recombines with homologous chromosomal sequences and can replace the endogenous cellular sequence. Boggs, 8 International J. Cell Cloning 80, 1990, describes targeted gene modification. It reviews the use of homologous DNA recombination to co«ect genetic defects. Banga and Boyd, 89 Proc. Nafl. 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 revenant 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 reversing a mutant phenotype to wild-type. An example of this type of reversion is creating a stop colon 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 ofigonucleotide would (analogously to an antisense oligonucleotide or ribozyme) have to be continuously present to revert the RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying 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 (Fi . 1 1) 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 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, Genesj1983 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 (tripl.ex formation) to double-stranded DNA, which is an established technique for binding poly-pyrimidine tracts, and can be extended to recognize all 4 nucleotides. See Povsic, T., Strobel, S., & Dervan, P. (1992). Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation.
J. Am. Chem. Soc. 114, 5934-5944 (1992). Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, A.V. & Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, ~ 152 1993) describe conjugation of reactive groups or enzyme to oligonucleotides and can be used in the methods described herein.
Recently, antisense oligonucleotides have been used to redirect an incorrect splice into order to obtain correct splicing of a splice mutant globin gene in vitro. Dominski Z; Kole R (1993) Restoration of correct splicing in thalassemia pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci U S A 90:8673-7. Analogously, in one preferred embodiment of this invention a complementary oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by antisense.
In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence.
This can be accomplished by activating an endogenous enzyme (s, ee Figure 102), by appropriate positioning of an enzyme (or ribozyme) conjugated (or activated by the duplex) to the oligonucleotide, or by appropriate positioning of a chemical mutagen. Specific mutagens, such as nitrous acid which deaminates C to U, are most useful, but others can also be used if inactivation of a harmful RNA is desired.
RNA editing is an naturally occurring event in mammalian cells in which a sequence modifying activity edits a RNA to its proper sequence post-transcriptionally. Higuchi, M." Single, F., Kohler, M., Sommer; B., and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GIuR-B: A
base-paired intron-exon structure determines. position and efficiency Cell 75:1361-13?0. 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 (e.g., an alkylatbr) 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.

This includes correcting a sequence instead ofi inactivating a gene but can also include inactivating a deleterious gene.
Thus, in one aspect, the invention features a method for altering ,j,n_ vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule. The method includes contacting the nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After formation of the duplex or triplex molecule a base modifying activity chemically or enzymatically alters the targeted base directly, or after nucleic acid repair in vivo. This results in the functional alteration of the nucleic acid sequence.
By "alter", as it is used in this context, is meant that one or more chemical moieties in a targeted base, or bases, is altered so that the mutant nucleic acid will be functionally different. Thus, this is distinct from prior methods of correcting defects in DNA, such as homologous recombination, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the transfected nucleic acid. This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.
By "functionally alter" is meant that the ability of the target nucleic acid to perform its normal function (i.e.., transcription or translation contfol) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.
By "mutant" it is meant a nucleic acid molecule which is altered in some way compared to equivalent molecules present in a' normal individual. Such mutants may be well known in the art, and include, molecules present in individuals with known genetic deficiencies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal expression of a gene, such as cancer, thalassemia's and sickle cell anemia, and cystic 154 ' fibrosis. It allows modulation of lipid metabolism to reduce artery disease, treatment of integrated AIDS genomes, and AIDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a "wild type" phenotype and/or genotype. For deleterious conditions this involves altering a base to allow expression or prevent expression as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant (i.e., non-human gene) to a wild type (i.e., no production of a non-human protein). Such modification is performed in traps rather than in cis as in prior methods.
In preferred embodiments, the oligonucleotide is of a length (at least 12 bases, preferably 17 - 22) sufficient to activate dsRNA deaminase in viyo 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, e.g., the mutagen is nitrous acid; and the oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to 5-methylcytosine.
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. cience 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, PCTlUS94/12976 in which entire exons with wild-type sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 - 3).
Thus,Y in another aspect, the invention features ribozymes or enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule. Applicant is the first to determine that such molecules would be useful, and to provide a description of how such molecules might be isolated.
Molecules used to achieve in situ reversion can be delivered using the existing means employed for delivering antisense molecules and ribozymes, including liposomes and cationic lipid complexes. If the in situ reverting molecule is composed. only of RNA, then expression vectors can be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes expression vectors.
There are several advantages of using such an expression vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that already express that gene. Furthermore, the corrected gene would be properly regulated by its natural transcriptional promoter. Lastly, reversion can be used when the mutant RNA creates a dominant gain of function protein (e.g., 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 dififerent 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 eukdryotes, very few Hi~As (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 -~ G). 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 A--~ I. Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.
In the present invention, the endogenous deaminase activity or other such activities can be utilised to achieve targeted base modification.
The following are examples of the invention to illustrate different methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific embodiments of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims.
Example 97: Exploitin4 celluiar dsRNA dependent Adenine to Inosin,~
converter:
An endogenous activity in most mammalian cells and Xenopus oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. L., 8 Weintraub, H. (1988). An unwinding activity that covalently modifies it double-stranded RNA substrate. ell, 5;z, 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 1 (G) 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 non-complementary RNA oligonucleotide was added to the dystrophinJluciferase 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 (G) cannot create a stop codon, so the ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a dystrophin protein made from an mRNA reverted by this method should retain full activity.
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 (5' to 3'):
CCCGCGGTAGATCTTTCTGGAGGCTTACAGTTTTCTACAAACCTCC
CTTCAAA (Seq. ID No. 1 }
Referring to Fioure 104j 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 111 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 (5' to 3'):
GCCCCTGAGGAGCGATGGAGGCGTTGAAGGGAGGTTTGTGGAAAA
CTGTAAGCCTCCAGAAAGATCTACCGCGG (Seq ID No. 2) . 158 This corresponds to base pairs 3649-3708 of normal dystrophin (Entrez ID # 311627) with a Sac 11 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 (0.5X=
25mM Tris (pH 7.9), 12.5°l° 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.. 8 Weintraub, H.
Cell 55, 1089-i 098 (1988).
The target mRNA at 500ng/ul was pre-annealed to 1 micromolar complementary or irrelevant RNA oligonucleotide by heating to 70°C, and allowing it to slowly cool to 37°C over 30 minutes. Fifty nanograms of mRNA pre-annealed to the RNA oligonucleotides was added to 7u1 of nuclear extracts containing 1 mM ATP, 15mM EDTA, 1600un/ml RNasin and 12.5mM Tris pH 8 to a total volume of l2ul. Bass, B.L. & Weintraub, H.
supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 25°C. Next, l.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. 1-uciferase assays were performed on l5u) 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, D.G., Valentin, V.V., Vafentina, F.Z., Lebedev, A.V. 8. Federova, O.S. Design and targeted reactions of oiigonucleoiide derivatives 1-366 (CRC Press, Novosibirsk, 1993) and Povsic,.T., Strobel, S. 8 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.

~ 159 Additionally enzymes that modify nucleic acids have been conjugated to oligonucleotides. (Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, A.V. 8~ 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, as described herein (see figure 100-104).
1. Deamination of 5-methylcytosine to create thymidine (performed by the_enzyme ,cytidine deami,nase .(Bass,, .B.L. "in."T.he RNA .._ lNorld (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).
Also, nitrous acid or related compounds promote oxidative deamination of C to be read at T(Microbial Genetics, David Freifelder, Jones and Bartiett Publishers, Inc., Boston,1987, PP.226-230.). Additionally hydroxylamine or related compounds can transforrii C to be read at T (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, inc., Boston,1987, PP.226 230.) 2. Deamination of cytosine to create uracii (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 5-methylcytosine 5. Transforming thymidine (or uracil) to 02-methyl thymidine (or 02-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, e.g., 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 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 exsmple, 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. !n this example an ArT' 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

i61 A Transversion Transversion DNA~.~3~RNA3 T(U) Transversion DNASiRNA7 Transversion C Transversion RNA2/DNA6 Transversion G DNA6iRNA6 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).
4 Methylation of cytosine to 5-methyfcytosine.
5 Transforming thymidine (or uracil) to 02-methyl thymidine (or 02-methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).
6 Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978)).
7. Amination of uracil to. cytosine. Bass supra, fig. 6c.
In Vitro defection Strateqyr 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, ,1. (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 cience 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.

~s2 ' The in vitro selection (evolution) stralegy 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) cience 2fi1:1411-1418; Szostak, J. W.
(1993) TIB 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 far 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. (i992) ci nc 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 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 vGriety 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 (RE). 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 ' 163 and transformed into bacterial hosts. Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques, Base modifying enzymatic nucleic acids (identified via in vitro selection) can be used to cause the chemical modification in vivo.
In addition, the ribozyme could be evolved to specifically bind a protein having an enzymatic base changing acitivity.
Such ribozymes can be used to cause the above chemical modifications in vivo. The ribozymes or above noted antisense-type molecules can be administered by methods discussed in the above referenced art.
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, i.e., 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.
U A 73, 2294). In addition, applicant has determined that that the RNA
20' portion of the R-laop 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 R
loop 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 firom 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 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 ari, see e.g:, Eckstein et al., International Publication No. WO 92/07065; Perrault ei aL, 1990 Nature 344, 565; Pieken ei al., 1991 cience, 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi ei al., International Publication No. WO 91/03162, as well as Sproat,B. European Patent Application 927 7 0298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules.
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.
These 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 37oC, but it is simply desirable in this invention that the R-loop structure exists to some extent at the site of action so that the expression of the desired nucleic acid will be achieved at that site of action. While it is preferred~that the R-loop structure be stable under ' . 165 those conditions, even a minimal amount of formation of the R-loop structure to cause expression will be sufficient. Those in the art will recognize that measurement of such expression is readily achieved, especially in the absence of any promoter or leader sequence on the first nucleic acid molecule (Daube and von Hippel, 1992 cien a 258, 1320).
Such expression can thus only be achieved if an R-Poop structure is truly formed with the second nucleic acid. if a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that transcription is enhanced.
In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by forming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or transcription termination signal such that once expression is initiated it will continue until the first nucleic acid is degraded.
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, e.g., an antibody, transferin, a nuclear localization peptide, or folate, or other such compounds well known in the art, which wilt aid in targeting the R-loop complex to a desired cell or tissue.
In preferred embodiments, the first nucleic acid is a plasmid, e.g., one without a promoter or a transcription termination signal ; the second nucleic acid is of length between about 40-200 bases and is formed of ribonucleotides at a majority of positions; and the second nucleic is 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, e.g., it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNAlDNA heteroduplex;
no promoter or leader region is provided in the first nucleic acid; and the R-loop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular transcription machinery. , 1n other preferred embodiments, the first nucleic acid encodes one or more enzymatic nucleic acids, e.g., it is formed. with a plufality 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 comr'lex An R-loop complex is designed to provide a non-integrating plasmid so that, when an RNA polymerase binds to the plasmid, transcription is continuous until the plasmid is degraded. This is achieved by hybridizing an RNA molecule, 40 to 200 nucleotides in length, to a DNA expression plasmid resulting in an R-loop structure (see fi4ure 106). This RNA, when conjugated with a ligand that binds to a cell surface receptor, triggers internalization of the plasmid/RNA-ligand complex. Formation of R-loops in general is described by DeWet, 1987 Methods in Enzymo~,, 145, 235;
Neuwald et al., 1977 J. Virol. 21,1019; and Meyer et al., 1986 J. Ult. Mol, tr. Re . 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 pofymerase. Applicant was told that crealion of an R-loop between the promoter and the reporter gene increased the transfection efficiency. Incubation of an RNA molecule with a double-stranded 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 f3-galactosidase gene. The R-loop was initiated either in the promoter region or in the leader sequence. Plasmids containing ,an R-loop structure were micr~oinjected into the cytoplasm of CAS 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 80 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, su ra .
~ O~ie 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 R-loop may aid in the recruitment of cellular transcription machinery. Once an RNA polymerase binds to the plasmid and initiates transcription, the process will continue until a termination signal is reached, or the plasmid is degraded.
This invention will increase the expression of ribozymes inside a cell. The idea is to construct a plasmid with no transcription termination signal, such that a transcript-containing multiple ribozyme units can be generated. In order to liberate unit length ribozymes, self-processing ribozymes can be cloned downstream of each therapeutic ribozyme (seg figure 107) as described by Draper supra.
Liaand Tar etina Another salient feature of this invention is that the RNA used to generate R-loop structures can be covalently finked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, eic.). 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 figure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 J. Biol. Chem. 269, 3198-3204). The RNA containing a fi carbon spacer with a terminal amine group is mixed with folate and the mixture is reacted with activators like 1-(3-Dimethyiaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). This reaction should be carried out in the presence of 1-Hydroxybenzotriazole hydrate (HOST) to prevent any undesirable side reactions. .

The RNA can also be derivatized with a heterobifuctional crosslinking agent (or linker) like succinimidyl 4-(p-maleimidopheny!)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 Pack 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 RfVAIDNA complex, thus increasing the accessibility of the ligand for its receptor and not interfering with the hybridization. These techniques can be used to fink peptides such as nuclear localization signal (NLS) peptides (t-anford et al., 1984 ell 37, 801-813; Kalderon et al., 1984 ell 39, 499-509; Goldfarb et al., 1986 Nature 322, 641-644)and/or proteins like the transferrin (Curiel et al., 1991 Proc. Natl. Acad. Sci. USA 88, .8850-8854; Wagner et al., 1992 Proc. Natl.
Acad. Sci. USA 89, 6099-6103; Giulio et al., 1994 Cell. Signal. 6, 83-90) to the ends of R-loop forming RNA in order to facilitate the uptake and localization of the R-loop-DNA complex. To link a protein to the ends of R-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 iminothiofate 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 dancer 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-Poop-containing plasmid efficiently using receptor-mediated endocytosis. The in vitro selection (evolution) strategy is iss similar to approaches developed by Joyce (BEaudry and Joyce, 1992 Science 257, 635-641; Joyce, 1992 Scientific American 267, 90-97) and Szostak (Barrel and Szostak, 1993 cience 261:1411-1418; Szostak, 1993 T I B 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) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their affinity to form R-loop and/or their ability to bind to a specific receptor, 3) introduction of a restriction endonuclease site for the purpose of cloning. The degenerate domain can .be .created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 cience 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 su ra . 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.

TA
Ch2racteristics of Ribozvmes Group 1 lntrons Size: -200 to >1000 nucleotides.
Requires a U in the target sequence immediately 'S' of the cleavage site.
hinds 4-6 nucleotides at 5' side of cleavage site.
Over 75 known members of this class. Found in Tefrahyme~a thermophila rRNA, fungal mitcchondria, chloroplasts, phage T4, blue-green algae, and others.
RNAseP RNA (M1 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.
Binds' 4-6 nucleotides at 5' side of the cleavage site and a variable number to the 3' side of the cleavage site.
Only 3 known member of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent (Figure 3).
Hepatitis Delta Virus (HDV) Ribozyme Size: 50 - 60 nucleotides (at present).
Cleavage of target RNAs recently demonstrated.
Sequence requirements not fully determined.
Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required.
Only 1 known member of this class. Found in human HDV (Figure 4).
Neurospora VS RNA Ribozyme Size: -144 nucleotides (at present) l~l Cleav~oe of ;~roet 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 5).

Table 2 HumGn ICBM HH Taraet sequence nt. Position Teraet Sequences nt. Position Target Sequences i C C C C=.GJC ~CC-~ JG 2 b 6 nC C v'GU '~C',;
C~::
~,C'J

23 C'JC~G'~J C C:JCUG:J ?~4 CUC~C'J C C: -C~.CG

26 ~G.."'L'CC',JC UG~'"L:~C'J420 C~CCC ~ C C;C'LJCL'U

Cv'CL~-CTJA CUC'-~G-':GS2s C'JCCC~J C LZ;Cz-CiG

34 L,'G.:~~C'JC F~G.=.GL'UG427 CCC:.'UC'JU G:~:AG~.C

40 UC_'-.GnGUU GC=ACL'C7 4~0 ~G~,ACCU 'J.~CCC'JAC

48 C~-,~,~,CCUC tiGCC'JCG 4~_ C~_CCULi .~C:.C'JACG

~4 UC~GCCU C G~;r:UG: 456 L'U?~CC::LJA CC-C"UGC~

58 cc,;c~,~ ~ UGGL--ccc 4 a ~ cc~accu c ~ccGUG;, J

64 L:-,UC~J C CCi:C-C<yG.10 L'GCL'G..'"UC CGUCz,C-G

6 CCGC~CU C CUG~JCC 564 CUG~.C~~U C =_C;~CC~

102 UC;: uC...':IJC CUG.: JCG 3 S 2 G~C~U C ? CC~UG::

108 UC~JG..:J C GCi-~~:C 6u7 ~C-C~.~,~,UiJUC'UCG'JG

115 CGC.C-:n~UC LwuC'CC EG8 GCCFAL'U U CLC'GUG.~.

'_,g C~JC'u'GU U CCC~C-::~ 60~ CC~L'L,'U C L;C~JGC;.

120 C'JCL'C-uDC CCAG~C c 1= :-..:~L'L'UC'JC G'.~GCCG~.

14 C~Gr.CJiU C UGUGUCC E = c" GaC~~1J U UCH
6 G'J C,~C

1 S2 UCUGJGU C CCC_~JCJy E'_- 7 ~~L;G',,'UU GCr_r~C~

158 UCCCCCU C nn:AGUC 668 ~C~CCLJ C Ciz.CCCC

l E~ C~,~-u:GU C nUCCUGC 6 7 GCCCCC'J ~ CCrC-w"LTC

168 ra-.GUG~U C C'JGCCCC 684 ACC~C~Ci1 C C~C~CCU

8 5 C-;~GG~: C C ~"L3G E 9 2 C? GACCU U UGL'CCUG
J

209 ~G~.~CCU C WJGUGAC E' ~CCW U GL:~CWJGC

227 CcCT~GU U GLL'GwC E96 CCL'L'UG'JC CIJCiCAG

2 3 :zAGULGU U G:~;~CAUA 7 0 r GC C:~CUC C C
0 C C~C~

237 UGC..~U A GnGrCCC 720 C~C~ACL1 U G'JGGrCC

248 hCCCCGU L'GCCL?~? X23 :-ACWGiJ C AC-CCCCC

2 S GL~ a GC A n.:-.~,~;G;~,7 ? ~ C C C ~ C CL~G.Cz:
3 ~J :,"J

2 6. ~=.C~Gv U Gw~JCCUG ~ 3 6 C-C,C-L7CCU? G=_C~.':UGG

2 67 nG'JL.'~G~JC CUGCCTG 7 6= CCG'JCvJ C UGu'LiCCs .
293 iu;C-.=JVJA 'v'Gf-.ACUG7E9 G,~-JCUGU U CCCL'GGA

'l9 r.G?.:yGAUr.GCC'CC 77G GJCUGUU C CC'UG:~.C

_' nUGJC-' ~ L'UC~AC 7 8 5 Cv:,CUGU U CCCAGUC
S J

3 3 GJC'''LTT~UU C~u-.nCUG 7 8 6 GC,C"LiGW C CC.=.GUCLJ

.38 UGC"J'rsULJC W-.~.:C"JGC'?92 UCCCT~GU C UCC~GG

'_ GC.,~C: C ~Cr C-..~L;7 9 4 C C=~GUCU C GC:.~
:GJ G.~...CC

367 r.~:Cr.G~:JA ~,CCUL,' 807 CCG:G.,"U C C=~CCUGG

374 r.:~:CCU U CCUCyCC 833 C~G~G;J U Gr_~CCCC

375 FA~:CCUL' C CUC~CCG c846 CCT~C~G'J C .~CC'UF.UG

378 CCtTJCCU C ACCGJGU 851 G'JGCCU ~,UC1-C.P
nC

E63 ."-.AC~C~J C CQUCUCG1408 UCCrG-'~U C UUGaG.~_~

866 GCJCCU U C'~.iCC~CC 1410 CAG:~LiC'J U ~~~

867 nCUCCW C UC'v:~CCr~ 1421 GGCACCU A CCUCUGLT

869 UCCL'QCU C GG..:.~G 1425 CCUACCU C UGUCGw 881 AaGGCCO C AGUCAGU 1429 CCUCL'GU C GGGCCAG

685 CCUCAGL1 C AGL~UGA 1444 GAGCACU C AAGGGGA

9~3 GJGCAG'J A AIJACUGG 1455 ~G.~.GGU C ACCCGV.G

936 CAGUAAU A CGG~~ 1482 AUGL'G.."'CT C UCCCCCC

978 'uG~CC?.U C UAC'-~CC~T1484 GUG...~'GCU C CCCCC~.u S80 ACCyUCU A CAC-W.JUU 1493 CCCCCy~U A UGAGAUU

966 UACAG.=J U UCCC,GCG 1500 AUGAGAU U GUCAUCA

987 ACAGC'W U CC~.NCC1 1503 AC.AL'UGU C AUCAUCA

988 C?G..~.IW C CGG..~CC1506 L'tSGUC?.U C AUCACUG

1005 ACGL'GAU U CQCACGA '_509 UCAUCAU C ACUGUGG

1006 CGi.JGL'U C UGACGAA 1518 CUGUCv--LJ A GCAG.~.CG

1023 CAG~J C UC~AAG 1530 CC~~AGQ C AUAAUG;

1025 G~JCU C AGAAC,GG 1533 GGUCAU A AU ~G~G~, 1066 CCACCCU A GAGCCAA 1551 C1~GGCCU C AGCACG'J

1092 aUG~~D U CCAGCCC 1559 AGCACGU A CCUCUAU

1093 UC-:T.vJU C C~,GCCCA1563 CGUACCU C UALTAACC

1125 CCCi~G.."U C CGG~~UGA1565 UACCUCU ?. UAACCGC

1163 C~"U U CLTCCL1GC 1567 CCUCUAU A ACCGCCA

1164 GCAG.'W C UCCUGCU 1584 GGAACaU C AAGAAAU

116 AG'~UtTCU C CCT~COCU? 5 9 2 AAGAF.AU A CAGACUA

172 UCCL7G._~U C UG~.rAACC'_ 5 99 ACAGAC'J A Cp.ACAGG

2 0 CZ CAG'"Q U r'.L~C~.C~.16 51 CACGC CU C CCUGAAC

1201 CCyG.~JU A L'ACACAA 1661 UGAACCU A UCCCGG

1203 :~C-w.'VJAU A CACAAGA1663 AACCVAU C CCGG AC

1227 CZ~::~G~~U U CGUGUCC1678 ~~~CU C UUCCUCG

1228 G::ACz~JtJ C GJGUCCU1680 GGCCUCU U CCUCGC~

1233 UUCGJGU C CUGUAUG 1681 GCCUCW C CUC'GGCC

1238 ~JCC~J A UGGCCCC 1684 UCUL'CCU C GGCCUUC

12 64 GACv::aU U GOCC'GGG 1690 ~ UCGGCCU U CCCAUAU

1267 G:~UUGU C CGwAAA 1691 CGGCCL'U C CCAUAW

12 9 AG.F.r.AU U CCCAGCA 16 9 6 UUCCCAU A UUG.~~UGG

.295 G.~W C CCAGCAG 1698. CCCAUAU U G.~~JGG~

306 G:..T~C1~CU C CAAUGUG1737 F.AGACAU A UGCCAUG

1321 CC:~GGCZ U ~~G.~AA 1750 UGCAG.~U A CACCUAC

1334 nACCCAU U GCCC'GAG 1756 UACACC'J A CCGG.~CC

1344 CCGAGC'U C AAGUGUC 1787 A ~~~ U U GUCCUCA

13 51 Cr~GtIGU C LRAAGGA 17 9 0 GCAUUGU C CUCAGUC

1366 UG:~CACU U UCCCACU 1797 CCUCAGU C AGAUACA

1367 G:vC'~CUU U CCCACLiG1802 ~ G'JCAGAU A G'aACAGC

13 68 G:~CLZ'U C CCACUGC 1812 ACAC~.AU U UG~wGCC

380 UGCCCAU C ~G.~AU 1813 CAGC.AL'U U ~~~
CA

.388 C-CPU C AGL1GACU 1825 CCAUG"~U A CCL~GCAC

1398 UCACUGU C AC"JCGAG 1837 CACACCU A AAACACU

1256 C=~CC-Ci;U C UG:~UCL'G 3? 89 UAUUL'AU U CnG'vGUC
861 ~'u'C'.'~GiU C L'W.r.GV.iC =~ 96 LT~-'..GUv"~J C L'UUUaUG
1865 G-':UCt.'GU :y GUC?~G.U 2'_98 'r.GLlG~ICp U L~JAUC'JA
186E C'JcwsGL' C ACr.UG~C 2, 9A GUGUCUU U UeAL'cJAG
1877 C=.iJG'eC'u 'r. AGCC'~G 2200 UGUCUL1U U AUCUAG~v '_ 9 01 C'~.G,CU C AAC.ACAU 2 2 O 1 GZJ~ A ~~
1012 aC:,Ln~,L' U G.T.~L;C-.~-.AU 1205 ULZJAUGU A C~'V'~,?.A
c 2 2 UG:z.= L'cJ U nA,=,CJC~J 2 2 . 0 cvTAG:~."U A =~~LT?,.~C
° 2 3 G'.~ :L'G'~TIJ y ~-~, C",TA 2 2 2 0 L'G? AC:,U A CuTC'JC'J
19 2 8 L'L~~-.; :cJ c U'r~ C UG 2 2 2 4 GG~LTAG""U C UCL'Gv:. C
c30 ~cvC;.v A C-C~..'GAU 2225 L'AG""'UC'.J C UGV,C~JC
1964 G=1~C'-.U A GC~~CAC 22?3 C','C~.CU C ?~;,C:r,G'~
1 ° 83 : ~CuC.,:~U =i C~CL,'~Cv 22 S2 C""vuAG~~[j C CC'-~GUCC
1 o c 6 C.'.~,,;_f.,L' A- - CLJC:nAAC 22 4 8 UCCCAGU C CA L'G'~1C~
2005 UCAi,nCU U G~:,JG.-'CU 2251 UCC'~L1GU C ?,C'yUUC'~
2 013 G'~LTGC C U .~ L JC-:~~UA 2 2 5 9 GUCAG~U U C '-~AGGUC
2 015 UGCC'uTAU U G;"=,L~,LJG 22 60 UCACAUU C AAG"~Z7CA
2020 sL'UGC-cJ A LT~uGAG 2260 UGw-,AC,"--U C ACC.AGv"U
20?° AGAG;,CU U AC~,G 2274 nCCAG.;~1 ?, ~GU
2 0 4 0 CnGAC"uTU A CyG~.G.A 2 2 7 9 G'uU G~GG, z o s ~ UGC-,: c cU c c=: ~..cac 2 2 82 c-,cuUGU a c~cc~.TG
2 0 61 C C uCCJ,U ~, GAC~,I;G,U 2 2 8 8 LTyCAG"--U U cJAC~.CU
2 0 71 G U cJ GU A GG»CAA 2 2 91 AG~~UUGU A CACUGCA
2 07 6 cur.GC_,;U C a-.F,:~,C~C 2 3 21 ~-.A~,GAU C ' '~' UG""
20-° % CCf:GaCU U CCL'GACG 2338 L'G~~ U ~~G
2 0 9 8 c~cacJU C C'.'Cr CGG 2 ? 3 9 GG::ACUU C Uc~UUG;~
21 1= C-C Cr;Gw~U U G;~...C=,CU 2 3 41 G:yCUUCU C A UL'GG CC
212 8 C'J G.: JG"J C L:.~CUGC 2 3 4 4 L-L7C~U U C
2130 G~~v'cJC'J ?, C'u'G~CCC 2358 CCL7GCC'U U UCCCCAG
214 5 G'u;CCC'L.' U G;.,UG,U~, 2 3 59 CL'GCCW U CCC'Cp.GA
2152 UG AUGAU A Uc~,T'r~UW 2360 UGCCUUU C CCCA~,A
21 5 6 G Lrt,'cJ A L'L JAUUC 2 3 7 6 Gf-~U U UUUCCIAU
21 S 8 Lr L'Gu~U U ,T L'L7CA U 2 3 77 AG'JGF,UU U UUCUAUC
21 5 8 Aucu~UU U A L'L:GULi 2 3 7 8 cJGAUUU U UCUAUCG
216 0 U GL~L~tJIJ A L'UCyUJU 2 3 7 9 L'GAUUUU U ~7CGG
2162 L~.:L'L~unU U CnUL'GGU 2380 GiWt]UZ7 C UAUCGGC
t? 63 A'u'L unL'IJ C I:L'L'L'cJU 2 3 82 L'ULJWCU A UCGw~,CAC
2166 LnL'LiCrU LT L?cJLi:L'U 2384 UL'UCUAU C C-vC~C~
2167 AL~JCr.L'L,'~ U cJ'uT'nLTJU 2399 ~.r°.GCACU A U?yUGGAC
2170 C :'u'uu'GU U ~:ULTuu'AC 2 4 O1 G:.~CLTAU A UGC:.ACUG
2171 AUL'UCJU A UUUUACC 2411 CAC'JGGU A AUGw,~UUC
2173 UUG~1'~iU U Lu7ACG,G 2417 L~t,AUGGLJ U CACAGGU
2174 UGLTurL'U LT LTACCAGC 2418 ~.r.UG,~L7U C ACAC,GU[7 21 7 5 GL~ a r UUL7 U- nC CAG''"U 2 4 2 5 ~ CA CAG~,'U U G:GAGAU
2176 L'L~L'L'UU A CCAGCUA 2426 ACAGv,''UU C AGAGAUU
218:: ACC~G.:.u A L~uuT't,L'LiG 2433 GGAGAU U ACCCAGU
2186 G'G"'~'U U Lr~L'L.'~GAG 2434 AGACAW A CCCAG'UG
AG: .,?~UU U AUUGAGU 2 4 4 8 -C iGGCCU U AUUCCUC
2187 GCTurL"W A UUCnGUG 2449 AGGCCW A UUCCUCC

2451 C-CCUL1AU CCUCCCLJ 2750 UAUG'JGU A Gr'.CfaP_G~.
U

2452 C~v'U'r~L'UCUCCCUU 2 759 A "G,nG.~U C UCGw."L1CU
C

2455 UAUUCC'J CCUUCCC 2761 AAG.~'JC'J C G.."L3CUG'J
C

2459 CCUCCCU CCCCCCA 2765 UCUC~.T.~U C UG'UCACC
U

2 4 CUCCCUU CCCCCAA 27 69 GCUCUGU C ACCCZICz, 2 4 GACACCU UGUUAGC 2797 GUGCAAU C AUG.~"LJUC

2 4 ACnCC UU GUUT~GCC 2 8 03 UCAL'Gw,"U U CACUGC_A

2483 CCU'JL;GU AGCCACC 2804 CAUG.~'W C ACUGCAG
U

2 4 CL'UtJGUU G~~CACCCT 2 813 C'JG'AGJ C UUGACCU-2492 GCCACCU~ CCC'~CCC 2815 G.;.AGUCU U CACCUUU
C

2504 CC ::C AU CAUUUCU 2821 L'UGACC'J U UUG:~."~J
A

2508 CAUAC=~U UCL'GC;~. 2822 UGACCUL7 U UG:~.JC
U

2 5 A L'ACAUQ CI7GC CAG 2 8 23 G?.CCUUU iJ GCV...~UC~

2510 UACAUUtl UGC.~.AGU 2829 UU ~~~tJ C AI.GUGAU
C

U

2521 CAGvGW C ACAAUGA 2840 UGAUCCU C CC4CCUC

2533 UGACACU AGCG.~~UC 2847 CC:~.CCU C AGCCUCC
C

2 5 C? G~'~G.~"UAUGUCUG 2 8 53 UCAGCC'J C CUGaGLTA

2545 GUCrUGU UG:~CAU 2860 C~J A G~~LJGG:~
C

2568 AC~AU A UGCCCAA 2872 G~C~,.,AU A GG,--L7CAC

2579 CCnAGCU UGCCUUG 2877 AUAGG.."'J C ACAAC~.C
?.

2 5 U'r.UG.~. GUCCUCU 2 8 9 G:,Cr,AAU i1 UGA

2 5 GC CU'JGU CUCUUGU 2 9 00 G:=-~F,AW U CAWtJW

2 5 UGGUCC'J UUGUCCU 2 9 04 AULZT~U U LTLJULTULJtT

U

2co6 CUCUUGU CUGJWG 2906 UUGAUL'U 'J L'WL'tJUU
C

2601 GUCCUGU UGCAUUU 2907 UCAUUW U UL,~JUt~U
U

2 6 UCC'UGUU GCAUUUC 2 9 08 GAUUUUU U UUtTW~,-U

2 6 UL'UG:AU UC~CUGG 2 9 09 AUUW W U UULW W
07 U ' 2608 UUGC~.UU CACUGGG 2910 LU
U

2609 UGCAUUU AC'UC-GGA 2911 tU
C

2620 C-~,~~U GC_'.~CUAU2912 UUtJLT~7LT~7 U UtILTUWC
U

2 62 UUG~CU A UUGCAGC 2 913 L~tJLILZTW U WUWCzI

2628 G.=ACUAU GCT~G~~UC 2914 UUWUUL1.U LZJWCAG
U

2 6 UGC~.G~~U CAGL1UUC 2 915 LU UUUCACA

2 6 CUCG'-.GU UCCL7G:A 2 916 UUUUUUU U UUC~GAG

2641 UCCAGJU C.CLJGCJiG2917 UUUUUUU U UCAGAGA
U

2 642 CCAGJUU CUC~CAGU 2918 WUUUW U CAC~G,~,C
C

2 6 Cz GUGAU :~G~'"UCC 2 919 UUUUUW C A --CAGACG

2 6 UCAGw''U CU ~G.~AG 2931 AC ~~~U C UCGCAAC

2 6 C CAAG.~~U UUG:~C~G 2 9 3 G:~C,.,~LTCU C GC~.ACAU

2 691 AAG.~''tTAUCr:AC-.GP.C2941 C-CA.ACAU U GCCGAGA
U

2700 G.C-::ACU CCUCCCA 2951 CCi.GACU U CCUUUGU
C

2704 ACUCCCU CGAGCUU 2952 " Cr.GACUU C CULZTGUG
C

2711 CCCnG,~U UG~.:AAGG 2955 ACUUCCU U UGC;GUUA
U

2712 CCAG.~L'U GGAAGGG 2956 CWCCUU U GUGJUAG
U

2 7 GAACZ-~''U AUCCGCG 2 9 61 UUUG'JGU U A GUUAAU

2 724 G.~,-.~''UCAUCC,~~~IJGU2962 WGUGW A GUIJAp,UA
C

2 7 UGUGUGU UGtJGLTAG 2 9 65 UGUUAGU U A AUAAAG

2 ~ c~.z~~~,ccr~a r c 2 c nG'u'Lrcs~,UA .'-.r'sGL.'"f.;L~
6 c c ~ LT'~"'~ O L'CLWC

2576 '~,G,,~ U

X577 .'-.:r.G~~,rC;t1C ~GSnC'~JG

2 S C-~ JL'LCUC ~CQGCC

Table 3 Mouse ICAM HH Taraet Sequence nt. Position .t argot Sequence nt. Position Target Sequence 11 CCCacG'J ac ~uUG 367 ~AugG~"'U cF,.3CCcg C a 23 CaGuGcU C;:CVGCU 374 eF.:.rCCU CCUccCC
a U

? LGcth:CU 6 375 =.~cCCUU CL'ScCCc C UG..'"ClcCU C

31 C'JCUG~~U CUCczca 3 78 CuacCaU C ACCGL,'GU
c 34 UuCUca U 386 ACCwGU A uU~'uuU
a r.G-c~GLTcG

40 cG.cncU GuAgCC'J 394 CcG.~-~_CU ucG~uC,1 U a 48 ccCACCO F~.'~CUgG 420 yCZC',lU CCCCCcg C C C

54 UgcGCCU GucAUGG 425 aCCCCU C ccaGC~G
C C

58 CaUccCU UaG~~UCC 427 CacC'JCU aC-:~Gug a c 64 cAcccCU C~.~a~GC 450 rl,~ACCU ACCCUgC
C AG c 96 C',:cscfiJ CUC~cCC 451 GAF,aCcU uCCrJuuG
C a -102 UgCcaGU CUGw~UgG 456 L~IJACCCU aGCcaCl1 t c 08 cuCUGCU - cuG~CcC 4 95 Cl~AcCaU ACCGLTGu C C

1'_5 uG.~"'~.vCUUGcUCCu .10 UG..~UC-..~UC~.'C-G
C C ;G

119 GcaaUGU zCC'AG:~?,'0'4 CUcF~G.~J uCc~,uCc c a ~0 CUCUGcU CsS~ccC 592 Gra.~.CaU ACaugGG
C C

146 CAGuCgU c~.,cuUCC E07 AC-:.~J~AU UCUCaL7G
C U

152 UCL'G'JGJ acCCaCu '008 G.:Cla.UU CUCzUGC
C U

158 UCCucuU A~cC 609 C~~UL"U C UCaUGCC
a 165 CAgAr.GU cUuuUGC E' ~ AAUtJtWCU aUGCCGC
a C

168 A?.GcCuU C?G.~CCC 56 ~JGU U UG'lcug C

185 C~:uG.~cU CG'JGLaG 6~7 f,G..~UCW G ;Gn,gA
C U

209 ccf~C~U CUcOGgC 668 cgacCCU a GGCCaCC
C -227 CaSAAGU GUUuuGC 677 GsCCuCU A CCAGCcu U

230 :~.nGG'LiGUuuGCucc 684 LuG.G~"'tJ CgGuCCU
U C

237 LTGuG..~sU GAGnaCu 692 CcG.l~:U cGauCUu a U

248 rzCCCa uCCVAAA U AGgaCcU c acCCUGC
c 693 253 ccUG.CU AggAaGA E96 CCUcUuU C CUGCCuc A

263 AcC-.~'LuU uCUaCtIG 709 cGCGSCU C CaCCuCA
c 267 AC-ccGCU C'UGCCUa 720 uACr.ACU uUCAGC~
C U

293 Ar.GcUGU ~ UCnSCUG 723 :~CL'UuU AGCuCCg a C

3?9 rC-cAGAU c~cAgCC 735 aCCaGaU C CUcCAGa A

335 cUCUG..~U UgaSAAC 738 LC-C-g~CU GuGaUG.;
a c 337 GUcC4AU CAcACLIG 765 CaGUcGU C cGcUuCC
U

338 aG..~UgUU SAgCL'Ga 769 C- GcCUGU uCCDGcc a U

59 GuGCAG'J cuCcGCU 770 uL'uUGcU CCUC-:~
C C

i85 ' C-C-cCUGUuCCuGcC 1 =53 . AGLC-cgU c~~GgUG
U c -786 GcCUGUtJ CCIiGeCU 1366 UeaCAcU a UaCaAC'U
a 792 UcgecGU UC~~,aG 1367 eGC~CcU c CCCACcu C

794 c,:cGgCU GGAGaCu ~ '! 3 GuACCigU CCACUcu a 68 a 807 CuCcGaU L.ACCL'GG 1380 UGCCC'~U GGVG
a C

ugg 833 CA~GcU c GAcaCCC 1388 GGaGcU C AGUGgCU

846 CCcuvGU ACCcuUG 1398 UGgCUG'J ACagaAc C C

Q5 G~c~,CCU 1 1402 ' UGUcc~~U GaCAeCU
c UacCAgC a 863 ~cCcnCU ~u CcLlC:.'gG 1408- cCGF.GP,U C cgGgaGG
666 G.acCC'J U Cc,:GcCC 1410 GAC,gUC~,7 c GgaaGgg 867 =.uLTCcUU a cCC~-acA 1421 ccCACCL7 A Cl~UuUGU
669 L:C~.:Uc~J C aucG~.G 1425 zCUcCW a aGUaGaG
881 =.uC-;~sU C ~.cCcGJ 1429 uCUC'JaU a GccC~G
685 C'JLJucGU a cac-GJGA 1444 G~ag~ C AaGaG.GA
°33 ct'auAaU c F.L~uC:~C-:~ 1455 G;:aAuGZ7 C ACCaGga °36 uraL'csU a CUG.~.~Gc 1482 iyguUC~l.~.U a LgC'sCCC
S 78 ~ Uar'.C~cU C ~C'~CU 1484 cUGuUCU a CCI~CauG
880 =.CacUCU =. CAcC'~'W 1493 C'ugt:GcU a UGAGAac °86 Lr:c~C'J U L'~:CaG~'~ 1500 AUGT,~.~t7 c aUcSUCc 68 7 ~:G'~C"uJ U uCaGC~C 1503 gGAcUzU a AUGyUuc X88 C'.zC'JLJ a CaC~.:CC .506 WaL'cuU a AUzACcG
1005 ACcaGAU C CUGcaGn 1509, cu.AcCAU C ACcGI3Gu 1006 uGaCrcU C UGccGAA ls? 8-- L:caUG"'"U c cC~GgCG
1023 ueG~~J C UCcG~G 1530 CuauAaU C AUucUGG
1025 G~G;JC'U C cGf,F,G;", 1533 ugGUCAU a gUG:~.;Cc 1 066 CG C.:C'J c at,~auAi, ~ X51 CAuGCCU a AGCA cU

.092 rc,:G:-aLT c uCnC-cCC 1«9 AG~,..;CcU c CCcaccU
1093 UGCaccU a ChG~~Ca~, 1563 CuUAugU a UALRACC
1125 CCC~:'U C uUc.~L?G': ?5c'S LTAucUuU A UP.ACCGC
_163 C~aF;C.'J TJ C'UuuUGC 1567 ugUuUAU A ACCGCCA
1164 Ga.=.C~JLJ C L~:urGt"L7 1584 GaAAGF,U C rlgG.AuAU
66 :_C-~JUCL7 a aUG.~"TJCL7 1 592 AgGAuAU A CAacuUA
_172 UCCUG~U a ~F,r,CC lcog F,C~aguU A CAgaAGG
1200 c,:C~Gt~' C C'.,Cv-~C~: 1 E51 CcCaCCV C CCUC,AgC
1201 cC',:C-'~vU a L;c~CAc 1661 caF,ACCU a UCCZ:uuG
12 03 r c.:L ~'~~U a Ct,CG =Gu ~ 6 63 AACCULU C CvuuGAa 1227 G~,:.t-.caU a CL~'~'cC ~ 678 AGGaCCU C agCCUgG
1228 Ga.~.C.C'u'U C uUuUcCU 1680 aGCCaW U CCUCuGg 1233 LuCGJuU C CcGacaG 1681- - GCCaCW C WCuGgC
1238 G'~aCC-G-J A UG..~-uCC~,: 1684 aCWCCU C uGaCUgu 264 G-.a GC-cU c Guc'~~G 1690 cCG~aCU U uCaAUcU
.267 L;r-~'..caG~1 C uC-C-C.cAA 1691 CG:~aCW a CcAUcW
1284 nC=r~F,U a C,:cAGCc 1696 Ue-CCG1U a eaG.;~lGG
-205 C=.ccccU C uC_=.G~TiG 1698 CcaAL'AU a ccUG~,ag '-_ 06 G=-'-.C:r:CL, C v.:cr.~.UG 1 737 cAC,ACcU c UaCCAgc .321 c~._C~~U c aG.CaGcA 1750 gGCcG~.~U c CACCUca '--'34 =.~CCC~.U c uC~~~A.Z 1 7 56 cAacCCU a CCuGCCC
1344 a.,:e=~G~.:~T C crG~GJc 1 78 7 gaGaCAU U GUCCcCA
-'S, ucAaUG'J a UT-~Fc,~uA 1790 G.=.r:WGU a C'UCliaau 1793 U_~~,.'UCCL' C QGc~eGF, 2173 WagagU U UUACCAG
179 7 C~cC?:GL' C F.cFL~.aA 2174 UacagW U UACCAGC
1802 ~cC=.Gr:U c C'uccrGa 2175 ~ acacL'W U ACCAGCU
1812 «.:GcrU c UcaGGCC 2176 cagL'UW A CCAGC~7A
1 613 Cr'.G~L ~' U a c c : ~ G 2183 ACC~GCU A UUUAWG
1 825 CG.cC~cU :~ CCUcJgC 2185 GGCLTpU U UAWGAG
183 7 C~.ucCCU a uACCuCc 21 86' - AGC'JAW U AWGAW
1845 ccAccC'J A GC-CCACc 2187 GCUAUW A UUGAGUa ~~6 c=~~csU a c~.LCVu 2189 L~Lnr~~L
~ -c~Wacc 1861 AeaUCAU a Ue: ~GLIa2196 ea,e'.~e'JeU
a eWgAUG

1865 cAc~UG'J G~'.:CAg 2198 gcaGcCU
A c G'UAL'G'Ju 1 8 Ca c c~:GU AG~UaAa 2 ~ 9 Gc c UCUO
6 C a UgUuUAu a 1877 CAUC~cCU AGCagcs 2200 UcJuccU
a c AUC-cAzG

1501 uAA.~,ACU AAG=cAc 2201 ~cUULZI
C A vGUc''.~GC

nuAL'agU GAUcaSU 2205 DGUAUGJ GGCcugA
a C

22 UGaFUGU a uAr.GJUa 22''_0 GcAGaCU
c AgUGgcs 1923 uGr':UGcU AgGZI2Uc 2220 c,:ccCAU ~:L;~CUC'J
c a 1 S28 Iiunc:~GU UuaCCaG 2224 csc~G.~~Li UCcauCC
a a 1930 ncF:G'JuU eCCaGcU 2226 L'cGaUC'J eCvCCgC
a C

1954 G_G:C~U a GuC'CCa 2233 C'JGaC~J c~G::AGg C

_, =G::.f:uAU C'"~L'ua 2242 uC~.G..~U gCgGaCC
83 A a c6 aG::.AcAU Cl3G~gcC 2248 auCcaU C C~UccCA
A J

2005 UC-cFrcCU GCgGaCc 2254 UCCAzuU ACAcUgA
a C

20.3 G'~LauuU WGaGJA 2259 aUCAC~U C.~.cG.~~Ug A U

2015 UGCCcAU c G~~cugG 2260 UCAC=~W acGGiJgc C

2020 ccUG.'~U UuCaG:~G 2266 ggAAuGU C~~.~G:~a c a C

2039 cCsGgCU a gCAGAgG 2274 AC~.~GaU Cl:GgaGa c 2040 C~CAC'cU CuGgAC-g 2279 GaT,ccG'J CUcrCAaG
c c 205 U~Gc~CC'J CAc~ucC 2282 GcUGU a ucaGcUG

206''_C.~eC:~U acCgUGU 2288 L?:uAeG'J aUggcCU
c U

2 0 CA c.:UGU GC cL'C? 2 2 91 c aGUgGU CuCUGCu 71 A g a 2076 CLnGCCU C AgAL't~',:22321 gAAAG~,U ~.C~aUC-G
C

2097 CaAC~CU U CuUG;uG 2338 UGaGAW c CQgcctrG

208 CzCi~CJU CcccCCG 2339 Gau,CcU GCcULIuG
C a 2115 G:.Cr.G..~U G~:aggaU 2341 GACcUW a caGcCu c c 2.28 CaGC"JaU LTF.uUGAC2344 Wuc=AU c uuCCAgC
a 21 cCUC'UuU CUGcC.:C 2 3 58 CCcacCU UCaSC~G
? c c 2145 C'-~AC~CU cuUG:yLlg2_59 CUGCVW U gaaCAGA
U

2152 UauUe.AU UecAgW 2360 ~eCCUUU C',:uuGAA
a C

2156 uucAL'GU ULJUAWa 237fi eG'u'GgU cUUWga A z U

2158 cAUC'-.~?~U UAUUaAIJ 2377 gGUGgW c DUCUaag U

2 i.UG'..~:UU ? 2 3 7 8 a gGgUW UCUAcuG
~ c U AUDaAIJLT c 2160 UGL~UW A WaADL'U 2379 UGcUUW c ucAUaaG

2162 LTAUU~~AU ~WUag 2380 aAcUUUU UgUCGGC
U a 21c3 ?UcL~W a AWaaW 2382 aUUcUCU UuGcCcC
A

2166 acUUCnU U cucUAW 2384 aUcCagU GaCACAA
a 2167 Auct:~:W cU~L'U 2? 99 AAzCACU UcUG~C
U A

2170 uAUWaU U AaBLTLmg 2401 eacCilgU UGagCUG
a _ 2171 i c WGW Ug cDcCC 2 411 uACUG.,~U AgGaUgC
a c 2417 cF.AUC~"U CAuAcG'J 2691 AAuGUcU cGACGcC
a c 2418 FcUGGaU C uCAGGcc 2700 CAaGcCU CCUgCCc a 2425 CzugG~''U gAGgGuU 2704 ~ gacCuW CCAGCcU
c a 2426 =.uuaaW a F.GAGuW 2711 ~ CCCAGCU UcegcaG
c 2433 uAG~.GuU uaCCAGc 2712 cagGucU C-~AGG:, U c 2534 ~.Ci.GUW aCCAGcu 2721 G~GG~~U cUcCaaG
a C

244E -C~GCCU U ccUgCcC 2724 GGuaCAU CCuGUGc a 2499 A~GCCW c cUgCcCC 2744 cGLGaGLI cG'JGcAG
c 2551 C-CC:"c,:UUC~ TcCCU 2750 'u~r.L;uL'aUaC?_guAcC

2452 C ~c,:W CWcCWc 2?59 cCgcaCU aUCGaUW

L45 C~ac~CU a5 2161 AgGaC..~..1CaCCWGC
CCTJCCCC

24cc CCaCzCLi UC~~Cc 2765 UuUuC-~~rJCUGcCct~

24 60 CaCaCW CCCCCCcg 2? 69 agUC',:G'JCAaaCr',G"

2479 G~.cACCU cUaccAGC 2?97 UGzAAU CAUG,"UcC
z 2480 uC~C'LU UGUcAuCC 2803 UCAL'G"~U cCcacG.~.g 2483 CCaaUGU anGCCT~CC 2804 ggUGGcU CcgU~AG

2484 C'~.'WcW caCCAcuc 28.3 C!:;cCgGU CcUC~.CCc 2492 acCACW CCCC~CC'u 2815 CAGUW ac'~CLW
z 2504 CCCiCcU AC'~L'L'LicU2821 c UG.CCL1 =cLG:

,agg 2508 uAUcCyU ccaLcCCA 2822 cCi,gCcU c~
.

c 2509 uUAgAg UuUaCCAG U L:eCC'JLZ3azC
- - 2823 C Ju e.:CcCA

2510 L'hgRgW aUaCC~Gc 829 UGGaW auAaUcAU

c 2520 C;:uuUGU UCcCF,AUG 2837 AcWGcU aCUuCsga 2'21 CAGca aACccUcA W UG?.gaW CC~gCC'Ug 2533 U--CnucC'IJCAGauaUC 2847 C CaAugU CaGCCaCC

2540 C'-.GCaGU CcccUcUG 2853 cCAGCC'J CuUauGUu 2545 CUgcUG'J aUG.~'UCcC 2860 cCcaF..GU AaCL-CuC~, -2 5 c,:G~oU cUGuCa.AA 2 8 ~ 2 G::ACCuU caGt 68 caAg L579 aLr'.r'Sl:U.~.ZiGCCC~.~G28 i7 LUCC'C-w."UaC~'.r'.llC_sC

2585 cucC.~aU 'JG'JuCUW 2899 GcAcuU UcGr',UcUU
c 2588 GCaL~t.,'GUaCUCUaaU 2900 uuAAuW aG:aUL'L'U

2'9 UcG'JuCU C? 2904 cWcAU UcUcUaW
UgcUCW
h '93 cUuCLJuU UGcuC'JGc 2905 c'JUc~L'U cTJcUeL'Li g 2_'96 CL'uLu'G'JaCccaaUG 2906 L'L'GF,UcUaLZ.ZaL'Ue 2601 acCcUG'J aUuCgWL' 2907 UCI~aUL'U aUt~~aUUU

L602 LJCCcGGU aCCAUCCC 2908 GAcCC'.uU CL'JL'UgCU

2607 cUc'-gAU aL'acC'LTGG2909 AccWcU UL'L~gcTJcU

2608 caC-:~,cU cCgC'JG~G 2910 UoUaUULI aUUaaLJW

2609 c-GarUg CACcaCuA U aUaUW aUU~aL'UU

2620 eC-~cCU caCcC'Jgc 2912 L'UcUUcU cUzzUoL'C

2626 UL'uCcaU cUUc'",~?GC2913 UUL'cUeU acL'ceUG

2628 GC:.Cac UGL:nGCca U UgcL'UW ccaUa 2635 L?uGC.~J CCcGDccu 2015 aUUUaW a.
aL'L7uAGA

2640 ScCC~GJ UUCC'JGCc 2916 ~'aL'UcgU UUcCcGAG

2641 cCCrGcL cuCaGCAG T aUL?c U
291; W

g cCcCAGa, 2642 CCuG~~TL1 CCUGCcuc 2918 ' L~JcgUL cCcGGAg uAcL C~.G;-aUcC L LJUcUcaU arC-cGuCG
Gc - 291c ~

2 E e~:~~c-~,7CcUG;~,F,G 2 31 ucGaC- CUCG
59 ~U

" cAAg 2689 C1~.P.uGU cUccGAGG 2933 ' GaC-.. CGe.~.Acg ZCU r.

2941 GacAC~U UCuCCccA ~
- -2 51 CC~,ccCU aCCUcUGc 2952 C:~GcagU CCgcLTGUG

2955 ~:gugacv cucJcJc~, 2956 uWCCUU UGaaUcAa 2961 UcUGUGU cAGccAcU

2962 aUGUaW aaUUAAUu 2965 UuUgAaU cAAUAAAG

266 GcUcC-cU A ccAe:,C-c 2 ~ ?.aUc~r'.U A ~AG1:L'UCT

2975 Lr"~aF~lU 'J UzcG'~C

2976 cAcGgUU U CGCuACZJ

2977 AAGC'LJgU a Uglsc~CLG

2979 uCaL'UCU C uAuUG~.C

?able 4 f-;;urr,en IC: M HH F;ibczyme SequEnces nt. Potion Ribczyme Sequence 11 C~C-'VJC CTCrC~~.~-".,~.u~G~.CG'1AACUC-.NG

~3 =_C~..r.GGC:."'~L~C~C~~CvCC~.~'nAAGCUCiIG

26 nC~y CLGtCAC-:~CC",.:-.i~GWAC-:riG."'LT
C'"anA

z, c~c~enG ~-e~L-~c~-c~ =~,~,c1-~c~AaG:~c.~.G

3 4 C-'~:CUC'JC' 'GJ,i~GAG.~.-C ~..'AGC~, C Gi- ',~,~,G~,,C
CG?.?

4 0 .'-.Ci C ~ GUG~G:n : ~,-~.i?iCL'C'"G~
vJGC Cv:. C -C.nA

48 C;.~C-C-L:JC:.'GUGnGC,CC~~C ?C-.~v'UC-v -CnA

4 C ;l. nC-CC'~; GLiCY.G.:-C AG.w"L~G?, C :: ~ "C -C~

~E C-Gi:GCCn CuGBU.C-:~:.C"'~'~,GC-CCGAAAGw .' G'~.,G

54 C".'C-C'JGGC'~'GIJGAG:~C"'~CuCC:AA:~"C~L'A

6 C-;~.C:AG C'~'G::L'GGvCC~._. AG'JGw.v"
ifiAG~~C~.~-'-.A

102 C'.-~'~~GG~GC''GL~nC-.~_-CC~'=.nAGC,CCACCr~.G:A
-Cr'~A

C-.C~ C C"~'G.'-. UGr.C-:~. AC~GGA
8 C C C".~.AACr,v. CG.~.A

115 C-:.ACA C"~~L'.f:G:.CCC~G:~Cf,GCCCCG
-CAi, c c, ~c~.cc ~;~ L~~.c-:.z c~.~,G:~: ~c~.
c -c.~A _.c~;Gc ,? c,~~ c c~ cl : L: c;~c-c.~ hA~GaG
o c"c-,~ c a~G:~ c;y-~

14 G:~:G'- C'~-G; : ~ G,G:.-C :yL'GUCGG
6 CA C"'.1:-~aG;~C C
CvaA

, r.r-. r ~ rrw 2 ,'Cr G:~.-aC,.'G L'GAC-:~...~~,..~r,AC.ACAG~

l~s e;:c.~;~ ccUL~~.;~c.:..c;.:_;."-.Ac~CC..~a~rr~_ 16~ G,:J-.C-:r;-.:UCC'CrUG~C~~~~C'="..ruai,GuCC:~~'1ACL1WZJG

1 58 GC~-.~-~~AGC..'C:;~L'~CnC-:..C~nUCACW
-aaar',C~~,CCGar lE5 Cf-.G:ACG C~'C:~-:L'.a-.C.C-CC'',:l,ri,C.;CCGnA~,GCCt7CC

2 09 GL.'G:~C=~GC:G uC::~C-.~.-CCG..:~AGGCCGrIAAC.~~UGCU

227 GCC~=~i~C C'.'CruC~..C-C-CCC~-.:-.CvCC:~AACULiGC,G

230 LT.~uGCCC C'uC::L'GGC-,:.C~,GV,CCAGACL'U
-G:A

2 3 G_-~u C'JCC'~ G C~C?:C-.:.-C hUGC
7 C;~~,GC-C C -CAA CCA

2 4 L1 L r.GGCC"~ GA L'GAC~C
E ~.a..-~C-.n.CC~,~. ACG'-.~Gp 2 _' FTC ' F'L'UC',C:r L ~.C~;.-C :~C-.:.:.~C
3 ~ ~;r..r,C~:,c CG.A

2 6 ' ~ r.:~C-CCL'. GnuG :C-C-C i~CUCCL'U
3 C G.a-AGw C -GA

y. ......
2 6 r CT~ G ~ ~ GC.C C P.G~AACU
~ ~-~-~-:G ~GC,C CC~

203 L.~-=~.-uCiaC.'G~'C.n~~"-CL''~.~vCCG'~AnC~.CCUU

31 C-~'-~.~t C:'Gr :.'~C. :CSC i:UCUUCU
c C-:~C CC=~~sC CC~ni~

1 ? .~ ~ W~'"~:.r~,C'~;:Y L.'~C".~GC~ A C-:ACAU
5 C :~l.,G:A CC.AA

33 .;=_C'-"..:~T~1GC'~~r:~~~G.:-C ~.',f-r.G.:~CCG~.AAU'~:GCAC

3 E C~.yG'~'W C'vGh i_'~C~-,Cv: 'u'~.'~GCA
wr''-':~.=~C~.:~C
CGAA

ACLGCCC
30C ~C-'aGJL CL'Gi::~C:rGC-CCL::.,~-.r ''~CGJ,F, 36 rhC-GL~~TtJCt'C:':i_'~C~:C-C-CC~~.GGCCCAAAGC'UGL1L7 ~

374 C-~vGhCw C'LC::-.i.'G:;G:~cCCr-'u.."C~CCv~~.AG:mT~JW

373 C i':LGAG C;'CrUGC-s..CCG.~-.~,C~;,CCG:.u~,;AG""LJW

378 ACf~CC~::JW.'Grt.'~'.;.:C-C~CC~-~:G.:~CCG~.ArG;~,i,G"

386 2:GvTC~=.GCUi:.':i.'G,G:~cC~G.GCCCAAACACG,,"U

.94 C~-~'Z:~CuGC'~Cf'-.U -Cs'-.G,:~.CC~-'~~~,G[;CCAG
T-.C, ;CCC"',~, 9 2 n~-LA GC~GCJG~ :CG-':CvC C'.l-.i-~r GC-C,.~''UG
0 :CvC CCZ.A

925 C"vGCC?A C't.'Cr.L' -G':C- F~CvC,G.pG
GCCG.~,CvCCGnn 427 C-.:a~"UC~CCL~GnU -CnC~~~.CG'r.Ai.G.~r~.CGAA
AG ~~~

SSD G'JAGCV'"U C'JCnDG"rCv~w~C'"'~CG?.A
AG"'"DQCU

S51 CG:.TAC-:~ CUGAU ' ~ CC:WC,CCGP.A
AAG.~"WC

456 G:,~ ~GCG CUCr -UC AC~CCC,AAAC~:~CC'.,~,A
AC~P, 495 CCACCv"Z7 CLiCu':UCv'sC-~v~~C""anAe~'.G~CCGr~A
AC'v"CUG~~

S1D CCCCACG CUG AUGAGGCC -' ~ CGAA
AGCAGC.A

564 UG..~-UCGL1CUGAL"~C~CCG~aAAC:~CC:
AA ACCQC~G

~ 92 C.~.."~TJCv~UCLiCa'rjWr-'.G.~ss.~CGFCCv~A
AUCUCQC

607 CAC~~ CL'GAL'C.ACG..~~"C GAA
AUL'Gw~LJ

608 C~CG~,G CUGAUGAGC~CC " ~~CCsAA
AAUQGGC

609 C:.~~.C~ CUCAVGnGGCC -" 'G:~CC~~
AnAUUGG

6.1 C' ~'~~C NGAL"'.C~~CC~.sAa AG'yArlW

656 C'UL~NCA CL'GAV"~:AC~CCC,AF~GGCC~~A
ACAGCrJC

657 vcnccC cvG;. -C~CGaA AaC~cCU

668 G~.-:~.C CGC~UG ;GGCCGAAAG:~CC~..?.A
AG.~"UGaL1 677 GAG.."LJGG C'UCn~GGCCGAAAGGCC~~?.A
~~ ;,~,C

684 r.G~~~?CUG CUCnUGAGC,CCC~AGGCCGAA
AG.~UC~"U

E92 C~C~. CC1GAVGr'"~CCG:~AGGCCGAA
AG~CUG

E93 G.:~C~~AC NCAUCAGGCCCAAAG:CCCP.A
F~AG~JCU

E96 CQC~."CAG C'tlGA -L~CCGAF~AGGCCCAA
ACT~AG.;

7 09 UGUCZ-.:.-s,C'JGQG.C-GCCGAAhG:~CC~~A
~.GUCGw.~U

720 G~~UGAC CQG.AUGAG~~~CC,AAAGC,CCGAA
AGUUGUG

723 G~w.."CT NGACGAGGCCGAAAGGCCGAA AC~,AGUU

735 CCQc :7AG CC1GALCGF~AGC,CCGAA ACCC~.~, 738 CCACNC CUGAUG?C~CCG~AGCcCGAA AG.~~CCC

7 65 G~.i:zACA CL3G?L'GF~GGCCGF~G.~~CCG?
A ACCT~CGG

7 69 UCCAC~G NG:~.t~F~GG~CCG~GC~CCGAF, ACAGACC

770 GJCCAGG CUGAL'GnGGCCGAAAGGCCGAA
AACAGAC

785 GF~NC-w NGT~LT~AGGCCGAAAG'-CCG1A
ACAGCCC

786 ACACiiGG CUGAVG'"raGvCCGAAAG.~CCGnP, AACAGCC

792 CNCCGA NGAU -C:~CCGAAAG:~CCGAA
ACL1C~GA

794 C-GCCUCC NCAUC~G GCCGAAAGC~CC -C
A AGAC~JGC, &07 CC~.G.~~L;GNCaU -C'rf'. "~''CGnAAGC~CCGAA
ACCUGGG

83. Ci-:.~,':?UCNCAUG~,C'~CCC,AAAGGCCGAA
ACCUCUG

846 CnL~G.~"U NGAUGAGGCC~CaAA ACUGLJGG

851 CWGCCA CUGAUGAG~vCCCT~AtaCvCCGr'~A
ACv~LJGAC

863 CG'.Gr.AG NGAU -C~.GGCC: AAAG:~CCGaA
AGL7CGUtJ

86E CvCC~.~G NGt:UGFaG iCCGT~aTsGC,CCGr'~..'~a AG~~sr~aGUC

867 UGC-CCGA NGGUGA~CCGAF~CvCCGAA AA
~G~U

869 CWGGCC NG'.UG.~.GGCCGT~AAC~CGAP, F,GAAGGA

881 ANGACU NGfiUGAGGCCGriAAG:~CCG,AA
AGGCCW

8 8 UCACACU NGALfi~F GGCC'GF AAGGC
S CGAA ACDC'~G

933 CGGJAQ NGAUC=~G GCCGAAAGC-CCGAA
ACUG~.J~C

936 UCCCCAG NGAUGF~GGCCGT~AAG.~CCGnA
AU~CUG

978 AGC'UCUA NGFiUCAG.~~CCGroAAG:~CCG.'.Fs AUG~~UCA

980 AAF.G~~UG CUGAUGAC~SCCGT~AAGC,CCGr=~
AGF~UGGU

S86 CGCCC-.~-~,CUGAUGACyCCGAF~AGGCCGa.A
AG~.~UGUA

987 GCG.CGG CUGhUGAGGCCGAAAGGCCG~.A
AAGCUGU

988 G:~CGCCG NGAUG.G.~~CC~AG:zCCG.AP, AAAGw~UG

1005 UC~~;C~G CL',~G.U';~C-:~CCGAAAGGCCC-.AA
AuC~CG'J

1006 UUCGUCA CUGnL'GAC~.CGAAG:~CCG.~
AAUCACG

1 G23 CL'uC"JGA CJG.nU''~~C -'CnAAGCCCGAA
ACCUC'JG

1025 CCC'JUCU CUG~UCAC~CCGAAAGC~CCGAA
AC-~' CC'JC

1 0 L u'Gv.: CL'GAUGF G~ C'"~GCn. C"~A
6 a JC ~.G:~..':L~'GG

9 Gu~"UGG CL'G:~UCAGuC C~~u~GG CCG?
2 A A C C:. C.AU

.093 UGC~~uG CUG~~C-:~CC~C -CnA AACCCCA

1125 UCAGCAG CUG;yL~nGC~CCGAAAG:~CCGAA
~.CZJC-G:, 1 6 GCAC-GAG CUG:w~,~,GC-C C 'G~A~ CGAA
3 ~.Ci ;1GCG

64 .'->GC~C-:~nC'uG;~LunGG:.CC~i~AGGCC~A
:-~Gw~JGC

115c .:G;~G~G C'UGnL~AG:~CC"~AnAGC-CCGAA
~~=.AGE~J

1 _ c-.~-.~-uGC~c~Gn~-.c-~ccc'~c~cGAa :
~ 2 ~-;~ r-:~

'_ 2 UG'JGJAU CUGAL'G?.G~~C CGAAAC,CCCGAA
0 0 AC-CTJG:~.

12 C UL'c'JGL'ACUGhU"~C~CCGAAAGG~"CC,AA
1 '~AG.."'tJGG

'_203 UCUUGUG CUGr.U-Cr'~G:~CCGFsAACv:.CC?.A
:.,tTF~SGv.,.'J

1227 GC:ACACG CLTGnUCAGGCCGAnAC~CCGAA
AGC"iJCC:.

1228 :~GGACAC CUGAL'GAGGCCGisnAC-GCCGAA
:i-~'VCC

;233 CALTACAG CUGAL'G~'~C:~CCGF~F~AGGCC'C~.A
ACl_CGAA

123 GC GG C CUGAUGhC-GC.CGF~.F~C~~CCGAA
E CA AC~C~?.C

1264 CCW.-:zAC C'.'C~FUGAGGCC~uFFaACvC.CG'ar~
iyUC'CCUC

12 67 UUL'CCCG CUG~-: -UCAG:~CGArr'~G:
CCGAA ACAAUCC

_ 2 U c.~~t c~~,:~t~;-.~.:~ cc~AaGG~ccAA
9 4 ~ G',G A~L~UUCv 12 9 c.~~~ sG,~c Tca.~L~cAG~ cc~G;~,: cca~
s raULroUC

_ ? G~C~UUG C'JG:~UGnC'~:~C C~ C GAA
0 6 AGuCUGC

_32. UuccccC c~~,_AUG.~ccc~.G~.c:~A AclcUCG

_334 cL?cG:Nc c~c,:~t~-.cc..ccG~AG~cc~:~A
At;c-G~,-U

3 4 CA C~CilU CuGnLW,G:~c CGAAAGGCCGAA

3 S UC~tr~p CL'Ga:UG.=.G.-C C Cz7=.AAC-:~CCGAA
1 z Ci.CLJL~G

_ 3 C =.UC CUGnUGnG:n C'GAAAGGC C G
5 ? C_ W A:y AGAG'yCU

_ 3 r.GUC-:~nACUG~L'C~CNCCCv~F,CW.C -CAA
6 o AC'u'GCCA

1367 CAC'JG'G CUG~L'C~G:~CC'"~nAAGGCCGAA
AF.,C-LiGCC

13 6 GCAG'UGG CUGAUCAG;~CC'C~GC-C CCAA
8 A.~.AGiJC-C

.380 AUUCCCC CUGL~GGCCGF~r.G:~:.CC~A
AL'C-GGC~

1 3 r.GUCACU C'~~GAL~GJ-.G~,CCG~,F,AG
E 8 CCGAA AL'UCCCC

'_ 3 C'~ C GnGUCLlCu :UC:AGGC C~~F F~GGC
9 8 CGA~, ACAGL1CA

0 r -G':UCilCC'~ GA L'GnG:~C CG'~:.C-GCCGAA
2 A G'~ACA

140E CCCLG;r, C"~'GnL~~GC-CCG~~nG:,CCGAA
AUCUCGA

1510 UG~.CCL1C C'ui~-.U"'~-.C~Crur'u'-.~GGCC
-G'~n AGnUCUC

1 S A C :GnGG CU~UCr'-.GC-C. CGhAr'~GGC
2 ~ C G~ :~G.:-JGCC

S25 CCCG?CA C'~~G:~UG.~~CG~.nAG;~CCC~
AC-~.TAGG

1529 CL'G~CC CL'GAUGG.=-CCG.F.i~AGGCCGAA
ACAG'~,G.;

1444 UCCCCUU CUC.?:UGnGGCC'GAAAGGCC'GAF, AGL'C,CUC

1555 C~C~-~"U Ct.'C~:UCf:GGCCC~F,C-GCCCAA
ACC'JCCC

S 82 C-C-:.~~-C-~~?CUGAL'CAGv~~.CuAr.AGC,CC~~iaA
.'-'.GCsCAU

S 8 C C C-:~C-GGCUG~ :UG-':C-:~C C G-.hAGGC
S C G:~ A GL.CAC

1493 t..::UCUCACuGnUG:C-CiCGAAAGC~CCG.~u~
ACCG:~GG

50G UGUCAC CUGZ:UG~C-~CCGAAAC~CCGAF.
ALTCLJCAU

'~ 503 UCAUCAU CUGYUC,AGC,CCGAAAG:~CCGAA
ACAAUCU

506 G=.GUGAU C'L'GAUGGGCC "'GGCCGAA AUGACAA

1509 CCACAGtJ CUC~UCF:G.~cCG?~GGCCC,AA
AL'GnUGa 1518 CC~"IJG:. CUGA~GGCC -C.AAAGC~CCuAA
A,CCAC~.G

530 CCWJLIAU CGGA~C~CC~~AAGGCCGAA ACL'GC~vv 1533 UGCCCAU CUG~AC~CC"~:?AF~GGV.~CG?~A
AUGACUG

' S51 AC~.nJG.~U CUGUG~~C~.~AACIZ "~A aCGCCVG

1 ==9 AL~G.G CUGALIGAGuC ACGUG.~U

1503 GcJUAUA ~~"~'~AF~GGCCG?.A AGc~TAC~v 1565 GWv"L'UA CUGAUGnGGCCGrr~AGGCC"aaA
AGAG"JA

1567 UG:~..w~U CLG:~UGnCw..~C~u~AC,GCC~~A
aUAGaGG

5 a auLVCUU c~a~c;~cccc~caA aUCU~cC
a 1592 UACJCJG c,;G;~L~cG.ccc-,aA,aG;~ccGAa aUwcfw _ _= ccJcwG cL-G.ccc~AaG;~,.~cGaA AGL'cLCJ

16=1 GZTJCi~GS, CLTGALK~C~-.~~CGAA rlG~~

1661 CCCC~GA C~iK~...~C~~AG~CC,?.A AGGUUCA

16 63 CLiCCCGG CDGhUG~."C'"'~r~AA~,CCG?.A

167 C~.G:~fi.A CL1G'~DGrIG:~C~ '~AAAC"~~CGAA
8 AGGCCCZl 1680 GCCGnGG CLKY.UGP.GGCC~~AAGGCCC,P.A
AGAGGCC

1681 C-:~CCG?G CQGn '' ~ , '"~CGAA AAGAGGC

6 8 G:~CNCC CDGA~C:~C C~~AAG.~CCCAA AG:~?

1690 FLTAUC-:.~, CUCALIGnC.vCCGAAAG~~~.C~"AA
AGG~~CGA

1691 F.F.UAUGG CU -C~LG~GGCC'".~-.F~,GGCC~.~A?.
AAGG.~CG

1 E CCACCAA CGGAI~GGw.'GGCCGAA ~~

1698 UGCCACC CQGAUG:~C:~~C~CGAA ADAIIGGG

1737 CF~UC~C~CA CBGF~~'C'GAAAGGCCC~.A
AUGUCW

17 5 GUAC.,~"LJG CCuAL""'~.i~.GGCC~~GCCGnA
0 AG."QGCA

1756 G~.-G,:.CGG CUGAUGAG~CC~.~AAGG~CGAA
AGGUGUA

1787 UGnGGAC CUGA " ~ CCG?~P. AUGCCCU

17 9 C.ACtJGF.G CUt~C-GCCGAP. ACTeAUGC

17 9 UCUGACU cUGAUG :GG~. -'~'CC~AAGGC
3 Cc~A :~CuACAA

1797 UcJAUCU CUGA'' ~ CGAA ACUGAGG

1802 GC'uGUUG CU~CCGAF~AGGCCGAA AUCUGAC

1812 C-:~cCCCA CUG;AIK~GGCC~~~CGCCGAA

1813 UG:nCCC CUGAU'"'w,r~GGCCGAA AAUG'~UG

1825 GL'G:hGG CUGAU'-.Y.C,~~CGAAAC~:~cCGAA
ACCAUG.;

1837 F.GUGUW CUGAL'GnC~CC~CGAA AG~~3GLJG

18 S CGL'C:~:. C CUGaUGAC-G~.~C"~F~GGCCG?.F, 5 I?,GUGUUU

18 5 CfiCAUCA CUGAL'GnC-GC CGAAA~C CGAA
6 AUG~~GUG

1861 G.ACLTACA CUGAUC':AGGCCC'~GG.:CGAA
AUCACAU

18 6 AL'cJGF~C CUGAUG~GGC C~~F.GGCCGAA

18 6 cJCA UGU CUGAU -'' '' CAA AC'UACAG

1877 CUUC-GCLJ C2JGAI~GG~~t'c~AAAGGCCGAA
AGUCAUG

'_901 AUCJCUU CUGAL~CCGF~AAGGCCGAA AGUCUUG

1912 AUCCJyUC CUC,AL7Gr.GGCCG~GGCCGAA
AUCAUGU

1922 F~GACUW CUGF~ -~"CGAF,AGGCCGAA ACAUCCA

c23 Up,C',~CUU CUGAUCAGGC -CCr'~G: CCGAA
AACAUCC

0 2 CAC:~~UA CUGAUGP.C~G C CGw.F,F,G:~CCGAA

1930 AUCACvC CUGAUG.CGCCCF~FaAGGCCCAA
AGACUULJ

1964 cJGC,G GC CUCAUG:.GGCCGT~AAC- GCCGAA
AUGUCUC

1983 CCAGUUG CUGAUG=.G~CCCF.AAGGCC'GA~.
AUGUCCU

1996 G~l~uCyG ~.,C.~LTG~C1.~C~~Z~C -C :A
aLTLILJCCC

2005 ~.C~.-:=-~C~C'T~LW C-:~.~C~c~:~CC".~
AG.1UUC~

2 013 .~ C... . .
L~ cc,.~A c~c~-e~.:~: ,~c..~,. ,-~,A
ac~,:~G;.

20~ c~.~nrLC c~c~UCAc-c~ c-.~rv.~.c--,~A
s AU~GC~

2020 CUCrr.CA COGL'G1-Cue. ~.f-r1-CCGAA
ACC~~'r.AU

2 03 C UUCuGQ CL'r -G'~~C-:~ C~~.AAC-:~CCC,hA

2 0 ~ UC~'t1C'JGCUG:yL -uAC-:n. C~G:~~CG'~A

2057 cvC"J'nLiGC'uC~nL'GAC-:~~'~G:.-:.CG,AA
aGwCC~

2061 ~:C:L'GvC C'JG.AC~G-ZCG~A AUG.~-.AG~, 2 071 L'L'G.AUG.~.C'GGaUG~-~Clz C ~'.~AG.=-CCG.?
A ? CAC? L:G

2076 G~G.~'UU C'uGAUGnGC-.:C:~AAG:~CG~.A
AL'G..~UAC

2097 C-'vG=LG CUG~aL~-.G~'"'C".~.AAC1.-.:CGAA
?G'u'GGC-G

2 0 C..W: a CL'C=.~aUC -'C.~a~aG:~ CG~.A
9 8 C.''~G AAG'v'G"vG

2115 AG'JGCCC C'LC~t~CAC-:~C~'..nA.'~CCC~A
AGCUGGc 2128 G-JC=~G'JACUG:,UGAG:~:.C "G.~AGGCCG:,A
ACAGC~.G

2'_30 C-:.-.GUC~GCUGU -CAC-C-'CG~AAC~CCGAA
AGACAC-C.

2145 LT''nIiCAUCCUGF.~CC.-CCGAAAG;~cCGA?~
nG:w~iJGG

2152 '~AUACA CUGAUC,AC~ ~"~.GCC''.y,A
AUCAUCA

2156 GnAL~ri~A CJGr~GnC-.~CCG.~aCCl1~CG~.A
rICALTALC

LISP !'SVL:.MV11C"V~~1~'~llCGr~ A

215 nnU -CAAU CCGnUG,:-s~. C G'~C~.-C CC~.A
9 i~AUACAU

~16o AhA~~;yA c~L~c-c-c~ _..c -C'~. AAAVACa 2162 nG'-.BUG C~~G-'-.L~GC-W CGAr=hGGCCGpA
AUl~a.eiUA

2163 ~C~AAU CUG:~L;CnG:~C'-~CuCGF.~.
AAUAAAU

21 6 ~L'~ACA CUG UG~=~GC,C C'~"..~AG:~
6 C'Cr~P. AU -Cr,AUA

2167 ~f-.n~AC C~L'GAC-:~.= ~:A:~C~.iC -CriA
AAU -C~.U

2170 G~~.f-.r.AUC-LJGALuAC-GCC~~C-:~CCGAA
ACAAAUG

2171 C-' ~.TAAHACUG:~L'GAC-C1 C'~~C~z C.~-~A
AACAAp.U

217 C'u'C-~'=LT'nACUG-',L%GAC,C~ C'".=Ani~~.sC
3 CG'1A AUAACAA

2174 C-GuGw:vIAC'uG-.L~~-'~GC-C~~Ar~C~,.:r~:
r~tRACA

2175 AGCCL'C-..~"UCCJC=~L~'~z~Cu~ AAAUT~AC

217 U ".~C-.."tJG.GCf~'G CGrr-:~~C"~:aC C~.:AA
6 AAnAUAA

2183 CnrUAFu? C"v'G=.L:~-CCCr'~:A~ ACn"L:~C~'-U

2185 C'~C~:LTA CUG;~tJGnG:~CC~C -CAA AL~.GC'~7G

2186 ACJC~.AU CUG:-S'~C,AG-CC~.~AAG.:~CC
-CnA ~-,ALTAGCLJ

2187 CnCt~Cr.A C'JCG~GCCC~CGAA nAnLIAC-C

218 G C :NC C'JCf-',L'~~G:.-C C"'~:AAAC-.=-C.
9 C -CAA A LTF~,UA

2196 Cl:L~nF.nACUG=~L~G:~c~:,i.AC-;~C~-~'A
nCr.CIICA

219 i.''~L=~L'AACUGAL'Cr.C-:~ C'-.~AAGC-C.
8 CG?.A ::CR., ACU

219 CL~1C= C"~ G :L_'G.G:W C:~F~AC-GC
9 L''t, C~.:r.A r.A CACAC

2200 CCLr'r.C_tUCOG,:~'"~ACG:.C"ViaAACi-CCG'~F.
AAAGACA

2 2 G:. C'.TACACUGAUG'r.GG..~'C'-,=~ C -CAA
O 1 AF~.F~,GAC

2 2 L'L~ JAGC COGnUGAGGCC'"v,.at,AG:~C
Q 5 C CGnA ACAUAAA

2210 GJUChULT CL'G=.L'.Gr:C-C-CC~~.G:~CC
-C'nA AGCCLTAC

2 2 .~G :GAC C'~,'GaCCi :C-C-.~. C~v:.Ai~AG.:aC
2 0 C C -GA nUGO[JCA

2220 C-:~C~.G:yC"JGnL;Cr.C.C~-C~AG.:iC~.A
:yC~JAUG

2226 Gr.G:~CCA CUG:-.L~GnG:~CCCAAi:G.:~CC
-C.AA AGACCUA

2233 C-GVCCGU C'JGrL'GAG:.i -C'C.~,C-:ACC
-CnA AGC,CCAG

2242 C-.=AC'OGGCUCnUGGC~CC~~.FJ~.AC:~CC
-GaA ~.C-CiICCG

2 2 UGaCaUG CC -Cr ~C-:yC C~~nAAG GCCGAA
4 8 aCUG

.254 UG,aUGU CG -C.:~G'r.CvCCGAAAGGCCGAA
ACAUGG?~

2259 G~CCULIG C'~'CAUGnC-:~CCGAAAG~CC~~
AUGUGAC

2260 U --CaCCW CL'G:~UGAG:~CCC~AAGGCCGAA
AAUGL~G?r 2266 ACCLJG.~"fJCLIGhUC,AC~CCC AA,AGGCCGAp.
ACCUQGA

2274 AC-'sACUG CUGhUGAGGCCGAAAGGCCGAA ACCUGGU

2279 CCUG'UAC CL~~GGCC"'~F~3'~AGGCCGAA
AC'~.C

2282 ChACCUG CQCALTu~.G GCC'GAAAG.GCCGr~A
ACnACCiG

2288 apGJF.C CGCaL'CAG~C~~.AAGGCCC~A
ACCUGUA

2291 L'C~GL~G CL'GAUG~.C~CC'G~AGGCCG~
ACAACCLT

2321 CCCAUW CG'GnDGACvCC~~AAGGCCC?.A
AUC'JUUU

2338 Clr'iUC.AGCCW-:.-CC 'C,ruaAGvC.CGnA
aGUCCCA

2339 C:_'-~AUGACG -G;DGAC.GCC~.~nAGGCCG~
AAGUCCC

2341 C~v.,~~"~.UCLICaUGAGGCCCv'-.AAGGCCG?.A
AGAAGUC

2349 GUi3GGCC CUCnLK,nC~~~.CuAAAGGvCG'~A
AUGAGAA

2358 CLlG.v::a C'~G?UGAGGCC".~AA~GrCG~.A
AG:~:.AGG

2359 UCUGC-.~G CUGAUG~GGCCGAAAGGCCCAA AaG:~CAG

2360 UUCUG:~ CL~GF.WanGvC:CC~T~AAGGCCG:~P.
AAAGGCA

2376 Ai~CJ;AA CUGAUGAC- GCCGAAAGGCCGAA
AUCACUC

2377 G. CUGF.LTGAGGCC"'.~F~AAGGCCGAA
aAUCACU

2378 CG~UAGA C'JGAUGAC-~w~CCnr"aAC,~CG'"r.A
AAF.UGAC

2379 CCGAUAG CL1GADGAGGCC'GaTaAGGCCG:~A
AAAAUCA

2 3 GC C~.~UA CUG:~UGAGGCC'GAAAGGC C~~AA

2382 CUGCCGa CUGA~GCCGF.AAGGCCGAA AGAAAAA

2384 UUGJG~_C CL1GF:UGnCGCCGAAAG:T CGAA
AUAGAAA

2399 GLiCC:~UA C'~~Cf~L'G :GC-CCGAAAGG.~.CGr.A

2401 CF~GJCCA CUGAL7GAGuCCGAAAGGCCGAA
AUaGUGC

2411 C.AAGCt.U CUGAI3GAGGC'C~' ~~CGAA ACCFGUC

2417 ACCUGUG CQCABCAGC-CCGAi~AGGCCGAA
ACCAUUA

2918 T~F.CCL'GUC'LTGAUCAGGCCGAAAGGCCGAA
AACCAW

2425 AUCUCUG CGGAUGAGGCCGT~AAGGCCGT,A
ACCUGLG

2426 ' nF.UCQCUCUGrLIGAC acC"~ATa?.GGCC~.~A
AACCUGU

2433 ~.cvG~,~~uc~GnUG~CCGaAA~cCGAA aUCOCVG

2434 c~cUCw c~GnvGacGCCeAp.AGCCCGaA
AAUCUCU

2448 G:-.G~.AU CUG:~UG~CG:~T,AG.3CCGAA
AGGCCUC

2449 CAA CiIGaUGAGGCCGAAAGGCCGAA
AAGGCCU

2451 :. "C~GC, CUG.UCAGGCCGi~nAGGCCG?.A
aUAAGGC

2452 ar.Gu;.G cvcz~UGAG~CGaAAG;~CCG~A
AaUaAGG

2455 C~GG CUG'aUGAGGCCC~A~.AG.~CCGAr~.
AGGAAUA

2459 UGC~,.~w CUCAUGnGvCCGnFaPC~~~.CGnP, AGGGAGG

2460 UUGG:~c, CUCAUGr.G,~~~~~~CAAAC~~.,~~CGAA
aAG~.,G AG

2479 Gv.~UAACA CUGAUGAGGCCGAAAGGCCGAP.
AG.~~UGUC

2480 GG."UAAC C"~lCf:UGAGGCCGAAAGGCCGAA
AAG,~~UGU

2483 C-.~~UGW.~UCUGAUGAGGCCGAAAGGCCGAA ACAF~GG

2484 AG.~"UGGC CUCAU -Cr.GGCCGAAAGG.~,CGAA
AACAAAG

2492 G:~.~"'UG.G:CUGAUCAG~CG yA,AGG~CCG?.A
AG.~~UGGC

2504 ACF.~iAUG CUGnUCr''~GGCCGAAAG:nCGAA
aUGUGGG

2509 CUGGCAG CUC:-RUC ;G3CC'~~,AGGvCGAA
F.AUGUAU

2 510 AC'JC-:-CACCG uCnC1-CC~~W.-.:C~.:nA
?.~DGLIA

2520 G=,L't:~GJGCL'GrUG:~.'~C~CC:~ 1C~

2 521 LTC_':L~GLICL~C~BG:C-uCCCGAA

2 533 G~CCC-...~UCL'G?L~x-'"t-.:.-: CGAF.r'aGuCCG'AA
ACA

2540 C'-~C~U CUG.~''u :C~CCGF~~.CGAA
ACC"~~L'G

2 S AL'["JCCA CUCr.UGAGC~.. _ A C~I~AC

2 5 L'UC-:~-:~CUGr:L~G:~:. CC GAA ADL
o E

257 C.'-~t~:~ Cu'GriWGC-CCGAF~.G-.:C~.~?.~1 :~G~JBG;

2 ' n -G'~G-.?C'uG'-.IK.~?.AAGu:.C~.~1A
2 S C ACy:.AVA

2 5 nC'-u.GAG CL' -Cr'.L'~=rt-:~ C~~f~-v.C
8 E GrlA nCA?.Gx~.

2 c r'.C=.:AG.iCU -Cr.C~i~CvW.~"~A =aCWs?
o 1 ~ C.'-~A

G 5 AC~.C-~:~CCL'G~C1-..-CCG,~A ~
r 3 2 5 C~ =. AC~.GCL'C~.u'GnC:~.=CC~,AGS,CCGAP, 9 6 AC'-GAG

2 601 ~.F.AUC~:.ACU -C:~L~AGGCCGAAAC'".":.CGAA
aC~CGAC

2602 GAAnQGC C'J-CALG:~.C-:~CCssAr~G.~aCCGrIA
,~?,f~Ga~

t 607 C''.~L.'.GACLIG:-.L,'~GnC~~CGAAAC.GCCG?A
AUCr:~AA

2608 CCCnCUG C'~JG"C'GAAA~GC1CG~A .~AL~C~,A

2 60 UCC"~' CL1GAL~~CGAAAC'~,aCCGr~.A
GL7 i~iaAI3GCA

2 620 ALTF.GLTG~_C'CC~LIGnG GCC: rnAGGCC:~'.A
AG~'17CCC

2 62 C~JC-C.AA C'CGr.I~nG~CC~C,G.."C iAA
6 AG'~GC~A

2628 Grt-.."UGCC'JG~G:.-CCGAAAC'~y:..CG~.A
:~~C

c 63 -C'".r~.nCUGCL'Gt"'~-C'CGhAAG:~."C"vAA
5 nG.~DGCA

2640 UC-CAGG?~ CuG.W. :C- G.:.C~.z?r'~AGuCCGr'~A
ACUGGAG

2 641 CLTC~-.~.GGCUGUG-t.:~CC'GAA~GC~:CGAA
A~.C'JGGA

2 64'2 AC'LiCCnG CUGnLGiGvC'C'.~'iAAGGCCGAA
AAACQG~v 2 653 G.:=.?,cccUcu'e'.: ~~-'.G;~CCGAAAGC-,:.CGAA
ADCs :: E CL'(JJCGC~aGC'~'CnL-C'CiaA~aGGCC"aAA
9 .~tCVLTGA

2 6 C. CUC C~JG:~'"'u~,G:~C CGnA~"~GCI.
8 9 C~.A CGr~F, AC C G'LJGG

2 691 GJCCUCC C'JG:,L~'~ :GGCC''CGAA
iiL~~CLlp 2 r UC~unGS, C'vGnL~JsC-GC C "~AGC,C
0 G C~u'~A AC~7CCUC

17 0 A~,G~~LTG:~CC7G .L~~C CGA~AC'wC CGAA
4 AG~~.SCT

271= CCUt,'CG. C',T'~L~:G:~CCC~'.AAC~c CCAA AGCL~GGG

L ~ CCCL'UCC CLL.nU '~:G~~.-~.~.CGhAACI~.C~.~A
1 L r,AGCLJGG

2721 Cue..-:~U C'JG="'C ''C,~AAGGCC~~AA
,ACCCWC

2 i nCr.C"J-CGCUGL~.GGCC'CAAAC,GCCGAA

2 ~ C"w~CACA CUG~-t-.:~CCGl~AGGCCGr~P, 44 ACACF,Cp, 2750 GCLIiGUC NGv~~C-GCC~~-~'AAG~.~~CG?A
ACAGAUA

2759 l:CnC-~~.aAC"uG.i,i',=.:-.C-~C~~.-.AAFt'n."CGAA
AGCQLIG'J

2 r AC'-~.Gr.L',w~CUGnL'G..':CvCCC,~JsAGG:.C
61 'Gw-.A r,GF~GCULT

c 7 G:.:GA CL'::hL~:.r,C~~CG~F~CCGAA
6. CA AG~~~~AGA

L % CCL3C-;~,~''Z;CUCnL~.~-t-GCC~~i~GGCC"'"AA
6 AC?.GaGC

2 r G.A CcT~U CUG. L~J-t.:~CC~: CCGAA
9 7 AL'DGCAC

2803 UC-CAGUG CUGAUG.C:,CC".3Ai~AGC-CCGF~A
ACCAUGA

2 8 CUG.~_r.GUCUG'~UG:~C-;~CCC~.AGGCCGAA
04 AnCCAUG

2813 AG.~JCAA CUG=:uG~C~~nAGGCCGAA ACUGC~aG

2 E''_ iv-aAG.~-.C'JC,t I:~.nG:~C C vFsAAG;~C
5 JC CGiaA AC~~CC~C

2 E21 AG~.CCAA CUCv:UG.rt. ;CCCZr'.AAGGCCGAA
AG~~UCAA

2822 GAGCCCA CUC,FaUCi:C-GCCGT~'~TiGGCCGAA
AAG,nUCF, 2 E2? U -C~~CC CUCAUCr:C-C,CCG~AAAGuCCGAA
A~AG.~"UC

2E29 AUCACW COCAUCAGGCCr' .-CG?A AG~_CC.AA

2837 G'J ~"-' CL'Gn -~C~AG~C.~~ .'~TJC~CLJU

2 8 4 GAC~~~'GG CL3GnUGAC:~C'J 'G.~iAC,~~CGAA
0 AG~.~:~CA

2897 ~G~~0 CUGnUGAGGCt'GAAAG~~~.C~.~'~a ACv"'JG~G
~

2E53 UACUCAG CUGADGnGGCC: AAACGCC~..~AA
AG~_-v."C1GA

2 8 6 UCCCAC-C' C9GnL~GGCCGAAAG~.~CGAA
0 ACUGACv 2872 c~C cvcA ~ ,.,~AA~~,~.~-as A Uc-~-CC

2877 GL3GWGLT CUGA ' ~~C~-~aAAG~CC~.~?.ea AG~_CQAU

2 Q99 AAAAUCA CL7 -CnL'CriG:~c ' ""CG~?~
AUUL'G.~.C

2900 AAAAaUC CU -Cr'~UC:~GC-CC'C~.AG~~~~C~~A
n?.WG~

2904 AAAAAAA CUG:-:L -' ,.~CG~.?.AGC-CC~.-?A
AUG'~?.?aU

2905 A C'JC.aUG'~GGCC~C~.-~ A.~UC'-~A

2 9 0 AA~AA CfJGAUCAGvC ~"CC~1 AAALICAPr 290? AAF~AAAA C'JGAUGAGGCCGA~~C~ AAAAUCA

2908 AAAAr~.T~A COCA '' r~CC~.sr'u~. iaF.f.F~.isL'C

2909 ~~AF~A7. CUGT,~,~',G~~~C"C~~.A 'Ar,i,F,AA(1 2910 AAAAAAA CUGF~UGAGGCC~~:FaAAG.~~~~CG?.A
?~Fu,AAe~a 2911 A~ CC~~C~GGCC,G:yA A.~A.AaA

2912 ' '' COGAUGAGGCCC,AAAGGCCG.AA AF~A.Arl~

2913 UGAAAAA COGnLT'v~GC~..~CC,F~AAC~C3CC'".yu~r .~F~aAAe~.

2914 C~ CUGhi~GC~CCC~ i.~r'.Ae~

2915 UCUGAAA CtIGAL'GFS,~C~CCG~AAAG~CC~.-,'~
'ruJir~AAA

2916 CUCUG~A COCA ~ .

2 917 UCtJC2JGA COGAUGF~C ' ~ CG~A AAAAAAA

2918 GUCUCUG CUGAUCAGGCCCG~

201 o CCUCUCU CuGhUGnGGCCG:~nGC,CCG?.A
~xAt~ra 2931 GUUGCGA COCnUGAGGCCG~?AAGGCCC?.A
ACCCCG'J

2933 AUGUL'GC NG:.UGAG:~CC~~ ACACCCC

2941 UcCTG~.~ COGAUC.AG~GCC -CC:~ ARGJQGC

2951 A ''GJSF~GG CLTC~L1CAG~c~CG~u~. AGOCUGG

2952 G.CAAAG COGAUGnGG~CGAAAGGCCCsAP.
A~JCOG

2 ~ 5 LTF ACACA CUG AUGAG:~CC 'GA~AG~CC~.-~,?.
AG~~'~AGT

2 5 CUAACAC CLiGAUC~.CC~CCCFu':~aG~~~~C~W
6 A 'rnCL~-T~r',G

2 9 61 AUtIAA CU CtICAUCAGuCC 'C~CC~uF.A
ACAC

2 9 62 LTF~UtJAAC cJG :UG.GGC C'CAAAC,C,CC~~
:yACAG~A

2 9 6 CUULJAUU CUGAUGAGGC c~.~AAG,~~C~a?

266 G..~UUUAU CUGAL'C~:GC.~_CC?.AAGGCC~~.
AACUAAC

2 9 6 AF.AG.''W CUCALJG.G~n C'~3AFv~Gv~CC~.A
9 AUOJyACU

2975 GLIUGp.C~ CUG~:UGAWCGAF~ACG~CGAA
AG~'VOZTP.

2 97 AGWGAG CUG'r.U -C. AGGCCCJ~C~C~-~.
6 ~AG..~OUCT

2977 CAGUUGA CUGF~UG~.GGCCGFhAGGCCG~.
F.AAGCtJU

2979 GC.cAGUU CL3CAUCACvCCG.AF~.AGCCC~.r.A
:~-GnnAGC

Table ~
Mouse lCAM HH Ribczyme Sequence nt. Pcsition Ribozyme Sequence c~.cG~J c~G~~G~~c~T ~ GGccc~A Acc~GC,~

23 ;yC-C.oGAG C'JGnt;~G/=~GV-'C~.-~ia~.C-:w~CC,nr1 ACC_'~.C',~G

25 A G~iC-:.~'~iC'TsnLiGnC~~~.C~' ,v~CCCz?JA
A 'CAACC~

31 UGuG;~G CL'GAL'GAG~C''.~G:~~C".~A
AGCAGaG

34 C~CCCU C'JGA ~ :~CCG~ AUGAGAA

40 ~.c:~.--uACc,~Gr.~ -chG:~c~cc:~AG;
ccr~ A~ucuGc 48 CCAC~CLT C'JGL'C,AGCr.~CGAAp.GuC'CG?.a AC,~"LJCC.'LJ

s4 CCABC~.C CU -C~JCAC-~CCGA~ AG.aCCCA

s8 C-::AG~~UA CL'G~LT~GC-~~C:,CCGAA AC-GCALTG

64 CUG.~GGG CBGADGAGGCCGnhAGCCCGnA
AGC-~ Gv~UG

9 6 G:~.GC CAG C'UG~~U~~,F.GGCC~CG? A
A G~GAG

.02 CC~:C-.~_r'aC-C'vGALGi~C~",~.CG:~FaFaGGCCC~
ACLJG~Cn, 1 0 C-:~.' CJ? CUGr'.L"".~GGCC ~ A GCAGP.G

s z~c~::nGC.~cJczU -cAG~ccc~cc,AA Ac~ACC.~

1 , UCCL~G.~'"CTC'JCAL1G~.G~C~.~~AGGCCGa.A
o ACALJOCC

12 GGC-CG'~G CUGALIC,AC~GC CG" '' ~,C~CAC;aG

146 C~.G:.G C'JGAUCnGGCCG?.AAGGCCGAA
AC:3ACCiG

l s2 AGL~~U CUi,nLTGAC-:~CCGnFu~G~.CG:aF.
ACACAGA

sa ~~~.~ou c-~GAU~cch;;AG~ccc~ AACAGcA

1 6~ G:~AnAC CUGn2,' -C.:~C-.GCCGAT~AGGCC~~.A
ACUUCUG

168 C-GGGCAG CUCAUGAC-C,CCGAT~AGGCCGaA
AAGGCW

l Es CUGCACG CL'GAUGAG:~CCGAAAGGCCGAr~
ACCCACC

209 GCCAC~:G CLJGT,UGAG~~CCG:~AGGCCGF.A
?.AGUGGC

L27 C-:=.AAAC CLGAUG:~C~-C~ " ~ CG~ AC~JUCtJG

230 C-a:GCAFa CLJGAUGF~GGCCCAAACri,CCCT,A
A~aACUU

237 AGv'UCLiC CUG AUGnC-C,CCGT~AGGCCGAA
AAGCACA

248 U'uLT"r~C~.C~.CUGAUGAGC,cCCAAAGGCCGAA
AUGC,.~~UU

2 53 UCLUCCU C'uGnUGF:C-GCC~T,GGCCCAA
A GC~CAGG

2 63 Cr.C'u .GA CLJCnUC~:GGCCGAA.AG~,CCGAA
AAACCCU

2 67 L~~C.:~CS,GCL;GAUC.;~-C-CC'C,nt"~AGGCCGAA
AG.:.CCCU

293 G:G..~UCA CuGf,UG :GC~CCG~GGCCGAA
ACAGCUU

319 C-GCLJCAG CUGAUG~GGCC"~fi.AAGGCCG~A
AUC'UCCU

335 CULiCUCA CUGAUC.YGGCCG'r,F,F.GGCCGAA
AGCACAG

3 3 C GUGUG CUCAUGAGCCCG:AAAGGCCCAA

338 L;C=.G'~UC C'JGr.UG:~C-GCCG:~F.G,CCGAA
~,AG~GCU

3 5 r G:. C-.~-,ACC'G.L.'.G C-.:-CCG''yAAGGCC~.~F.A
9 ACUC,CAC

367 CC-C-~:~JUGCL,'~G=.UGr:GC~C'',-~.AGGCCG?.F, AGCCAW

379 GC.~-:i.GG CL'CnUG~:C- G:.CCi,F..AGG~CCC~.A
AG: CUL1C

375 GC-CMG C'JCALG~.Ci-CCGAAAG:~CCGAA
AAGGCW

378 nCACG.~"tJ CUC.:-.UG-'~GC,CC~~AAG:~CCGAA
AL'GGUAG

3 8 t =.AC -CnACUGAUC:~GGCCCAAAG ;CCGp.A

3 94 ~. -G:UCGA C'JCAUG G.~C C ~~.AAGC,CCCYf, A G'JCCGG

42 C ~-~.:W C:.'Ci L.'G C-C-C C,~-'~.,~-.i.,CvCCGi.'r.
0 , =.r GUGUG

425 cJ~C-..~c;C-;~C',;C:AUGG;~CCG:-u-.AC-CiCG,=.
AGC~"wuG

427 CaCL'Gw~U C'vCiLT-_'r.Cv~CC~-~F GW.CGr~
~GlaGC'~TG

45O C-~''zGC.v~UC'sGAUC~'G~~~.Cv~~r'aF~eiGCcC~w'~A
.~Cv~UCCU

4.1 Crai-EGG?.CUG'nL -"G'aC~C~-'~' aG~v:,CGr~A
aG"' uCUC

456 .'-_GJG.~~~UCGGaU -CnGC,CC -'Cru'~aGvC.C~VaA
?.GC-C~JAr1 495 i:CnCGv'v C'~.~JC~?.L"GaGV,CCGn?l1G~v:.C~.a?.a . e~.i:G.~~UPl', 51p CCCC?CG CUGAUG'~G G: CGAAAGGCC~.~A
?~G:~.GC.~

64 ::YLG,~-.acUGnceacl-ccc~ac~lc~,~aa~
ac~JG~~

?2 CCGaUG'J CL:C?.UCnC:~CGanhCt-:.C~~ae1 ai;C'JUGC

607 Cr:UCCa C'UG'~UGaC-~v~~C~.~n~C~CG'a~
. aL'CiGVsC'J

608 GC?UGAG CUGrVGaGC,CCC-aFaaC~.CGe>~a .'-sr.LVC"-.,C

sp9 c-;~vca cUGavcacvCc~.-.aaAGGCc~~
anavcG"

011 C,: Czt~U CUGnL'CnC-GC CG~aaaCvC CGr~?r r1 -C~'ar~AUU

656 C~.GCLJC~ C"uGAUG?.GV,CCGAT~P.GGCCG?.A
isCAGC'JU

657 L'G~C~."UCCUGnUG~GGCC:~AAGGCC~~ AACAGCCT

668 C-GJC-C C2JGAUGG:~CCGAF.AGC~CC -G'~
:C ?Cu."UCG

077 _~'VGG C'UGa.BC,~CCGMAG:,CCG:~, rG?~GGLiC

684 AC-.::ACCGCUG aUCAC-C,CC -G'~'~GG:.CGF.a AGL~CIG~, 692 r.~GUCG CUG~L'GnCvCC 'CGAA '.~JCCG

693 G~jG:n,~J CUG~UGr~C- GCC~~F:GGCCGAA
sG~~UCCp 696 CAG~_-:~G CUGALTGaC,GCCG~AAaGGCCGnA
'r~nACAGv 709 L'~.~C,~.~-UGCL'G'.UC'~GG.:.CG'ruiAGG.~.CGaA
AGCCG~.C

7 2 t:C-CZJG'iAC'JG~UG'~G.~r:. C~.~?.G~~~~.
0 CG'~A isG'JUGUa 723 cc~-,~~u c~GAUCac.~C~.'~G~ccGaa :,'~aGw 735 UCUCCAG C'~:CAUC GGCCC?.F~aGC,CCG~.
AUCCiGG'J

~38 cc~ue~e eJGauGG,~.~ccaAAGG,:cGaa aG;~.ee~

765 G:f-.F~GCGCtJGaUC.~sG~.C.~-~P.GGC
C~~nP, :.CC?.CUG

769 Cw~C~ CUGAUGAGC-CCGA~sF~GGCCGT~P.
AC~GGCC

77p uVCCAGG CUGF~GTGGCiCGnr.FG.~_zCGA.~
~G.~1A?~.

7 E Ci-:~ G:~.CUGAUC:.G:~CGF.~AC=~ CG?.?.
5 ? C=.GG:C

706 ?-Ci-:~GG C'L~CAUG:.GG.:C:~.?.Ci,CCG?.?~
AaCr~GGC

752 CL'UCCGa CUC,T,UCnGGCCGF~.G:~CCG.~A
ACCUCCA

794 ?-CVCUCC CUGAUGf-.GGCC -C~-.e:Ct-v~.CGAA
e'-GCCCaG

807 C':C-..':L1ACLGr.UC:~C-C,CCG.AAGG:.CG~A
AUCC:?~G

g33 G..v"tJG(iCCL3Gi-.UG?.C-W.CGT~nG isCG~Pa nG.'-'W~7G

a46 c ~ccvJ c,?cAVCac~c;~.~.aG~cG~,a ac ? ~~

851 GCi,IG~:JAG'JGAUCAc.~C~~AG: CCGAA
r?G.~'"UCL'C

ss3 c~Gr.GG cc~cavcsac~,~.ccaaAGccc~
acvGGcu ass c-~~G~ cvGnv -c~ccc~GG.ccGAA AGCCUVc 867 UCUCCCv CL"GAUCnGC-s.CG'nr'1F~.G:~CGAA
F.nCGi~AU

g69 CWC-;~.U CVGAUGAG~C.~-.F.A~:G:~CCGAA
AC-:SAGA

8$1 :~CC~JU CUGAUG?Cv.~.C~~nAAG:~CCGAA
~r?GCCAU

885 UC~CCUC CUGAU -CACz-CC -Ca~AG:~CCGAA
FCCp~.GG

933 CC~aGrr~U
CGGT,UG~G.~.zCGA~WG:~CCGAA
AUUAiJA~

936 C~ACC?.G CCiCYIVGaGC-CCGAAAGGCCGr'~.A
AUGAUUA

CLJGr'iUGACvCCC~GGCCC~A
ACUGUUA

980 ra~GUUG CLGAVGaG ~CCGfiF.AGGCCGAA
AC?.CUGU

9 8 ~ C-.."IJ C'~JGnUC'~.Cv.~, CG.AnC-vCC.~'aF~
6 -G'~'- AGUUGUA

S o C :C-~~'C?CL'GrI7G CW-CC ~r.AF GWCG.a.?
7 ?_ G'~uGJ

.cc v:jC-W,:G ' C'vGAUGCI~CGi ::C~cC~~
i=::C'~'CiG

1005 UCUCG~G
C',.~~UGAGGCC'"' ~ C-GCCG~u~.
AUCUG.~"U

1006 'u'UCCCCA
NG:~UG:yG:~cCG:v~~CCGAA
.~CUCLTCe~

1023 c,~~,~cce~
c~GnUGhG~ccGhA:~GG~~'GAA
AccuccA

C'JGnUG'r.G:~CC'"~aGG.=CGAA
AGACCVC

1066 L'C;rL~LJW
CL'CAL'GAGGCCGAAAGGCCCAA
AGAGUGG

1092 GG.~_CDGA
cJGALGAGGCCGAAAGGCC~.3AA
AUCCAGU

1093 L'UGGCUG
CUCnUCnG~CCCi~AaG:.-~CG?u'~.
AG.~"UCC'~1 11 2 UC'-~AGAA
CUGAL -'GG.-C
C:~AAGC-~
~'".3AA
AGUUGC,G

1163 C-C.'-~AAG
CUGAUGAC.GCCGAAAGGCCGAA
AGCUUC;~

1164 AGCznAA CUG_UGAC~GCCGnaAGGCCG.~.P.
AAGCWC

1166 AGyGC~A CLTGAUC:~GCCC~.-~AGGCC'".~P.
a 1172 G.:JL'UUL7 CUCnUG~-~~CC,AAAGGCCGAA
AAGAGC,A

1200 L'G'JGGnG CUGAUC,AGGCC'"~'-.AAGGCCGnA
AG.:AG?G

12 01 CUC'JUCA CUGAUG~:GGC CG''~r~AGGCCCr"~A
P,AGCAGC

X03 AC:.'~G.~~JGC'JG:yUGAGGCC 'GnAAGGCCG~r.A
AAAAAGU

X27 C-C.ACf~CG CUGAUG.AC~-CC~C~.:AA AUCL'ACC

'228 AC-Cr.AnA CUGr.L'GhGGCC"~GGCCG?.A
?~'UUC

X33 CUCJCCG C'JGnUCnGGLCGi~AG:~CGAA
AAACGAA

1238 AGC_ACCA NGAU -G'~GC ,,C'Gr~AAGGCCGAA
ACAGCAC

12 64 CL'UGCC C"JC,nUGnGGCCGF~AGGCCGAA
ACCCWC

7 L'UCCCC_~ CUGP.UCnGG...~C "' GGCCCAA
ACUCUCA

1294 Ci-cJC.:~G CuCnUGAGGLC~.AGC-CCGAA AUCUCCU

1295 cJG~-'~UGA C',JGAUGr-.GC,CCGAAAGGCCGAA
ACCCC'UC

1306 G_uUuCA C'~~AUGrGGC.CGnAAGGCCG~.P.
AGUCUGC

1321 UCCJCCU CUGnUC,nGC,CC'"~GGCCGAA
AGCCUUC

13 3 UL'LT'r~ cJGAUGAGGC CGnAF~GGCCGAA
4 AUGG""UtJ

1344 CACUCJC CL'GAUGnGGCC~CC~A AG~~UCAU

1351 'r~.cJUA cJG'r.UGAGGCCGnAAGGCCGAA
ACAUUCA

1353 C~C~JUC CUCAUGAGGCCG~AGGCCGAA ACCCACU

13 66 i-.G'Jt'Wi.'ACUGnUGnGGCCG'r~i~T.G:aCCGnA
ACUGWA

1367 ~:C-._'~.'C-;~GCUCAUGJ-.GGCCGr~AGGCCGAA
AGuJGCU

1368 ~GAGJGG c~~UGhGGCCGi~AGGCCGAA ACAGUAC

1380 c: ~cccC cJC.hL.'~G::GGCCGAAAGGCCGAA
AUGGGCA

1388 AGCCAcJ C"~~CnUG~-GCCGAnAGGCCGAA
AGUCUCC

1398 CWCJGU C'LGAUGF.C-:~CCGAAAGGCCGAA
ACAGCCA

1402 :-~GLTJCL1CCUGAUGhGGCCG'nhAGGCCGAA
AAGCACA

1408 CcJCCCC CUCAUG::GGCCGAAAGGCCGAA
AUCUC'GC

1410 CCCJUCC CU FsUGr.C-C-CCC~GGCCGAA
AGACCUC

1421 AG.~.:~~G C"JGAUCr.C~.-CCGAAAGGCCGAA
AGGLJGGG

425 cJC,.~~ncC cJcAUC~GG:.cGAAAGGccGAA
AGG~Gu 1429 Cf:C-C~-;C CUCAUCAGGCCGAAAGGCCGAA AUAGAC~

1444 UCCUCCU CUCAUCAGGCCGAAAGGCCGAr~
AGCCUUC

1455 UCC'JG.~"U CLICAUGr.GGCCGaAAGGCCGAA
AC~UUCC

14 82 GC.-:AGCA C"~JGAUGr~G:~CCG :AAGGCCCAA
pACAACU

1484 CAUG~-~GG CUGAUGnGGCCC'.~AAGwCGAA
AGr.ACAG

14 9 G'vJUC"~JCAcJCAUGAGG.~_ C C,AAAGGL
3 C GAP. A GCA CAG

1500 C-.:=?CCr.UCGCAUGAGGCCCi.~.GGCCGP.A
AUUUCAU

1503 C=?_UCAU CUCnL'GC-:nCGr.~_C-GCCG_~A
AUAGUCC

~ 506 CC~ WAU CLiGAUGhCi-C.C~~_GGCCG ~
At~C.F~UAe~

. 193 1509 ACACGGtI CUGAUGr.G~CC -G'J~CGAA AUC-~~UAG

1518 CG.~.CQG.G CUG'~ -UCAGGCCG~AAG~CCGaA
ACC~UGA

1530 CC_~G~nU C'CIC~GaGC-CC~.~CGAA AU~UAG

1533 GGCCCAC CUG?.UGAGGCCG~CCGA~. aUG?~CCA

551 AG."UCr.~U CL'C~~aGC,CC _ ,G~nnGaCC~vr~t~
a ~G.~At~a 1559 AG.~'"Z7GGG NC,aL'G~C-G."C=" '.~n~C~~CGr~.A
.'-~G.~"QG~.~U

1563 G.~JCTAUA G.i~L'C,~.GG..' ~'=" ' ..,.'..~
ACS

1565 GC~JUA CUG~L'Gi.C.GCC~-' v.."CGaa :~C_~.UA

150'7 UCvCG~;J CL'GAL'GnGC.-:.CC~.C-~vCCG?~.
.~~r AACa 1584 AUAUCC'J CUGAUG:~CCG Ar~G.~CCG?~ AUCUUVC

1592 vAacw,G NcavGf~G~.:C~~aG~~CC,aA av~.UCC~

15 99 CNUCUG CLG?.UG~~CGAF.AGGCC~~? A aACUl7G0 1651 GNCAGG NGnUG~.C-.GCCG~aFanG: CCG?.A
AG,~UGGG

1661 C=.AnG:~?,. CU -CnLGaG.~~~.CG~CZ,CCCAA
:,C,w,~UUUC

1663 UUCAr.~~ CUC~UG?GGCCGA~CvCCGAA AAaG~'"W

1678 CC?.C~CQ CUGAUGaGG:.C'"~GGCCGAA AG"~UCCU

1680 CCACAGG CZJC.nUCi-'sG:~:.C "G.~i.G~CCG?A
AGaGGCQ

1681 GCCACaG CJCia _ ~~C~-' ~CCCsr~?~ 'nnGUGGC

1684 ACAGCCA NGAUGAGG.~CG~r'~GG.~_CGA~.
aGGnAGU

1690 AC~UCG'~ CUGAUG~GG..~C'-'~'.GG..~CG?A
AGDCCGG

1691 ArGAUCG CUCAUG~GGCCG~C~CC~.~AA aAGQCCG

1696 CC_~.CCCC CL;GAUG~C~GCGAT,~~GCCGAa aUGC,GCA

1698 CUCCAG G CGG~BG:~GG:.C'CA~'u'~C~CCG?.F.

1737 GCUG.~~JA CUGa.U~~AGGCCG?sPI~CG:~
AG.~~UCUC

17 5 UGisGG"JG NG.TaUG.GC-LCGAe'aAC-CsvCCa'~A
0 : _GCCGCC

1756 GC-:~.p.G~, CUGnUG?Cw.=CGAA'r~G~~CCGnr~.
AG~aCWC

1787 U ~'"' C NGAU -C AC.GCCGAAAC-GCCG.~,A
AUC~iTCtJC

1790 AUUAGAG NG:~UC,AGGCCGAAaG~CCC~ aCnP.UGC

1793 UCCAGCC NGAL1G~GGCC -"Cn~G:~.C:~A
AC-:UCCA

17 97 UUVAUw Nc"vcac-;~ccc~~cGAa aNC,~,~c 1802 UCUCCAG CUC,AUG?,GGCC:~G~CCGF,A AUCUGGU

1 8' GGCNGA NGAUG ;G:~,.~CG?.F~.AGC1CG'-~A
2 AUC~AGiJ

1813 UGr"-.G~-,~"t) CL1GP.UC.?.G:~: C"~GC'~CC.~A
i~UGv.~UG

1 E25 C-:.F:C. GG NGnUGp.G:~CCGAAAC- GCCG~?, P.CCGUGG

1837 GGAC~'"ITA C'uGAUC,nC~CCGA~.ACvCCC,?~
AGGCAUG

1845 G.~"UGGCC CUGAUGGC,CCGnAAGG:.C.~A
ACCCUCG

1856 Ap.GAUCG CUGAUGAGG~CC'GAAAGGCCGAA
AaGTICCG

18 61 UACLIGGA NC?~UGGC-CCGAAAGGCCGAA AUCAUGU

1865 CUGAGGC CU ,CnIJG:~GGCCGAaAGGCC''~A
ACAAG'UG

1868 UUUAUGV NGAL'GrGGCCGAAAG: vC:.ap.
ACCTG.~"UG

1877 AGCUGCU CUGAUGAC-:~CCCAF~AGC-CC~.~
AG~AUG

1901 GUCCCN NGr~UG~sC-GCCCsnF~r:CuCG~, AGL'UNA

1912 ANGAUC C'UGAUGAGGCCGAF~G.~.-CCG?A
. ACUAUAU

922 UAACUUA CL3GAUGAGC,CCGAnAG GCCG'~A

1 02,3 GAVACN CUGnUG?.G~T~.CGFar~.GWCC~.A
AGCAUCA

1028 CUG.~~UAA CUGAUGGGCCG.FJ~ACv.~.CG.'~.r'1 ACLICrJAA

1930 AC-NGv~U CUCnL~Ci=.C-CSCC~-fir' "~C-v.-v.CGn?a ~ ~=.C'JCU

1064 LC~"'' .C NG~LiGs?Cz-CC~~-r~~-C1-w.G~l.
:.~~~UC'JC

1583 U?.ACL'UG CUGU -Cr'sC~CGirC~,iC~-~
.'-.u~UCCLT

19~
c 9 C~"L?C~G
6 C U~~-~"
UGAG.-C
C C"r~GC-C
C ~.:?~
AUCUCCCT

2005 ~:JCCG~.
CUGAUGnGC-CC~AAC-:~C::AA
AG."UCC~

2013 LT~hCtiC?A
CUCAL'G?~:.iCGAAaC-W
CA?. ihALTACi 2015 c;~cccc c.;GnUC,~c;~.;.~,-~ac~;.c::na aUG'..~,~_.~

2a2o c~.-C~.c.~A
c~cA~e~c:~_:~~.~ic:~.a AACC~Cc CGCsnUC~,AGC,CC~-~"~u~C-.~sCC~.-~~sr1 AG.CnC-v 2040 CCUCC~G CUGAUG1C-C,.=C~G:~C~.l.A
aG.'=iJC'S.G

2 0 C-.~-.AUGvGC'~JC~L'C.A G:zC C.:r.?~GCi 57 C a.?~ AC~GC~.

2 0 aC~C G~J CUGAUGAG:.1 C ~~-r~ 'n~.C-GC
61 C ~,;rfs AUG~"Lr'lG

2071 CUGAG:~C CUCAUGAGGCC~?.GGCC~~ .~C~,aG'u'G

2076 LJAG~."UCUCJCAUGACv:.CC:r~nC~CGl~ AGC~"UAC

2 097 CAUCAAG CUGaUGAC~c C~'~GG.~. C ".-~
AGAGJL'G

2 0 C C~-C.:~;CUGAU GnC~c CCAAAGC~ C u'~, 9 8 AAG'JGL'G

2115 AUCNCC CUCAUGAGuCCC.~.AAGGCC:.-.Ap.
AG~~UGGv 2128 CUGy:UA CL~L'GAGGCC''.~AAG:~CC -CrA
AUAGCUG

2130 Gt.C-C.CnGCUCaUGAG:~CC -C~.r'.C~-:.C
-CAA '.~P.C~GG

2145 G'.UCr.AG CUCAU -G.C-GCC"'=" "' ~CCC=~
AGAGLn;G

2152 AnCLTC"JA CUGAU -CnGGCC'G~-.r~G~vCC''.:r.A
AUUT~'UA

2156 U'rr?L~.'rACQGAL'GnG~CC~-.=C~.:?A ACAUCAA

2158 hULr.AUA Cu'CAUGAC-:~CC~C;~A AUaCAUC

2159 RAUL~'i,AUCUGAU -CAGGCC~-v~AAC~~a.~C~~'a~
Ar3.Ue~.Cr'~.U

2160 AhnUUAA CUGL: -G.C-G.:.C~-~anC-GCCG.A
:t.AUrlC_A

2 ? CL~.u-.HL'UC'uG~=aUG~aG:~ C~~G;sC C
o' GAA A ' " IT?.

2103 ~.nLTiJ?~'aUCUGAUG~aGCtCCC~~r'ar~ AnUAC~U

2106 nr.'v'~.GAGCL3GAUG.G:~cCG-'~AaC~CC~..nA
AUGr.ACaU

2167 AnL'U'r.AUCUG?UGAG~~cC -G'~nAGC,:.CC.
iA AAUACAU

2170 CL'AI-.~:UUCUGAU 'C~CC'",~AAC-C-CC~.A
AUT~.AUA

2171 GC-GAG: CL1G=.UGAC-:~CC -'G~ACZ-CC~.:~A
AACAaCU

2173 CUC-.uP.A CLIGhUGAG~CG~J~:C1-C.C~r~
hCUC'L'?.A

2174 C-CJC~"L7aCL~GAUGr'1-GvCC -"CnAAG~cC
-Gsn AAC'JCUA

2175 ~G.=,.'G~"C7CUGL1G-'~GGCCC~AC-GCCGr~
r.I.ACUCU

217 L~ GC"JGG CL~GAUG:~G:~C CC~G:~C C.~~.~.

21 83 C:.~:LT'~-.F~F~CUGAUGi:C-C-CC -Gn~CvC.CG~?
AGw~UGGLJ

2185 CUC::nLTA CUG AUGAC-:~CCGJ~C-CiCCAA

2186 ACUC'r.AU C'JGnUC~-C~CCCr'~'rrCi-C.C~.-t'.r~
A,r',UAG.~U

2187 L'ACUC~A CUGAUG=.G: CC~'CCG:~ AI.F_UAC-C

21 8 G.:..?F CUGAUGAG:~CCG.i~AGGCCGAA
9 CUC ALTAAP,UA

2196 CALTCr.F.GCUGAUGAGG.=CGrI~AGC~.CC?.A
AGAGUUG

2198 AAC~L~r~A CC'C,AUGAGGCCGAAAC~C -CnA
AGGCUGC

21 00 .t,Lr~ CUGr~UGAGGCCG~_G.=.CC.=.rte ~?C~ ~-.r"~.GAGGC

2200 C'CT..~C-CAUCUG'=.UGAC-CZCCi.AAG GCCG=A
AC.GAAGA

2201 GCCC:ACA C'UGAUGAGGCCG-'~nAG: CC~,,~.A
A"nAACW

2205 UCAGGCC CLTCAUG,:.C-:~CCG~AGC-CCG~'.?
ACAUA~

2210 AGCCACU CUGAUGAGGCCGAAAGGCCG~A AGiJCUCC

2220 AG.CAAC CUGAUGAC-:~CCGAi~C-GCCC?.A~AUGCCAG

2224 G._.AUGGA CUCAUGAC-:~CC~~AC-C-CCGrJ' 2226 GCGC~CCU CVCAUGAC-GCCC-'-lrCllC ~~=.
ACAUCC~

2233 CCUCCAG CUC_'_UG.C-C.-:.C.~-_=i_.CZ-CCC=.=_ rC-.~UCiG

22x2 CvJCCGC C'LJC-r'yUG=.GC-:.C.~-r".r_.GC-:.~.~_.
=.GC'LiCC:=

2248 UCH: UG C'JGAU -CnGGCC~~GGCCGAA
AUG:~.UA

2 2 UCAGUGU CUCAUC-~ CCAAAGGC CG'1~.
54 r'~F~UtIGGA

2259 C.~CCGJG CUGrBG?t-:~CCG:,AAGGCC~.~
aUGUG?~U

22 60 GCACCGLT C~JG:.UGAG:~: CGAAaG~CG~
AAUGUGA

22 66 UCCUCv--U CLC~?.i.'. _C~CG ;AAGG:.C
;:11 ACrUUCC

2274 UCUCC-'~G Ct7G?;U -CAC~C "GnAnGGCCGr~r~
AUCiJG.~"U

2279 CUGGC~C CuGAU -C. AGC,CCG~.AG:~~C~.,nA
ACCCUL'C

2 2 c~G.,~UCA CUGaU~t-.~C C:~1? G:~c.
8 2 CGAa aG~GCUU

2288 ~ AC- GCC_~.UC'JCAU -C.AG.~CCCAT~AGGCC.~A
ACUUAUA

2 2 .'-t-CF CUCAUCAGGC C -CnAaGGC CGnA
91 G".G ACC_~CUG

2321 CCC-~LTGLTCOCA -DC~"-.GCZCGAT~AGG.~.C~.:?.A
AUCUL'UC

2338 CAC''"" CUGnUG~G~.C~"CGF~ AGJCUCA

2339 CAF.AC-:~ CUGAU -C~CG~FGAA AG.~"LTUCC

2341 AG:n.~JGG CUGnUGAC-:~CCGF~PtvCCG?.t~.
AGAG~"UC

2344 GCVG3AA CUGAUG~t~:~CC-CnAAGGGCCAA
AUCGAAA

2358 CUGC~ C'JGAUG.GG:. -CG:~P. AG~~UGGG

2?5c UCVC-DUC CUGAUGnGGCCGAr~.G.:n.C~~
Ar'J~1GCAG

2360 UUCnF~FaG CUG'~DGr'~G:~CGrn~.G~~~.C
-C~'u~ Ar'~GGUU

2376 UCAG.~G CUGAL7GAG:~CCG~u'.e~GGCCG?.~.
ACCACCU

2377 CUCAG~ CUGrL3G~GGCCGF~GGCCGAA AACCACC

2378 CA~~ C~~~CG~AC~CCGa.A AAACCCLJ

2379 CUL7~L'GA CUGAUGAG:~CCGAF~,~GGCC~~
AAAAGCA

2380 G.:CGACA CLGAUGAGGCCGAAAGGCC~~?.A
AAAACUU

23 82 G:~C"~'" CUC~UGnC~CC:~AAG~~CC~~ AGaGhAU

23 $4 UUGUGUC CLGAU -CAC~CCGAFuIG.~CCG?.A
ACUG ?.U

23 og GJCCACA CL1GAUG:~C- GCCG-'~GGCC
GAA AGUGWU

2401 C~~ N~C~~~ AC~GCW

2411 ~GCAUCCU CUGUGAG:~CC -CAC- CC~.~?~
ACCAGUA

2417 ACGU'r~UG CUC,AUGrt-GCCC,AAaGC~CCGAA
ACCAUUC

2418 GGCCUC-~ CUGUGrGG:.C -CAF~P.GGCCGAA
AUCCAGU

2425 AACCCUC CUGAUGAG~C -C~AGG: C~.~A
ACCCAUG

2426 AAACUCU CUGAUG~.G~CCGSAAGGCCG~.A
AAUUAAU

2433 G..'"UG.~"LJACUGAUGI.C-;~CCG~.F.F~GGVCC~, AACUCUA

2434 AG.'"IJG.~"VCUGi~UGnGGCCG~.FaP.G:~CCGr~~' AAACUCU

2448 G.;~CAGG CUG.~.UGAG:~CGAAAG GCCGAA
AG~~LTfJC

2449 ''G. ~~G CUGAUCCi:C- GCCGT~AG~~CC:~,A
AAGC,CW

2451 AGGCAC-G CUGAUG~CGAAAGGCCGAA AACAGGC

2 4 GAGGCAG CUCAUG.T~G:~C C -G=.AAC-vCCG?.A
2 AToACAG;

2 4 C ~-~cAGG CUGhUCY.C~C CGAAAG:~ CGAA
5 5 AGG.,.~UUC

2 4 GC~~G CUG'~UGAC1-C C~.~AC11CGAA

2460 C ~G.~GGG CUCAUG?~G~: CCAAAG.zCGAA
AAGUGUG

2479 G.~UG..~.LTACUCrUGAG3.~.CGr.AAG.~.-CC.~.-,~.a AG.~"LICUC

2480 G GAUCAC CUCAU -CAGCZCGAT~GGCCG~
ACC,.,'"UGA

2483 G~"UGGCU CUGnUGAGGCCGAAAGGCCGAA ACAUUGG

2484 GACCTGv~U CUGAUGr~GGCC.:AAAGGCCGPJ~
AAAAAAG

2492 AGGUC-Cv CVGAUGF:C-GCCGAAACv~CCGAA
AG,~UGCU

2504 ACA'r~.AG CUC?sUCr.C1-CCG=~C-GCCG..A
AG.,~UGGG

2506 UCz-C:~:UGC~GF-i.'~C~.CizCCA.aC-CzCG.i.
=.UC-:QUA

2509 CUCZ'=tlA.~.CUCrUC=.CW.-CC~=~ ~CZZC.~-_=~
ACUCUFs~.

c 510 C-.=vC-' C'(.~CnLG.C-:~CCC~_G:~CCGAP.
17F. AACUCUA

2 52 C .UL'GGG CUGAUGAGGC CC~~AGGCCGAA

2521 vGnGC-._~-UCUG~ -LlCnC~.:~:~G.nnGC~CC"L:?A
:~:L'C,CC~;

2533 CnL''"rsCC'JCUGn -UG'.cGCC"~~.nGGCCG~
AGC=~DCA

2 5 C =~Ci_GC CLOG=~UGt-' CCr_~.C~. CGaA
4 0 G ACT~'Gw~GT

2 5 AG::ACC_T1CL CnUG CvC CG''~anG:zC
4 5 CG~?.A AG~Cu~.C

2568 L'UUGAC~ C'JGUG'.G.~C~~-t~CC".:AA
?CLGC~.C

2579 CAGGCC_~ CLC~UG.?.GG.:CC-.~~C:~A
~CUQAU

2585 ~c~c:-.ac cc~~L~G~Cc -c~..c1-;.ccaA
a-~GCc~c 2 5 hL'L~.G CUG:-.L; Gi.GGCC G~AGC1 8 8 G CGAA AC~-~.DGC

2 5 aG;~G;~. c~~ caUCaGC-cc G~AG~;.cGaA
g 1 ac~ACCA

2593 G~G'~C~ C2.'-GnUC,AG:~CC,~:G:.-CCG~
~.AAG?.r~G

2 5 CAUUGC-G CUG.=.UG:yGGC CGnAaC,GC
9 6 CGAP. ACAAAaG

2 6 A~-.nC~.-~C'UGAUG~GGCC~~.G.GCCG?.A
O1 ?,C~ C'~w"Q

2602 C~-",-.,.-'UGGC'uGnUGnC~C -'Cn~GGCCGr'~a AG..'RGuA

2607 CC:G..JA CUGhUCl'-_G:~CC~:G.;~CCG'~A
AL'CCGAG

2 608 G~CAC-CG CtiGUGt-:nCG~nCvCCGrZr~.
ACUG..."~JG

2609 L,'CC'~Gu'JCL'GnUGAC-GCCG"~G:~CCGAA
AC~DLJCC

2 6 C-C..':GC-.~~UC"JG UG?t~C CGA:-.r GGC
2 0 CGAA ,~G' JCCU

2 6 G."L~ C-GA?.C'JGAUGa.GC~ CG:~-.r CvC
2 6 C~..~A aUC~P.A

2628 nG:n."UaC C"JCl:UGnGC-CCG~-.nG:~CCG?.A
~GC

2 635 ~.G:;ACCG CUGUGr:G.~ C~~.~=.~G:~CCGAA
AG~'21G~

2 6 G..-CAG.~-~Ct.'~G? L'CriC~.-C C -G'~AnCvCCG?

2 641 C'~GCt CC7G.nUG.GGCCG~ ~GGs.CGAA
GA AGE

2 6 G~GC-G:G CLTG.L'G GGC C Gr.~.C-GC
4 2 C GAA AA.AC?~GG

2653 G.=AUCC'J C'UG:,UGnGCCC~A:~Cv:.CG?~?.
ACCAGUA

2659 C'JUGCAC C"JG::UGAG.:,CCGW '-.nCv:.CGr'~.A
ACCCWC

2 6 C CUCC-:~.CUC~UG? C-GC C G~CvC CGr.A
8 9 AC~UUAG

2691 G~CUCG C'UG~UCu:C-C.-CCC:~.:CZ-CCGAA
aCACAUU

2 7 G'-C-~:C~ C'CG~UGhGC~CC~C-.;vCGAA
OG AGC~UC

2704 ~_C-:~CUG:~CL:~G-~U -G',G:~CCG~t~CGAA
AGAGGLJC

2711 CUG''"L~Cr'~C'UGnUGAG:~CCG:~.nC-GCCGAA
AC~~UGGG

2712 CCCL'UCC C"~GnUGG.:~CCGn~:G:~CCGAA
AGaCC'UC

2721 C"JUGCAC CUGuGG.:.-CCGnr.~-~C~.CGAA
ACCCUUC

2724 C-:r:C=_CGCLiGAIJGGaCCCr.F~GGCCGAA
AUGUACC

2 7 C'~.TC-C~CGCUGAL?GnC-;~CC''-~ :G.:.~.CGAA

2 7 G.: JACUC C"JCAUGnG:~C C -CAAAC-C,C

2759 ACAUCGA CUGnUCr_G:~CCGT~F~GCCGAA
AGUCCGG

2761 C~~:GC~"U CUGAUCr.C-:~CCG~AAG:~CCGAA
AG.~~UCCU

2765 :GCGG:.A CUGAUC~GC,CCG'.yArt-GCCGAP.
P.G~A.F~, 2 7 C C LTGJW CUG=.UCrt-GC CG.i~.r~C-C-C

2797 C~CCAU CUC~UCiGGCCG.rrCI.zCGAP
AUUUCAU

2803 CC-CCUGG CUCAUG.nGC-CCG~~-.AC~CCC~'P.
ACCAUGA

2804 CUGCACG CUGAUGAGGC -CCr~GC~CCGAA
ACCCACC

2 813 G:N~LiG.G CUGr.UG.~G:~CCC~=.nrlG~~~~CGAA
ACCGGr'iG

2 815 =rf:GJUG CUC~UCG? C-C-CCCI=.rr Ci-C
CGAA AG AC'JGU

2821 CCVCCr'1G CUGnUC~?G:.-CCG.=_.r C1-v.CGhe~, AC~s~UCaG

2 222 _ _ .GUCCGCUG_'=L.'GG:.-CCC-'-: rGC;.CG,'.,p, rGCi,;CC

_023 JCiuC-C C'UC=L~G_'11CG==t-=CCG?~
r_.r?Civ.

2E29 ~UGr.U~rA
cUGaUG~:G~cc~AGC,cCGaA
AcuCCAG

2837 UCACAG CL'C~UGAGGCCCAAAGGCCG?.A
ACCACCU

2840 C=GC-CAG C'LiCF~UCAG~CCGnF.AGG CCGrI.A
AG'JCtJCA

2847 G~JC-GC'IJCUGAUC~GGCCG?.AF~GGCCGaA
ACAUUGG

2853 ~C~L~r CUGAUGAG~CCG~GGCC:~A AC~~UG~.
A

2860 UC_ACnGU CUG:~UGnGG CCG~GGCCC~A ACtJLSGGC

2872 CUUGw'U C'JGr~UGAGC,CC~'~n~AG~~CCGaA
?.e'~Gva~UCC

2877 GJGAUGG CUGAUGAGC~CC~~FCGAA AG..~G::aA

2 8 Tx.G:~UCG CUC~UG.GGCCG~GGCC'".z?A

2 9 F~~ACUC CUGAUGT.GGCCGAAAGGCCGAA

2904 F.F.UAGAG CUGAUG .GC~CC~.~Fa~AG~."CCr~
AUG:u~GC1 2905 CAAUAGA CUGF.UGACGCC~' ' ~ C~.~
AAUGAAIG

2 06 LT'nPUp.AACUCnUGr~.GGCC".~AAGvCCGAA
ACAUC'aA

2 9 F~UUAA CUCriUGnGGCC 'G~C G?.A AT~ALTACA

2908 :sC~~AA CUGnUGAGGCCGAAAGGCCG?.A
AAGCUUC

2909 AGG:AA CUGrUGAGGCCGAAAGGCC-CAA
AG?AGCQ

2 910 F.w:UtJF~ACUCnUGAG~vCCGAAFaGGC CGr'aA
AAFaUACA

2911 ~:~.UtlAA CUGAUG?G: CC~AAGGCCGaA AAAUACA

2912 GCAUUA CUGAUGAG: CCGAAAGGCCG?.A
AGF.ACAA

2913 UCl.CCAG CUGF.UGr.GGCCGAAFGGCCG?~A
AGAGAAA

2914 CUUF:UGA CUCr'~.L1GAGGCC~~.AAG~~~.~.~CGAr~
?~'ar'~AGCA

2915 UCU''t,F..AUCUG?UGrGGCC 'GraAAG~~~.CGara AAUAAAU

2916 CUCCC~A CUGAUGnGGCC".~G~.CGAA ACGAAUA

2917 UCUCCGG CUGAUGhGGCCGaAAGG.~.CC,AA
AACGAAU

2918 NCUCCG CUGAUGAGGCCGAAAGGCCGAA AF~ACGAA

2919 CGaCCCU CUGAUGAC-GCCG~AAAGGCCG?.A
AUGF~GaA

2931 CL'UCCGA CuGF~UGAGGCCG:~AAGC~CCGAA
ACCUCCA

2933 CCCUUCC CUGr.UGAGG.~_CGAAAC-~CC,~1 AGACCUC

2 9 UC~:~,C CUC,AUGAGGCCGAAAG;~CCCAA

2951 GC'~GnG~v CUGAUGr.GG.~.CGr~AG:oCCGAA
AGCGUGG

2952 CzC~GCG CUGAUCAGG.~.CGAAAGu'CGAA
ACUG.~UG

2955 UC.~.CACA CUCAUGAGGCCGAAAG:~CCG?.A
AGUCACU

295c UUGAUUC CUGAUGAGGCCGAF.AGC,CCGAA
AACuAAA

2961 AGJGu',"U CUC,AUGAGGCCGF,RAGGvC~s?.A
ACACAGA

2 9 AF:UUF CUGAUGAGGC CG?.AF~GCCGAA

2965 CUUUAUU CUGAUGF:GGCCGAAAG:~CC~~?,A
AUUCAAA

2969 F~CW CUGAUGnGC,CCGAAAGGCCGAA
AUUGAU~3 2975 GCUG.~'"tTACUGAUGrGGCCGAAAGGCCGAA AACUCUA

2976 nCUFGAG C'UCAUGAGGCCCr~G.~CCGr'1A
AACCCLJC

2977 CAGCUCA CUGAUG~GGCCG~GGCCGAA ACAGCUtJ

2979 GC~UA CUGAUGAGGCC
-C~AGGCCGT~
AGnAUGA

v ~ c~ ~ ~ ~ a ~ ~ a v U ~ a U U U
W J v ~ W
Z- U U U U a U C7 ~ ~ ~ ~ a ~ ~ U t~ ~
r C.~ ~ ~ ~ ~ ~ ~ ~ U
C~ ~ ~ ~ ~ U ~ ~t ~ U U ~ ~ U
a ~c a a ~
UC:.'~J~JU~~~UU
U U U U U U U U U U U U U U U U U
~C ~ ~C .2 .G d d d ~C 4 W2 d ft it ~G ~t a j ~ ~ a U L U V ~ 'uJ ~ ~ U U U ~ ~ ~ ~ L
V ,~ ~ ~ ,~ a a a a a a a a a a a a a o _ .~ .~ o 0 0 0 0 0 ~ a o 5 o a a O
4) ~ U U U U U U V U U U U U U
U ~ ~ U

5~~~~~~5~~~~
a ~ U U U
,uc c ~ ~ ~ ~ ~ a a a a a a a a a a c~ ~ :~ c~ a U c~ ~ ~ ~ a c~ ~ a ~ o ~C ~ U U
y_ U U .~_. U J U ~ ~ a ~ ~ ~ a Q7 .~ C7 C~ G~ C~ C.7 C7 C9 C9 C9 C7 C~ C7 >,= a ~c a a a a a ~a d a N ~ C7 L L O C7 C7 C9 C7 C7 C~
o a ~c a a a a a a a a a a a a a ~c a ~~~c~~~~~a~~~~~
U ~ ~ ~ C? C~ U' ~ C?
~a~a~~~va~a~~'~aa T
Q
U O r, ,., ..~ e, m c ~n N ~o c CD ~ ...r ~ C vC r ~ t~t1 D ~N O t~ N C\ O f1 N 1~'1 r '-i ~-i .-i ~-i N N N N N N
'= a au~c~~~
».
a dc~c~~'~c~aaac~~~~'~~c c~c~c7Wc7c7~c~c7c7c7c7t7c7c7c'7c7 o~ U U U U V
cn O C~~~~ ~ c.~c~
~~~~~~~~~~~~~~~t'~~
N=
O
a c~ a ~ a v c a a a d a ~ a ~a a a ~ t~ ~
V , .~ a~ uwn o er ~ o I~ .fir yp C' N ~ 00 G~ a ~ rWD ~ ~ N U1 Y~'1 t11 CD
m ~D tf1 ~ '-1 er O ~ rl C~ N N H'1 t~1 tt7 CD A
ri N N r~1 a W CO C1 C1 r,.~ rl r.l ri e~i rl ri O
a U a ~ U ~ ~ ~ ~
.. D U
.a ~~~~~~~c~c~~c~~~c~v~~~'~
U rt ~ ~ ~ ~ U
U ~t a C~
C

v v v v v v ... ... .. ,.. .. .. .r ... ,.. _ _ _ _ c j, Uc~~c~c~c~c~~~~a~~c~c~c~c~c~3c~c~J

a a a a a a a a a ~x a a a a ~ ~x a a a ,c_ p ~~ ~ C7 a~xa~xaaaaaaaa~~s~xa~a~
a a U c~ c,~,9 ~ c~ c a ~(~~~~U~
c~ c~ c~ a ~5 c~ ~ a 'o..
= C
N t~ t~ N CD L~ N r'1 C1 _ !l1 01 t~1 10 l0 Q1 t!1 ~ O Ilt CC 111 CD e~-1 O f'~1 ~ N e'N7 c ~D O~ D Ot ~ !~f M c''1 QJ D O r1 tf1 ri ~-1 ~-~I ei v-~1 e-I N N N
c ~o Table 9: Rat ICAlI~I HH.Ribozyme Target Sequence at. H8 TaxQet eequeace at. 3H Target Sequence Position Positioa GaUCCAAU U C~CACQGa394 GuT~~'G..."U~ ' CAG
U

23 ~~ C ~.'~A 420 ~G,f,CCCCU C;.AG..~G:.A
C

26 G~ACDG~~U C UUCCUCUO425 C~JC~~ U C;,GC~CC

31 CCUC~ C CUG.~"tJCCQ427 L'CCTGUU :,AAAACCA
U

34 cuGr~GCV c AcavAtmC45o AACaacw avc~G..~
c 40 ~~"'Q A C:AAGC'CCC 451 Gw"G~rC~J CCCC~~
C

48 ~CCU C G:~CCL~ 456 CL'C~~~L'U L'C~C~C~,~, C

54 CCCCG~~CU C CCUGAGCC495. GCC~CC~U
C

58 CCGUGC'CV U UAiGCTJCCC510 GCGCCTGC.v C
C

64 CAAUGG~."U U CAACCCL,U564 ~ U C~~C

96 CCUCLJG~~I7 C CL3G.~~UCCU592 GC~U C CC

1 p2 CUCC~~U C CQG.~~UCGC607 CAGCC~U DCL'CAUC-C
U

~ 08 G GAC'JG."TJ U GC~~608 AGCC~UU C'CCaLG~~LT
'J

~ 5 UCCtmCCV U U~GWCCCA609 C~.C'-,AUOQDC~DGCL~
C

9 cr.CACVCJ c cccAACUC~ 611 caavUUCV aUC-~~WC~
c 0 GWGUCAU C CCCGGGCC E56 GuC'~CUGV Ci~,AL'G
U

46 CCT~GaCCV U G::~ACVCC657 UCACL~'JU ~~ --CAAL'G'J
C

152 ACCCG ECU C CACCDCAA668 G:~ACUGC'J L"CCCLiCW
C

158 AWQCDUU C ACGAG~ 67? GC.ACCCCLT C:?~~.G:~
C

365 UGnACAGU A CWCCCCC 684 AG~.'~.G.."QCC~CUUU
C

168 G~AGCCUtJ C CQGC<vCGE92 CCAGACCU CZA?CUCC
D

185 GG~'"JGGAU C ~ E93 C'GGaCLW GDCVOCC
C

209 CAGCCCtv A ADCUGACCE96 GCCUGL~U C
C

227 GnCCAAGU A J~~AA 709 G~C~.L'L?p CCCCUCAC
A

230 CAAGCflGU U GUGC,C,AGG72'0 CLC'J U a ' 237 CUr~AAGCU C C,ACaCCCC723 CAACUL~TQ AG.."'L7~CCC~
C

248 GGCCCCCU A CC'UVAGGA735 CUCCIJG3U CuCvJCGv C

253 CACUGCCO C AGUGGAGG738 UC'~GCCIT Czv"~CGG;, C

2 63 GAGCCAAU U UCVCADGC7 65 p,L~GtJC,CVp -~W
U

2 57 -G.~GCCULT C C'UGCCUCG7 69 UCUUGL;GU CC
U

293 G~GCUC~l U C~AGC'U~C,A770 CUUGJGW CC's-::AAG
C

319 CGuAGGAU C ACAAACGA785 AG~CC'UGC7 DCCDGCCU
U

3 3 5 ACUGQGCV U tIGAGAACO7 8 6 G:~CCiK~7U CCCzGCCVC
U

337 t7CUGCLJAU A UG.~~UCCtIC792 C~JCCUG.~~UCVG,-UCGC
C

3 3 8 AAGCUCUU C AAGCL~GAG7 94 UCCUGCCU LG.,Gw.--pC
C

3 5 9 CACGCAGU C CUCGGC 8 07 G..~v?CAGr~aUL~~C~TC~, UV A

3 67 CAAtTGGCV U . CAACCCGU8 3 3 CCUGG:r.~-UC~ CACUA
U

374 WACCCCU C ACCCACCU 846 CUCACAGU nLiu~.UUG
U

. 3 75 AG~AGCCV v ccvGCCVC851 GcvCACCV vAc-~GCU
U

378 ACCCACCU C ACAGG""'Lm863 CAAVGG CU C~.~srCCGU
U

3 8 6 CGC'UGUGO U UUG~u~Gtv8 6 6 CCAUG~~UU CL-CL,CaC,A
C

867 GAC~''~CCU C CC,~~C"~,1421 G~CpU C CCCCAGGC

E69 CL7C'DUCCU C U~~".zAAG1425 ACC",~CU CUCVGGV.~U
C

E81 AAUG:~'W C :zACCCGtrG1429 ~,A ~~~

885 G~CCza-~.GU A ACL'G'v'G?.A1444 ~-sGAAGC,~~L7~AG
C

933 UG'JGi~,ULl C GWCCCAG1455 GGGAGUAU ACCAG~~
C

936 G=AGAGnU U UUGUGUC~, 1482 AC.~ACU CCC'CCAGG
U

978 UUGAGAAU C ~ACW 1484 ACTu'G~~UCUC~JCUUG~
U

980 GACAAUCt7 A CAACJp~'(,1493 C~G:~,~,--UCr;~.GAC27A

9 8 C'JACnAC.'U U UL1CAG'~CCL~ 00 C'J'.JG AUG.,~UCAA

987 UACAACvU U UCAG~'VC~ 1503 Gr,A~UGU C~'AACC~C
U

9 8 aC'~AC~UU U C iGCUCCC15 06 UG;";UC,AU ,~

1005 CLJC~U C GUC,: CGGC 1.509 GCC.~.CC~,UAC'JGUGUA
C

1 006 GJGG.AGZT A UCACCAGG 1518 G'JCCUGGU ~~~U
C

1023 CCGGAG,~U C BCAGAAGG 1530 ACCUG,GU ALmAUUG'J
C

1025 G~.GGUCQ C ~~ 1533 ~~U U GCGuGC~

1 0 CCCD~CCQp U GUUC'CCAAL 51 C~G;~.,~UCUUGC UCC~~1 1092 ~~~U C UCAC~CA.G?~ ~~59 AGU C CC~L~TA

1093 A~~~";AAU C CAG'~CCCp1563 UCCLmCCU UGULICCCA
U

1125 CCCCAACU C U x.565 ULmCACCQ UUACCGCC
A

1163 ACC:ACG."U U CWUI3GC[T'_567 AC~CCpAU ACC'GCCAG
U

1164 C'"ACGCUU C UDC L 84 AG ;AAGAU A
C

G~,UALDr, 166 ACGC"~Cff U LrCGCOCL1G1 592 G~G

CA~U A CA:~C
,172 CWQUGCU C UG..~GG..' 1599 UA~pU A
~U

'_200 AUCCAABp C p.CACL~AA 1E51 CCCCGCCLT
C

CCDGAG;
12 WG~.~W C UCCAC~G;, 16 61 CLU C
O ~

GCCCOG., 12 G~"tlDCU C GEC ; 6 63 U

GAACAGAU AAUC",ACA
1227 L~TGC;AC~7 C CALJG'JGCQ C

1678 C,AGAAUCU GGCCUG~~G
2 8 G~~G:~:~ C
~W C ~
U~

. 16 8 GG:~.."t~CUCAG;G"'"LJC
' , 0 C

"' CUCC
' "

_ ~G, 1681 GGCCLTGUU CC'UGCCUC
iJ C C U
t3Gu UCGC

1238 EAU A UG.~"UCC~1C 1684 CtJG~~~
A

~pCUC
64 GGAAAGAU C ALIF.C'~.,~U1 E 9 CCCCACCU CAIJACAW
12 GZJC~C'~ U CAAi" A
67 AAtlG

.z 1 E 91 C CGC,ACUU CGAUCUUC
12 C~.GaC,Am7 U UGUGL~G U

1 E 9 CUC CUGGU C'OCr,~UCGC
12 AGAGG:r,~p C UCAGCAGA6 C

1 E 9 UCAGAUAU CCUGC,AGA

~ 17 3 GADCACAU CACCn,"UGC
' GAC~7 C L~CA~ 7 U

-321 AACAGAGU C UG.:~.AAA 1 750 GUCCAUUW CACCUAUU
A

13 GUAUUCGLT U Cr'CF.GAGC17 5 CCUCUGCU ~

UG~
1344 UCG,~~UG~~U C AG.~'"i.JADCC1787 UCCU

GAGAACW G~GCCUGGG
C

13 UCAG~"C'U A AGAC~:~F,CL717 9 GACACL1GU

CCCAACUC
1?53 ~ C AACAAUGG 1793 AUG~JCCU ACCUGGAC

6 :

~ 17 97 UCCCUGpU AAApp ~GDACp U CCCCC~G U CCA

1367 G~~uACQU C CCCCJ~GC 1802 GCUCAGAU , A ' UAC
13 G~UG...~UGa C CCG~GCC1812 AA CUGGA

CAO;AGU UGG.:,CApp, 1380 C'UGCCUAU C G; C
=AUC~

~, 1813 GCC~;CUU GUGAUCGU

; :~AGACU A ACUGGAUG 1E25 GCCACCAU ACUGUGUA
' C

98 CUGG~~UGp C ACAGGACA 1837 ACCCACCU

, C ACAGGGUp, 402 CffGZfiCUU U O;AGAA~ 1845 A

GAC,C,ACU GGAGGG(,,C
1408 UUCGUGAU C C~JGGCGUC 1E56 C

CCCCUAAU UGACCUGC
1 410 C:~P,CL7~1U C CAGUG C

A

1865 UAUCCCyJ A -Cr'~CJ~G 2198 -CAaL'G'JCU C C~.~G~
C~, 1868 UCACG~G'J C :.L~ITA~J2199 ~CUCUU A C~DGCCJiG

1877 ACAGL~CU U CCCCCAGV 2200 GG.,~UACUU C CCCC~G,~.C

1901 W~s~U C 'M'C~.~'LTACr'12201 GCr~sCZILiCU C CACACv~vTC

1 g -Cr'.ACA~U C A?.UG:~C~22 05 UUUGGJG'J C AG~~CACGG
~

1922 aUGU'r~?.GU U .~UL'GCC'LTA2210 LT~C,ACU A aCCGC,ALiG

1923 LT~CG."'J C :~CCWUpG 2220 G?.G~rICCCT C G:~CWGC"

1 a2$ CiJC~ -G'~U ?i ~?ACCCK1G::?,2224 e~.C.~L~nG~.U U
CC'.r~ICCUIJ

1930 L'G.;~G?~C'J A AC'.;G.:~UG2226 CUC~,aCCIT C AGCiC~

1964 -1,~G~~i~? U GUG'JC~.GV2233 UCAL'G~"LZ7 C ~1.?~C?~1~C'J

1983 -C~:?.C~J C C-C-:.C'"'G:~G2242 ?~C~.C~C-.."U C
UC~1U~1 o c L'C,~.C-.."tJ C UUC~~J22 4 8 CL'CCGG.3U C CL'G
6 ~-UCC-v .

2005 =~G~G'J U AUUG~'"CiTA2254 AUCCAAUU C ?~C?.CQG~A

2013 C".~~~'G.~J A UC~.N::~.L'G2259 G?.UC~CAU U CAC~,vGC

2015 C~CCO~U C ~~G~.:~L1G.~~J2260 AUC~UU C ACGC,UG,-~U

2020 UAWC:~,U A CCCVG~C 2266 AUCAG~~U A ~AGiJCI

2039 C'G ;?.G.:.AU C ACA.AACGA2274 G?.G:_~.G~"U U AAA

2040 C~CGaL'C'J C CUGC.~G.,~U2279 G:~GaU C AUAC' 2057 CGG~"L~CWJ C CAr.UC~C:LT2282 ACAG'v'Oa.U U CAULTi'-~C'aU

2061 G.~,GVCC:U U U~.C'~CCL?~2288 GCCCUC~J C CUC~.~ALG

2 071 AUACVGG'J A GCCUCAGu 22 91 G~,UAU A G~C~C

2076 L~~J C AGC-CCi~ . 2321 ~C~AAGaU C AL1A~CGG"~U

209? C~..:~.CUC'J U GUL'G1UGU2338 L'C'G~~~ULT C UCC'~CAGG

2 09 C'UGAC.: J C CUGG~J 2 3 3 G:u~UACL'U C CCC
8 9 ".~GC-C

2115 LZiCCG?1ZT A GCw~UCCUG2341 G:,-:~CCUG'J C Gw,"L'G..~(1C.~

2128 r.C-JG.."GGU A CCAUGAUC2344 CCGC'LTCGU A G,?,C'CDC:GC

2130 GCCUGUGZJ C CUC-CCCiC'J2358 CCCL'Cz.C'U C CGCCCACa, 2145 CC~ACL~C'J U G"vL'. 2359 CCAUCCAU C C'C~aCAG~
-C~sLIGU

2152 LAG-'~,G~U C UAGACULT23 60 CWGLCW C CCUG"A,~,G

215 UCAG~tJ A L"CUAUaGA 2 3 7 C,~ACUGCLJ C UUCCUC

2158 L'GrUG~~U U U1~IJ~AL'U2377 G?,C'ULTCCU U CVCUAWA

215 G'! UGJ'riUU U AL'~aAWC2 3 7 GCiiGAUUU C UUUC'~CCA

2160 AUGUAUUU A UU3~.UUCA 2379 CUG~"UC'W C C'LTCULiGCG

2162 AC.~UUCCU A C~'INGUU 2 3 80 UGAUQL;CU U UCACGnG'J

2163 L~LTULJAW A AUGCAGAG 23 82 AWUCOUU C ACG.GUCA

216 UG'~UGUAU U Ut ULD~AUU2 3 84 UAUCCC-.~"L7 A GACACAAG

216 G~.UGURLZJ U .~UL'''.~AUUC2 3 9 UAn~.UACU A UGL'GC,?,CG
i 9 2170 GUAUUUAU U AAUUCAGA 2401 UGUG..~U"nU A UGGUCCUC

2171 C~G'JCIAW U AUGG:,GUp,2411 CAAUULJCU C AUGCWCA

2173 LGUG."LJAU A UG.~"UC'CUC2417 AUCJ;GGaU A UAC'~p 2174 UCUC'OAUU A CCCCUGCU 2 418 UCAUGCW C AC~GF,ACU

2175 nUWCUUU C :~CC~C~CA 2425 WAUUAAU U CAG~UUC

217 GAAAUGG' U CG~ACCAC 2 42 6 CC ~~"'CT U

218 UG.CAGJU a UUtTaUUC,A2 4 3 UCAG .G'JU C L'G?~G',G'u'U

2165 ACnCUtTAU U U?.L'LCGG'J2434 C ~- -~-'U C ACArACGA

218 Cr.CUCIAW U nL'L -"C~CLTA,2 4 4 UGAAC'-aG~'J p, 6 8 [~CCCCC

2187 AG'JUAUUU A UCiG?.~,C_ 2449 C,'rAGCCUU C CUGCCUCG

2189 UUAUUtJAU U GAGLTACCC2451 Cz,CCUG'JU U CCGC-CCUC

2196 CLC,?.CnGLJ U AUUL~r~UUG2552 GCC'UGL'L'U C CUGCCUC'J

2455 AG;UUC'CLJ A C~ 2761 C~C G

AUCWCC
2459 C'CUC,~CU C CUCC:AC~.2765 "
' CL
2460 CC~CCW U GWCC~'AA 2 7 69 WUC~
U C UG:WCCU

WCUCUaU U ACCCCUGC
2479 U~CnCCO ,~ WACC~~C

279? CGUGAAAU U AUG.;JC?,A
2480 GUC"~CCvU U GUGAUCCC

2803 CUCAUG..."U U C~~C
2483 AC~~7 U CCC'-ALJG'J

~ 2804 UCAL"G..~UU C AC'~GAACp GUU C CC~C 2 813 GCUCCC'~U C CLGACCCU

2492 GACCACCU C CCCACCDA 2815 C~~,7UU C GALTC?~CC

2504 ACCLg,C~1 A CAUUCCUA 2821 CCUGaCCU C

CLGuAGW
2508 AG.I~C~U U CC~CCUU 2822 UAC~T,CW U UC~~uCC
2509 C~UAG,UU C C
TACC~-U

" 2823 C~.AC'~JULJU C AG~~TCCCA
2510 G';CC:~L~GU A G
C~L~J

, 2829 L'CGG,'GCU C AG.;JAL,7CC
2520 ACCGL7L'W U CCCf , 2837 CACAC~.;~~U A CUZ,CCCCC
2521 CC'JUf 'GUU C C

2533 . 2840 G~~,CCCCU C CCAG'GC~

, ACAGGyLI7 U p,CCC~CA 2847 UUACCC'CL7 C

ACCCACCU
2540 UCG.,~~"C~.~p C AG,,,~UAUCC2853 WCGaUCQ U CC~~CC~1G
2545 AG~
-GCp C CG~~(. 2860 CU CCCL'GGAA
2562 UpU
' ~GAGAUp U Ur;WUCAG C
G

, 2579 CCUGC~ U UGCCCUG.; 2877 , ~~ C
U

G.3AGUCU C CCC
2 5 CUGCiIC'GU A GACCUCQC2 g c 85 g GCU C CGC~C~
2588 UGCC'JCCU C CG~CAGCC 2900 GGC

2 5 CUCWCCp C UUGC'-SAG 2 9 04 GAACUG~.~p C UUCCUCW
2593 tlCtTCL~',UQ A CCCCL~CLJ

2905 G:~~~CU U CCWC'UCU
2 5 CUCCUG"'Z7 C CUG
9 6 ~LfiGC

, 2 9 0 G'JUCAUG'J A UUUAUUAA
2601 UGGG'~'LmU A UG 6 ~UCCUC

~ 2907 CL'i~~L7CW C CUCWGCG
2 6 GUCCUG~1 C GCS

2 9 0 UG~,UG~U U ~UVAAUu 2607 GJ ~c~.,~~,~ A BCACC~GG8 .

2909 CAACUGC~7 C WCCUCUU
2608 CL7CL~,C~U C CC'G'UG~
GA

" 2910 ACWCC'W C UCUAUUAC
2 609 IaGuAGACU A ACQGGAUG

2620 DCAGAL~1 C UGAC~
G~7 , AWQAUW A

CQCUCAGp A GUGCUGCp 2913 UGLIG~W C GWCCCAG
2628 LmCAACUt7 U U~~

2 63 UC1~CT~AU C C~

,AU~JCAC 2 915 UAUUUAW A AUUCAGAG
2 64 G~"UCJaC~,~Q A UCCAUCCA.

2916 C~CUUCCU C WGCGAAG
2641 CCCCACCO A CAL~CAUU

2917 CWCCUCU U G~.GAAGAC

2918 AUWCUW C ACG~Cp, 2 6 CCACAC,"~U C AGC

'CT

, 2 919 UUUUGUGU C AGC C~CUG
2 659 .
AGAACG"~U C C'tlC.CAF.
GC

. 2931 GAUG.,~ilGU C CCGCLTGCC
2 6 AC'JF~GG.,~p C C~
8 9 p~

; 2 9 3 Lfi.GAGL7CU C CG~GCACC

UCAGC.'~Cp A pC~CU

2941 CAGL7ACW C CCCCAG;C
2700 AC~CU U CCCCCAGG

2951 ACCAUGCU U CCUCUGI~C
2704 GACCACCCT C CCCAC'WA

2952 CCGGp,CUU U CGAUCWC
2711 CCCL1AC'CZ7 U ' ~.~C~0 ~U
,~,~

, 2 UGH-WCCU C UGACAUGG
2 712 ", 55 A G
~

UG 2 9 5 CUUUCC'LTCT U GAAL7Cpp,U
2721 W,~, 6 C~-~,C~U C AUACGGGU

2961 L'UL1LG'JGU C AGCGa.CGG
2724 AAGAUGa.U A CG:riv"JUG

2744 Guu~UGGAU C CC;~G

2 7 GUCCC'C1GLT U UAA

2 9 69 C~F~,UCAAU A AA GUtWA

2975 L ~' ~U C L'QC~G..~p 2976 L~.L~DG~~U C CGCACCUG
2977 -C~AGCtICU U CAAGCL~?, Table 10: Rat ICA11I HH Ribozyme Sequences nt. R.at EH
Ribozyme SeQuence Pos:.t~on ,1 UG~l~uG'~~GCUCu-UGnG:~cC~'.~GC~.C"wA?, ALZG::AL'C

23 LnGaG:JyGCUG~~JG:~G~CC~-.~?.GG..'~'",~
AAG'.:C~G~.

i6 ~~AA CUGAL',C'~'"'~"-'--~=....~

31 nG~~C,~"-~GCUGAUG~'"C'-An~GvCC.~-~'~'r a.G~C?.C~G~u 34 GL~AL~UCUCUC~L'GF.G:~ ~ _. .,.,"~.A
: LG.,.~'J GCAG

40 ""G~.~CVt7GCLJGhUGA ~ ~. '' "~C"~?.A
ACCJUGAG

48 CCC~C~,CCCUG~L'GhG:~CCG?.AF~G~CC~.:Aa AG.w'UCLC

54 G~CZ3CA~',CJGnLTG'nG~~ .~CG'nr~~LGi,:aCCC,~A
s ACvC

58 "Gw~?~C(1ACLiGAUGnGGCC:~AAGv:.C'~.,a~, . ~. ..

64 AC''.N.~~OUGC'U"'~FJCG~'~AGGCC'~~1 AGCCADL1G

96 AG: ACCAGCUGAI1G~~.C'GAAFC~CCG~A aG.~_~GG

102 G.."GaCCAGCLT~C~~AAGG:.CGhA ACC'~GGAG

108 hG~.~L7CCCC~G:~UGhG~CC~.~AAGGCC~,~A
A ~ ,JCC

115 UG~Ch CL~t,F,IJGAG:"C'".~AAGG.'"C'".-~
AC-.~~~

119 '~ .. ' ~ .r.AAC~G."CGAA ACAG'~'GUC

12 0 G:~~CCGG;~~' :."".~r ~r A~rG'~.G~AC

146 G:3AG'JUCCC'CTts~iUGkG:~CC'C'CGP~ AG~~?C'UGG

152 L'LJC~G~"QGC'JCAL'GAG:~.C~-"~..w~aG:~CCG~-.:e AGCC:r,..~-U

158 UCACLTCGVCiJG~~IJGAG:~CC 'C~GG~~.C'~,,=.,?~
~D

163 ....~... CUC~nDGA.C.,GC~'~aF~AC~G:.C"a.A
G ?C"~'C'v'OC'~

1 68 C~~G:T:.AGCT7C~UGnGGCC -C iAAGC-:.C'"~, A~,G;"~WC

185 CCUGCACG CUGhUG~~CG:~AGGCC'~,.sAA
ADCCACCC

2 09 G~~JG.C~UCLT~UCZF~G:~ 'C"'CC'GAA AG~~CQG

227 UUCACAGU CUGALTG~GC~C'."~T,AGGCC~~A
ACQGG~3C

230 CCQCCCAC CUGA ~ . ~ C:Aa aCAG~,"~GG

237 G:~GGQGUCCUCJ~UGAC~CC"'C~u?A AGC'GpCAG

248 UCC't.TAAGGCL~UGAG~.~'C~FGG CC~~A .
-..... C

253 CCUCCACU C'vG~~UG:~GGC ~l~GGCC~.s:~.
: "-~G.~AGZJG

2 63 G:~U -C. CL'G~:L'GF~G~CC~AnC~C~~ ;A
FGA AUUC:~"UC

267 CGAGwAG C"JGF.UG:~~G:~'~'~~CGAa ~~'WC

293 UCAG~'"ULIGCL'GaUG;yC-:~CCC -G iA A

319 UC.'GUUUGUCUGn " ''~C~CGA.A AUCCUCCG

3 3 5 AGLJCIC"JCACLTGI,UGAG~ C",CC'GAA AG:
AC~GLT

337 G~.ACCA CL~GAUGi~GCCC~~AAGGCC~A AL~.Gt'~.

3 3 8 CtJCAGC'WCUGAUC,AGG.'"C"G~GCCGAA AAG
;GCW

359 AAGCCGAG C'~TGAUGhG~CCG:~'CG~A ACUG."GUG

367 ACG:.-'JUGCUGAUGAGGCCG"~AGGCCCAA i.GCC~,huG

374 i.Gu~L1G:~JC'UG~UGi~C,GCCCi-'v'zF~GGCCGaA
?.G:~:r,"'LTAp, 375 GnGGCAGG _ CUG.~-UGAG:~C -Cr'u'~isGGCCG~A
AGu'~UIjCU

378 UACCCUGU CUC~UG~CG~.isGGCCG~.A AC-GLJC~o.a~U

386 AG'~UCCAACUGAUC~GGCCG:,i~.P..GGCC'"..-.AA
?.C~,C,e Gr~G

394 Cu~UUC~G C~IGnGG~.~CGiu~G:n."CGr~
AG:~CCAC

420 UGCG~."ffGG.'' ' ~ C'GAF~GGCC'~A AC,:~,~7GC

425 G CGG' ' "~CGAAAGGCCGAA AGCC~~GG

427 UCv~DDDUU CQGAUGr'~G~CCC~v~CCG?.A
AAGyGC~~' 450 U ' .AAA AGuLJIJCOt7 451 GCCUGwG CL'GAUGF~C,GCCG.~'1~F'~~CGaA
AAGJACCC

456 UG.~"'u"'C-ACACDGAUGAGGCCGAAAG~..'~GaA
AAGCCGAG

495 UAC~CAGLJ CQGnUGAG~C AUGuv'GC~
.

5.0 UUCC~~1CG CU'~~CG~.F~AGC~CCGr'~A
7~~C~CAC

564 ~-~'~ ' ~"CGa~~~ ACAUUUUC

592 DCCCUG..~-CC~ -'G~G:~CGAAAG:~CC"aAr~r ALQ,CQCCC

607 C~L'G~ CLT~GAGGCC~'~AG~CCGAA ADG'GG..~UC

608 :~~ '' ~ ~"CGAA AAULJG~.V

609 ~AU~ CUG%~G~7C'0=~y~AAGGCCGAA
AAAUt~GC

6.1 UC,FJaGC=.UCLK~UGAGGCCG'nA~~G~~CGAA
A~AUL~G

65o CnDDC~G C~UGAG:~CCGAF~AC~:~CC~A
AG:GUGAC

657 ACAUUCW ~

668 AnGAGC,AA C'CtC~AUGAGGCCGAAAGCCCGAA
AC~AGLTGC

677 DG.~G.."DGGCL1GAUG."~CCGAF,T~GGCCGAA
AGGG.,"UGC

684 AAF~GUCCG CDGhUGnC~naCC'GAAAG~GCCGAA
AGCUGCCU

692 G:~GDQCC CUC~AUGAG:~.~C~ ~ AG"~DCL7G

693 G:y~ACAUC C'CGAUGAGGCt'GF~AAG~.~CGAA
AAAGUCCG
~

696 ~ ~
.~AAAGGCCGAA AAACaGGC

709 ~'~ CDGF~GAG CCCGF,F,AGGCC~.~?.A
AAAUGCVG

720 GAG'"DGAA CL'GAU"~G;~CC~ AGUUGL1AG

723 U ''G~GCLJCflL~AG~~CGAAAC~.~CGAA ~,AAAGWG

i35 C,~~G.CCAGC'LT(:~ADGAGGCC~uAAAGGCCG~
ACCAGGAG

i38 UCCACCCC C'L~CGAAAGG...'"CGAA p,GuCAGGA

i 65 AGJUCUCA ~GCCGAAAG~~CGAFa A,G;~CAGL7 769 DUCCAGGG ~ ~ AC7~.C~AGA

770 CDDCCAGG ~ . > >CCGAA p,ACACAAG

?85 "A~ ~ rICAGGCCU

7 8 G,?.G:,CAGG' ~ ~ ~CGAA Ap~,GGCC

792 G~'".~ACCAG~ ~ CGAA ACCAGGAG

794 -C~"LTOCA CUGAUGAGCCC~CG~A AGGCAGGA

807 UCCAG.~UA CL1GAUGAG~CC'GAAAGC~CCGAA
AUCUGAGC

833 UAGCJCUCC CUGAU"~,AC~GCCGAAAGGCCGA,A
ACCCCAGG

84 CAAUAAAU COGF~UG:~GC~CC~~CGAA hc.'UGUCAG

851 AGCQGCUA CUGAUG~GGCCGAF~AGGCC"~A
AG,~~UGAGC

863 A~ CUG~sIJGAGGCCGT~F~AG~~CGAA
AGCCABLJG

866 UGLJCAGAG C2JGAUGAGGCC~.~F~AGGCCGAA
1?~AGCAIJGG

667 LTAG~'UGGGC'C~C~aIJGAGGC~CGAA p,G"~L7~C

869 CWCGCAA " GT~AAGG.~CGAA AG~~AGp,G

881 CACG:N"W CUGAIJGAGGCCGA~'~AGGCCGAA
AAGCCAUU

885 UL1CACAGU CUGAUGAGGCCGAAA~GGCCGAA
ACWG,~UC

933 ~~ SAC CUGAUCACa~vCCGA.~iP GGCCGRA
AAiJACACA

936 UCACACAA CUGAUGAGGCCGAAAOiCCCGAA
AUCUCUGC

978 AAGDtJGUA CUGAUGAG~CC ~ AUQCQCAA

980 ~F~WG CUGAUCACvCCGAAA,GGCCGAA
AGAUUCUC

0 8 GnG~"GG~P. CCG~DCAG~ _ -"Gy~F~C'~-CC".~1 6 A.GUi.'GUzIG

087 GnGC'vCA Cu'~-C.CGr'~A ~J~G'JL7GL7A

988 G;u~G.."GG CLJG~DC-~GGC~~'GA:~AGGC'C~.:~A
~,.~AG'JUGLT

'! GGGGCCAC CIJGAUG~GG.~CG~~GG.~.CG~1 005 AnG'~CG~

1006 CCDG~' uG.~CLTGAUG~1G.~ '~C~r~GvWCna ACCCC~...r~C

1023 cc~ovc,JCa cvc~cG~~C~,.~a 1025 CC'~'C',JGCOCL~~AUGGG'C " 'G~C"~1 AGaCC?CC

1066 C CUGALG~CG~GCZC~.~ .~.?1~,~1GG

102 LCVG~~cGa C~-~~aG~cc~~cGaa AcCCCCcv 1 OS3 :~G~:~"CG CL7G?. -UCr.GC~ ~ ~-.C-.~??r AL~C

;, aL-cnac~ cCGat~ -~~ ~a:~a~:~CCC~aa ~5 aQUG~;

~ , ~t.." , CLJGADGAG~CC~.-~.~ ~"GLCGC1 53 . G

1 , . .." . ~~ 'CG~ a~G.."C~LC'C

11 G. -G~~C~ CtICACTGAG:~CC.a~G~. ~r'A
66 AG?~

1._172aC~CC',~C.'ACLTC~.LIGnG~.'',CC'G~A~G:~CG?.?~
s~C~aArIAG

12 ~CAG'~ ~UG~-'"~"CG~A AAL7L7GG~,LJ

Ol CCQGUG~ CL;~C~ A ?~J~CCCAA

1203 GACCt ~iG CQGaUGAG:~CCG~i.~'~C,C,CCGIa AG'.AGCCC

X27 aGC_yCr.UG CUGAU -CnG.~cCC~.>~CG~?~
AL~CCC?,A

:~ r.C~~UCAC CQGAUGr GuC C G~C.GC C~~
2 ~G.: C C~.~C

1233 G~.nCC:~G CUGAUGAGGCCGC~.AA ?~~G

1238 G~G:~.C: C'CtG~UGAG~~'AGG:.CG~?~ ~?~
~.

1264 ACCC'"..rT'1UCC~CG:~aGGCC"'.~ AUCLJCDCC

.267 CnL'UC~uUG CDGFatJGaC~CCG?AAGG:.C~.-~.
?.CaGv'G?.G

1294 CL'G~GYC~ CUGAUGC~~~.CGiv~~C,~~~.C~.z?..~
A?.DC'L'CLTs 125 UC~-~~ CtIGaUG~C~C ' 't-GCCGAA AL~CCUCQ

_306 G;~L'G'u;'.AC'C~CC~-.:SAC-GCC".Aa, ~C~."U

1321 L'UtlCCCC~ CU~"C"'aT~AT~GCC~~A AL'UCDG~

334 G~JCQGw CLTG'r~DGe~G: C ~

1344 GC:~.L~.CCTJCDGApC~G~CCG'~F.AG~.~C'G?A
AG~~CCGA

~?5, AGvICCGCQ CDG?.L1G?C~:CCG.'~FGGC'CG~1 ~CUGA

13 C CaUL'G'.JUCUGAUGAG ' ~ C?A AGCt?CCI~

13 C C~JC~F~UGAC,GCCGP~,F~AGGCC'v:aA
6 AGUACCCt7 o 1367 GC'C~JG,~:~:7CUGAUGAG.~~CC~''~,nG;CCGaA
A?.GLTACCC

6 2 G,G;r.GC CUC,~UGAG: CCGF~F~.:C~,CCG~, G:~ ACACC~UC

3 8 ACG~UCCC C'JGADGAGGC ,CC.aAAG.;C CG~A
0 ABC-:SAG

'_388 Cf:UCCAGL7 C~UGT,Gu'CGFi~.?t-CSCCGI~a AG"uCUCCA

13 L'GJCCOGLJ CUGABGAGGCC GT~1AGGCCGaa, S ACAGCCAG

1 sot c~,c.TUCUC cv~UCAGCCC~3~G,.~G~A AAC~C~CAG

408 GaCGCCAC C'OGFUGAGGCC'G~AAGG.~CG~A
?~CACGAA

1410 GJCCACQC CZTtsF~UGAGn~CCGCCGAA AL~'aQJUCG

1421 GCCLG~_-w CUG~ABGA.C.GCC'GAAAGGCCGAA
"~CCC

142= :~CiG~GAG CUGAUGAC~:.CGAA:~GvCCG?A
~.G~'G:~1 1529 CC'~GG:~ CUGAUGaGGC 'CC' ' ~,CCCirl AC.i~F,GUAU

14x4 ct;cczccu CuGavchG,;,~.CC~aA~C;,tcc~
aG,~.CVtscv S. UCCCLiGa~U CUGnUC~G'~CCv~.Ar'.T~.rCCGna ~ AL~GUCCC

1482 COLT~. . C'UG.ADGAGGCC~CGA?, ~.G~ACCCU

1 C G~AP.CAGG CUGAUGAGG.: CG~ARtvCCG~.A
8 A C? C-Ca GU

1493 LTaGUCVCC CUGAUC,~GCiCGAF~C~.-CCC?
CCCC=.GG

1500 BUC~.CCAU
CUGAUC,r.G~~~CC~A
ADQ<TCACG

1503 GJGG'UUGG -CU'CAI1GAGGCC~.~~CC'".~.~A
Ac~WIJtJC

1506 CCAAG~AU CUGAUG~GGCC'GP.A,AGGCCGAA
AUGACCCA

.509 L~G~CAGU CUGnUG?.G:~CC 'G~1GGCCGaA
.3.LT.~uT~ aC

1518 1~ C'JG~aDGaC'~c,CCGAelAGGCCGAA
ACCJ~G~.~AC

1530 ACAAUUAQ CUCF.U~GaC~ -'~AA~C~a~CCGAA
ACCCAG.a"iJ

533 AAGCCCGC CUGaUGF~GGCC'C~A~GGCCG.~A
.3.DGnUCAC', 1551 DACG~ C'C~C~~?.AAG:,CCG~ AGwCCaC

1559 ~CAGG CUG:~UGAG~~CC,AAAG~~CGaA
ACDtJGCCA

1563 ~~ CLlGr'1UGAC,~,C.Cu '~G~~~~.C~~' aG~AG aA

1565 c-:~c~~,~ c~GavGaG;~C~,~aG~zCG~A ~GG~~aA

1567 CL'G:~CG.~~UC'UGAUGAGGCCG~.AAGGCCGA1 A~RGGUGLT

1584 UAL~UCCU CUGAUCAC,GCCGAAF~~ ADCUUCCtJ

1592 ~CWG CUGAUGAG~C'GF~AGGCCGaA AL~UCCC7G

1599 Gv."C'(JCC'U~2CC1GALIGaGC,C ' ~ CG~a h~ACWGCTA

1651 GG,.~QCAGGC'CtC:AUGAGG~. ..

661 A~G~"~GC ~GCCG~F.AAG:,CCG?~A CAG

1663 UC;JCCAULTCUGAL~GAGGCC~ ~ ADCVGUUC

16?8 CC'C~C CUCAUGAGGC ' ~ CG?.A ~JVCUC

1680 GACCOGLJG CVGF~DGAGGCCG~' '~J~~CCGAa -AG~r~.GCCC

16 81 'G~GGCAGG CUGAUC~C~."CG~AAC~CC"~AA
AAC~G~CC

1684 G?~C,AG.~"iJCCUGAUGAGGC~'"AAAGG.."CC,AA
AC~

1690 ~.BGUAUG C'CK~"CC,AAAGGCCGAA AG.~
~~~

1691 G~GnUCG CL~j'C ' ~ AAA AAGtICCGG

~ 696 G~~~~.CCAGCUGAUGF~GGCCGFaAFaG s: CGr~A
AC~...FaGGAG

1698 UCUCCT,GG CDG?.UGAGGCCG~AAAG:~CCGAA
AUAUCUGA

1737 C-:ACCGUG CUCAtTGAGG:.C:GAI',AGGCCGaA
ADGUGAIJC

1750 AALI~G.~"'UGCU~C ' ~~CGAA AAAUGGaC

17 5 AG: ACCAG GAAP.GGCCGAA ~ AG~AGAGG

1787 C'CCAG:,CCCL7GA ~ ~~CCAp, AG~JVCpC

1790 GAGBOGGG C'C~CJ~1GAGGCC'G~AAAGG:.CGAA

1793 GWCAG.~"'UOZn~DGAGGCC'"~AAAGGCCGAA
AG:,ACCAU

1797 UG.~"UDDW C'C~CGAF~F.GGCCGAA p~~

1802 W~"VA CUGF~UCAGGCC'G~F.AGGCCGAA
1?~fiUGAGC

1812 UWCCCCA NGAUGT,GGCC'GJ~GGCCGAR ACUCUGUU

1813 AC~~CAC CUGAUGF~CCGATLAGGCCGAA AAGCCCGC

1825 ~ NG~UG~GGCCC~AGGCCGAA AL'G.~~UGGC

1837 Z7ACCCUGU CUGAL1GAGGCCG~GGCCGAA A:G.,"LJGGG~T

1856 GCAG~'"UCACUG1~UGAGGCCGAAAGGCCGAA ADUAG:~GG

18 61 G:~CCAL~ C'I1G~1GAG~C ~ "AA AG:ACAUG

1865 CUUGLJGDC CUGAUGAC~CGAAAGGCCGAA ACCGGAUA

1868 A~UAV CUGAUGAGGC -C~GAA ACUCGUGA

1877 CCUG~~~ CUGAUGAGGCCGAAFlGCCGAA AGUACUGU

1901 UGUACC'OU CUGAUCF~GGCCG~1AAGGCCGAA
AGUUUUAG

1912 UGUCCAW CUGAUGAGGCCC:~'~AAGGCCGAA
AUCUGUtlC

1922 UAGGG~AU CUGAUGAGGCCG~AAGGCCGAA~ ACVCTACAU

1923 ~ C~1GAUGAGGCC'GAAAGGCCCAA
AGCGUCCA

1928 UCCAG~"C~ CUG:~UGAGGCCCAAAGWC~~A AUCUGAt~C~

21~
930 CAUCCnGU CGG~G~' C~~l~ CC".~A AGUCDCCA

964 GCL~ -Cl:CACC'C1C'~AL' .~CCG?A, AAAIJCUCU
'G.,G~~~CG:~A:,G

1 gg3 CC~'F~GGCCC'CTC~ ~ AG~70COC

o g ~L CL -' .. -'CGAau~l.~..~WCCA

2005 ~~~U ~~~~ C~A AC~CAB

2 013 C=~L'CCCGACU '~"C.2aAaGGCCGap, A

2 015 :~CCi~UCCCCOC,~~~ CCGAA AUAGGCAG

2 02 G'.1AC,AGCV" ~ ACr3CAAUA

2 03 U~ ~-~'O C~ ~~1CCUCCG

2040 ACCUCCnG CG' ' aG.~~UC~

2 0 ? Ci CnDLIGCC -G'~C~G~ ~ a~CCAG

2 0 iT~G~..'~ACGGADGAC'r.~AG~.~CCGaA ALIGuaCGC

2 071 CCUGAGGC CGG~~tK~GGC ~ ACF~.GL~U

2076 Ut~G~"~ C'Cttj~UC,?.GG..~~ ~,G;,~~C~C~

2097 ACAUCAAC Cv'GAI7GA~~CCi?.AAf;GCCGaA Al 2 09 .~CCUCCAG AG~CAG~, 2115 G'~.CCC AC~CGGP,p, 2 3.2 c~U ' ~ C~occCG~AAAG uCCGAA ACAGCACU

2'_ FLAG C UGJ.UC'~GC~CCC,AAAGGCCGAA AAAC'AGGC

214 nG~UCAAC ' ' 2152 :~:~GG'GGUA' AU~7C'QCAA

2'_ CC~A CQ~ ~ AA~3C~ICA

215 ~ ' ' ~ :~~CC~A atTACAi~, 2 ~ cr,AUUAAU aC

21 5 L'Gn~LTL~AC'JC~UG'~G:~
0. CGGCCGAA ?.AP.UACAU

2162 "~ C~UGT~GGC ' AC~ALIaT
' 2 _ cJCLGnAU CU _' ,. ~~CGAP. AALg,AAI7p, 216 RF~UtRF~C~CI?GAOGAGu.. a~CAUCA

21 67 c~.ULIAAU CL7 ' ~ AA C, GCCGA,A
RAI~C~C

2170 BCUGi.AW CUGA ' ~ AA AUAAAL~

2171 UACUCAAU ' ' 2173 C,:,GGACCACQGn ' ~ p 2174 ACCT: G C'OGnBGAGGCC'GP.AAG~~'CGAA 1~

2175 UGACQCGU ' ~

217 GDG~JUGG '' p,Cppp~7pC

21 8 UChAUAAA CUGF.LTG:~C App, 2 ? ACQCAADA CUGAL1GAGG.~CGAA'nC,GC:CGAA

2 ~ c~cJc~AU cJC-s~c~c~CC AA~AACC~

2187 GL7ACUCAA CGGAITGAGGCCGAF.AGGCCGAA AAAI174ACp 2 ? G:~.~"~C CtTGADGAGGCCGAAAGG.~CGAA
2 9 tTC AUA.AAL~p~

2 ?9 cAFsUAAAtJCvGAUGT.GuCCGF~AAGGCCGAA ACQGUCAG

219 UGACCLTCG CUGALrC,AGGCC p~ZJ~JC

219 CL~UG CUC~UGAGGCCGAAA.G~GCCGap.
9 AAGAGpCU

2200 G~.~CLG~,NGC'UGU 'C.;GC~CGAF.AGGCCGAp, ~,A~CCC

2 2 G.a C
O1 C'JGUG CUG:oUGAG~CCGAAAGGCCGAA
AGF,~CCC

2205 C:.G'JGGCUCL7CAIi - r~ "~ ~ Ararnnna C

2210 CAUCCi.Gi1CQGAUGAGG.~.~ CCGpp~ p,Q7CUCCA

2 2 CCChGGCC CUGAUG,AGGCCC p G"~TJpCUC

2224 ~.G''JAG.~,CtJCI,UCAG:r.CGAAAC~cCGAA AUGUAUGL1 2226 UC~?G~:.CQCQC~sUG~G~M ~C~a~A ~TCCaG

2233 A~C~ W~~-~~-CC~ 's~~'GCADGA

2242 ACUACDG.A C'C~u~!'~CCCG~ A~~UGCGn 22 4 G~GACG~G CVGAUCA~CGA.~AG~: C~.~1A aCG'~G~,G

2254 WCF~1G~7 C'JGADG '' _ ~~CGAA AaDL'G~~1U

2259 GCACCGUG CUGnUGAG~~~~.CGAAAG~CCGr~A AI;Gv'G~sDC

2260 , AG=AC~'GLJCDGnL'GaG~,.'"C'~"r~.AACa~~CC'".a~.A
nauG.JG~U

2260 AACUL7GLTACUGAUG'~G~vCCG~AAG~CC~~e1 AZCCL'GaU

2274 UACABG'.JQCI;~~C"'~AAG~CCGAa ?~CC'JG.~(~

2279 aCCCGUAU C'UG~~QG~GC..~C~~l~.:CG~A ALCL'LTCCC

2282 ACUCAA~A C'GC~L'G~G~C ~'" ~~C~.y~r1 :~t~CUGU

2288 CAWG~~nG CUGAUGAGGCC'G~AAGw"CGaA nC~ ~~~

2291 Gi~C~I~G CL1G?S3~C~.~AAG~CCGAA AL~UCG,~

2321 accccuAV cuC,~UC~~C~,~aAAG~C~~~aA ~tc.~~cvC;.

2338 CC'C1GUG~~aACL'GnL1 ~ ~.aAAe~"~._~C"sAA naGCCCA?a 2339 ~ '"' ~G~C~ ~ :~?~GiTACCC

2341 UGAC~CC ~ ~ ~,G~G~CCC

2344 G~GAC~JC CUGADG~~'C'G~AAG~.~CGAA iC".~GC~G

2358 UC-~JC~::AGC~JG~UGhCuCGAAAG ;CCG?.A x .., ....

2359 WCUGtTGG CUG~rUG~~C~~AAAGG~~C~.~AA ALG.?,UGC, .

2360 CWCCAG~ CUG~DGF~.."C"'.:~AAG~CCG?~ aa~lCArlG
.

2376 F,AGAG~~P.ACL1CAL1GAGGCCGAAAC,~CV"a?~A ' ~,~.GUUC

2377 UAAUAGAG ~ aAA ?t~~GUC

2378 UCGLTGAAA CUG:AIJG~G~CC'G~AG~CCCaA ~flL'CAG~.

2379 C". =~GrIGCUG:~DGaGCCCGCGaA ?.?~ACCAG

2 3 ACLJCGUGA CZR~ AC'.,AAUCA

2382 UGACUC3L1 CUG;~~IJ -G'~GGCC~CGAA ~.AGaAaU

2384 CflUGUGUC CDCAUG~G~CCGAAAG~CCGA,A AGr~".~~UA

2399 CGL7CCACA CU~~C~.~AAG~CCGAA AGJAWUA

2 a GAGGACCA cUGa.UGAiGGCC'~;~AAAG~~CCGAA AtIPtt~
o1 2 41 a cvc,~UC~cccaAA~ccc~A a -cs,AA~
~

2417 AACUUGUA CLJGa~DGAGGCCGaAAG;CC'GAA AL2CUGAU

2 418 AGWCUGL7 CVGAflGAG~C~CGAA ~UG?~

2425 GAACUCUG CQGAL1G~~G.~.,~CCG~ A.AG~CCGAA ALZTAAUAA

2 42 UAGUCUCC CUGF.UGAGGCCGA~aAG~CCGP,A ACCCCAG;

2 43 AACQGUCA CUGAUGAG~ CCGT.AAG~CCGAA AAG'i;COC,A

2434 UCGUUIIGU CUGAUGAGGCCGnAAG~CCGAA AUCCCiCCG

2448 ~ ~ CUGAUGT~GGCCCAF~AGGCCGAA ACuG'JtTCA

2449 CGAG~cAiG CCJG~AUGAGGC'C.'CaAAAG~CCGAA A1~G~CZJUC

2451 GAGG:AC~G CUt;~.BGAGGCCGAAAG~,CCG?.F, AACJ~G.~C
~

2452 a~r~c cvc,AUCA~~ccan~c~CC~.~ A,~.ca~C

2455 AACAAAGG CUGAUGAG~.~'C~G~AAG~.~CGrlA ~'~,L'GLJ

2459 UGtiGG~G CL1CAUGnC~,C.CG~TaAC~CCGAA ACv~~Gv~r.~, 2460 UVC~AAC CUGAUG~G~CCGr~G;CC sAA ArtyJAG"
.

2479 CvCG.~~JAACLGAUG?~C~CCG:~.GC-CCGAA AG,WGIJAA

2480 GwAUCAC CUCAUC~G~...~CG~C-GVCG~~ ACW.CGAC

2483 ACA ~~ CUGAUGAGGCCGAAp.C,GCCGAA ACrnAG~~(J

2484 GACAUUCG CUGAUGAG~CC'G~AAAG:~CGAA AAG,A~,C~~-.

2092 UAC-v~L'C,~~',C'UGnUC,r'~ ~C,~~C -Cr~r~'.AC-GCCG'PA
ACvv'Gw,~UC

2 504 '= '' c~'-~n~'.CGaF. F~JG~C~~U

2508 :u C'C~CGAF.AGGCCGAP. AUGt~DGLT

2509 'VAG C~~C AAUGi~UG

2510 AAUAGw'G CDG~~GCCGAAAGC,~~C"u AA
AAADCGaC

2520 AG ~ ACAAAGGU

2 521 GAC~UQCG CU'GAI~GGCCGAAAGGCCGAA
l~,ACAAAGG

2 .3 vcaw:~.~--uc,.-~a~.G~CC~~a~AGG,.~ccaa 3 a~cocv 2 5 G:~UF.CC'C C~CAGCACCGA

2545 A~CCG C2~C~"C"~ AGC'L1GGCU

2 5 CLTGaCAC~ tzJG?~GGv."Ct'y~AAGGCC:
6 ".~P.A aAUCUC~tJG

25:9 C ' ~''~ CU'C~~UGAGGCCG~?~AAGGCC~~AA
ArUG~

2585 GAG~.~''L1CC'DGaDGA~G.~CGaA ACG.AGCAG

2 5 GG.~oGOGG C2tC~AIJGAGG~~C~.~AAAG~."C'GaA
8 l~

2 591 CUL1C~~A ' ~ ~,C~

2 5 AL~.GGuG AADO~GAGA

2 5 G~.CCAG C'C~C~AiJGAGG..~CGAAAGGCCGaA
9 AC~.~GGAG

2 601 G.A~~~ACCA

2 602 ACAnCG~~ CDGAI1GAGGCCGAAAG~CCGaA
h~GGAC

2 607 CCVG.~~UGA CQGF~UGAG~~:.C'GAAAGGCCGAA
ACUCCG''~C

2608 UCCCACGG cJGnDGAGGCCGAAAGGCCGAA
A~.AAG

2609 G~UC'CF~GL1AGL1CDCCA

2620 AF~UGQCA ~ ~ CG~1 AACQC(7GA

2 62 F.GCAGCAC cJGaD GCCGAA ACQGaGAG

2 62 G ' ~~C'GAA AAG~G~

2 63 CUGAAtJDG CDGF~JG' ~ ~,~p, 2640 UGuA~A C~ ACCUGAGC

2641 AhTJG~IJG CUGAUCAG~"~CG.~.A AG.~, ~~

2642 A~ ~ .A AAACAGC,C

2 653 AC~~.ACCCO " ~ A~G

2 659 GCLTOC~C.AGCDGADGaGG(.'C~~AAAGt~CCG:~A
ACCCUUCtJ

2689 AGC'~G

2 691 AGOCCCCQ ~ A~~,C~

2700 CCUGG.~~G ~C~J

2704 L''"G.~~~ CUGnUGAC~ AG~~UGGUC

2711 ACCWCCL1 ' ~v~A~ AGE ....

2712 ChCCOUCC CUGnDGAG~.~'t.'GAAAGuCCGAA
AAG.,,~UAGG

2721 ACCC~'OAU CUGAL'GAGJCCGP.AAGG..~CGAA
AUCUUUCC

2724 CAAACCCG CUGAUC~GGCCGAAAG~CCGAA
AUGAUCW

2744 CCUGC'iCG ~ ~ AUCCACCC

2?50 G~1UWUA ~

2759 CCACUCGA CU'GADGAGGCC'G;AAAGuCCGAA
A~pC~C

2761 G~AGADC CLJGT~IJGAGC,CCGAAAGGCCC,AA
F.AAGUCCG

2765 F~G~CC".~ ~ ~ A

2769 c,C~c~:~,~-acUCa~GaC~ccG~,cccccaa AUAGACAA

2797 UUGACCAU CUGAUGAGCCCGAAAGGCCGaF, AtRJUCACG

2803 GJUCOGUG CUGALT,AG~C~,p, ~U~

2 8 AGWC't7GQ CDCAUGAGGC ~ p ~~J~, 2 813 AG.~_~JCAG CL7GF~ ~ C,GCCGAp, AI:~GGAGC

2 815 GGAAGAUC CUGAUC.nG GCC'GAAAGGCCGAA
~~AGUCCG

2 821 AC:.'UCCAGCQC~.BG~~GGC..~ 'G~,~G~C".~,A
, AG~"UCAGG

2 g22 ~G -,.~~UG?~NUL'GAC~,.~C~'.~Pt~CC~.~,A
A~LTC

2823 U ~.' C~~- '~C:G '~CCCAA AAAAG?L'G

2 829 C:~Q'ACCU CGG~.UGAGGCC~"w~GG.."".~~'il r~GCACCGA

2837 ~,.~'= C~-'~-~'~"~A ACCNGuG

2840 UG:.C~"LJC,~vC'u'GAUGAG:~C.. -.. ..

2847 AG.wGC-:~J~-:~_CG'r.~lsCw~CC~?.A

2 E CTJAGL'CCNC'u'G-'iDGAGGC.. - . ...

2 8 UUC~.~GGG CL~"CG.~CCG?~a ACAC.=.aGa t 872 L~:~1C.~.CCC~'G~GAGCZC~-"~Cv.,."CGr~
ACraC"yCCC

~ 8'77 C~G.'.GG.~C'CG~L'C,nG~...C -\ \G~GGCC,..~
AC,?,CJCCA

2859 AAAGZ7CCG cJC,AL'G~ '~CG'~GGCCGAA Ar~'JGC~T

2 9 ~G ~~-"CG 'ArJ~ AGJCAG:.C

2 9 A~AC-:~A C'uG~LC~G~~C~~GG.-~C~~ '"' 2 9 ~~ CQ~B~"C~~CCzAa nGUG~.C.CC

2 0 C~t~,F~AAAC'C~BGAG~"CGA ' ~ CG?~ ACADU1AC

2 9 CG:3,AGAG C'CG~JGAGG.- ~ A,F~

2908 'IlA C'GGAUG~CCGAAi~G~."CGA~i Ai~C~aUCA

c 9 ~~ C~C~~C: " ~~CC=~ AG~UC

2 910 GL~I~G~ ~ . \ . ..CC,~r1 '~,AG:~AG'J

2 911 GC.~"'CAAUAC'L'GAIJGAG: C'C~C"~AA 1~~AG~AA

2 912 U~~A~ NCAU~G~ = ' ~ ~ A~~C~J

29.3 COCv:AAC CGGAZJG: GC,CCG " ..

~ a Uc~G~vQ cvcavcxC;~C,. ' r.~cr-.~ap, l 4 Atm~at~C

~~15 ~~~~ ~J'C7~\. ' ' 'VC~

2 S NUCGCAA N -CAUGAGGC ' . ' .~.CG

2 S 6v'CWCG~. NG~DGF.C~CCGr':r'al:.GGCC".~AA
17 ~ -~CAC,~~ACa 2518 L'G-'~CUCGUCQGADG~GC~C'G~LAAGG:.CGAA
AAAC',AAAD

2919 CAGL7GG~U CDC~DGAGG...~'C.'GA?.~'.GGCCC7~,A
ACACAAA~A

2 931 GG:AGCG C~CGaAAG~..~CGAA ACACCALTC
;

2 S33 G.~".JGC'L~GGC'CfC~3~DCsAGuCC~' . CGAa AGrIG~CCA

2 9 G~CCUGwG CG ' ~C 'CG ~~C C'GAA AAG~CUG
< 1 2956 GUCAGAGG CU _' ~CCGT~,7~;~C,GCCC~AA
AGCAUCGC

252 -C~.CAUCG C~.T-C~'GA~C~C~.AA AAGUCCGG

2955 CCAUGUCA NG?~UGAGGCCGAT~AC~CC~uAA
AGGI'~AC~

2956 r.UtJG~ULJCNCG~A~GGCCC~ F,AGGP.P.P~.

2 S Cr.GLTC. NCsUGAGG'.CGAAAC~CCGA?. ACAC~IAaA

2 62 CLTC NGADGAG;CC.'C,? A>;Gw~tGAA

2 9 ACUUUAULJ NG~UGA~GCCGF,AAGGCC~GAA ADQCAAJ~G

2966 r'~.G..~UUGr'~r1NGnUGAGGCCGAAAG~..~CGAA ACa~~WC~.

2969 ZTAAAACDU CL3GAUGAGGCCGAAAG:~CC'GAA
AWGAWC

2 9 AG..'ZTtJGaACUGAiJGAGGCC'G~',,AAGGCC'CAA
7 5 AC~~WCCA

2 97 CnC~"UGAG NGUGAGGCC -CAA7~GGCCGAA ACCAUAT1A

2 977 VGGCUUG CL7Cr~UG?.GG~~CGAAAGGCC~~?A
n -GGCU(TC

Table 11: Human LIrS HH Target Sequence at . HH Ta.r~et SeQveace at . BB Target SeQuence Poaitioa Position 8 AUG~CU U UCUUDGC 245 AAGAAAU UUUC_3GG
C

9 UG:aCUU U CUU~,CC 247 GAAAUCTJ 7C~C
W

G~~CL'ULT C UDUGCCA 2 4 8 A~UCUU U CAC-G:~~

~aCWUCU U UGCCAAA 249 AAUCUW C U

13 CUWCUU U GCCJ1AAG 257 AG:~U A G~CAC

36 AG~ACGU U UCAGAt'~C 273 G:zAGAGU AAACL7G'J
C

37 C~ACGUU U CAGAGCC 291 ,~U ,~, ~,T~

3 8 AACC~DU C AGAGCG'~ 3 05 AAAG? CLJ U~1~A
F, 5 6 G:~.L'G.."'U U 'CL1GCAW3 07 AGACi7AU C?,AAAp,C
U

57 GAUG..''W C UGC.AWtJ308 GAS C ~A~

63 UCUG~~U U UGAGUUU 316 AAF,AACU GUCC'Wa U

64 ~ ' W U GAGWUG 319 AACUUW C QUA

69 TJUL7GnGU U UGCUAGC 322 UUGL1C'CU AALTAAAG
U

70 UUGAGiTLJ U GL1~AGCQ323 UGUCCW A F,U'r~
.

7 4 GUUUGCU A GCUCCJlr'G3 2 6 CCUUAAU AY~G?
,~ p~,U

7 8 G~"'~.GCIJ C UUGGAGC3 3 4 AAGAAAU CAUUGAC
A

80 UAG.."UCU U G.~AG~~UG338 AA~U U G~CGGCC

91 G~~UGCCU A CGUGLU1U 380 G~GAGQ ,~, i.ACCAAU

97 u"P.CGUGU A UGCCAUC 388 AACC.AAU CCUAGAC
U

104 AUGCCAU C CCCACAG 389 ACC?AW C 0'UAGACU

116 C~AU U CCCACAA 392 AAWCCU A G,CL,TACC

117 AC~AW C CCACAAG 397 CAW A C

13 0 AGUGCAU U GGUGAAA 4 09 CAAC,AGp L1CWGGL1 U

145 GnGACCU U GGCACBG 410 AA~;A~ U ~

155 CAC'UG~.'"U U UCUACUC4I1 AGAGUW C L~JG..~~UGU

156 ACUGCW U CQACUCA 413 AG~1WCU GGJGVAA
U

157 CUGCUUU C LU~CUCAU 419 UUG,3GU ?.UCAAC.3.
A

159 G:.~1UBL:U A CUCAUCG437 AGOG: AU ALTAGAAA
A

162 UUCLRCU C AUCGAAC 440 G:~UAAU G?.AAG'JU
A

16~ UACJCAU C GAACUCU 447 A~,W U ~~~A

171 UCG :ACU C UGCLK;AU 454 UGAGACU AF,~,-.U
A

179 LT~t7C~D A GCCAAUG 462 F:,~~C'CGGUUGUUGCA
U

192 UC,kGACU C UGAGGAxT S 63 ACUG~~Up CL~G~
U

200 L;~G.~~U U CWGUUC 466 G"~~7W U

201 GAG:~.W C CUGWCC 479 CAAAGAU L'UG"AGG
U

206 WCCUGU U CCUGUAC 480 AAAGF,W UG:=AGG~
U

2 07 UCCUGJU C CUGUAC~. 4 81 F~'aGAUUU Gv:,r~_C,~.,G
U

212 WCCUGU A CAT.1AA.AA 497 AGCAGa,U G'tJACUGC
U

216 UGUACAU A AAAAUCA 498 GGAG~W U UP.CUGCA

222 ~~AAU C ACCAACU 499 GACAUW U ,~~n~

500 ACJ~WOQ A CQG~GO 684 ~GpUU U UCWAUU

531 ~~ C A~UU 685 ACUQC10U U C'CLTAUfJO

538 C~~ U AAU~tJC 686 COUUQUU C UUAUpDA

539 aGGCCQQ A A~ 688 UUUQUC'U U AUWAAC

542 C~LTAAU U UOCAA~r 689 UQtJOCUU A L10C7AACU

543 CUUAADU U UCAAI~D 691 L1UCUCP.U U UAACUpA

544 UUAAUOU U Cp.A~ 692 UCUUAW U r,~p, 545 L~AUWO C AA~UAA 693 ~ CQ~1UU A ACWAAC

549 UWC~AU A L~ADC10A 697 UUtIAaCU U AACAUUC

551 UC~AI1AD A 698 UUAACOU A AC.~1L1L7CU

554 AUAUaaU U UAAC00C 703 UQAACAU O CJGUAAA

555 L'AUAADU U AACQQCA 704 ~1AC~ C UGt~lAp~

556 ?~.AUUU A AC~G 708 AUUC'UC;LT A AAAUC~JC

560 UtT~~ACU U CAGAGGG 715 AAAADGU C UGUpA,AC

61 ~707sA~ C AGAGuGA 719 UGUCDGU U Ap,CU~

573 ~AAGU A AAIDSDDU 720 GGC~U A ACUUAAU

577 AGUAAAU A UQUCAGG 724 GD~ACU U AA~C~, 579 ~VAU U UCJ~'~CA 725 UD~,ACUU A A

580 AAA~DO U ~ 728 ACUOAAU A

581 FAUAUUC1 C AGGCAI~ 731 A U~g,UGA

588 C~.GGCAU A C~CAC 733 AUAGUAU U UAW

597 LiG~C~ U UGCCAGA 734 ,~U U AWAAAU

598 C~CACDO U GCCAGAA 735 AG~UUU A UGAAAL7G
~

611 AF~G~U A ~DQCUw 745 AA.ABGGU U AA~,AUU

6? 6 AITAAAAU 0 C~AAA 746 AAUG.~~W A AGAPUUQ

617 L~AAADQ C UCAAAAD 752 UAAGAAO U DG~A

619 AAAUC'CU U AAAAQAU 753 AAGAAUU U C,~AAQ

620 AADLJCUU A AAA1~DA 757 ALmt~ A AAUOAGU

625 UD~AU A UAUOUCA 761 G.,~CU~Ap,U U AGLmUpU

627 AAF.F~J A UCOt~GA 7 62 GUAAAUU A GUAIJ~

629 A~UAU U UCAGA$l 765 AAUUP.GU A OQQp,UpU

63 0 AUAUADQ U C~.C'~A~1U 7 67 WAGUA1U U UAUQUAA

631 UAUAUW C AGA~C. 768 ~UU U AUUt~IAU

636 DDCJ~GAU A UCAGAAU 769 AGUADUU A UU~,AW

6 3 8 C ;GAUAO C AGAADCA 771 ~UU~p U U

644 UCAGAAU C AUCGAAG 7?2 AUQ~ U AAU~UA

647 GAAUCAD U GAAC'UAD 773 UpUAppO A A

655 G:.AGUAO U UUCC~CC 779 UAAUGGU A OGUUGUG

656 AAGUAW U UCCQCCJ~ 783 GUGUiUGU U GU~UW

657 AGaP~DW U CCOGCAG 788 GUp~U U C~UAA

658 GLD~UUUU C CQCC~GG 789 UU~,~UU C

661 UC1C~CCI7 C CAG~GCAA 791 C~lpCU A p,,, 672 GCAAAAU U GAUAImC 794 U~7~ A ~A~AA

676 AADUGAU A UACOOW 805 G~AAAAU A GACAACU

581 :~~JACU U L1UWC'Op ...

21 fi Table 12: F3uma.n IIrS HH Ribozpme Sequences nt. EB Ribozyme SeQueace position 8 G:.~J.AGr~C'uT~LnAC~CC~~.nr~GG.."~.~~..~
AGvCC3U

9 G~.~AG C'L"GAI~GwC'.AAaGG..~C~.~?.
:AC~r'GC?, o U ~G~:~A cL~L-GnG~~.:~a~.GVC~-"aa Aaac-~c1 12 UL'L'G:~ CL'C,AUG~C:~.AG:~C"'.:AA
.~G~G'J

13 CUUUCvC CUCUG :GG;. C C:Ai~IGC-v.
~.:?.A ' -' '~", 36 G..'"UCUGaCL1G~UC~G.:~"C".~?.AGCZC~~.A
AC.~.JUCLT

37 GGCgCt7G C'LJG:~~v:.C~,~.~?.A~C".~,A
.'-.e~.C~GC

3 8 UG:~~UCU C~UC~ C'Cr':~A A?,ACGLJD

56 AAL7G.CAG CGG ;~~C~..,Ar~AG;~CC~.-.Aa AGC~I7CC

57 AFAUGCA CaGAI~~G~.~C"~?~AGuCCGrA
AAG~UC

63 AAACDCA C'UGALIG~GuCC'-'..~G~C'C?.A
AUC.:AC~

04 G'~T,AC(1CCUGACGJ~.G:~CCC~AG~C~,~A
AAL'GC?.G

69 GGVAGCA CUGnL3~G ;CC~'..~,AGGCCC.A?, 70 nG,~UAGC CUC~,~.C'''~CGAA AACQCaA

74 C~AC~GC CL'G-'~L1GAGGCCGr?.?.Cr""~C~,:AA
e~~?~.aC

78 G..'"UCCAACZJG~-.~,G:~CC~-'~,?.~C~.-~
AGCQAC-C

80 CAGCQCC C'DGnt~,.~C~~.:~~.a AGa,G,_'-tTA

91 ALTACACG CUG:~UGnGuCC~.~C~~C~??~
?L~~,~cAGC

97 GAUGGC~ CUG;,UG~C~_'~.~C~.~,a AG~CGLa 104 C'JG'JGG.;,CUGnUC,~.G:~:.CG:Y~.. -'.?.
A ~~~U

116 UUGUG:r, CUGr.U~~~GGCC -'C~:,C;.~~,A
AUUUCUG

117 CUQCUGG CUG?.UG~,G.~~cCC~p,G,CC~~,Ap, p,AUWCU

13 0 DLJQCACC ' ~.nAC~CCG?.r~ AUG.:ACU

145 CAGL'GCC COGi~LT~C-C,CC::~AG:,CCG?.a AG.~7C'JC

155 -ChC~.RG~ CL1G' '" -~CG~?.AGGCCC~A
AGCAGL~

156 UGAG~AG CGG::~C~CC'G;n:~G;,CC~.3?.?.

157 A~"UA CDG:-.L'G ;~C~C~ ' 159 C~"f,UGAG CQG:~CCCG~A AGAA?,GC

152 GJtJCGAU CVGL"'.J~C~CC -G'~?G:~CC
-C. AA AG'JAC.?~A

16 5 ~'UIJC CL'C; :~C~ C'=' ',l.~CC?
A AUCAGLJA

171 AUC.~.C~CACUGAL~GGCC'=' '.~hAC~CGA_~
AGUUCGA

179 CAUUGGC CG"~,UG~GGCCG~AC,GCC~,,_,?,P, AUG"~GCA

192 AUCCUCA CUGA ' ~ .~ -~,AC',,C-CCG3A
AGUCUC~, 200 GAACAGG CL~C -C ;A,AGGCCG?.A AUCCQCA

201 GG?.ACAG CUGAL~AG:~CC~AAGGCC~ AAUCCUC

206 GUAC:.G~G CUG~UGAG~C -G'~AGC-CC;?.1 J~C

2 07 UGJACAG C'~1G::UGF~C-C,CC~-~GC,CCG?.?
AAG~C"A

212 L'ULZ"'rAUGCClG:yUGACvCCG~GGCC -G'A
AC3G.~~~, 216 UGAUUW CUGf,UGAGGCCGnAaG;CC, ?.?, AUC~LTACA

222 AGJUGGL7 CUGaUGnC.:,cC ~C.~GC,CCG?.?.
AUWIJ~, 245 CCUGhAA CLiG~UCnG~ _ - ' ~~C~.s~
AUUCTCUV

21?
247 vcCcJGA c~vGAGuCcc~~cGaA AG~-wC

248 WCCCUG CUGA 'tlCAGw' CGAAAG:~CCGAA
AAG?.UW

249 :~UCICCCU ,CU'G~GAC~.,"C"~AAG.GCCGaFa AAaGAW

257 GJGUGCC CQG~CGAAF~;CCGAA AWCCCU

273 ACAG~fJ CUGALSGaGGCCG~AAGGCCGaA ACUCL1CC

291 DC~CAG ~~C~ ACCCCCD

3 05 UQUOGAA CUGALT~C~G~".~AAAGGCCGAA AGDCL1L70 307 GJUUUUG CUGAUGAGGCCG~AAnGGCCG~1A AUAGUCU

308 AGJUUUU CUGr~.L7G?.GGCC~CGAA nAUAGVC

316 SAC C~'GCC~~P.AG~CCGAA AGWUW

319 UAW ~~CGaA ACRAGL7fT

322 C'LJtIUAUd CDG:~?.GG..~C"'~AC~vCCGaA
AGGaCAA

323 UCU~U CUC~L~GG.-"CGAAAG~CG~A AAGC~CA

326 ADWCW CDGAUG('': CCGAP.A~~CGAA AtILTAAGa 334 GUGAADG C~L~AGGCCGaAAGGCCGAA AUULJCIJU

3 3 G~ CGQC CT~'"CG~~A?.GuCCGAA AUG~W

3 80 ADt3Gu"W CflGA~AG: ..~~~vGAP~ ACQCOCC

388 GUCCT.GG CDCJ~.T3GAGGCCGAAAC~CCGAA
AUUC,GtJQ

3 89 AC~CL~G CDGAL1G ~CC,AA AADUG~

3 92 GGTAGflC ~AA~CCG?~A AGGAAW

3 97 CUGADG~.-'"CGAAA~CCGAA AGUC'~G

4 09 ACG"-.F.GA Cfl~C~TiDGAGG..~C - . ACUCULTG

410 CACCAAG CDGA ~ ~ AACUCULT

411 ACACCAA C2~C~AGGCCGAAAGGCCGAA AAACfJCU

413 DUACACC CUGADGAGGCCGAAAGGCCGAA P.GAAACO

419 UGJtICAU " ~ C'GAAA~CCG?~A ACACCAA

937 WUCQAU CUGnVGAGGCC~CG?~A AUCCACU

440 AACW~JC CUGAL~AGGCCG AAAG,~~CCCAA
AULD~WC

4 47 ~CUC CU~CGAAAG GCCGAA ACUUUCLT

454 ACCT,GW CUGAUC'~GGCCGAAAGGCCGAA AGUCUCA

4 62 DG:~ACA CDGAL1GAGGC ~ ACCAGt7U

4 63 CC3GCAAC C'CfGO~UGAGGCCGAAAGGCCGAA
AACCAGU

4 66 DGGC'UGC C~IC~ADGAGGC~CG?~A ACAAACC

479 CCUCC~A C'C~COCCGAAAGGCCGAA ADCUUL1G

480 UCNCCA COGAUGAGGv.~CGAAAGGCCGAA AAUCVf~

4 81 C'DCCQCC CDGATJGAGGCC'GAAAGGCCGAA
AAAUCt7U

497 G:~GLTAA CUGA ~ " "~CGAA ADGTJCCCT

498 UG~.~. C'L1GADGAGGCCG~AAAGG~CCGAA
AAUGtJCC

499 CQG:AGU CUGAUGAGGCCG~.AAG.~~CCGAA
AF.AtIGOC

500 A,C(JGCAG CUGAL1GAGGCC'G~AAAGG~CCGAA

531 AAGGCCU ~ ACL7C'~JW

538 GAAAAL1Q C'UGADGAGGCC~~AAACGCC'GAA
AGGCCUG

539 DC-~T~AAAU CUGAL7GAG~CCGAAAGGCCGAA
AAGGCCLT

542 VAUQGAA CL7GF.L;~GF.GGCCG~'~AAGGCCGAA
AWAAGG

543 AUAWCA CLIGAiIGAGGCCGAAAG GCCGAA AAWAAG

544 LTAU~:WG CUGAL'GAG~~CCGAA~.G.;CCGAA

545 UL~t~W CUGAUGAGGCC '"G.~AGC,CCGAA
AAAAUUA

549 LT~aeaAUt~a CtJGAtIGAGGCC'GfiAAGGCCGAA
AUUGAAA

551 GUtRAAU CUGAUGAGGCCGAAAGGCCGAA AVAL1UGA

554 c~GUVA cJe~e~c-~,..~CC,~~G~cCCaA
h~~AU

Uc~:~ACJU c-cGnUGr~.~c;~..~cr~GGCC~aa AAU~A

550' CU~'Q C~U"~~CG~~AGGCCGAA AA.ADt~,U

6 CCCUCL7G CUGAIC'GAAAGGCCG'r.A AG'CLTAAA

561 UCCCUCU CUGAL~G GcC'G~AAAGGCCGArI
AFB.

5 i3 F~ CUOuCC~AAA;C~CCG?.P. ACWQCC

5 i7 CCUGr'~AA CQG?.Z~~F.AGGCCCAP. AUUL'ACLJ

579 UGLCUCA CUGAUGAGC-CCGAA.~vCCG'~A
AUAUUUP, 5 8 AUGC C _' ' . CGAAGC,CC~ A~LJL'U

581 UAUGCCQ G~AGGCCG~A AnAIJALJU

58s cJ~cAG cJ~~a.~;~CCCAAACG~~ A~G;.CCC

~~7 Uc~G:~.~ cUCaCC~,aawCccaA AGcGUC~

5 9 ULJCUGG CUGnUGnGC~CCGAAAC,GCC~.~A
8 C .aGUGCC

611 AGAAUW C'UGAL'GAGGCC~' AUG._"CW

616 UUUL:~.AG CCGnUGAGGCCG~AAGuCCC-~A
AD'LJL~U

617 r~UWLTAA CUGAUGAC~GCCGAAAG:sCCGrIF.
AAUUiICTA

619 ADAUUUU CUGALT~C~CCGAAAGGCCGAP, p,GAAUpLT

620 L~AI~UW C'DGAi7GAG.GCC'GAAAC~CCGaA

625 UG~FF~tJA CffC' ~ ~ C~ Abp, 627 UCUG~A CUGAUGAGGCC'' ~ AL~UQpp 629 L~UCLiCA CUGAUGnC-GCCGAAAG~,:.CC~
A~tJAUU

630 aLTAUCUG C~ 'UC,AGGCC'Gn.AAGGCCCzAA
AAIJADAU

631 GnBAUCLT C~DGAG:~C~"AF,AC~CCC,AA
AAADAUA

63 6 :yUUCQCA CUGAUGAG~~'CC~.F~AGGCCGaA
AUCUGAA

638 UCABQCU CUGaUGAGGCCG~AAG~,CCCAA
AUAUCUG

644 CULTCnAU CUG~CG~~1AGGCC""?,A AUUCUGA

647 y.UACL'UC CLGAUG~aC~,CCGAAF.C'~.~~~.CGAP.
AUGF,UL)C

653 A CUGnUGAL'~CCGAAAG:~CCG~.
ACULJC~F~

555 G;,~.G:~A CUGALTC~G.:~CCGA~AGGCCC~A
AUACUUC

656 UGGAG~A CUGLT~GGCCGAAAGGCCGAA AALTACUU

657 CUGAUGlsGGCCC,AAAGGCC'v,AA
p,AAUAW

658 CCUGGAG CL3GFiD~AGC,CCGAA ~A.~C

661 UL~CCL7G CLIGnUGFaCri,CLI;sAAp.GC~CCGAA
p~,~-.AAA

672 GUALIAUC CUGA '~ GCCGaA AUUUUGC

676 ~ CUGADGAGGCC'C~AAA~CCGAA
AUGAW

678 AAF~:~AAG CUGAUGAG.~,CCGAAAGGCCGAA
AUAUCAA

681 ~A CUGAL7GAGGCCGAAAGGCCGAA
AGUAUAU

682 U''.~AGAAACDGACCT,.F.A(',:~CCGAA
.AAG~LTA

683 F~(~AGr~i~CUGAUGAGCr;.C'C~~GGCCC~AA
AAAGUAU

684 Fv~LRAGA CUGALTGAC-GCCGFap.AGuCCG?.A
AAAAGUA

685 ~AUAAG CUG~L~F.GGCC'C;;4AAGGCCGpp, ~

686 LRAAITAA CUGAUGAGGCCGAA,~~CCGAA
AAAAAAG

688 GUL'A~AU CUGhUGhGGC'CGAAAGGCC~A
~p,~A

6 8 .=sGZJUAAACUGAUGAGGCCC~GGCCGAA p~P.C,F,AAp, 691 L1T~CUUA CUG:~UGAGGCCCAAAGGCCGAA
ALTAAGAA

692 L1~GUU CUGnUGAGGCCGAAAG~,CCG_3,,A
t,pt~.AGA

693 GULTAAGU CUGAUG:~GCCGAAAGGCC:~P.A
AAAUAAG

697 --G:AUGUU CUGJ~UGF,GG~CCGA,AAGGCCG~.A
AGU~,p~

698 ACAAL'GU CUCAUGAGC-CCGAAAGGCCGF~A
AAGUUA.A

703 L'C'JACAG CL1GAUGnGGCCGT,~.AGGCCGAA
AUGvUAA

704 'uG~.C~. CUG~.LRzr':C~:,CC~v'r.FuaC, sCCG~A AADGO(~, 708 ' ' C'" ,~ . ...~,. ..

715 CCiGr3.UGAGuCC~~aAAAGuCCGAA ACAD~D

7.9 ~ CU~~CC~.aFAF~CC~.~AA ACAGACA

720 AUL~AGQ CUGAC~~CGAA AACAGAC

724 UAC~UQ CL'G~C~~AAAG',..~C~~1.A AGCUAAC

725 A~CUAU CUGADGAGGCC'G~AAACC'CGAA AAGQtmA

728 CDGaI2;AGGCC~ ' ~ AUQAAGO

731 UCz~C~CCGaA ACUAUUA

733 L'WG.UA C~i~AG~' ~aAAGG.~C~.~A AID~L7AU

7; 4 ALZVC~.U c.,-~a~cCC~,~AxAGV,.~,~ AAUAC,~

7.5 CAL'UCCA CL'GAD"aAGGCC~.~AAG~CC~AA
aAA~

7s~ A.~CVU cucca~"AA ACCAVw 746 'nAAWCQ CC~Ci~~GAG~~aCCG~P,AAGaC:CGr'~A
AAC"~~

752 UUQAC~A CL'GAUGT,GV...~CGAAAG~,.~C~AA
AWCOiJA

753 AUOt~CC Csr"CCAA~~CCGAA AADUC00 757 ACCAAUQ C~ADGAGG~'"CGAAAGG.."CGAA ACCAAAU

761 A~ C~U~G~"C~A AUDQACC

762 C CZ~~~"CGAAA~~CGAA.AAUOUAC

765 ?~~P. CLT,~,GGCCGAAAG~.~CGAA ACL1AAL1lJ

7 67 L'OAAAISA C'C~C~KAG~CCC~AA~CC~.~AA
A~CC~,A

762 AL1C~U CUG~'~GAG~C~c:CGAAF.GVCCGAA
AAUACUA

769 G~SAA t'DG-'~DGAGGCCGAAA~' C'GAA AAAL~C,D

771 AAC~ C'~,'tGCCGAAAf '"~.~CCG?~A AVAAAUA

772 U1~CAUU C(JGADGaGC'~CCC ~,F,~.AAU

773 ~TAAC~U CL'c~C'"AAA,GGC'CGAp. AAAUAAA

778 ACAACAU C'C1C:AD~GGCC'G~~7~AG~~CCG?~A
ACADCmA

779 CACJ~C'~ CUCA~GGCCGA~''~AGGCC GAA AACADUA

783 AGACAC CCC,P.DGAGGCCGAAAG3CCGAA ACAUAAC

788 ~G " ACAC~AC
.

789 ~ ~...'~CCAA p.ACACAA
~ C~

791 CL~UUDAU CC1GAUGAGG~~CG~J~i~CCGAA AC,AACAC

794 UC70~T CQGAL~GGC'CGAAAC~CCGAA AUU7~GAF, 805 .~C~UGUC CGGABGAGG._~CG~AAAGGCCCAA
AULI~7QpG

Table 13: louse Ilro HH Ribozyme Taraei Sequence at. HB Ta.rQetSeQuence at. 3H Ta:Qet Sequence Position Position 8 c''~'l~CtlUCUUCZG..'~253 AG~,> ;gcJ GaC.~.uAC
C A

11 uCr~LTc~J UG,~ SAA 259 vzSAG~.U C'JGaagA
U a C'~'UcCtJUGCugAAG 269 GaAGI~U C AAPC'JG'J
U

36 G~cacU CAGAGuC 269 GaAGaaU c aAaCugU
U

36 GaAcAcU cAgAG'Jc 269 CAAgaAU c ~.c:7gU
a 37 AAgac;JU AG~C~. 287 a "G.~.,~U CGC~GGp, C A

43 QcaGaGU AUGAgaA 301 AAAugCI1 WC~~AA
c A

58 G,=ALG..~UCDGCAcO 301 AAAugCL7 uOCCaaA
U a 59 GaUGCUU UGCAcUO 303 3UGC1~AU CCaAaAc C a 59 gADGcUU uGcAcUO 303 AucCUAU U Cc~AAC
c 66 CUG:.AcU CAGUgOu 304 , ug-CCTAW cAPAACc U C

82 UcAc,~cU aGcUGUG 315 ~CcUGU C aULTF~UA
c 91 GcUcL7GU uggGCCA 318 cL'GJCaU ?.hL~AG
c . U

112 ucG?.gAU CCCAugA 3 ~ L'G'CiCaUU ALA
U a 113 SG;~cAUCJ CCAugAG 322 CaUL~ ~U AAGAAAU
C a 141 G:~ACCU GaCACaG 330 =.nGAAAU CALZGC
U A

141 GAcACcU GaCAcAg 334 ~UACAU U GACcGCC
U
-158 cUCc~ C AcC~C 334 ~~.UaCaU CACcgCC
a 167 cCGAgCU L'Gut7GAc384 AgcCAgU U CCUcGau C

196 UGAGGcU CCQC~JcC 385 SSCASUU C CUgGAuU
U

197 GAGGcUU CUGUcCC ? 93 C'JeGAuU CCQG:~A
C A

197 gAC~U c CUGuCcC 405 C~,AGaGU cCUL7G,--U
U

202 L'UCCtti~JCCUacuC 406 :,r.~GAGW
c c 202 UUCC'GGL1 CcUAcuc 409 ~~L~ilcCU G,~JGUgA
c U

206 UG'JCccU cuCaUAA. 481 LtczCAAU UAAgUUA
a a 212 UACUCAU aAAaUCa 4 82 c.~cAAW U Ar.SUUaA
a 212 Uac~CAU AAAAUCA 483 :,cAAUW ~, AgUUaP.a A

218 UazxzaU aCcAG~.~U483 =.cF.~.VuU aGL'IJAaa c a 218 ~AAU C ACCJygCC1495 '.~UUgU c AAc~gAU
-218 uAAAnAU acC~aCL1 :53 G.."'UGuuU CaUuUAU
c c 232 uaUGCAU CvaGAAA 557 UuUcCAU U UauaDUU
U

241 cAGAAAU UUUCAGG 564 UUauAuU a aUgUCCU
C

241 gAcAa.AU UUucAGG 564 ULauaUU a AugUcCLT
c 241 cacA.'-,?,UUWCAGG 565 uaUAUL'U ucUCCuG
c a 241 gAcr~AU UUUCAGg 565 L?UrluUt~ USUCcUg c a 243 g~>;ucU UCAGSGg 569 UUuAUGU c cUGUaGU
U

243 -G'~,AUCU UCAC~G~g 569 LL'L?~UCU cUGUagU
U c 24.4 AAAUCUU C1~GGGgc 613 AFAGuGU a uaaCCUU
U

245 AAUCUUU AGGG9cU 614 AAgUGuU a a_aCcUW
C

620 UUAACcU a uUuG~U 1407 cC AgUW ,~ CJcCAGg 793 caAGgCIT a UGuGcALT 1407 c~~ a ~C

816 CUGagW n UACQCcc 1410 gUWaCi7 C CAG ;aAp, 818 G~guUAU a cUCCcuC 1434 AUgCUpO U aUuUaAU

825 ACUcCcU c CccC'QCA 1434 aUgcUuD U AUUIJAAu 825 aCDccCL1 c CcCcUCa 1434 aUgcsW a AuUUAAU

839 AuCc,~cU U cGU~ 1435 L

~ a UuUaAW
840 uCcucW c GWGC~IU 1435 ugcUUW a uOt~aW
863 c~St~D U cC 1438 ' UuUUAL
U U AAuUcug 864 AAgQAW c C~G:~g 1438 uUUUAW U AAUucU

g 864 :~Gi~UD c caggCug 1439 ULZJAUW ~, AUucUgU

913 gAaCUCU U G.~~cCaG 1443 L'WaAuU c UGuaAGa 917 UcLiuggU c CAGAuGG 1447 AWCUGL? A AgAUGUu 957 WagcAU c COUUcUc 1458 ugWcaU a UV'r~UUUp, 960 GCAuccU a UcUc~A 1458 ugUUcn,V A uUAUVUA

960 GcaUcCU a uCUCcDa 1460 ~~ a A~ug 962 AUctvuO c UCcUaGC 1461 UcAUAuU A UDL~UGP, 975 gcccCQU a AgAI~gA 1463 ~,~~Q U ~~

g 987 aGaUGAU A cuuF,AUG 1475 AuGgAW

c aGUAAgU
990 UGAuACU a AAugacU 1479 AWcaGU A AgL'tJAsU
1000 UCACVCU c UugCuGA 1 483 as ~~UW

1027 CcgaGCU U cCUgCUC 1483 GLTAAgp U AaUAUW
z 1034 UCCUGcU C CUaUcuA 1484 GUAAgW A aUADWA

1037 UgcLTCcU A UcUAACIT 1487 agUC~AU a W
' A
UA

1039 cOccuAU c LRACDUC 1487 u u ~.~aU A ~Wa 1039 cUCcL~U c UAACppc 1489 UU'~.,AUaU U uAuVAc~

1041 CcUAUcU A ACWcAa 1489 W~U a ~Wa~

1051 UUcAAuU U AAuAccC 1489 UUAaUAU U U'r,WacA

1148 uCAcUW a cUuaUGU 1490 L~~UeW a ?alt~cpc 1213 GCUoGaU a UUGGAaa 1490 UAzUAW U AUuAcAc -213 ocUG.;AU a uUgGAAA 1490 UAaUAW U AUUacAc 1214 c~aGT,Dp U UG.;~an, 1491 AAUAUUp a uuaCAcg 1215 ugGAUUU U GCAaa.p,G 1491 AAITAUuU a UuAcAcg 1234 gGuAC~U c UccuUGC 1491 AaL~ A U~cG

1236 GACAUcU c cuUGCAG 1491 AaUAUW A U'ilacAcG

1275 ugC~,CCU U AcWcUC 1494 AUuUAW

a CAc LmU
1276 gG ;;.CW A cUUcUCc 1502 g cACGtIaU A UaauAUu 1280 CUUAcW c UCcgUgU 1502 cAcgUAU a VA.AUaW
1298 UgAACUU a AGAaGc~

, 1507 AUAUAaU a WcUaaU

g cAAAGL1 a aAuACcp,15 09 ALTAAuAU U CUaAuAA

1310 GC~AgU a aAUAcca 1509 aUaaUaU U CUAAVAA

,?10 GcaAAgU a AAL~ccA 1510 UAAuAW C UaAuAAa __50 AAAGCAU A AAAUggU 1510 UAAuAW C UaauAAA
' _358 AAAUG.,~V U ggCAugU 1510 tT~:AuAuU

c UaaUAAA
370 UgUuaW C AC-gUAUC 1510 UaaUaW C UAAUAAA
1375 WCAGgU A UCAGggU ~ 1512 aUaWCU A AUAAAgC
1377 CAGc~UAU C AGggUCA 1515 WCUAAU A ,~pg~gA
1383 UCAGggU C AcUGgAG

1405 cccCAgU U DACIJcCA

m c~

c~
m v c m N

O
N .G

~i O R'"

wi y x c ..

c:

...

N

. 0 GL

U

C~

V

d m r C m v N

O

a a arc ._ ,e~ o ~w~~i ~B'c-ll~~e~-I~N~'p'7~~~~O~f ~l ~

~ m r r-i r1 ri v-1 W

~~~~~&FE~~~~~~~~~~~
~ a a ~

B

o ~~~ ~ ~
~~~~a5s ~
~ ~
~~~
~
~~

~g F
gg ~
g g ~
g q ~
kk '2YZkYi'e~Y'2Y~'Z'dk'2'2Y'd'd ' ~' ~

, , e5~F9~'G~R~'sR~~w2aa.:

ro E~

Table 17 Mouse re! A HH Target sequence nt. Position HH Target Sequence nt. Position HH Target Sequence 1g AAUGG~.~U a caCaGgA 467 cCAG~."LT_ c cur~.uUCg 22 aGCOCcU a cG'J~GUG 469 ~ 3aGCc.~4LT a AGcCr4Gv 26 CcUCcaU a GcGgACa 473 UuOgAGU C ?~GauCAy ~3 CAuCt7G0 U uCCCCGC 481 ?~ ~z~U C C.'~G1CCA

94 AuCIIGt3Q a CCCC~1 503 AaCCCCU v? uCAcGC?CT

100 UuCCCCU C AUCUGuC 502 ?.ACC....,, a C.~Utfi 103 CCCOCAU C UUuCCcs 508 ULC~rcGLT U CCL'~.UAG

105 C'JCAUCU U uCCcuCA 509 uC.~cGUQ C CUAUAGA

106 OCAUCW a CCcuC'-~G 5~? c~LCC'J A UACAgGA

129 C.~'1iU C UGGgCCIi 5:.4 UC,~CCOAtT A G

138 C,GgCCI~U A L'G7GV?l 534 fz~CU A uG~UG

148 UGGAC~AtJ C AUcGAtC 556 L'G..~GcCQ C UG."Of3CC

151 AGAUCAU c GAaCAGC 561 CrCOGCU U CCAGGUG

180 ,ADGCGaO U CCGC~Au 562 UCUG4"W C C~~GGA

181 UGCGaW C CGC'UAuA 585 aPr.,C~~a,U a AGcCAGc vUCCG,.~U a uAAavG,~. ~~e G~Ccccu c cLCCUGa 204 G GG..~G~.~U C aGC'G:~613 CcCCUG? C CUcsCaC

217 G~~i~liAU a CCuC-GCG 610' CL~GUCCQ c uCaCAUC

239 C~GAU A CC~CAA 617 SucCCUU C CUC~gC~' 262 CCACCAU C ~UG1 62J CCULCC'fJ C ~rC'Caug 268 UC~AGAU C ~.A~ 623 UCCUgeU e: CC.AL'CfJe 276 ~AL'C~CIJ A CACAGGA 628 ALiCCgAU a UL'~JGAuA

301 VuCGaAU C UC ,. ~ 630 CCSAUuU U i.'GAuAP.c 303 CGaAI3CU C CCUGGQC 631 C~AUuL'LT U Cr~uAAcC

310 CCCOGV~LT C ACCAAGG 638 UGgCcAU a GvGI~uCC

323 GGcCCC(J C CUCcuga 661 CCG?1~C:LJ C A~UCU

326 uCCaCCD C ACC~~GCC 607 UCaAGAU C L'GCCGaG

335 CCGGCCU C AuCCaCA 687 CGgAACU C UG~gAGC

349 AuGAaC~T U GUS~A - 700 G.~UGCCL7 C G.~~v'G.7GG

3 52 AGaUcaU c Ga.A~cAGc 715 AUCAGAU C WCIiUgC

375 CAUG~~J. a C'UAZJG~"~G717 GAC?wcC1 U cwgctTG

376 AUGGucU C UccGgaG 718 AGUCUU C uUSCUGU

378 G ~CL7aCD A UGAGG..'U 721 UucUCCLJ c CauUGcG

391 CDGAcCU C UG.~CCaG 751 AaGACAU U G?~C~~UGU

409 GCaGuAU C CAuAGcU 759 GAG.,~~UGU A UL'UCACG

416 CCgCJ~Gt7 a UCCAuAQ 7 61 G~GUAU U UCAC~wG

417 CAuAGcU U CCAGAA~C 7 62 GUGUAI7Q O CACGGG?, 418 AuAGcUU C CAG?~ACC 763 UG'JAVW C AC~~~

433 LT"~,gAU C CAGUGUG 792 ~ CGAGGC~T C CZ'UUUCu 795 G~.,.~UCCU U UUCuCAA 3167 GAUGAGU U UuCCcCC

796 G~"'JCCt7U U UCVCA1G 1168 ?.UGAC,ZJLT U v:CCcCCA

797 CUCCUW U CuCAAGC 1169 L'C~GLJW a CCcCCAU

798 UCCUUW C uCAAGC~T ,' ~ - - AUGcUGU U aCCaUCa 829 UG: CCAU U GUGLJUCC 1183 UGcL)GUp a CCaUCaG

834 AUUGiIGU U CCGGAL1~ 1184 G
;

. CUcCUGa 835 L'UG~L1LT C C'~,~,C~C1187 ccccU C

GVccCIiU CUcaGCc 845 G c ~CCU C CgUACGC 1188 UUaCCaU C aG;"C~G

849 CCUCCgU A CGCcG?,C 1198 G:,gA~U a A~~Ga 872 cCnG:~~tT C CUGUuCG 1209 CAGcCCU

a caCCUUc 8R3 UuCGaGU C UCCAUGC 1215 cuGGCCU U aGCaCCG
885 C~",zGUCLJ C CAUGCAG

1229 GG1~CC'CLT CCvcAGc -9D5 GC:NCCU U CliGAuCG a 1237 CCCAgcU C CUG~.CCC
906 ' CG:~ 1250 CCAGcCU C CAGgL~C
~ C uGAuCGc 919 GcG:~GC'Q C AC~GC 12 68 CCCaGCQ C

CuGCCcc 936 AUC~gU U CC~C 1279 ~

CC~UG" cL~uCc,1 937 UGLAcUU C CnC~ Q c l . 1281 gOGGgcU C AGCUgcG

-UUCCAGU A CuDGC~
A

,,. 1286 AUgAGuU a UccCCC_8 053 GCCl~c~lU c CAc~
GA

u 1309 Cud a CgA~C~
962 AGAuGAU C GcCACCG

1315 cCCCAGU a CUAaCCC
965 CagUacU a cCCaG?
c . 1318 CAGUuCU A aCCCCgG
973 ACCG;~U U GAaGAGA ~

1331 gGG~CW C CcCAG~C
986 GAr~ACcU a cAAGa 13 u g 34 6 ClzuUuCLJ AaGCUGa C

AGGACcU A DGAC~liCC 13 89 A~ C

1005 9~GCC
~W U 14 ~

U UGAUGcU
1006 AGACCUO C AAGAGuA

1414 UGCJ,GUp ~U~UG
1015 AGAG~AU C AtKAACp U

, 1437 ~W U

,AGACU C CUW~

1031 -CAGUCCU U BCAauGG 1467 ~a~ O

1 032 AG'UCCUU U CAeu~A 14 68 C~CAGAC
' 9aG ACAGACC
1033 GUCCUW C AauGGAC UGUU C

1482 CUGGCAU C uGUgGAC
1058 CCGGCC'U C CAaCcCG

1486 CuUCgGU a GggAACU
1064 UaCACCU a GAucCAa 1494 GaC~IA~ C aGAGUUU
~U U ~C

1082 ? 500 UCaGAGU U UG'~GCAG
UGUGCCU a CCCGaAa 1083 1501 CaGAGUU U CAGCAGC
aaGCCUU C CCGa A;Gi1 . 1502 aGAGUUU C AGCAGCU
1092 CGzAaCU C AaCpt -CU

, 1625 9GuGCAU c C
1097 CUCAaCU U CUGUCCC CUGUGu 1566 ADG: A(v CCCUGpa 1098 UCAaCUU C UGUCCCC A

1577 LA UA~~

1.579 AaGCUAU A ACUCGCC
1125 CAGCCCU A caCCUtlc 1583 UAL~p,W C ~CUgGU
1127 GCCaUAU a gCCUL~
C

r 1588 CUC17CCU GaGAggG
1131 cAUCCCU c agCacCA A

1622 CCCAGCU C CUGCcCC
1132 AcaCCUU c cCzgCAU

1628 UCCZ.1GCU CggUa~
1133 UCCaUcU c CagCVUC a 1648 ~~ a CC~UG
~U a AgCgCgc 1660 cUGaCCU C ugccCAG
cCaQCAU C CCUcAGC

1663 cuCUgCU U cCAGGuG
1153 GCACCAU C AACUuUG

1664 uCUgtW c CAGGuGA
1158 AUCAACU a U'GAUGAG

1665 ' CUCgcUU cG:,AGgU
1680 GAAGACU U CUCCUCC a 1683 C~CUUCU C CUCCAUU

'_690 CCUCCAU U GCGGACA

1704 Avc;~cU U Cu~sc..w l7os vc,;~acw C vc~~,.-~C

1707 GACiIUCU C uG~'vCQu 1721 uuUGAGU C AG~UC4G

172 GUCAC,AU C AG~~UCCU

17 31 AUC''~G..~LT C COAAGGt~

17 3 AG..'ZJCCa a AGv"vGcU

1754 CaGIigCU C CCaAGaG

T able 18 Human rel A HH Target Sequences nt. Pcsition HH Target Sequence nt. Position HH Target Sequence 19 AAUGG.~U GUC'UGL~.4 67 GC~C~'v A
C QC~GUCA

22 G~~UCGQ C UGH 4 69 AGGC'GAU AGUG1GC
C

26 CGJCOGU A GUGCACG 473 u'~UCaGJ
C AGC"'.~~U

93 C'~ACLJGV CCCCCL1C 481 AG..~GC~U CAG?.C'~
U C

94 AnCUGW C CCCCUC~1 501 ?.~1CCC:.v CC~Lr'U
J

100 UCCCCCLT AUCUUCC 502 ACCC'C'uv CAAGUGC
C C

103 CCCUCAU C WC'CCGG 508 CCCA?G'-~7 CCLTAL~G
J

105 CUCnIICU CC..~A 509 CC'~Gvv C CUALTACA
U

106 UCAUCW C CC".~C~G 5.2 AGUL'C~J LaG~Ga ?~

129 CAC,GCCU UGGCCCC 514 QGCCGAU A
C

13 GGCCCCD A rJGLIGVAG534 G.~.~C~J CGP.CCL'G

148 UGGAGALJ AULJGAGC 556 T;GCG:~J UGC-JUCC
C C

51 AGADCAU O GAGCAGC 561 C'JCVG'~U C~,.~.G.w~~G
v?

18o A~G~~ccU ccccQAC 562 UcUCCtv c caGGacA
U

181 DGCG~"W C CGC'~CA 585 G~.CC~,,~U AGE
C

186 UUCCG.~U C?~GUGC 598 G.:~C'CCU CG:.~GC
A C

204 GC~"CCtT CGCGGGC 613 C 1CLGLT CQL7CCUC
C C

217 GCAGCAU C CCAC~CG 616 CLGG'Cw U C~C~.UC

239 CBCAGAU A CC?.CCAA 617 UGVJCCL'U C'JC.'~1UCC
C

262 CCACCAU C AAGAUCA ~ 620 CCOUCCU C AUCCC~U

268 UCAAGAU C AAL7G~GC'U623 UC~,TCAU CC~UC'JU
C

276 AF.UG.~~ CACAGGA 628 ALCCCAU C L'tJL'C?.C~
A

3Ol UGCGCAU C UCCCQGG 630 CCCAUCU U UGA~?U

3 03 C'',~CAt7CU CCUGGUC 631 CC =UCL'U C~.C~AUC
C U

310 CCCL'G~'"U ACCAAG 638 UC~CT,AU G'JGCCCC
C ; C

323 G.ACCCLT CUCACCG 661 CC"AGCV C AAGaUCU
C

3 2 CCCZTCCLJ ACCGGCC o' 67 UCaAGAU C UGCCG?.G

335 CC".~CCU ACCCCCA 687 CG~ALV C L'GGC~~C
C

3 4 ACGnGCU U GUAGC'~A.A7 0 0 C-~~Z7G.

352 AGCUUGU A GGAAAGG 775 2,UC?.GAU WCCUAC
C

375 G~DGG.."U CUAUGAG 717 C?G~,UCU CC'LTACL'G
U U

376 AUG:~..~W UAUGAGG 718 ~UCUU C CU'r,CL'GU
C

378 Gu<WCLT A UGAGGCU 721 UCL'C7CCU CUGZ7GL~
A

3 91 CUG~GCV C UGC'CC'GG? 51 r.G~CAU U G:~GGUG'J

409 G'''"JGCAU CACAGUU 759 GnG.;JGU L'WCACG
C A

416 CCACAGU U UCG~GAA 7 61 G.,~LTG~U U~~, Q

417 CACAGW U CCAC~AAC 7 62 G'JGUAW U C~CGGCA

418 ACAGQUU C CAGAACC 763 UGUAUW C ACC-:~G?C

433 UGG:~AU C C:T~TGUG 792 CC CL'UWCG

7 9 GGCUCCU U UUCGCAA 1167 ~ C? LG? L'CCC1~1CC
GLi U

796 G.:JCCW U UCGCAAG 1168 AUG?GW U CC~~,CC?

797 CUCCUUU U CG:~AGC 1169 UG.G'JUU CCACCAU
C

798 UCCUUUU C ~ 1182 AUCv'vG'J UCUVC1C'J
U

829 UG:~CCAU GUGUUCC 1183 UG.a~v'GUU CCUUCL'G
U U

834 AUUGUGU U CCG:zACC 1184 G"~Z,-C;~ ~-U~,.C~
C

835 UUGUGJU C CGGACCC 1187 G'JUUCCU U CUG~~C~

845 --CACCCCU C CCUAC~vC 1188 UUUCCUL1 C UG~G

849 CC'UCCCU A CG:AGAC 1198 GGC~U C AGCCAGG

872 GCAGG.."U C C~1GCG 1209 CAGGCCU C G~CC1UG

883 UGCGUGU C UCCAUGC 1215 UCG~CU U GGCCCCG

ses c~uc,~cu c cz~G:~ 1229 cGCCCCV c cccAACU

905 GCGGCCU U CCGACCG 1237 CCG'-.AGU C CUGCCCC

906 CG:~"CW C CGACCG a 1250 CC~CGCU C C?.GCCCC

919 GGG~"Q C AGLJGAGC X68 CCCUG..~'J C CAG.~,CAU

936 aUG:~IU U CCAGUAC 12?9 CC~' U A UCAGC(JC

937 UG~AUU C CAGUACC 1281 AL'G;JAU C AG~.~UCL.'G

942 UUCCAGU A C~CA 1286 AUC~ICn.~LT C UGC.-CCCA

9=_3 GCCAC"s?.U A CAG~CGar1309 CCCCUGU C CCAGL7CC

962 ACACGAU C GUCACCG 1315 UCCCAC~7 C CUAG.~_CC

965 CG?.UCG~? C ACCG ;AU 1318 CAGJCCQ r1 GCCCCAG

973 ACCGGAU U GaG GAGA 1331 AG:~..~CCU C CLTCAG

9 8 GAAACGU A AAAGGAC 13 3 4 CCCUCCLT C AGG C'tlGU

996 AC~ACAU A UGAGACC 1389 ACG~~UGp C AGAGGCC

1005 CAGACCQ U CAAt'~AGC 1413 CT ~ , T U UGaUGAU

10 nG~CC UU C AAGAGCA 1414 L1G~W p G~UG

1015 A -CAGC.3.U C AUGAAGA1437 ~~~~W U ~~~"C

1028 -GAAGAGCT C CUOUCAG 1441 CCUUGv.~U U ~C,~CAACA

1031 CAGUCCU U UCAG~"G; 1467 GCVGUGZ7 U CACAGAC

1032 AGUCCUtJ U Cr'~GGA 1468 CQGUGUU C ACAGACC

1033 G'JCCUUU C AG~."CGAC 1482 CUGGCaLT C CGUC~"~,C

'! UCCACCU C GACGCAU 1494 . GACAACU C C~~,GU<Jt7 1072 GAC~~~U U GGUGi7GC 1500 UCCGAGU U UCAGCAG

1082 UGL'GCCU U CCCGCAG 1501 CCGAGUU U G~G~~GC

1083 G'v'GCCULI C CC'GCAGC1502 CGAGUW C AG~?,G.~U

1092 CG:J~~CV C AGCL'QC'Q 1525 AGGCCP,U A CCQG'.IG

1098 UCAC~C'L7U C UGUCCCC 1577 DGAGGCC A L~.ACL7CG

1102 cuUCVrw c ccc~A~c 1579 AcccvAU A Acuc~"cc ,125 CAGCCCU A UCCCUUU 1583 UAUAACU C GCC'UAGU

1127 G~.CCUAU C CCDUUAC 1588 CUCGCCU A GUGACAG

1131 UAUCCCU U UACGUCA 1622 CCCAG.~U C CUG~.~UCC

1132 AUCCCW U ACGUCAU 1628 UCCUGCU C CAC'UGGG

1133 UCCCWU A CGUCAUC 1648 ~~~V C CCCAAJG

1137 UQCTACGU C AUCCCQG 1660 AUG: CCU C CWUCAG

114 ACGUCAU C CCUGAGC 16 63 GCCUCCU U UC~.G
0 ;AG

1153 GCACCAU C AACC~DG 1664 CCUCCULT U CAGuAGA

1 680 C~.ACACU U CUCCL1CC

1683 C~-'~CWCU C CUCCAUU

1690 CCUCCAU U GCG~ACA

1705 L'G:~CUQ C UCAC-CCC

17 GACWCL7 C AC.C,'CCVG

1721 GCQGAGU C ~CAG

172 GUCAG~IT C nG~."flCCL7 1731 ADCAGv."Q C CU~'~GGG

1734 AC'aCUCCU A r~Gw:~~LT

17 CL1G..~CC~ C CCCAGAG

Table 19 Mouse rel A HH Ribozyme Seauences nt. HH Ribozyme Sequence Sequence 1,9 UCCDGUG CffGAUGAGCZC"~AAGVCCG?,A AGCCAW

22 CACCACG C'UGAUGAGGCC'GAAAGGCCGAA aGGAG.~U

26 UGUCCGC C'JGAUGAGGCCGAAAGGCCGAA AUG~~GG
.

93 GAG~GA CLrcaAUGAGGCC:~.~AGGCCGaA aG'~GALJC

94 C~GAG~CUA~AGGCCGAA aACAGAU

100 caAACAV cvG~UC~c; :.C:~AAC~cccaa a ~~~

1 p3 aG:~AA CUGAUGaGGCC~.~AAiAGGCCGAA AVGAGGG

105 LIG.~.GC~GA CUGAUG?.G: CCGAAAGSaCC".~AA
aGAUG'aG

106 CUGAGGG CL1GAL~' G~"CGA'rIAC~~,CC~.oAFa ~.AGAUG?~

129 AGGCCCA CUGADGA~CG CCG.~'~AAGGCCGAA
AAGCCL~G

138 CUCCAC~1 CUGAtJGAGGC.. , ~~CG~A AAGGCCC

148 GWC"uAU CUGAVGr'aGGCCG~AGGCCGAe~a AUCUCCA

.51 GCUGUOC CUGAWAG~CGAAAGGCCGAA AVGAUCLT

180 A~GG CDGAUGAGGCC'GAAAGGCCGAA aVCGCAU

181 TJAtTAGCG CUGAUGAC-GCCGAAAGGCCGAA AAUCGCA

186 GCAVUQA CUGAUGAG:CC~.~AAAGGCCGAA AGC~GAA

204 G.~CCGCIJ CUGAUGAGGCCG AGCGCCC

239 U~~GG CL~CG~UGAGGCC'G~AGGCCGAA AVCUGUG

268 P.GCCAUU CUGAUGAGG.~_CGAAA,C~CCGAA
AUCWGA

276 UCCL~GUG CLJG~UGAGGCCGAAAGGCCGAA AGCCAW

301 CCAGwA CQCALTAGGCCGAAAGGCCCAA AUC1CGAA

3 03 c~a.CCAGG CUGAUGAGGCCGAAAC~,CCGAA AGAWCG

310 CCWG~'"IJ CQGAU ~CCGAA ACCAG~G

323 U '' ~ CUGAUGAGGCCGAAAGGCCGAA A ~~
C

326 G GCCG.~~U CUGAVC~GGCCG?.Ap.C,GCCGAA
AGC~JGC",le, 335 VGL1G:~AV CUGAVGAGGCCG~?,~IAGGCCGAA
AGGCCGG

349 UCCCCAC CUGAL1GAGGC'C:cs~,F.GGCCGAA
AGWCAU

352 GVUGUUC CUGALTGAGGCCGAAAGGCCGaA AUGAUCU

375 cucavAG cucAUCAGCCCGaAACGCCCAA AGCCAVc 376 CUCCGGA CL1GAUGAGGCCG~F~AAGC~CCGAA
AGACCAU

391 CUG~~: A CUGAiTGAGGCCGAAAGGCCGAA AG.~"UCAG

409 AGCQAUG CUGAUGAGGCCG~AAAGGv,~CGAA AUACUGC

416 CL'AUG~A CUGAU GGCCGAA ACUGCG~v 417 GUUCUGG CUGAUGAGG~CG~AAC~GCCGAA AGCUAi~G

418 GGUQCUG CUGAUGAGGCCGAAAGGCCGAA AP.Gw~UAU

433 CACACL7G CUGAUGAG ;CCGAAACGCCGAA AUCCCCA

467 CG?ACAG CUGAUGAC~CCCAAAGGCCGAA AGCCUGG

469 GCUG:~CfJ CUGAUGAG: CCGAAAGGCCGAA AUGGCW

481 UG.~"UCUG CUGAUGAG: CCCAAAGGCCGAA AUtTCGCIJ

O ~C ~ ~A cu~cAUe;.~:~C C G'~G~;.
1 C Gr,A A~:~,~~uU

502 CAAC~G CL"'~UGAGGCCG~AAGGCCGaA
p~

508 CCAUAGG CUCAUC~AGGCC"'.~?.F'~AG~.~CGAA
ACG'L1GAA

509 DC~G CDC-~UGAG.G.."C.'GAAAGG.."CC,AA
AAC

512 L~CCUCLA CuGL~.GC~.:CGAFaAGGCCC_AA
AC-GAACG

514 G."QCCUC CuGAI?GAG :~~C'=' ' A ADACuAA

534 CAAGJCA CUGAL'GAGGCCG?.AAGGCCGaA
AGUCCCC

556 G,3AAGA CLG~UGAGGCCG~CGA~. AC,:~CGC~.

561 CACCUGG CUC~UCzAC~CCC'GAA AC,~~C~G

562 UCACCBG CL'GAUGAGGCCG~AAG~CCG1A
AAG:AGa 5 8 G.~UGG~."U Cu'GAt3G? G:~C CGAAAGGC
5 CG~.A AUGC,C UL~

598 UCAG~G CUC'.:AIKnGG:.C~.~.aAGC-CCC~AA
'r,GC,GGCC

6.3 GL -TC~C~.GC.,nC-'~UGAGGCCG?.AAG~CGAA
AG~GGC~

616 GAUGJGA C'CJ(y~JGAGGCCGAA.AGGCCG~.A
AGGACAG

617 G:~C'JGAG C ~ -.~A?.GS,AAGG~C

620 GaUGGCQ CVGAUGnCGCC~-~' AAGGCCGAA
AG:y4AGG

623 GAGAUGG CQGAUGAGGCC'G~AA.AG: CCCP.F.
A

628 UAUCAAA CUGAUGAGGCC'GAAAGGCCGAA
AUCG:~U

630 GUQAUCA CU~CGAAAGGCCG?.A AAAUCGG

631 G.~"L7LTAUCCL~CCAAAGGCCGJ~A AAAAUCG

638 ~C".,~~ACACCt7GAUGAGGCC~~AACw,CCG?~A
AL'GGCCA

661 AGAUCUU CQG~UC,T~G:~cCGAnAGGCCG~.A
AGCUCGG

667 C'u~CC~C~ C'LT~UG.~'aG:~cCCAAAGGC.CGAA
AUCUUGA

687 GL"C1CCC~ CLTGr',UGAGG,:C~~AA~~,CCGAA
AGULICCG

700 CCCCACC C'BGAUC~GGC~CGp~ AGC.GAGC

715 GJ~iGAA CUCiAUCAGGCC"~AAAC,C,CCG~sA
AUCUC_AU

717 ChGCF.AG CUGAUGAGGCCGAAAGuCC'G?A
AGAUCUC

718 AC.~G:~A CUGAUGAGGCC''-~GGCC:'~A
AAGAUCU

721 C"~"':,AUG CLTGAUC,nGGCC 'G~nAGGCCG'~1A
AGCZnGAA

751 ACACCUC Ct7G'r.L'G?GGCCGAAAGGCCGaA
AUGUCUU

759 CGLJGAAA CLJG:~.UG~~GUCC ' ACACCVC

761 CCCGUGA CCC,A. ' ~ C'C~AAC,GCCGAA
ALTF,C~CC

7 62 UCCCGUG CUCy~I7C;AGG~CGAAAGGCCG~
AAUACAC

7 63 GUCCCGU CUGAIIGnGGCCGaAACC,CCC,AA
AAAUACA

7 9 ~.C~AF~AG CUGAUGAGGC C'C~F AAGGC CGAA

7 9 L'UGAG~sA CUGAUGAC~CGaAAGC,C CCAA
5 AGuAGCC

7 9 CUUGAGA CUG:yUG:~GvC CC~i~',r'~G:~:.
6 CGAA AaCrinGC

797 C-CUL7C~G COGAUGF~GC-.CC'CAAAC~CCGAA
AAACuAG

7 9 AG~~UUGA C'u'GAUGAG~,CC'f'~' AAG~CCGr'~'s.
8 ~iFaAAGC;A

829 C-,~AACAC CUCAUGAGGCCG.AA~.GuCCGAa.
AUG.GCCA

834 AGUCCGG CGC,AU ~ ~ CCAA ACACAAU

835 GnGJCCG CDGnUGAGGCCG~AAAGGCCGAA
AACACp.A

845 GCGUACG CLIG~UGAGGCCGAAACGCCGaA
AGGAGUC

849 GUCGC~CG CUGhUGAGGCCC~T,AG;,,CCC?J~
ACGGAGG

872 C''L:~,ACAGCL1GAUGr,GGCCG~.AAGGCCCAA
AGCCUGG

883 GCAUGaA CUGAUGAGC,CC 'G~AGC,CCG?.A
ACUCCAh 8 8 CUGCAUG CL1GAUC,F.GGCC~C.~-_AA AGACUCG

905 CCAUCAG CUC,AUGAGGCCGAAAG:~CCG?.A
AGGCCGC

906 GCC,AUCA CUC,AUGAGGCCGAAAGC,CCCAA
AAGGCCG

of g GCUCAtV
' ~ C~~F~GG.~,CC~A
AC',CZTCGC

c 3 GDACDGG CU ~ ' . '"'C'~~A AC<ICCAZJ

g3 ~ AG'~ C'~'.IJGAGC,CCGAAAGGCCC~A
AACDCCA

942 D .~ ~~ ~

g53 DCAUGDG COGAUGAGGCCGAAA~ A~AGGC

9 62 CGG'JG~~ CUC~JG~GGCC~' ~ AUCADCU

9 65 GUCUGGC C ~ AGL7P..CUG

9?3 DC'OCUQC CLTG ;UG~GGv."CG~AAGGCCG~
AUCCGCR
.

S 8 ACQCODG C _tGADGAG~~CG~GG.."~A
6 AG.~"DCL1C

006 GuJCLJC~. CCJ'G;F~I7GAG:~CC~.~AG~~rGAA
AC~CCU

1005 aCUCQGG G:~AGGCCGAA AGuLICUC

1006 Li~CUC'LJQCGGADG~ ~ "AP.

3 015 UCGiJC.~U CG'G~'~DGP.GGCC".~AAG~"CGaA
AUACDCU

1028 L'L'G~AG CUGAL1GAGGCC'GAAAGGCCGaA
ACUCIJUC

1031 CCAUUGA CaG:~GAGC,CCGAAAGGCC''"AA
AG.~~ACL?C

1032 DC~~G ~ ' ~CCGAA AA~ACD

1033 GDCCADU CWAUGTaGGC'C~AGCr.~CGAA
AAAGGAC

1058 CW:~LIG ~ ~ AC,GCCG6 1064 ~'C~ '" AC,~

1072 G~J~C~.GC CtJGADGAGGCCGAF~AG~.~CGAA
ADACGCC' '! 082 DULiCG~ CGGAUGF~.SGCC~~AAGGCC~"AA
?~~CACA

1 083 ACWCGG cJGT~UGAC;GCC'"~AAG GCCC,AA
AAGGCt7p 1092 ~~ AG~CG

1097 GG~~CAG ~C'CGAAAG~~C'GAA AGODGAG

1098 ~G~C3~ CL3Cp.DGr':GG~C" AAGUDGA

1102 Gt"U~7Gw C.'UGAUGA ~ ~~C;GAA ACAGAAG

1125 GAAGCDG CUGADGAGGCC~.~GGCCGAA AGGGCVG

1127 c~c cvG~~~Cc,ccGAA Atmz~GGC

31 ~Gu~UC~~U c ~ '~:,G ~CCGF~r ~~

32 AVGC'OC~ CVGAOGAGGCC~CGAA h 1133 GAAG..'ZJGAGADGGFa 37 G'~cc~"CV cvc~:UGAGGe~cGr~A ~,~, 1140 GCVGAGG C~.~CCGAAAGGw~CGAA ADGCLA',G

1153 CFJ~GW CUC~F~K~AG~~CCGAAAG:~CCGAF.
ADG~JGC

1 '_ CuCAIICA CUGAUGP.G~~C~GAA AGQI1GAZ7 116? C~~"~,Ga,ACVGF, ~ GAA AC(JCADC

,168 L .-.. CL-GA . ~ CCAA AF CQCAU

1169 AU ~GwGG CUGA ~ CCCGAA AAACDCA

182 UGsUG.~~V CG'C,p, " CGAA ACAGCAU

,183 CDCAUGG CUGADGAGGCt~GAJi~F~ AACJ~GCA

1184 UCAG~AG CGGAU ~ ~ CGAA AGGGGCC

11 87 G:~CtJGAG CUGAUGAGGC,'C'Cl~AGGCCGAA
AAGGGAC

6188 CUGCCCO C'CGGC~CCGAA AI7GG~1,A

?198 UCAGACU C'C~CGAAAGC~CCGAA AACUCCC

..2 GA?.GGUG CG ~ GGCCGAA A~GGCVG

215 CG~'UGCU CLiGF.VGAGGCCGAAAGGCCGAA
AGGCCAG

'!229 C,CUGAGG CGCAUGaG~CCGAAAGGCCGAA
AG~GACC

_237 GGC-GVAG CUGAL'GAGGCCGAAAGGCCGAA
AGCL?GC,G

1250 GAGCCUG CGG'~.UGAGGCCCT,AAGGCCGAA
AGGCUGG

1268 ~,.".' CC~CG?.A AG.~UGG

1279 AG~GG CDGAUC,AG:~ "CC~G:~..~CGAA
ACCAUGG

1281 C~~0 CL~C-:~~C~~ AGCCG?~C

1286 -... CL~t,..CG.'~AAGG..,CG?.A
AACUG'~U

.3 09 AGF~CUCG CI:~~t'CAA?~C-:~C CGAA AC~G:y?
G

13J.5 GG.~~L~G CQGhUG~GGCC'GAF~?.GGCCG~
ACL3G~.~, 1318 CCG''~~ C~7GAUGAC:~CCG..ranAG~~~G'~
,"'O AC,~CUG

1?31 GACL~ CVC~UGACv:.C~~G.ZCG~A AGGaCCC

1334 UG'..GCT.1LJC(3Gp.DC~IG:~C~.~G.~.-CCGAA
,~.G

1389 G~"LNCC CQG?.UGaG:r.:."'~ AG~GGGU

1413 AG~J~.UCr"sCUGaUC;AG:r.~~~' CvC~~G-~r~a 'r~CuGC?.IG

1414 ' "' C CuGi C'='W C"~?,A ?.r~CL.'GC?r 1437 GCG.:~GC C~JG~CG?.GGC;.~'.~AG~"'CG~A
~.G~.CC;.' 14 41 UGUUG:. CL'G.'.. - ~ ~C GAA ~~,~,GG
C

1467 GUCUGOG COG"CG~.ACWCG~ ACACQCC

1468 CC~7CUQJ CUG~LJG~C~.~CCGnaAC~CCG?~A
AACAC'LJC

1482 GL1CCACA CUGAUGAG~"'CG~AGGCCG?.A
ADGCC?~G

1486 AGUOCC'C C~~7~GAGGCCC~nAAGGCCGAA
~,~C:CGa~G

1494 AAACQC'IT COGaUGAG~~~CG~AGwCGaA AGUUGL7C

15 00 CUGCUC~A CL1GABG~GGC CGAA.ACuC C
G~ ACUCDGa, 1501 G.."'G'GCUGCflGT~L7GAGGCC~~G~CG?~.
AACUCGG

1502 AGCCLJGCU CUGA~G'~G:~CC~-~'J~aG.~C'~G~
.~r'...aCt7CU

1525 ACAC~.GG CL~~C~.~A.AGC-CCG~A AL'~CC

1566 UUG'zG~vG CUGATrGAC~CGnAAGaGC".-~?.
ACUCC~iU

1577 CG~7UA Ci:~~C~~GGCCG~:A AC-CULJGa 1579 G~~GAGU CL~C~UGnG:~CCCAAACz-LC.~_-?~
AUAG~~UU

1583 ~CCAGGC CL'G:~DG AG:~CCGr~G.C-v."CGaA
AGvILJALm 1588 CCC~CUC CUGT,~G:~..~CG~u~AG:~CC~~:?.A
~G?,G

1622 ~C.G CUG~L'GAGGCC"'C~.~A ?.Gw.~UGGG

1628 CCUACCG CG'GFaUGaC:,C " "~C"'.~.
AGCAGGA

1648 CAUUGGG C~QC'~ '~'CC~AAG~C~.~A AGCCCCG

1660 CQG~ CDG:~UC,AGGCC'G~,~~G.~-CC~.~y?, aGGUCp.G

1663 CACCUGG CUGAUGAGGCC'G~AGGCCGAA AGG1GAG

1664 UCACCUG CUGhUGAG:~ _~~CGAP. p~G:~G?, 1665 ACC'JCCG CffGaC~C~GC,CCC:?.~'~AC,~:~CCG?.A
AAGCGAG

1680 Cr;AC~G cJG.=.UGAG~CC~CGaA AGUCUUC

16 B "UG.~G:~ C'CJGAUGAG~C~~~AC~C.1 CGAA

16 83 Ar.UG;s? C'JGAI:CCu'lA AC~~7C
G

1686 CG,IaAUG C~1GAUCv'~C,:~ -C'GFanG:,CCG~.A
AC~?C~, 1690 L?GDCC:yC CLJG~UGnG.~~CC~~ACi-~~CGAA
AUG:~GG

17 04 AGCAG? G CLTGAUGAGG~CCG~GGCCGAA AGL7CCAU

17 05 G~...~GA CLTGr,U ' 1 Ap,~C

1707 AAGAGCA CLTGr~ZfGAC~CC'rlr.?.G.a~CGAA
AGA~GVC

1721 CLJG~UCU CC~UGAG ;CCGAAAG: CCG.Ar?
ACUG~.AA

1726 F~GaG~."U COGAUGAG:~C " ""CC~A AUCUG~:C

1731 ACCULTAG CUGAUGAGGCC~-GCCG ;A AGC~TGAU

1734 AG~~CCLT CUGAUGAGGCC ' ' ~ CCAA AG.3AGCU

1754 CUCZ1UG~a CUG?.UGnGGCCGr'aFaAGGCCGr.'~.
'~C,CACUG

Table 20 Human rel A HH Ribozyme Sequences nt. Position HH Ribozyme Sequences 1g UacAGA,c cvG~GAGG,.~r-,~AaccccaA AGCCAVv 22 CACOACA C'CGnUGAG~~.C".~AACvCCGAA
AC~.~GCC

26 C~JGCAC C'OGa~rGaGGCCG~.AAGGCCG~r AG~t;?~CG

~3 GA ~G~ CUGnUGAGGCCCAAAGGCCGAA AGaGL'ZJC
~

94 UGr ,.,. CflGAVGaGGCCGF~1GGCCGaA AACAGOIT

100 "C -~U C'~GAC,GC'C"~AGGCCGAA AC-C.vCGA

103 cc ~c~.,~~ c~GavG:~Gcc~cc~a, AvcaGw 105 'LG'.~C~ v ' " ~ C""VAA AGr'IUGrIG

106 CUGCCG a CUGr~UG~.G~.CGr~AAGGCCGr'1A
AAGr'~.UGA

i 29 GGGGCCA ~ _~ ....CGA?, AGGCC'CG

138 CUCCACA CC7Ga.UC'l.~C~..~'C.'GAAAGGCCGAA
~~~ C

148 GCDG'-,AU CI7GnL'GAGGCCGAAAGuCCG?~A
AVCUCC?~

151 G..~GGCDC ' " " ADGAUCQ

180 GUAGC"'~G C'C~CJ~ffGAGGCCGAAAGGCCGAA
AC~CGCAU

181 C~AGCG CL7GnUGhG~CCG~AAG;GCGAA AAGCGC?a 186 G:ACWG CU~C~~GCCGAAAGGCC~.~A AG.~~uGAA

204 GCCCGCG C'C1GAUG~GGCCGAaAGGCCGAA AG~_GCCC

217 CGCCOG:, CtJ~CGA~J?~~CCGAA AUGCUGC

239 UUG.~~tiGG CL7GA~GGCtGAAAGGCC".~r1 AUCCTGUG

2 62 VGAUCW CL3GAt;~AAAGGCCGAFa AUGGUGG

268 AGCCAUQ CUGAUGAC~.GCCGAAAGGCCGAA AUCUUGA
' 276 UCCLTGLTG CUGADGAC~CCGAF~AG~~CCGaA
AGCCAW

301 CCnGGGA CL3GALIGAGGCCGF3~GwCGAA ?~UGCCC~, 303 C?CCAGG CDG~.L~AGGCCG~AAAC,GCCGAP, AC~.UGCG

310 CCUUG~"Q CUGABGAGGC ~ ACCACGG

323 CG~"OCAG CUGFsU - ~ CGAA AG:~.,~LTCC

326 GGCCG.~"U CUGAUG~C' CGAA ~' G

335 U ~c~GGGU C9GA ~ ~ CGAA AG ;CCGV

349 UQCCLTlIC CC1GAUGACuCCty~AGGC'CGAA
AGCUCGU

352 CCL1WCC CUGAC~AAAGGCCGA?, ACAAG~~V

375 CUCAUAG CUGAUGAG.~~CCGAAAGGCC'GaA
AGCCAUC

376 CCC1CAUA CUGF.L1GAGGCC'GAAAG:,CCGAA
AAGCCAU

378 AG..~CUCA CfJGAUGF.CGCCGAAAG GC'CGAA
AGAAGCC

391 CCG~GCA CUGAUGAG~CCGAAAG~CCGAA AGCUCAG

4 09 AACUGUG C tTGAUGAG GCCCaAAAGGCCGr~A
AUGCAGC

416 UOCDGGA C'C~GADGAGGCCGAAAGGCCGAA AC'~1GQGG

417 GWCUG~v CUGA AGGCCGAA AACpGUG

418 GGWCUG C'LJGF.UGAG~~CC'~AAAGGCCGAA
AAACVGU

433 CACACUG CiJGAUGAGGCCGAFAC~GCCG?~ AUUCCCA

467 UGaCUGA CUGAL'GA ~ CCGF~A AGCCUGC

469 GCUGACU CUGAUGT..C- GCCCAAAGGCCG~.A
AVAGCCU

473 AUGCGCU CUGAUGAC,GC'C:Cs?.A.AGGCCGAA
ACUGAUP

481 UG~"tJCUG CUGAUGAGGCC~CGAA AUGCGCU

501 AACUtIGG CVGAIJG~GGCC"~AAAGGCCGAA
AGGGv,~UU

X36 ' 502 G-nC~UG
CUG:~UG~C-:~CCC~-C-:_CC,~r?
aAC~,'U

508 C'cUC~UGnCNCCC;~A.'~G:~CG:
A ACJL7G:~A

509 Ueur~L~IG
CZGAU"uAG:~.C"'CCGAR
AAC'OUGG

512 UccUC'~
c~~~c~~aa.~ccG~A
Ac;GAACtT

51s G,.~UCJUC
c~.lcaccnaAC~ccc~A
A~G~, 534 Chc-.~~LicG
cJGn -'' ~CccAA
AGUCCCC

556 G:~F~C.~.
C'L~vnLIGF~C:~.CG~AC""CCG?A
AC,CCGCA

561 CACCJG:a CUG ALTGAGGCC"~GG.."CG?~A
aGG'~GAG

562 UCACCUG CCG~UG~G~..~CG?..'~AGGvCGAA
aaGCAGA

585 CCL'GCCQ CtiC~UG~CCGAAAGGCCG.~.A
~"aC

598 ~i Ca~CC~~C~

613 GAC~G CL7Cr.UCaC~C"'~n~C'~."CGAP.
?.C~GGCG

6.6 GnUGAGG CUC~.U~G:tCCGAF~AC-~~CCAA
:'~

617 c: .~UC~.GcL-ca~e~G: cc~raaAGG:.cGaa, a~ccac~

620 AL'GCUAU CuCAL'C~GG~~'C'G?~.AGG._~CGAA

623 '~UGG CUGn ~ -ZT~.G:T."CGAP.AGGCCr'.~A
AUGAC',:,Fa 628 UGUCAAA CUG:~.UGAC~~'CGAAAGGCCG?.A
AL~.;VAU

630 AL'UGJC.A CDG~.BGAGG'~ AGAIR'~.;G

631 GAC~JC'JC CUGAL~GyGG~~C'".~AAF '~
AaGAUGG

638 C-Gc.-GCyCC'C -CADG.F~G:~.CG~r.AAC~GCCGiaA
~aDCGLICA

661 :-c~UCUU CuGnUGACvCCGinA~GGCCG'Aa.
GCLTCGG

667 C'uCC,C-:~.CLIGAUG<aGG._'GAAAGGCCC~'~.P.
AUC'LJ~

6 a 7 GC'L7GC CUGi~LT~.GGC C'G7"LAAGGCCG?.A

700 CCCCACC CUG:,L'C;~G:~cC'CAAAGG."CGAA
AG:~CAGC

715 G-~?~G: C~ ' ~ .~DCDCALT
AA

717 C=.GJAGG CLG~U -C~GGCC~~GGCCGAA AGA~C

71 a :~C.~GL~G CUG:sUGAGc.-~~CC'~Fv,Fac~"CGaA
AAGADC(1 721 C?CGC~G CVGAUGAGGCCG~CGAA ?.GCAAGP, 751 Y.C~Cf~C CUCr.L'GI~GGCC'C~FF~GGs.CGnA
AUGUCCU

7 5 9 C GUG~AA C'~'GAUGr~"C ' ~ ACACC'UC

761 CCCGLICA C'UGAIJCAGGCC~.~AAAGGCCGAA
?UAC~1CC

762 UCCCGL'G -C~C~CGAA ?,,F.UACAC

7 6 3 GU C C ' '" ~F.P~G~"CGAA AAAL7ACA
CGV

792 C''.~G C~ZK~~C.:~CCC,AF.AGGCCC~AP.
F,GCCL3CG

795 L2GCGAA CL~C'GAAAGGCCC~.A AGuAGCC

7 9 6 c,,~~Gc cJC,~cc;.~G:~ccGA~~GC,.~ccAA
GA AACua~C

/97 G~J'LG'"G CUGhUC~G,~CVAAAC~CCGAA F

7 9 8 r G~~L'UG.~_C~UG''~G~.;~CGAA AAAAGGA

829 C~nc~.C CUGAUCAGGCCGAAAGGCCGAA AUGGCCA

834 C-.~JCC'G.GCUGAUC:.:~CGCCGF~AAGGCCGAA
AC.ACAAU

n35 G:NJCC'G CLGAUC~-CCGAF.~'~GGCCGAA
AACACAA

845 G~.GJAGG CL'G:~L~AC~GCCw,FLAF.C~CCGAA
~~ , a49 GUCUGCG CUGAUGAGGC'CGAAAGGCCGAA
A,GGGAL,G

a72 cc-cacAC cvc,~UC~G:,cccaa~GGcccaa.
AG;.cvcC

sa3 c,:~UC-:~acuc;..UCaGCcccaaacGCcc~.
AcACCC~

ass c~G~UG cL~:.,L-cr.GVC.cG~G~ccAA
AcAC~cG

905 C 1JCG.;~ CVGnUGr.GGCCGF~AAG:~CGr~?.
AGGCCGC

906 CCCv"UCG CUGr~.UGAGGVCG'~FaAGGCCGAA
AAGGCCG

919 G~~UCACU CUCaUGAGGCCCAAP1GCCGAA AGCLJCCC

936 cJACUGu CuGaUGaGucc~~C~~
aUUCC_aU

937 G.~"'~.RCUGCUG~UGAG~C~~AC~CG~ iaL'CTCC.3 042 UG~u~GG CCiGAC~~CvCC~,.~?a ACCG~s?~
' ~

053 UCG'JCUG CC~JGr',G:,w~CC'~F~C, sCCC.?aF~
ABCCGC-C

062 C"w"~?GaC :.UGnUGAC Gc~~~F~AC"~s.~.CG'~.A
aLiCGUCLT

065 aUCCCVU ' .-C~' .CC~a aCCaUCG

973 UCtJCCQC C9GAUGAwCC~.~Al'~AC,~C~a?.~1 AUCCG.~~-J

986 GJCCUW CUG~UGaG~CGAAAG.;CC~v:?~, ?.CGiJULJC

6 G,~~cvcA CvGa~c~GCa~a~ac~cccaa~ aL-~oC~J

005 Gv.'"UCWG CDG~LJGhG: CC~' ~CC~,.-.aA
:,G,.;JC'JC

1006 UGv.~uCCTf3COGADGaG~~CGAAAGGVCG~A :~T~G.,uCJ

1015 UCWCaU CuGAUG~~C~,iaP.3'~.~cGra aL'G..~UCV

1028 ccc:~aG cJC-~UG;~cC~ac~~ -tea ~c~c~rvc 1031 CCGCUGA CUG:~UGaGGCC'G~AAG aCC"._~.A
AC-.~-~C'~?C

1032 UCCGCUG CUGAUG~GG: C~~aAAG~CGaA
AaC,GaCV

1033 GUCCGv.~U CUGaUGAGGCC~- ~ ~G?1r1 'r1?.?G
GaC

1 058 C~~GGJG CUGnLIGaG~~~~~~GnAAC- vCCG~A
r~~~CG;

1064 AUGCGUC CUGAUG;GGCCGAAaGCICG~ .~G.,~L'GGr1 1072 GG~CAGC CQGaUGAGGC~CG?~ AUG.~GUC

1082 CC1GCGGG CL1GAUGhG~CG~FsAGGCCG?A
aG~,~,Ca 1083 Cn~UGCGG CU -C'r.IlGnC- G: CGr'1A'nCZ,CC.~-~'.A
'.~aC~~sCrlC

1092 AC, ~AGC'UCUGAUGAG~CGAAAGGCCG1~ :~G..~UG'G

1097 "G.~ACAG CUG~UG~C:~CCGAFJ;GGCC~.:AA
?.G..~L'G ;G

1098 G:~GaCA CvGA ' ~ CC~ :~aGw'ZC~

1102 G. ~~ CUC,~UCAGGCCG' : ~ CG?.A
?,C_~GaAG

1125 AF.AC,vGa CQGAUGr'~.GGCCGr " C~.-'fir ~~"L'G

1 ~'1 WJA~ CUGF~UGAGu'CGAAAGGCC~~ AL2.G~~

1131 L'GACGQA CUGAUGnG.~~CCG~AAGGCC"aA
~G~GaUp, 1132 AUGACGU CUGAI1GAGGCC~..nAF~GGCCG?~a :,?, ~G~.~-~IJ

1133 GAt'~GACG CflGAUGaGGCCCGAA ".~AG:,GA

1137 CAGGGaU CUGaUGAG GCC ' ~~CGAFa ACC,'Vr~1 1140 CCZJCAGG CtyGAUG?~GGCCG~P~~AGGCC".~AA
aUCACGU

1153 Cr.T~GULJ C1TGAUGAGG..~'C'GAAAG~~~~CGaA
i~L'Cw~UGv 1158 CUCAUCA CDGAUGAGC~C -CAAT~GCZCCAA
aG'JUGAU

1167 G.~''LlG::sACLTGAUGAG.~~.:.CG'~F.AGGCC~.:.aA
AC'CJG'~UC

1168 UG.~"UG:~ CUGAL1GF~G:,CC'G~AF.F.GGCCG?~
; ;,r~,CUCALi 1169 AUGGUGG CUGAUGT~C~CCCa~AaGGCCG?~r iz~.ACL1CA

1182 FGJ~F:GGA CUC~UG~GGCCGT~AAGGCCC?~A
ACACCAV

1183 GG~nGG CUGasUGAGGCCG'~aAGGCCGAA
?.AG~CCs~

184 CCAC:iAG C'UGaUG~G.GCCGAAAGGCCGAA
AF.aC aCC

1187 UGCCCAG CQGAUGAGGCCGAAAGC~CC~.~A
AG:~P,AC

1188 CUGCCCA C~UGAGG:.~CG?~, aAGGAAA

1198 CCUGG'"Q C'CGaLIGAGGCCGAAAGGCCGAA
AUCUGCC

1209 CFaAGG.~C CL1GAUGr'~C~,.~~CGr'~AAGGCC~.anA
ACvCCUG

1215 CC~vGGCC CUGF~UGAC,GCCGr'1AAC,CaCC~.,:r~'r_ ~C~~,~~CGa 1229 ACWG:r; CUGAUGhGG.~.C~CGAA ~'~~~C

1237 ~G.~c.~G CDCAUCnGGC -CC~AAGGCCG~
ACZJUGGv, 1250 G~.~~UG CUGUGAG~:~CGAAAGGCC -C~:, :~G.~CUG~

1268 AUGGCUG C'QGAUGnGGCCCia~'~AG;CCGn?a GC,C, X79 -G'~G..~GGAC~UGAC~~'C'CAAAG~CGAA ACCAIJGG

1281 CAGaG..~0 C~~~'C.'C~F~AG~CGaA ADACC.AIT

1286 CG~~CA CL7GABG:~CCC~AAC~CCG?~A
h~GC~1 13 09 G,~-.ACGGGCQCADGnG~.~CGAAAGC~:C'CAA
ACAG:~ ;G

1315 G~~G'PG CQC,~.~DGAC,GCCGAAAGGCCGAA
ACtJGGG?r 1318 CL7G:~Gw CL1GA0 ~ ~ AGGACUG

13 31 GCCUt's CQGF Z~l~sAG~J
:G

~~34 AGyGCCU C'~T1G~~r:C~AAG.GCCG?.A
.ACG1G:~G

13 89 GGCCQCO C~LGF.GG~ '" .~A AC~GCG'J

1413 ABCAI7CA ~ .AAA ACQG~G

1414 CADCADC ' ~ " ~ AAC"LJGC~.

1437 G~~C~.AGC CQGADGAGG~~t"GAAAGG~CGA?.
aGGCCCC

144' UG~?L'GCC C~GnG~."CCaAAG:~"CGr~r 1 AC-Ci~ACv 14 67 GJCLTCL~G CL1C~.QGAGvCCGaAAG.~~~.
C~.ar'1A ACACAGC

1468 G.~'"tlCGC~CUGADGAGG~~C''~?.T~.AGG
CCC~ ~ACAC.J~G

1482 GUCGACG '' ~ " AUGCCAG

1486 AGLIUGUC C~AUG~~~tlt~AAAG~."CGAA
ACGGAI~G

1494 AAACQCG ~sAC~~'"'CXy9AAGCCCGAA

1500 CDG'."QGA CDG AiIG~GCCGAAAGGCCGAA
ACUCG~~, 1501 GCUGCOG '' '' ~ AACUCGG

1502 AGC"UG~."UCLJGUG~GG~t~rGAAAG~:~CC~.~A
AAACUCG

1525 CC~CAGG Cl7Gn3JGAGV~CC'GF.AAG:yCCGaA
AUGCCCLT

1566 CUCAG:~G C0'GAUGAGGCC'GAP.AGC,CCGaA
ACUCCAD

1577 CGA~ CO~U'GAGGw~~..~LGAA AGCCQC~r 1579 GC.~"CAGO CDC,ADGAG~~C''.~AAAGGCCGaA
ALmGCCU

1583 ACL7AGGC CUGADGF~GG~~C.'C~AAGGCC~.~, ~'.G~AITA

1586 CUGUC~.C CI?GACGF~nAGGCCGAA h~GCGAG

1 622 G:zaGCAG CC1GAUGAG~.~CGAAAGGCCGAA
AGCLJGGG

1628 CCCAGUG CQGAtTGAGGC'CG~.AACGCCGr~a AGCAC,GA

1648 CA~GGG C'OGAI1GAC~C~"CGAF, AGCCCCG

16 6 CLTGAAAG CUGADGAGGCCGAAAGGCC'GAA

16 63 CDCCC1C,A CDGnUG:, ~ GAA r3~CC~GC,C

1664 UCUCCUG G~ AA~,G

16 65 AUCQCCU CDGAL1GAGGCCC~AAAGGCCGAA
AA~'~GGAG

1680 GGF~CuAG COGAQGF,GuCCGAAAGGCC'~.A
AGUCtIOC

16 81 ~AG:~A CQGAUGAO;C.C ~'G~AF,AGGCCCAFr AAGDCUO

1683 AAUGGAG CLIGr.UGAGGCCGT,AAGGCCGAA
AGAAGLTC

1686 CGCAAUG CVGAUGAGGCCGaF~AAGGCCGAp, ~:GGAGAA

1690 UGUCCGC CDGADGAG~:.CGAAAGuCCGaA
ADGGAGG

1704 ~ CUGAUC~C,GCC'G~AAGGCCCAA
AGUCCAU

1705 ~G.~ CCVGACUGALC'CO~AAGG~CCGAA AAGUCCA

17 07 CAC~CU CUGAL7GAGGCCGAAAGGCCGAp.
A,GAAG~

1?21 CUGAUCU CLTGF, ~ ~ CGAA ACUCAGC

17 2 AGGAG."U C'UGAUGAGGCC'G;F~JaAGGCCGAA

? 731 CCCLTtIAG C'cJGAVGhC-GCCGAAAGGCCGAA
AGw'ZTGAU

1734 ACCCCCU CUGAU-CAGuCCGAAAGGCCCAA
,~.C-;AGCV

754 CLTCUG:,G CUGAVG.AC~CC'G:A3',AGC,CCGAA
AG ;C,C~G

U ~ ~ ~ ~ U U
a ~ a U ~ ~ ~ ~ ~ ~ ~ ~ ~ U
~C W C V ~C ~ V
d a Ac a a a a .s a ~t .s a ~t ~t a U U U U U U U V
.C 'C sC a d it iC ~C a sC iC iC iC ~C iC
a 5' ~ ~ ~ ~ '~ '5 5~ ~5 '5 a ~ a ~
c~5c~uc~c~u5555c~c~c~5 a a d a a a a d a a a a a ,~ a U U U
~c'~c'~t"~c'~~c~c~c~~c~taJc~c'~c~
~~5~5~~~~~~~5~~
0 ac'~~ c~c~
a~-n c~c~c~~~c~~c~c~~c~c~c~c~t~
ACC UUC~c~c~~c~c~c~ c~
m c ~ a a ~ a a ~ ~ ~ a ~ a a ~c a ,.
U U U C~ C9 5 ~ C~ ~ C'J
~ CU7 C~7 ~

~c~'~~' ~'~U5 'a a c~ U
z ec -o N a :~ ~ ~ l~ tf7 C o tn ~ ~ o ~ O
N ~ ~'~ f~1 v .-t tn l~~

w f--~
c C9 ' U_ a S~aU
C7 ~ ~ C~ ~ U ~ ~ ~ ~ ~ ~ U U
U ~ U U U U U U V ~
~c~'~c~c~c~c~~'~c~
(j ~ U C9 ~ V
st ~ ~ a ~ ~ C9 ~ a a ~ ~
U ~ U U U U U U ~ U ~ ~ ~ U U U ~ U
V ~ a a U U
a~~c~~~t~ c~3 ~~~~~~~~~~~~~~c~~~~c~
cna~ c~c~~5c~5u5c~5555c~55c~c~~
C a a a a a a a a a a a a ~ a ~ a ~ ~ a c9.~ ~~.U~~. ~C~j,7~~ ~ ~~ UUU
~ U C~
N a a a a a a a a a a a a a a a a a a a U U U V U
U
~~~c~~5u~~'~a~ ~J~a'~~a ~a c~ c~ c~
Z
_.
N ~ -O o W .-i u1 .-i o~ e~ o~ er rt a~
N v) c- r-~ r, vo r-, vo c ,-~ o o vo co ~-, c o o N r~ u~
r-1 N fh P'1 ~G ~ CO D "'~ N r"f f'~1 C' aT C7 O ri N f''1 .-~ r-i ra .-i ri r-1 N N N N
O

Table ~3: Huma.n TI3F-cc HH Ribozyme Target Sequence nt. ~ Target Sequenc. at. 8H Target Sequence Position Position 28 C,:~CACv"V U C9C~CC

29 C~?G.~'W C UCiJLVCC~321 G'JC~1U C AL'CL'UCU

31 AG.~JL'C~J C ~DCCL1CU324 AGr~UC.''~U C L'C;CCr'C.~,?r 33 CWCtICCI U CCUCUCA 326 AUCAUCp U W

34 UQC;~2W C C'UCQGAC 327 UC_~,UCL'U C UC~.~,CC

37 L'CGUCCQ C UCACAUA 329 AUCL'UCU C G:~CCCC

39 UUCCUCV C ACAUACU 352 ~:G~."CUGO A GCC--,~,UG

44 CQCACAU A CUGACCC 3 61 CCC.T~UGU U GL7AGCAA

58 CACG:,CQ C CaCCCVC 3 64 aUGL~GU A C

65 CCACCCU C UCUCCCC 374 ?~CCCU C :~AGC'fJGA

67 ACCCQCU C UCCCCaG 391 C~G~.~p C C.~C

69 CCL2'UW C CCC~A 421 ALGCCCU C CpG~CCA

106 GC_~UGAU C C ~GwACG449 136 AG:~w~LJ C C~ ' 468 GUGC CAU C .~~G~G:,~
- ' 165 CAG~:~."U C C.~C~s 480 G"CCUGU ,~ CCT3C'-RUC

177 CG.~''UGw."Q U G'JUCCUC4 84 UGQACCV C AUCZJAC'(T

180 UGCUGGV U CCUCAG~. 487 ACCUCAU C UACUCCC

181 G..~L'LGJU C CUC?.t',CC489 CGCa.UCU A CUCCCAG

18'4 UGWCCQ C AG~.COCU 492 AL'CC~CU C CCAC~C

190 UCAGCCU C UUCUCCU 499 CCCT,G""U C CQCUtJCA

192 AGCCUCU U CUCCWC 502 ;,G"~Z,CW C W~~

193 GCCQCW C UCCWCC 504 GLiCCUCU U CAnCGGC

1 c5 ~ C 505 UCCUCW C F.AG~GCC
-198 UUCUCCU U CCL'GhUC 525 UGCCCCU C CACCCAU

199 L'CVCCW C CQGADCG 538 AUGUGCU C CUCACCC

205 UCCUCAU C ~ 541 UGC'QCCU C ACCCACA

225 CCACG."U C v?UC'UGCC553 ACACCAU C AGCCGCA

228 ACGCt7CU U CU"v..~CUG562 ~ GCCG:AU C GCCGUCU

229 CGCUC'UL7 C UGCCQGC568 UCGC'CGU C UCCUACC

243 CL1G~PCU U UCGAGUG 570 GC'CGVCU C CUACCAG

244 UGC~CW U GGAGLIGrI 573 GUCUCCU A CCAGACC

253 --CAGLJGAU C GGCCCCC586 CCAAG"~U C AACCUCC

273 G:,AGAGU C CCCCAGG 592 UCp~W C Cp 286 G~ACCU C UCUCQAp, 595 ACCUCCU C UCUC-CCA

2 88 G'~CCUCTJ C UCVAAUC597 C~,'CCUCU C UGCCAUC

290 CCUCUCU C UAAUCAG 604 CCGCCAU C F.AC.AGCC

292 UCUCUCU A AUCAGCC 657 CCCUCu,.U A UGAGCCC

295 CUCUAAU C AGCCCUC 667 AGCCCAU C U''...UCL1GG

302 ~:~C-CCCU C UGGCCCA669 CCCAUCU A UCL, ~~p~

671 C~UCUAU C UG.:,-~.GG 960 UC-.~.~UU C ~ LG

682 GJ C DUCCT.~GC 1001 AACC_AC'J ,AAGAAUUC

684 G:~~UCU U CCAG.."L1G 1007 L~'r~G~,AUU CanACUG

685 G:~.~"'JCJL7 C CAGC'uGG1008 ~,GAAW C , ? AC.. " C AGCG...~UG 1 021 G:~C-GCC'JC CMG
09 AACt1 721 cv-r~A~ C AAUCC~C ~ a29 cAe.AACU c a ~~

725 CABCAAU C G:~CCCGA 1040 GGC,:,CCU A C?,C~,~UGLT

7 CCCGACU A UCL1C".~AC 1 04 6 LTACAGCU U UG?~UC.~.C

737 CC~ACVAU C UC'"~:ACL~J1047 ACaG~"UU U CAUCCCU

739 ACUAUCLJ C GACWUG 1051 CL'LZ1GAU C CCUGAC_~.

7 CUC""uACLT U UGL"C"..=AG? 0 6 C'JGACAU C L'G~?.AL'C

745 UCC,AC.W U G~~CGAGU 1067 C'JC-::AAUC U ~
C

7 GCCG? G'J C UG:~:~.AG10 8 5 G:~GC C(? U L-C.,G-,JUCU

7 C~,CAG.~~U C L1ACUULTG2 0 8 GAGCCUU U G.;

765 CAG.~~UCU A CUUUGGG 1090 CUUZJC'",~UU CL'GGCCA

7 GUCQACU U UG~~v~AUC 1091 WUCv'W C UGC,CCaG

7 UCCIACZJU U G;~AUGA L? 3 CAGGACU U C

775 WG;~:~U C AUOG'~CC 1124 AACACCV C aC~

778 GGADCAU U GCCC~GU ~~29 CUCACCU A GAAA~

8 CCAACAU C CAACCUU 113 5 ~CAAAU U CAGACAA

808 CCAACCU U CCCAAAC 1151 UC-:=ACCU U rGCICUU

809 CAACCUU C CCAAACG ~ ~ 52 G~~CC',7U ,~,GGCCUUC

820 AACGCC'0 C CCCDGCC 1.158 UAG~,CC'(7U CCUC~,-W

833 CCCCAAU C CC~.D~ ~? 59 AGGCCW C CUCUCUC

837 AAUCC'CU O UAIJQACC 1162 CCL'~CC'J C UCUCCAG

E38 ADCCCQLJ U AU~CCC ? 1 64 L'UCCVCV C L;CCAGAU

839 UCCC'UUU A ULTFrCCCC 1166 CCVCUCU C CAC;aUG'J

841 CCUUL1P.U U ACCCCCt7 1174 C:ACAUG'J U UCCA
GA
C

842 CUULIAUU A CCCCCUC 1175 . aG?~L'GUU U C~yGACU

849 ACCCC'CU C C'QOCAGA 1176 G~:,UGUUU C CAGACUU

852 CCCUCCU U CF.GACAC 1183 CCACACU U CCUUGp~

863 ACAC'CCU C AACCVCL7 1187 ~ ACVOCCU U G~CAC

869 UCAACCU C WC'DGGC 2208 CACiCCU C CCCAUGG

8?1 AACCUCU U CUG,~,,~UC 1224 C-:.C~.G..~~7C CC'UCL~1U

872 ACCUCW C BGG.'~Ca X28 GCtICCCU C UAUUUAU

878 UCL'C;~Cfl C AAAAAGA X30 LCCC'QCU A UUUAUGZT

8 AGAC~AAU U G~,:~.~U 12 3 2 CCUCUAU U L'AUGUUU

898 G.~~~"" ;CQ U AGC~CG 1233 CLCVAUU U AUGUUUG

899 GGG; ~~UU A G:~,~JCGG1234 UCUABUU A UGUWGC

9 tJ C CGAACCC 12 3 8 UUUAL'GU U UG:.ACUU

917 C'C's U AGAACUU X39 ~,U~ U CMG

918 CAAGCW A G,AACUUt7 ~ 45 UUGG'~CU U G'JCAUUA

9 LTAGAACU U UAAGCAA ~ 51 ~7UG'LG U i LWr~W

9 AGAACUU U AAC-CAAC 12 52 L~GUG~L'U A L'L'tThUUa 926 GAACL7UU A ~.GCAaCA 1254 UCAUUAU U L~.L'tJAUU

945 CACCACU U CGA~',ACC 1255 C;AL'UAL'UU rJUAUUU

9 ACCACW C GAAACCV 12 5 6 i WAUW A L'UAUUUA

9 CUC~AU U CAGGAAU ~ 5 8 UAU~, U U ;~~,,~W

12 ~ A UUL~LW 14 4 0 uG7L'UL'U U 'LUAU

61 ~U U tTAUL'@.U 14 41 G,'OUUUU A ?,A~UAUU

X62 ~'L'~ U ADaLmL"Q 1446 "U A DG~UCL~

12 ~UW A L'~I7~1 14 4 8 "LUAU U p,G'CuC,~,U

L~U U UAUtIF,Dp 1449 .~L~W 1U A~ UC~UU

12 AULTD~,L1U U ?.UiRDLIU14 51 :yLT~.L'UAU C BG.~.IJL~A

o 67 UO~.D~ A UUAU~ 14 5 6 AUCL'G?~T U U

X69 LRUtIt~.D U AU~J~CT 1457 UcJC.~.vQ a ?.~,wct;C

27 a~ A vuuA~v 14 61 a~-~~J a ~-~cr..,aa o X72 UUAUL~D D i~DtJGaU 1464 :~GLJ~,'GU C L~?.CAA

1273 UAU~sUO U ADGLTAUU ? 466 GL'L'GL'CV r7 ,~C'-,,RUC, X74 ?UUAUCIJ .~ UULRL~Z,T1479 CG.."L~G1U U LT~.,T~,C

i27 I~U~.U U UAULZTAC 14 8 0 Ci JG',L'U U ~..~,CC

X77 AU~W U AUOL1ACA 1494 Cr.?CLiGU C i~CLiC~UU

~7 L~RWU A ULJiIAC'aG 14 9 8 L~C~C'J C ~"'(~

8 0 LTAL'QiIAO U UACAGaU 15 Ol C~.C UCAU U G~"GGaG,~, 1281 f~~UU U ACAGaUG 1512 C~G,~C~J C L'G..~~CCC

X82 UCD7~DL~ A C~G~ 1517 CUCflGCU C CCCAG~;, 1294 UG?.~~ A U~F.UQO 1528 ~~ U ~, 1296 AAUG~U U UAUQCGG 1533 GJL'GLtGU C UGt~,UC

1.297 :~UGJ'r~J U AUUL'G~, S37 vwcLTcJ a avcC.;~CC

x..298UG'~O A UWG:~:~.1 1540 CLGiIaAU C C-C-:.WsIC

1300 TRUQLJAU U 1546 UCH.-CCU A CUALZ1C~

1301 ADU~DC U GG~C~C ~ ?549 GCCL1ACU A WCAGL,'G

1315 CCCv:~,"C A UC ~~ 1551 CUACL1AU U C'~GvG:aC

317 G~:~.~"L7AU C ~~''~ 15 52 VaCCAW C ?.GL't-:~.G

3 3 CC~L'GU A G:~GCDG 15 6 6 GaGaAAU A ~C-.~
4 ~JC,~

3 4 GC"CGC ~ J U C~GCQCAG157 2 L7~.rl.G~--U U
5 GC'L-Qp W

3 5 CUL1G~.."Q C AC~AL'G 157 6 G.~~-vUG~~U U aG~.AAG

359 GACAI3GD U LZJCC~~G 1577 GJUG~~,7L1 a ~C,~r,p,Gp, 1360 ACAUGUU U UCCC~GA

1361 CAUGDOU U CC'G~1?~

13 ~W C CGtIGAAA

1386 GAAC~AU A GC~p 1 ? F~C~ U CCCJIDaJ

1394 GC,~'"t.iC~LJ C CCAU
.GTA

.401 CCCAL'GU A G:.CCCCV

1414 CUGw'"CQ C UGUGCCV

.422 LGIJGC'Cp U CWtniGa 1423 6JGCCW C LIUiJLJGAU

1425 GCCt~UCU U WGAUL~

1426 . CCUt7CW U UGAUI~1U

1427 CWCZJ~1Q U C,aUUALTG

1431 UQUUG~U U AUGUUUU .

.432 UWCAW A UGUUUW

14 AUUAUGV .U L~r~.

.437 UUAUGUU U L;~JtJAAAA

1438 UAUGL'L~J U CTtTAAAAU

Table 24: Human TNF-cc Hammerhead Ribozyme Sequences nt. 3H Ribozyme SeQueaoe Position 28 G~.G?.G CUG~UG?.GGCC~CG?~A ACCLIGw"'C

29 nGC~.ACA CUG yUCaGC~CCGAAr.GGCCGAA
AACCUC'aC

31 __-C~G;:~ACL'GyL'G~,GGCCG~F~GGCCG~A
AGaAGCG

33 UGAGAG3 C'LT"~~ -' GGCCGA~AGGCC
GAA .~~GAG~AC

34 GUG.nGAG CC;C,?UG.GGCCGAAAG:~CCGAA
~AGaGAA

37 ~JGA CL'GAL~'~GGCCGAA1G:~CC~~A
AC,G AAGA

39 AGwTA~IGQ CUGA -UCnGGCCGAAAGGCC~.~A
AGAG~~,v~A

44 GG~"DCAG CGGAUGAGGCC'GAAP.GuCCGAA
AUC~G

58 GnGC,uzrG CUG~!~IJGaC~CCGAAAGG."C'C~A
AGC'CC~G

65 C~~~' A -CGC~CCGAF.F.GGCCGAA AG:~.~~UG~

67 G~G:~:1~ CUGABG~"CGAAAGGCCGAA AGAGS,G~

69 UC: ~GC~G CUGAU~~.G~G~C~CCGAA AC,F,GAGG

106 C~JCCCG CL'~~UGAGGCCGAAF.C'~:.C.~-~A
AUG:UGC

13 6 UC~JL7GC~,CL'GAUGaCCGCCGAAnGGCCGAA
AGE-"GCCLT

165 CC"~CCUG CUGn "~GG.~CC~Ar~fC~CCGAA

177 C~G~AC CDGAiIGaG.~CC"'~~C'.~CCGaA
AG'ACCG

1 ao cc~lc~ caG:~.UGAG;,cc~CcGAA ACAAGCa 181 G:~~G CUGAL~,GGCCGAAnGC,~."CGaA
AACAAGC

184 rG.~.G~."UCL'G:~UGAG~C ~ '' -- CGAA
A "G.nACA

190 nGC~GAA CUC~UGACG~.~CGAAnC~CCGAA
AG:~~OGA

192 G~G~'~G CUGAUGAG;CC~.~AAAG.~CCCAA
A~GAGGCU

193 G~G::P. CUGA ~ ~CCGAAAGGCCG?.A
AACAGGC

195 G~-GAAG CUGACGAAi~GG: CGAA AGAAGAG

198 GAI1CAC.G CUGnLTGAG~CCGAAi~G~CCCAA
AGGAGAA

199 C-.~UCAG CUGAUGT~C,GCC~~A:~~GGCCGAA
AAGGAGA

2 OS CUGC CAC CBGI-.LIGGC,C CGF~aAGGC
CCAA AUCAGGA

226 C-:~CAG'1AC'UCAUGAG: CCGAAAG~~CCGAA
AGCGQGG

228 Cry C'JGT,UGAC-:~CCGAAAG.~CCGAA
AGAG~GQ

229 G~.C~ CUGAUC~.G:~CCGr"aF,AGGCCCArI
F.AGAGCG

243 GEC-~1CCA CL~G~LfiAG~C -G'~C.GCCG~.
AGUGCAG

244 UC:-.CUCC C'uGAL~Gr-''.G~~CCGF.~~aAG~CCGAA
AAL~'UGCA

253 C-;~:.-;~cCCQGhUC~C-:,CCGAAAC-GCCGAA
AUCACOC

273 CC~G C 1~C~CG~.A ACL1CWC

286 U'JACy,GA CUGA -L1CAGC,CCGAAAGC,cCGAA
AGGUCCC

288 CnUUAGA C'.1GT,UC'J,G;CCCr'vSAC,vCCG~~, AGAGa"UC

290 CLTG-'-.UCTACUGF~UGAG:~CC "~G,~CCG?~
AC~GF,GG

292 Cz-,.~UGAUC'UGUG~C.~CCuAAAGGCC -CA.3.
rIG.G~GA

295 -CnGC-C~LJCUGA -UCi.GC,cCG~GC,CCG
aA AUUAGAG

302 UGC~C-CCA CUGAUG-'~GGCC'GAAAGGCCGAA
AGGGCUG

321 ' -nCnAG?~U CU -CAUGAC~CCGAAAGGCC~.~'ie1 AUCDGaC

324 UCGAC v~ CLiC~UGAC~: ~'~G:~CG1~ P.DC,A

326 c,~%c,~-.~.~ cve~~=.c-;~ccGaaAG~ccc~A
A~:~aUraU

327 G.~"UOCGe'~ CQGA~CC~~rCG'~A AACAUGe'~

329 C-uw"WC CUGr'1C.~."'.:~ACWavC~.,a?~
U

352 C:~IJ ~G:~:,C CL1GAUGAGG..~CGAAAGGCC:~1 AC~W."U

3 61 UQG.."LTAC C~L'GaGw"C'"~s~AAGG."C"~A
AC?LlGGv~

364 G.~~UUL1GC CGG?aLKa~G:sCC~alaA~~.CCv1'~
ACAAC?iU

374 UC~~UU CnGaC~.~AGtIC~.~ AG.~JL~J

3 g G~~C.~CDG C'~AG~..~C".~AAGGCCGAa ?.C-..'"UGCC
~

423 L'C~CCAG CT.1G~AaAG~CCG~ A ~G. ~~.~LT

449 ~~G.."L'G,.'~ CL~i~CvCC~.~?~AG'~.C.~.-~~1 AUCZTCC~C

4 6 G:. C CL'CU CUG~.L'G?.G~~CCAAAG~C
8 ~""~ AUC~C

4 so c~UC~acG c~c.~~aGuccG~AG'-cccy,A c 484 i~GUAG?~U CDG?.L~a~lGw"CGC~.~a?I a 4 87 CZ-::AG9A CUGAUG?~G':-v.'"CG?~~.~.CG~
ABGAGGQ

4 a9 cu~:~~G cvcAt~GAGC,CC~~a~AGucc~..-.a.a Aca~AG

492 C~CCUGG CGGAUGAGGCCGAAA~C.~~ AGi~U

4 og UCAAC.AG C'.lGaDGAGGC'C~,.~?.AAG~~~~C",~rl ACC~GC-:~

502 CC'UL'Cr-'~A C'CGAI1GAGGCC"~' At~CCGr~r1 saGCACCfJ

504 C-CCCLJUG C'CG?.UGr'~G~~CCGaAACw~"'C"~.r'1 AGAG~AC

505 CvCCCW CLJG?~LCGAAAGu..~CG?.A ~

525 r'~L7C~.~"UG CL1GCCGAr~A~CCG?~ .~G~~~vC.~

538 G:~~7GAG Cff~GAA,A~CC.~-~A AC,C~CAU

541 U~Gw'"t1 CL1GAL'GAGGCC"~AFa~CC".~~.1 AGGaGCA

553 UGC"u'"D CUGnUGAGGL"CGFCC~~ AUGv~Cs~J

562 ?.GACGGC CUCrL'C?d> >CCGAru~GGCCGAA
AUG."GCC

568 C~JAG.~-A CGC=AUGAG~CC' -Cue' AAG.~~~~C~w~.
ACGw~,w~G~a 570 C:.G~JAG CU~CGAAAGGCC~.~r aG:~GGC

573 C-.~CL1GG CVCAUGAC.vCCGAA,AGGCCG?.a 1?~GGACv~.C

8 C~F~G.~"'UtJ CUGADGP.GGCCGAAAG~C"~ps~.
6 ACCWG:

592 CnGAGAG CUGACCGAAAGuCC~~l~a AG~~flL7GA

595 il~C~:~GAGA ~'GP.C~G:~CCGAAA~CC~~~1 AGGAG.~~7 7 _. ,... ~CGAAAGGCC~.~A AGAGGAG

604 G:~~UCW CQ -G'.UGAC,GCCGAA.~GC,CC~~A
ADG~~AG

657 G:~"UCA CUGAUGaG~C'C'GAAAGGCCGaA ACCAGG

667 CC~VA CUGAUGA~G:~CCGaAAGGCCGP..'~
ALJC,GCCLT

669 UCCCAGA C'v'GAL1GACC,CCGAAAGW."C'GAA
AGAL1G~~, 671 ccvcccA cuc~UC~~c~~AAAC~-y:.ccaa AvAGAUG

ss2 G'rv~caA cvc~r~AC,~caccaA AccccUc 684 CAGCLJGG CUGAL7GAC~CCGAAAGGCCGAA AGACCCC

685 CC~~UG ~ GCCGAA AAGACCC

709 CP.GCGw"0 CLIGw~IJGAGGCCGAAAGGCCG?~A
AGUCGGU

721 ~ccc,~tro ccr<~c~aAAC~ccaa A~cvcAG

725 UC'.~:~CC C('G~.UGAC,GCCGAAAGGCCG?~

735 6JCGAGA CUG-'~UGAGGCCC.-'-~GGCCCAA
?.GUCGGG

737 :~F.GiICGA CLGAL'G~CGG~ACvCCGAF, AUAGUCG

739 C.~G'JC CUG~.UGF.GGCCCJ~J~AG~CCCv'~a ~?~LTAGU

744 CVCC~1 CGGAUGAGGCCGT~fi~rGGCCGP.?~
AGUCGIAG

745 nCuCG:~C CCGr..Ur~GGCC~'w~rra' "G~w~C~.~ar1 '~,.p.GJCG?.

;53 CLi~CCCA CG 'G~'G~~~C~'~'"G~u~~C~~r~
ACLCGSaC

763 C CUGnDGGC~CC~CC -C~ .~CCCGCC

65 CC~~G CL~F~UGAGGCCGF~r.Ci-~~C~.~1A
AGACCJG

r 68 GnUCCCzI C~JGADC'~A~GGCCG~. AG'JAGAC

769 UG?.IJCCC CDGAI~~GGCCr~F~AGGC.~_G~
' ,, 775 C~~~~' CUGr'a~aF~CCGnr'~~ AUCC.~r'~A
aU

778 C C~~~ ~UCC

801 "~"WG CtTG:~ ~ '"CC~.~ AL~WCG

808 GWDGC~C"s C.'I7C~CG?.nACvCC".,?.?i ~Cv~JLJG~

8 09 C~~JQGGG '' ~ "~,CC~,y~ ~,?.G.~"UL'G

E20 G:.-~.G:u CUCAC~i~T~C~.,CC"'.~?. ?~G~GJU

833 AL'AAAGG CLT3A ""CG?~. ?rL'C~ GGG

837 C-;~JAAUA CUGAL~GGC C'GCC?~r1 AC-C~UCJ

838 G.:~"DAAD CQGF~I~F~GGCCGAAAGGC~ ?.~.G.~~
G?,U

839 G:~;~"CTAAC'CfC~LIGAG.GCCC,~F.F.G:~...~C'".-~
. ..

841 :.G~"U CUO;ADGAGGCCG~AGGCC~.~ ~RAAGG

842 CAC C~I~AGuCC~~AAAC',GCCGAA
paUAAAG

849 UCL1GF~AG " C"".~1 ?~GC~

852 G1GL7CGG CUGADGAC~CCGAT~F~GGCCGAa.
~?~C~..nG~G, 853 C~,JGUCR CDC~L~C-:~CC 'GAn~~,CCG?~r~r n?G::AGv 863 F.GF~JU CL1GAUG~C-GCCCA~C:~CCG~.?.
a~~UGLT

869 G~.~'".J~AACUG:~ ~ ~ =~C-~3G~

"071 C~C~C'"'.J~GCGGAI7""uAGGCCGAFaF~C~,CCCsrl~.
A,G?.G.~'W

872 UGAGCC:A CtIGADGAGGCCCAFu~GGCC"..:A?, a 878 UCUL'WU CUGAI~GGCCGARAG:,CC~~.a.
.~C.~""~AGA

890 FGCCCCC CUCriU~~CCGF,AAC,:~CCGa?r ?~'v'tJCZTCU

898 C~:ACCCU CtIGALIGAGGCCG~G.;CCG:~
aGCCCCC

899 CCGaCCC CU~"C ' ~~C~~~?a iaAC~CCC

904 G:N"DOCC CUGADGF~CGAF~~,CCGaa ACCL"L~1P, 917 A~Ci1 CUGAL~~GGCCGAAAG~CG?~ aGC~JC7GG

916 FJ~AGDQC CL AGG:.C~.~rl ~.~G~~WG

X24 t~~~ cuGA~r~G;~-~~c:~~cccaA a~wcvA

S25 GWCCW CDGAL~,A~GG.."CG~AC~.CGaA
AAG<JDCL7 926 CIt~JDG~~UCZ1C~~G~CCC,T~AAGGCCGaA
A~F~

945 G~TUCG CZtGAIIGAGGCCGF~ACvCCGaA
AGUCr,~t,'G

946 AGu~DWC CUGAC~" ' " CG~1 F.i.CZGGtT

59 nULJCCUG CUO~CGF.A~vCCGAA rIUCCCAG

960 CAUUCCD CUGAUGAC~CCGr'~AAC- GCCG:~' iar~LiCCCA

1001 GAF:UUCLT CL1GAL7GAC~CCGCGAA ?GLJG~JL7 1007 CAG(JUUG C'CfG~~I~GF.GGCC'GAAA~CCG:~A
aWCWA

1 008 CCAGZ1UU CUG~~I~sA.AAGw~CGAA Aa ,L~JCL1U

021 nG~COG CUG ' ~ CG ~AAG:~CCGAA n~CCCC

1029 CCCCAGL1 C'DGAIJG~.C~CC'GiT~AAGvCCGr~.e1 AGL'LJCUG

2040 G CUGAUGAGGCCGAAAC~CG~A ~.G.-CCCC

1046 G:~ s~aUC.~CUGALTGAGGCCGAAAGvCCGr~.
:sC-~"UGUA

1047 " '"~ UC CtIGAUGAGGCCGAAAGG:.CG~A
AAG~.~LGU

1051 UGUCAGG CUGALSCAGGCCGAAAGGCC~~.A
AUCAA.AG

1060 CAVtICCA CUGAUGAGGCCG:~.AGCZCG?.A
Ai,'GUCpl 1067 GLCDCCA CD -CAL"'~C~- , i-.~,CG?.A
AWCCAG

1085 e'aG'~ACCACDGr~UC~C~,CC~u~GCZ~CG'Aa aG~vC't?CC

1086 CAG'~ACC CUG3LT~C~C -CA~GG..~CCz?.a, AAG~.,~UC

1090 U~~ ~~ ~~

~ 091 C~JG~..C.ACOGr~IGF~aCCGaAAG~ICC~1A
AACC'~Ae~a 1113 UCUtICUC CUGnL - ~~CG~nAG~CG?.ar AGUCCUG

12 4 OCLAG.~"U C'0~"C".:ArIAGGCCGAA AG~7CCW

1~9 CAAUQUC CUG~DGF~G:~ :~1AAGGCC~.~A
AG~TGaG

1135 UU~7GLIC CDG~GGCCG:1AAGG..~CG?J~
~ aUUtnCUA

1151 :~.G:~CIJ CL~C~CGAa AG.~"UCCA

11,52 G~AGuCC CUG~~GGCC~C~~ A~G.~~UCC

1158 A,G'~C~GG C'JG'~G?.~"C'',J~AAG~C'".-~
~C'LTA

1 1 -G~n~' ~Ge~ ' ~.~"~W.: ark u~~~.CU
S~ -rya 62 CC~'~~-.'r.C~TGA -UCH' ~"aCCGCCe~A
-Cue' AG ar'aAG~~

1164 AUCD~"C~A C~JGGGCCGAAAC~CCG~A AG?.CGAA

1166 ~ ACAUCL'GCL3G~C~.~AAC,:~C -CAr1 AG~GG

1174 WCUG.~-~~ CL'Gr'1I~GGCCC,aAAG.;CCG.~

175 ~CDGV ~' CCGAA AACAUCCT

11? AAf~COG CZrc~AGGCCCAAAG~~CGAA AAACA~7C

1183 C'CC~.C' CQC,?.DGaGGCC 'CuA.A ~;~CCG?.A
.~., AGDCUGa 1184 UCUCr'ar~aG' ~CCGnA~'~~CG.~A AAGUCUG

1187 GCGiJCUC C'JG~CG~?.~A, AC,AAGL?

X08 CC~UGGG CU ' ~ CGAAAC.GCC~.~A, ""

224 :~L~G~G C~UGAGGCCG:~r~AGGCCGAA
AGCI7C~GGC

122 ~I~AALTA CLTGAC~CG?.A AC~AC,C

'_230 AC.~AA CI,n~~C~CG:,a ?~

1232 AAAG~UA C'C~C'C~AGGCCCaP, AUAGAGG

X33 C-T,AACAU CT~C-CGCCC~A AAUAGAG

234 C:~ACA CZtC:~C~CG~1 AAAUAGA

1238 AF~G~'G:A C~TG~DGAGGCC'"~AAAGGCCG,?.a, 1239 CAAGnGC CUCnL7GAC-GCCGAAAGGCCCaA
AACA~A

12 4 UAADCAC C'Cit~AGGC~CG?..~ AGt~F, 1251 F~UAAP.U CO'C~GGw~C"V?.F~AGGCCGrIFa AUG'~CAA

'_252 LIP.nDAAA CUGF.~AGC,CCGr'~AAGGCCG'aA
AAUCACA

X54 r'.~At7A C'JGA~vCC"CG?.A AUAAUCA

X55 AAAUAAU CU " '' ~~'C.'C~AA~CCGAA
AALJAAUC

2 5 L'~AUA CUGF.L7GAGG~CCG'r.AACGCCGA?~
6 A " ' U

i 258 A~UP.FaAU CUGJ.I7GFaG.~~~C~.:~.AAC~CCCAA
AUAp.AUA

2 5 ~UAAA CUGAL'GAGGCC'G~AAGGCCCAA
9 AAtmP.AU

12 61 AUAFAAUA Ci7GAi;~C~~i~GCCGA~'~CCGAA
AUAAUAA

12 62 AAU~AAU CQG3.L7GTaGGCCG~AAAG~~a~CCGAA
An~AUA

1263 ~AU~r AA CQG:~UG~GGCC'GAAAiGGCCGAA
AAA~AU

1265 QUA CLTGAt7GAGGCCGAAAGGCCCAA
AUAAALJA

12 6 A:~F.UAAU CUGADGAGGCCG?.~AG~CCGAA

267 L~UAA CUGAL1GP.G~CC~CCAA AAAUAAA

12 69 .~L~.AAU C'tJGAIJG~G~CG~'~AC, ;~~CGA?a AUAAAt~r 1270 r'~AUAAA C'UGAUGAGG.~.C 'C~AaGGCCG~.A
nAUAAAU

1272 AUAAAUA CUG~UGAG~CG~.r"~AGGCCGAA
AUAALTAA

1273 AALg.AAU C'DGAiIGAC.G CC ' ~~CGA?, AF,UAAUA

.274 :~A CUG~UG~CZC~-Y.AGGCCC-~ A~ALTA~rJ

12 7 G'.RAnUA C~-.~~. CGa~'aaG;sC C -Cnr~
6 nUAr'~e~LTA

1277 L'G,TAF~U CGCAUGAG~C~~aGGCCG?~?~ aai~AAU

27a c~c~A cvcaac=J~cGaA~aGCCCGA.~
A,~az~.aA

1280 AUCQGIJA CUGaD~.GGCCC~GGCCas AUAAF~UA

81 C_'~TJCUGUCUGACC~~~A'r~C~CCG~ ?~L~Ae~aU

1282 UG.VCUG CLTG?.UGAGGCCG?~hi~GGCCG~1 arZAUAAA

1294 ~LTA~A CG'GAL'GAG:~cC~CG~ ~.G~.UUC~

1296 CC'~LTA CUG?.UG?~-:ACC~.~.GGCCG?a AJAC~UU

1297 CCC~U CUG:~UGAG:~CCGaAaGGCC~.~?, ~L~C~U

1298 UCCChAa CGGaL'GAG:~::CGA~G.:zC~.:
~?. ~AL~CA

1300 U ., ~:A C~'c~.L'G:~c-:~Cc~.~cvcccaA
avap.AVa 13 O GuC'JCCC CTTGaL'Ci? C-~C C G3~C~sC
1 C". :~.rl ABU

1315 CCCaG~~r CUGAL'GAG~C -'G~C,GCC~~
aCCCCG:

1317 CCCCCaG CUGAL'GnC-Gv.~CG?.r~~.GGCCG?.A
AUACCCC

1334 CAG,.'VCC CLiG~.UGAG:~..~CGAA~.CvCCC_~.?, aCAtJ~;

1345 v~- CCC ' ~ C'GAA.AG:~"CG~P. ~.G:~.G~

13 5 C~G)CQ CQG~a~CGaAAG:~~CGAA aG.."CAA~G

13 s c~GGaA a~aL~aCr~,,.~CGaa ac~uc 1360 OCr~CG.~~ C'UC,AUCAC~ sCC~~ArIGGCCG~, r~.CAUG'LJ

1361 L''vC'~.C:~CGCaU " ~~CGr'u~r'r:,CCG~a i~.ACAUG

13 62 L'L'C~C~CGCUCAUGAC~.C -CGr~. A~.~.At~AU

1386 AACJ~GCC CGCAL7GAC~CC~:aCC"'~ ALltr~WC

1393 AGy.UG:~G CLIGnBGAGC-s.C~~GGC.~CGnr~, AC~GCCU

1394 UACaUGG CUGaDGnGGC ~ AACAGCC

1402 >G~:~C CQGAL'GAGGCCG~C-:~CCG'..a aGUGG.;

1414 CUGAL'G~C- GCCGA~:~CvCCG:~a AG:~c CAG

1422 UC_'-.?.SAGCUGALCG~AG:~CC -CA.~ :~G.~cACA

1423 :,LG's~Ap CUGA~C,GCCCne~r'~CvCCG?s~.
'-~nG~~.C

1425 UA~UC'J~A CQGADG~~C~.~:~AC~CCGaA AC~AGGC

1426 :,L~UCA CZJGat.r ' ~~C'C~F.AC,:~.~CG~
:,aC'~aGG

1427 CAUAAUC CUG~UGAGC-CC -CJ~.A.=.CvCCGAa AA~.CAAG

1431 'A.~.isCAUCUGnL~GaC,.~....~CGAaAG.:~~C'.,~
i :~UCAAA.A

1432 A~.~.ACa CUC,~,CCCa~,ac.~,~.cG,>~
aAVCaAp, 1436 Dt7~,~',~ACUGaLR3AGGCCGA~.1G"CCGA~, AC3iLIAAU

1437 U'JUL1AA.ACUGa " ~ CG''nr'u~.GGCCGA?, aACAUAA

14 3 AL'atJtTAP.CLJGAUG.F~C-G.. -CCi,~,F,CT,C
8 C -C:~?. ~.nACALTA

1439 t?~W~, CUGaI3GaG;~:.CGr~AAGGCCGa.A
AFa~.ACAU

1440 hL~LUUn CL-GAL7GaGGCC ' 'G~.,r,GGCC~~.
?.AAp.ACa 1441 :~.LTALW CUGaUGnC~GCCC~liAaGGCCC:Y, '.~.~F.AAp,C

14 4 CAG~sUAA CUGaL'GAGvCC'GAAAC,C,C CGaa, 14 4 T~UCAGAU CUGA ~ C'GAA AUAIJUW

1449 AaUCAGA CQGAUGA~' ~ CGAA AaUAUUU

1451 Ut'..T,AUCACUGAL1GAGGCCGAAAG:~CCGAa, AUAAUAU

1456 i~C~aCUU Ct3GAUGACvCCGAi,aC~,CCGr~A
.'-.UCJ~GaU

1457 GAG~CU CUG.~L'GyGGCCCaF~C1-CCG?~, ~AUCAGA

1461 L'L'LTFaG~CCCiCyaUGAG.;CCC=~.T.GGCCCPA
i_CWAAU

1464 L'UGU-uZTACL'GaTJGC-v.CCG'~AaCvCCGr~?', ~?CAp,CU[7 14 66 CJ~L'UGUU CUGAUGAGGCCC-'-AAA G'C-CCGP.A
: ~GaCAAC

1479 G?CACCA
C'GGAUGnC-GCCG~r~C-G..~CGAA
AUCAGCA

1480 G.~"'JCACCCGGAI7GaGGCC~CGAA AAUCAGC

1494 AAUG~ CQGADG~C"'~AAG:~~CGAA AG'sG'1UG

1498 CAGCAAU CffG:~ ' ~ ~ ~~CG?~A AGUGACA

1501 CCUCAGC CL7GI3GnGGCCGAARGGCCGAA
AUGAGUG

~~Gv~CA CUGnUGr~G.~~~~CGAAAG~~a~~CGAA
AGGC'CG'C

1517 CCCQGw CUGADGAG:~CGT,AAG~~C~~AA
AG~GAG

1528 CAGACAC CUC,nI7GA,G.:CCGr~~AAG.~C'CGAA
ACUCCCO

1533 GADUACA CUGADG?.GG.C'GAAA~CCGIPa AC~CAAC

1537 GGCCGAU CLIGAtIGaG:~CGAAAGGCC~.~A
AC.

1540 GVAGGCC CLT~C'G~AAAGGCCGAA AUUACAG

1546 ~UAG C'~.DG~'"~'C~AAA~CCGAA
AGG.."C".~

~ S5Z CGCCACV C'UGADGAGG:.CGAhAG GCCGAA
AAA

5ss c~accw cvGAVGnc~cGa~AAGuccGaA
Avwcac 1572 CCUAAGC Cn~~GGCCC ~ ACCUO'QA

576 ~ ~G~~G"CG~A AC~ACC

1577 UCUOOCC CTtC~AG:~CCC,AAAG:~CCGaA
AAC~AC

Z5~
Table 2b: blouse T.NF-a HH Tasget sequences nt . HH Ta.rQet SeQvencent . E8 Target Seqveaea Positioa Foaitioa 60' VcG~AU a GcucCcA 324 Gg~U C GCIiCCCC

101 G~G~"U U CUgBcCC 347 GaGAagU a cCCAaaU

1 O1 c~:~GgU u. CuGUccC 3 64 CCVCcCU C Uc~IDCAG

02 C1-~CvJU C US~UcCCO3 6v LC:CCICU c ADCAGuu 102 cCaGgUU c ugUCCCO 360' UcCC'GCU C auCAGuU

106 GUUC'UgU c CC'UuUCA3 69 CUCUc~U C AC~.~:uCUa 110 UgUcCCU a UCALI~cA 376 CAGuuCU a UC-GCCCA

111 gUCcCW a CaCDCAC 390 AgACCCLT C AcaCQc~

111 gLCCCuU a C.AGIiC~c396 ucaCAcU 'C AGaDCaIT

~ ~ 2 UcC'C'QuU C ANcA~CO401 cUCAfAU C AUC'WCp 116 LJuUCACU C :~cUGgcc404 AC,AUG'1U C UUCUCaA

137 G~CaCAU C uCCcUCc 406 ;,UCADCV U CUCaAAa 139 caCAuCU C CCUCcAg 406 AVcADcU U cUcaAA~, 177 GCAUG;~U C CGcGACG 407 UCAUCUU C UCalAau 207 AC~CU C CCCcAsA 409 AUCZ7tJCU C aAaauuC

228 G:~.:~C'sU C CAGF~ 409 Au~uCO c AaAAUUC

228 G~.'1iU c C~.GzacU 409 aUcUUcU c AAAauOc 236 cac-~cu c c~.-~;~ 432 acccoGU A cc~,cc 23 6 C~GaAC(T c cAGgcGg 249 G..~-ugCCU a UgUCUcA

249 G.~"~GCCU a UGucUCa444 AcGUcGU A GCAAACC

501 AcGCCCL1 C COCA

2 61 UC,AGCCV C UUCUCaU 5.60 gGgUOGU a CCZJuguC

2 61 UG~CCLJ C UL"C'L7cau5 6 0 GGguUGU A ~gpC

263 AGCC'UCZJ O CUC~1L~C564 Dc.~GCp a gUCOACU

2 63 AgCCVCU U CUcruUC 56? ACCUugU C UACLTCCC

264 GCCUCUU C UCaUUCC 569 CUugUCU A CUCCCAG

2 64 gCCLICUJLJ C UcauUCc572 gUCUACU C CCAGGLJu 266 CUCUtICt1 C aUUC:.UG572 GUCUaCU c CCAGguu 269 UUC'UCaU U CCUGcDu 572 GuCOacU C CCAgGUu 270 UCUCaUU C C"JGcUuG 579 CCCAG.~~U a CUC'WCA

276 UCCUC-cU a GJC:.~:~G580 CC~IGguU c uCWcAa 297 CG~C''~CLT C UUCL~C580 CCaGGuU c UCuUcaa 299 ACGC'JCU U CUCI:CDa582 ~1,C~Up~ C egg 300 CGCQCUU C L'Gl~CUaC582 AG.,~UuCU C WCAAGG

304 CUuC'UgU c uAcUGaa 584 GUuCUCU U CAAGGGa 306 UcUGUcU a cUgAAcU 585 UuC'UCUU C AAGGGaC

314 CUGaACU U cG~eGUG 608 CcCGaClJ a CgugCUC

315 UGaACUU c GGgGUGA 615 aCgUGcU C CUCAcCC

315 uGaaCUtJ c GGGguGa 615 AcGUGCV C CUCACCC

324 eGCUGaU c GgUC~CcC 618 UGCUCCU C ACCCACA

63O ACACCgU C AG'~ =au 94O GuCLTACU z cUCAGaG

630 A~CCAU C AgCCcaU 943 'ur'~.C'UccU C AGaGcCc 638 aS~6AU a uG~~JaUc 972 UC'Ja,aCU a AgA?.AGg 643 aUUt7GcU a uCLIG~uP.972 ucUa~CV a AGAa.AgG

645 UuG~~~aU C UCatTACC 973 CQaACuU A G?~AgSG

647 GCuaUCV C aLTACCAG 984 ~GgAU U auGC,cuc 663 agAAa~ C AACCQCC ~ 984 AGC~gaU U atlC~IJc ' 669 UCAACCU C CUCUCL'G 985 G:,:~auU a LC~cUCa 669 Uc~AccO c cDcL~JG 997 UcaGac~U c C3Acscu 672 ACCUCCU C UCL'GCCg 1010 CuguGCU c AGASCW

674 C'JCCOCU C UGCCgUC 1017 c_CAgC'J U Uc~.aC~

681 cUC-CCgO C AaSaGcC .018 aC?~5C'L't? U c~CAJ~C

681 CQG~.~CgU C AAG~GCC 1019 GAS~CLyJ c ?~C3s?G1a 6g1 CUGcCgU C aaGAScC _073 USC:~.~.CLT c ucyUgCA

734 CC~"U A ACC lOg6 A~.GgAcU C ?.?AugCy 734 CccUG.~"U a ugaGCCc ? 106 aCGwcU U ucc~.~AU

744 AGCCCAU a L~cCDG:a '? 07 UG:.-:~cUU a ccGAAUu 746 CCCAUsU A cCUG~r "08 GGgCIaW c cGaaUUC

759 GAgG~U C uuCCAGc "'_5 Cc~-..AauU C ?~CUGGaG

759 GAGGaGU C ULJCCAG~. ~?.33 CGAAugU C CAL~iCcU

761 GGaGUCU U CCAGCUG 1164 cacLT,gU c AgGJUGc 762 GaGUCW C CAG.."UGG 1180 UcTJgUcU c agaAUG?, 7 8 ACC~ACU C AGCG~~OG 12 03 zz.G?s:CU c AGC-CCUtJ

798 CUGAGgU C AAUCuG.~ 1210 cAG:CLJ U C=L7acCU
~

802 C-gU~B C uG.~CCaA 12? 1 AGC-~.' ~UQ C CUacCUu 8u CCCzAgU A cuUaGaC 1214 C~'LCCU a c~JuCAG

816 AgUA~U a GACUWG 1218 CcvACcU a CaCACCu 821 uUaGACU U UGCgGAG 1218 CC.:aCCU U C~CnCcu e22 UaGACW U GCgGAGU '?18 cC.:F.CcU a cngACCU

E30 GCgGAGU C c "G.~AG 1218 CCUacCU a C~G?.ccU

840 ~'"U C G 1219 CuaCCUU C ACACcuu 842 CA~'~ A ~a ~?1 g C~Ac~UI1 c agACcUU

842 CAGgucU a CWucG<1 1226 CagACC'J U uCCAgAC

842 cagGuCU a CQUOgG?. 1226 CAG~ccU U UC~AGAC

845 ~C~U U UGGagUC ?227 agAC~,~ a CCASACu 846 UCUACUU U GGagUCA X27 AGAccW U CC?.C_BCLJ

852 UUG: agU C AUUGCuC '_228 GAccWU C C'~G~.CUc e55 GagUCAU U GCuCLJGU 1238 gACL'CuU c cC'~'~G,AGG

8g7 AUCCaUU c ucLRCCC 1.262 CAG.."'CIaU C CuC~caG

891 AuucuCU a CCCaGC'C X83 CCCCccU C uaL'LJUAU

905 CCcCaCU C UgaCCCC X83 cCcCCCLJ C UAUUUAU

905 cCCCacU c UgACCCC '1285 cCCCUCL7 A L~7UAUaU

905 CcCCACU c uGAccCC 1287 CcsCVAU a UauAuUU

914 CAcCCcU U uacUCUG 1287 C~JC'UFaU U iTAUaUW

915 ACCCCuU.u acUCuGA 1288 CUC'JAUU U ?UaUUUG

919 CUUUAcU c ugaCCcC 12 89 UCLA u'L'U A LTaL'L1GGC

g28 GACCcCU a UaUugUC 1293 UULT',.,UaU U LC-~:CUU

g2g gAcCCCU U UAUUguC 1293 uWaUsU a LTGcAcUu g32 CCUUUAU U cuCuaCU X94 L'tJ'T.~tiaL'U U GCACUUa 100 WG~ACQ U aUuAUUu 1462 aCCuUGU GCCsCCU
a 1303 CAcuUaU a AuUuAW 1470 GccuCcU UUWGcU
C

1304 acDuAUD A U~sUQ'A 14?2 cuCcUCU uZJGcUUA
U

1306 UuAUt~U U UADUAW 14?3 uCcUCW U UGcTJLTAU

1307 uA~I7U U AULTADUU 24 i4 CcUCUW U GcUUAUG

1307 UaWaW U AuuADuO 1478 UUUUGcU AL'GLJULIa U

1308 AU~UULJ A UU~~UOA 1479 UUUGcW a UGuuuAa 1310 UauUuAU U AUUUF.LZ7 1479 UUUGc'"1U UGUUUaa A

1310 UA~U U AUUL~DU 1484 UUAUGW U a.aaAcAA

1310 UAUDDAU U AUUUAW 1498 AAAuauU AUCL7aAc U

1311 AUUUAW A UUUAUUU 1511 Ac c cA~aU GUCUuAA
i1 1311 :~aoaW A ~~ ~ 514 c~WCw c vuAAuAA

131? AuuDAUU A UuUauW 1516 aWGUCU a AAuF.AcG

1313 L1DAUQAU U UAWUAU 1529 CgcugAU UGG~GAC
a 1313 WADL~U U UALTUAU 1529 cG..~UGaU UG~3GaC
U

1313 u~U a UauUDAu 1530 gCUGAW a gGUgacC

14 U AUUtIAUU 15 3 0 GC'UC,AW G"~UGaCC
U

1314 U AWQADQ 1563 UgaAcCU UGcOCCC
c 1315 AUUAUW A 1563 ugaaCCU UGCUCCC
C

1317 BAUD~U U ZIAUUAW ~ 568 CUCUGCU CCCAcG
C

1318 AUULIFsW U AUUAI1W 1589 UGaCUCU AUuGcCC
A

1319 UUiRUW A UQAUUUA ~s92 CUGUAAU GcCCUAC
a 1326 AL'QADW A UGUAUL1U 161? GAG:~AU AAGaUcG
A

1328 IRUWAU U BAUWgC ~ 1623 UAAAGaU G~~UUAaa c 1329 AUUUAW U AULIUgCu 1633 UUAazaU a.aAA,aCC
a 1330 UUUABW A UWetl:u 25 AcGSaCU gCCagGA
- a 3 2 LTr~UQLmU U UgCuuAU

13 AUUUAITU U gCl:uAUG

1337 auUUG."U U AuGAAuG

1 3 uQUGCL7Q A uGAAuGI~

13 L:C~.AIJGU A UUUAUW

13 AAUC~U ~ L?AUUUGG

13 P.UGUAW U AWL7GGa 1350 UG~DOU A DWG~GaA

13 uAUuLTAIJ a DGGaAGG

1352 UAU~U U UGGaAGg 1353 AUUUAW U GGaAGgC

13 G;~:~.~~UgU C CUGGaGG

1398 gCUguCQ U cAGACAg 1398 G,.'"UG~CU U cagaCAG

1412 GACAUGU U WCuGUG

1413 AcAUGW U UCuG~A

1414 CADGUOLT U CuGL3GAA

1415 AUGUUW C uGLJGAAA

1415 AUpU(nT c UgugAaA

1438 gaG~."UGU c CCCAccU

1451 CUGGCCU C UcUaCCU

1453 ggCWCU C UaCCuUG

Table 26: 1'Iouse T'~F-a Hammerhead Ribozyme Sequences Mouse ~ Ribczyme.SequeaCa Pos'_t'_oa 2~ LCCL'GGC CG -C~:~.C~ '~C~~.,~~CC'r~1 aGUCC';."J

60 C'IJGAI~C~CCG~.rIaGGCCGAA
A~7UQCCA

GC~1CAG CLT?~L'GC~:a...' ~",r~.~'.aG.~~~CG?.rl ~CCu'G.."'r' G.~sr~G~GCDGraLiG.~G..1V -"C~?~Gu~."C:s?~e1 ACCDGv."'C

.C2 ~ C'JG1L'G~C~..":.'~.~.aAGGCCGaA

i 02 AGGC,AC~ CUGAtIG?.GC,~~~CGau1 A1CCUGC

CC~a:~ CUGUG.~'~C~'CG1~1 '~G;C,?~rC

'.0 ~G~GJCA CL'G?iLTy,GGCCCv~r~C,~,"CCG?.A
AGu:~C?r GCG~,GOG CGG?.UG:~GGC ' ~ .~IGGGaC

GL~.r'-'~aJGCL~~.DC'v'~C,:sC -'CGn~G~.~CG?.A
Ar~~ S~?~C

..2 '~v -~GLJCVC~GnG:~CC _CAaAGGCCGAA
AnAGGG~

GC,CCJ,GQCL7Cs?.U -C~'~C".J~l",hGGCC"~
AGL'GAAa ~~7 G:~C~A C'JGnL~~C-v,CCG~A~GGCCG~
ACGCGGC

35 CL'GG-'~GGCv?G?.L -'G'~~~vCC~u~r~G:aCC".ylA
AGnDC~L'G

/ 7 CGJCh. CUGn ~ ~ C~"AA AUC~.
V

207 ~~,w CL'GAL'C,?~GG..~'-'~:~GGCCGaA
AGJGCCU

228 ~JG CCGI.~G:~. ~ w ' AAG.~CCC

226 :~GJUCUG G:~.~CGAA AnGCCCC

236 CC''w,:.CUGCUC,:.~-CC~CGAA AG'v'UCUG

236 CCC-CCRG CLiG~L'C,aG:~~CC?v~GGCCGr~/~, AGUUCUG

249 LG~G~G'~ CL'G:~L'G~GCICGInnG~.~CGna i~C~' CC

L4S ~GAG.~CA CL'G~.L'GaG.~C 'GMAC~CCGA?, AC,.~.~CC

2 51 AL'C'M CL1G:~T~AGGC 'C'G~AGG.~CC,AA
AG~'ZJG?~

26. :~L CQGALG?.C:~.,."C".~:~?.C~CCGAA
AC,G..'vG?~

253 -C~F,BC,AGC~G:aL~~GC-CCG~AaGGVC~.~A
AGaGGw~U

253 G:~.T~QC~GCUG:~L1G:~GGCC~.~J~AGGCCGAA
AG:,GG~~U

26C G:~tGA C'CGa~ICG~AC,C,cCG.~ AAG.~GVC

264 G:~ADGA CL'G?.iTGAGC1~ "Grv~C,.GCCGAA
AAGr~GGC

26c C.AG:~.AUCUGnL'GnGW.~~'v"~~CCC~AA
ACvaAGr'~C', 269 CZ1G:~L~C-.;AAAGGCCGaA
AUG?.GAA

270 CJ~AG.:.:~GCUC~L~GGCC 'C,~.G~CCGAA
AFB -CC:~

276 CL'GCC.~CCL'GAL~C"~:,CC~~AAAG:~CCGA1 AGCAC,GA

2S7 G CQGAL3GaGGCC " ~ AGCGUGG

2S9 ~ CGG:.CG~AA~CCGAA AG sGCGG

300 GUF~C~CA CL'GGG~CCG'aAAC,GCCGAA
~'~JaC~,CG

3C4 UL'C~'~GIJACL'Gr~1~CG/~r1'~CCG?u~
ACAGAACs 306 AGv'L7G,GCGT~L1GAG:.-:.CGA.A?.GC,~.~CG1A
e~C~GP

31C C?CCCCG CUG.ALT~GCiCGAr~GGCCG?~A
AGUUC~.~G

~s5 LCACCCC CUG.~LIGr~'C11CG~'a 'hr~v'CG?~P.
Ar'~Gu'CrCr1 LC;ycCCC c~~-~r-CCap, a:~uC~

324 G~.;~CC C',1G:..UG~G:~:.C".~,G:~cC~aaA
WG~CCC

3 2 C~CC C'UC'~F~C',~GG.."C"~:~Fu,Gu..
4 ~ GaA .'~'C~CCC

347 nL'L1L'G:~C"'u~-.~~~CGF,r'J~G~~ ".AAA
AL'~CQC

3 64 C"'-'W- Cuu~D"~,nC,G..~C",1 '".~A
?~GGG~C,C, 3 60 '.~r'sC'vG%~sIJCL'G:-.L'GnGG..";:Gi-u~.'~GGC~..:~.r1 aG~,C~

3 6 tu~L'LJ"~UC':~G:~~CG:,F~',Gu.."CC,AA

3 69 ~CmCU C UGF.UCy,G:~CCC~:~AGG:.C"'~A
?.RGaGaG

3 7 UG:~ C?. C~~C": ,~A AG:~AC~G

U~~V~ ~U~~~~1 W

3 0 FCC C~ . ....C..

401 U CL'GAUG~:..~.~AF~CC~. aI7C~G?l 4 04 UL~~-' C'JG~G:,..~.~:v.i,C~~.
'.GnF. ~.:~A aL~J

406 L'LTtn~G CUrGAL'~G:~CCG~1G~~C~..:?A
aGaDG~U

406 UtILJL~G CUCGAAAGC~C"~A aG:,UG~LT

407 nUUWGa C~'C".~:~F.nC~._"CGr~.
F~

409 G~I~t7W CL'GF.DG;~G~~CCGaF~~C'.~A
.~'.aAGaO

4 09 GF AUU~ CUG%~I,~"'CG%,AAGGCC".~A
AGaAG?~D

409 G:~L'L7DV CUG:~T~"C~aAAWCL"'.~A AG
aAGAU

,32 CGUG:~ C'U'~:~G:,CC~~el~uuCC'C~A
"O

Y44 WJL~GC CVG~'~..~A aCGaC~J

01 UCNC.r,G CUG~UGG.~..C~J~C"~CCGAA
aC~CGLl 360 Gr.C'~ CU"C~VAF~GG..' ~GAA AC~CCC

9 6 G:zCJ~.F.GGC~UG~G:~ "CG~ ~ACCC

364 C CDG;,UG;~G:~"C;~,:~.:~G:~CGr.A
AG.~u~C.~

6 C ~~' ' CL'G~L~~r'aGw C '.~.r~G:~C
7 G~ ~'GaA r~C~IC,~.~,J

3 6 C'JG.-uAG COGAUG:~C~'~C C~~F~C'~"~
9 ',.~A F~G~C~IaG

.72 F.F.CCL'GGCL~ :L~C:~F,F.GGCC".~ AG'~rlC

7 2 ;~,:~c'UGGc,JG~UG~G;~.:."~=.aFu,.~crcaa AGCAGAC

5 72 ~C'CL'G.G CZ~~CGA:~AGC'".C~.~A T~C

579 LTG:~AG?~GC~G,.~1JG:~G:~CC~CG?p, ACLU

a o L2-~;~;~a cvc~v~ "aA apccvG

9 8 L~AGA CUG1.U~AG:~C'G~~GC~CC""ap, 0 ~lCCipGG

c 2 CC'JQGnA CLT~L~nC~ CGr'~aF~,GC,CC
GAA ACnACCU

~82 CCUUC'~A CL'Gni~C,GCCC,a'CuAA AGnACCU

304 UCCC'JQG CUt:ABGAG:~."CG~AC,GCCC,?.F.
AG:,GAAC

525 GuCCCW C'.fi~nL'GF.Gu;.~.~ni,GGCCG~A
F~FaGnGAA

608 G~G~CG CUG~L'C~G:~CCC~.F.AG:~:.C~.~,F.
AGJCGw 6? G'-,,~~.G CUG~U"'~'~G~CCC~F.C'NCCG~.
S AGC~CGL7 6.5 GG...TG CL'G:~L~GnGGCCGAF,F,G:~.:C~~A
:G AG.:.ACG'CI

618 L'G~JGG.~"UCUGF.LIGnC~.CGT,F.T,G;,CCG?,A
AG:~~GCA

630 F~UCG~CLJ CVGF.L'G:~Gu'C".~:.u,AGGCCG
AA ACC~.~"'UGU

630 F.UCG:,CV CUC'y~IaGAGC~CCGAhF.GC,CCG?.A
ACGGLIGtJ

E 3 G~IJAGCA CUGF~~C".~F.~AC'~CC'~A

643 L"nUG:yGr1CUC,AL'GAG:~CCGi'aAesG:rCCGrIA
AGCAAAU~

6S5 G.~uT?~UG~.C'~'G:yUGAG:~:.C~.~.=.AG:~CCG,?.F, ALTAC~A

04 CUG.~''IJAUCUGr.UGAG~CCGnMG~CC:~.A
7 AGAUr.GC

663 ~~"W CZ1G~.UG~.G.:,~'"C " ~,C~:~1 aCLJ01'CU

659 C~CvaGaG CLGaUG:~G:~C 'CG~G~CCCalr1 elGw'~'~?~

663 'C'~rcCGF~F~r:G~CC~~.1 aC~""G~

672 C"~~~~~CC~.rI AG~:~G.,"U

674 G,:~C"~ C'L~GAL~G~CC~~AiC.:~.."AiGAG:~1G

681 G:~JC'Ut7 C~CC~AAG~.."C~',arrel aC~w:rG

681 GG~"LJC'D'U COC~UGACHCC'GAAAG~.."~A
AC~.~G

sal c;~wc~oo cvc%~ca~.~~aa~CC~;a ac~"',~

734 G:~.."CCa C~C~~G~CCC~ aCCi',Gw 734 COCA CUGa ~ ' "~CG~1 aCC.~.C~

744 CC~CG~ CVGAI~C, a~"'C;GA~AG'~..'C"~11 aU~."'U

746 ~~ "~

759 G~c,JC:~L'G~C~~'CG~i~aG~~CG?~A aC'~~~

7.9 GC'L3G~.A ' , ....C..~ ~..'C

7 61 CAG."fJGv CUG.'i ~ ~CCGr~ AGAL V
CC

7 62 CC~~JG .

7 8 C:~G~~''.~~U CUG:~au ' '" ~ "CGrla 6 ~J

GCa~itJQ CUG:a~a~'aWC'C~'v~.AG~CCG~A
aCC'CGG

a oz v~-~;~cA c~vcawCC~~.aIG.~CCCaA az~"~cc a, wcv~ac Cuc~v~rcc-c"~~ac~..~ .~ce~;~
z 816 CTu~.GLJC C~CG~ ~AG:,.:.CG~1 a~C'J

s21 c.."CCc-C a~ Cv~,UGaw,.~c"~:,~-"cca~
acvc-~"~.a s22 acucccc c~,cc~aa~:~;.CCaa a 830 C~~CCG CL~GAU"~.G~CCG~F~AC~C~,~CGa~
aCUCCG' 840 CnAAGiTA C~a'~~G:,CCGaAA~~.~.CC~e1 .'~CCL~."C

842 BCG~AAG CUG~DGACG~.~CG~G:,CC~.~?s.1 AGaCCTG

842 UC~'..,F.AG CUGIsDC',r~G~CCG~aAAG~.,."CG~?, AG?.CCr:.G

842 UC~~ CUCu~LIGi~GSaCCGAT,?.G:~.CG~1'~
aGACC'uC

845 ChCUCCA CLIGF~t~GGC~C~A aL~~C

84 UG?.CGCC C~uGnl~a'nGw~,~~CCv'JaAG:,C.CGAA
6 .'.~,GTi~ra 852 ~v cv;~cawCCCc~~ acvcc ;a ass ~:~cc cvc~rc;~~a~cc~a :~czc se7 c;~,.u~cA c"~,n~r~cc~r-.~ ' 891 G~.."DG:~, C17GF~iIGT~G~CCGAAAG:~CCG~r1 AGaG?~1J

905 G~:~.~'RC?~ C'D~OGAG~CC'GAA?~G~CCC~A

905 G~:N~UCA C'CiC~"CGAAhG~CC".~AA ?.G~, 905 G:~.~~UCA CIIC,FaDG:.GCCCGT,AAG:~CC~~Ar1 ....."

914 CnGnGUA CZJGAL7C'a:aGGCC'GAAAG~CCG?~A
aC~u'C

915 UCAGP.GU CUG~UGAGGCCG?.AAG~CC~.~.?~
' ~,.

919 G:~:~"UG'1 CUGAUC~?.C;~CC'GAA.AG~CCGAA
aGLg,AAG

S28 GAG.AUA C'J~UGAGs~.~CGT~T,aG~CCC~r1 :~.~. vC

9 2 ~C~~ CUC,~UG~~ ;CCG?.F~ACuCCGAA
8 :~G:~C

932 ~C~3'.C CDGFaUGAC,G: C.'Cv':AALHCCGAA
AI~AAG s 940 CUCOGAG CUGAUG~CGCCGAAAG~CCGAA C

943 ~ GG:~.."'QCU CUGADG' "~ ..,.C:~AA
A;~AC~

972 CCUWCU CUGA ' ~ AAA~..~CGaA AGQ<TAGr, 972 CC~CU CU~UGaG~C~CCGF.F~G~CCC~A :~G:, 973 CCCUUUC CUGAIIGFaG:,CC~C:~CCGrIF, :,aGLJtTA~G

984 GAGCC.~U CUG'ri~;GGCCGAAAG~CCGaA
aUCCC'''J

c84 G'~.,C'"~nD
CU~~nC"CG1A
raUCCCCO

9 8 L'G~GC. CA C~G~'~G~.,~.~.r~J,C~'",1"'.~A
'r~rDCCCC

og7 .-~G~GCDG Cv~ "CG~CG.~.A .aC~JLZGrI

1010 ;~-acv c~.v~-~~c~~cc~,a ~

1 Ol? L'~GCQGA C'I7G~L~G'.~GGCC~~F.A~~CC ""?~A
AG~'~CUG

1018 ~'"'uG~CG CilG~.UG:~G~.~C".~F~G:,CCG
;a. ~~UC'J

1073 UG;aBG1 CQG:~CIGAGGCCG'rJ,AG:~"C'.~F' ?~G~.CC~

1096 CC~~ CDG'.~ ~CGFu~AG:~.:,.~,~?.P.
aGDC'CU<7 1106 ~L'CC~~ CiJG~~ G~'"C"~.~GOaCL"Gar1 ~1G."CGaU

t 107 :-,:,DpCGG CD~ C'CvCC".yl~, .~G:.C"..A

1 ~ G~a :UDC CL1CCCa~"CGr'1A~'~.'"C"'.i?.A
Q$ .,C

111 ~'CC,'y~ r'a GV.~.~C~~ar'~!~~av.C.~..v~
~~.oV

1133 G CVC~UG~.G~~~P.AGG.."C"..-.~

1164 G:A~CCQ C'~LW CC~"C"~A lCCaCLIC

1180 CCT~UQCV C~G:~L'CAGGCC'w'.~1 :aC~CaGn 1203 1,:,G~"CU C~ C~.'1A AG~CW

1210 C~ -~C~.~AA aC,:~.,."C9G

1211 i~F~G.~~QAG GGCCG~AAG'~..~CGaA
CUG~'~ .~.C,:~CCU

1214 C'~A~G:~ C~~ GGCCGAAA~ AG~~AGG

18 F~C~CLiG CUG:ai~s~',GC~CCG?..'~
AG'~1GG

1218 :,G.~"QC'GG ' "CGAA AC'~:~GG
CU ' 1218 ~G.~"~?CL1G sCaC,CC:~AAG s~.'"C"u CL3GF.L7GF iA 'wa~

1219 ~~ CLIGnL~ G~.' C'~Fv~CCG ;A
~AG:..~G

1219 ~AG.~"'CCO CL~GA. "?.A A?~Cu"UAG
' 1226 Gv7CDG:~?. CUGFL~.G:~CCG~AG~CCG?A
aG~CG'G

1226 GUCL3GG?. CUG.=.L7G;G~~~~CGAAA~CC~.~nA
AG.,u"'CT~G

1227 nGOCL3G~a ~ ~CCG~1A ~C~DCO<

1227 '~G'vC'JGG CUGAVG:,GGCCGA:,AGGCCGa?~
~1G,~.1CU

X28 G~CUG CL'Ga ~ CCG~Fa A?.AG ,CUC

1238 CCtJC:~G:~ C~GAi7G'' ~"C:G?.a AAG:,GC~C

12 62 CL~JG? G '" A~G.~C~G

12 83 ALB,AAUA C'CfC~;C,:aCCCa~AAGW CGaA
"""

12 83 r~Ai~TlA CDGA ' ~ CCGAA AG:~G

12 8 F~~A NG~BG ~C~GCCGA~J,GGCCGAA
5 AC,T,G~~a .2 87 QA CUG~.UG F~LNCC'C~J~.AAG:~C"~AA
F~GG

'_287 iuaAU'nffA CLIGF~t~,GCC'G:fi~FaaGGCCG?~A
~AGG

12 8 Cry' raL'AU ~nGGCCG:v'~AGGC CGr'ir~r 8 CUCALIC FsFaI~iGaG

1289 G~F~ CUGFL~ '.~GGCCGAAAGGCCG1A
AAALmGA

'_293 :~.GUC~.~.A "~C~~.~ F~?,~~
CDGZ

1293 ~~A C'CGAL7G AGGC FJ~AA

12 9 UF.F~GQGC AAI~A

1300 '~L.~r~U :~AAAGGCCGAA AC~CAe'~

?303 'FnI~P" .U C'CGnUGr~.G~CCGnAA~CCGAA
FatTP.AGUG

1304 L~AA CUGAL:IG p~CGCCGAAAG:~~CGaA
AADAAGQ

13 0 ~F L~nAUA CLTGAUGAGG: CC;aF,FaGGCCGaA
6 AITF,F.ITAF, 1307 a,ML'T~FU CvCALIGnGGCC~~.~n~a?.G:,CC~L.u1 r~',UhALTP, '_307 ~Lr.~U C'JG~UGAG:~CC
' ' GGCCG~?, r~UAAt~, 13 08 U''r'.r~~' L?~A CL7GALu:~G~CC~~GGCCGr'1A
naatTAaU

_310 :EAU c,~c~,.~cG~~:~.~C~.:.aa a~.Aazu~

.310 inLRF~.U CUG?.~aGaCC"vaF,rl~~GGCC~va:v~a .310 : '~~ "~A A~A,r~Vi4 13, :~F. CLTG~~UC~.~~CG: P.AG~CG?.A
, aAI~.AAU

;,A;~rr.~a c;,c~L-~;.~;~;.C~~,a~CC~aA
'~~t =a :~AAU~ c~~c~ - ;.~ccu aaz~~

313 i~i~Al~. CL1G:~L'G:,GaU~~~G:~.~C~~' :,Ue~?.~1 '.?13 :~LRAF~~ CuGaDGAGGCCGA<.?G:~CC'',i:~?, ?.~

13.3 AUAAAUA CUG~,LJGaGG.," u:,.~GGCCC?~
~L'AA ~.

13 i1 nAUAAaU C',JG~UG:~GaCC "GnrvaG~CGr'1?r i,.lUP.AI~

:~IR~.U CLT~i~Cv.."UG?J,~Cv~~CGr~
1r'.'t~~w~

s~15 Luv.UFa~A CQGnL -GSC,v~.CC,n?.~C~-vCGl1 .lrz~lLIF?.D

i~.7 :antJAAUA CUG~L7GhGw~GGCCGaA e~U'na?~.1 ~ O '~~ ~G~~~~ f1 i? 19 UAAAVA~ CL1G?.~C~.~AAC~CCG?.A

1325 :-.F.:.~ CUG:~UG~G:~CC~.anA.',G~aUCG?ar1 ?~A13~IT

?..3 G:=-~ CL~C 'G.~AAG:~~CG?~?r .~L~J4~

.329 AGCAAAU C~L~'~GG.. .AAA A..UAAAU

330 f.AG~A CUCT,L~GnC,vCCCG?.r1 .~iv~~

1332 ~c,TC~~.CC~A at~ADA

3 3 CAUAAGC CL7GALT.AC~:~c ~~G:~nC~CC".~?~
3 :~IRAAO

1337 C~CGU CQGAL'GnGC~CC"'~nnACvCCGnA
r~C~~r'~AU

~~38 :,CnDOCA CQGAL'GaC~CCGAnnG~"C"~?~1 :~GC1AA

,346 F.F~~A.A CDG:~L1G~;C,:~C~~GGCC~.~,r1 .1C~

3 4 C~CQGn~F G:~CCGAAF~"C".~?.A ~t'~.CAW

1349 L1C~~U C'LJGAL"~G:,CCr'~F.F,AG:,~~CG~
AhU~U

1350 WCCAAP. CW,?.G~G~CG~u~AG:~CCG?.A
:~.~P~1C~

1352 CCUUCCA CUGaUGAGGCC'".~AG:,~.~CG~

_2 CCLTL?CCA CUG:~UG~G:~C ~.'w'~~',G:~CC~.~:.u1 :~U'AT.aLm 1==3 G:~WCC C'~JG~LK~G~CC~.~A:~AGGCCCAA
?~U

1309 CCQCCAG CUGAUG:,WCC'G~J~A ~.~CC~~
.'~C~.CCCC

'_398 CLZ;LJCUG COGAUC",AGGCCCG1A ;GACaGC

1395 Ct7G'JCUG CL~CG'~CCG:~r1 A.Cr~CAGC

1s12 G'~CFaGAA CL7Ga.L~,hGGCCGAAAC,:,-~.~.a~A

s 1 v~~ca c.~;..vG;~;~CC~,~a~,c.~cc-~"aa ~ :,acaUw 1414 UDC~.CAG CL7GF.LTCCC~CCC,?~T~~CC,A?.
aAAC,~IUG

1 s1~ L'WCACA CUGhLIGAG.~:.C'GAAAGGCC;~1P.
:v,.'~ACAU

's'_5 L'UC1CACA C'JGaLIGnC~.CGAiu~GwCCv'~'a r'.r'aAaCAU

1438 :~G.w'G;N CL1GAUGAC,:~.CGnFai~Cw~cCG?.A
r1C?G~"'QC

14=1 ~~UAGA CuG:~L7GnG.;CCGnAACvCCCaA?.
~CrIC, 133 CAr.G.~"'~i CUGAL~CCC~.GGCCGAA ACC

1 s ~AC~.cc c.~t~Ac,G'~r-.~~AAC~cc:~a s s ~~c.~

14 62 nGGAC'~: C CDGAL~~ ~ .~:,A ACAAG~~U

1470 ACu.F.T,Fa CUGnLIGAGGCC'C~,AAGGCCGAA
?~G~~GGC

1 C72 Ll~' ~~AA CUGAUGAG~,CCGaAAGGCCGnA
isGnGGAG.

1 s ALTAAGCa CL'GAUGr'.CGCCGAAnG~~.:C~.-~'1A

1 s Cr.UAAGC CUG:~L~Cz,C C~~AAAG:~:.
i 4 C~.A ' 'EGG

1C i U'nn~C~U CL'C,?.UGACv.~.CG.' '~'.G:~CCGnA
2 ~G:.'~AaA

1079 ~sCA CvJG~L'GaGC..~C~AaAG:T.CGaA AAGC~A

1479 t7L~s:~CA CLTG:~LICnG:~CCG~',~a~G~..' ~"'.~?.A AAGCF.~1A

1484 UUv~7CU~1 C'JGAUC~G:~CC'C~AA~CGAA AACAIJAA

1999 GutRGAQ CQGF~LT~.y~.C~~CC~ ~ .. AAUA~J

1511 vCRAGAC CCGA~nG~~.~C~uAAAG:aCCG?~a AUK

1514 L~'u~DL?aACUGA~G:~ ~ ....CC~?rA ACAALTGlG

1516 Cw'L~.QU CBGALGT~G:~CC~aAAAI~.~CG.~A AGaCAAD

529 G~CC1 CLJG~'..~ ~"CGaA ALTC~GC:G

1529 UCCA C'JC~UG:~C~C~~AG'"VCCGaA AUC~;CG

153 G.~G~CC CGG:~~~~CG:~AG;~~C~..~A AADCAGC

1530 G:,uCICC CL'GArGTvGvCCGAAAGG..~CG?~1 AAUCAGC

1563 G:~GCA CUGAUG?C~.~~~C~.a~AAC~aL'C"?~1 AC,~JUCA

1503 G~:.~.G;..~CL'G'CG?~.AG:~.C'~1A AG.~"'CUCA

1 ssa cc~;~ ccc,~,~;~ccca~a~:.c~~ arc 1589 C,~:~C~U CUG:~UC~C;:~CC'G~C~"'C".,~ ACAGUCA

'_ .~ G:~. C~CGr.AAG~CCG?~A AUUACaG

g 1 617 C"~:,UCUB CVCADGAG~CL'G~~AAG~~CC~A AWC7CQC

1623 UCOAAGC AUC~, 1633 G:~WOU ~ AUDU~1?r V ~ ~ ~ ~ C9 iC ~ 4~ yC n!
C9 a ~ C~
b m c~
c v ~n ~c~c~c~c~c~c~c~c~c~c~c~c~c~~c~c~c~c~c~c~c~.c~c~c~c~c~v'u 5~~~~~~~5~~~5~5~~5~~g~555~~~5~5 c a ~~c~c~c~c~c~c~~c~~~~c~c~~c~~~c~
':. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ U ~ ~ V V
:_ r c~ ~ a c~ a r wr ~"i C
. "~ ,n .,, o a ~r vo r e~ a r~, ao W vo e~ .e vo o ~ N m m N o e' 0 0 y y ~~" ~ O O 1'~1 P'1 ll1 01 r'1 ~ O 1f1 r~l tf1 ~O !~ D O N N~1 N ~ ~-1 ,~.~
N~ N N P1 f'1 ~ w'1 ri N N N N N f~1 e'~1 a aT IC1 1~1 ttf tf1 ~ f~ !~ !~ m ,."p! ~i v-1 ..;
,.., ,.q rr ~~~~~~s~~~~~~~~~~~~~~~~~~~

m ~r c m d cn m d C
a a n ~, ~~~~~~~~~~~~~~ _ H
:, ~ y'1 ~O N '~-I tlf O t'~1 ~ ~ 1G G1 ~ f~1 ~ ~ ~ G~'1 ~ G~'1 ~ ~ t~11 l'~'~, N
t0'1 ~ P~~
N N e0~1 ~N'1 f~~'1 t0'1 r1 c ~ W f1 ~D v0 W O ~ ~ Q ~ ~ C1 O

Table 29: Human bcrlabl HH Targei Sequence Sequence E8 TatQet Sequence LD No.
ZO ~LL~ pIJA ?L
?1 C CLU C?C.GJ
A~ ~Z
b3-a2a2 s7tz~_ct~ on T T
C~r~~.n ~~~a~x uvc ~c-~n~

Table 30: Hun bcr-nbl HH Ribozpme Sequences Sequence ~ R:.bcryme Se~ence ID No.
26 G:~"L10C'DUCCLJ C'JGnDG:~ ~'.~:,G~..a;?. T,~JGG~J~"0~
27 aC':,~ ~.:,,G c~~~"'.~c;~..~ -..:,a. a~:~;~,.~cc~C
2 8 LT'nCuG:~. CG~"'J C'~ G: L'GnG:~. ~ ~? AFaC,.:,..4.~r. ~nG:aO,CWCW
29 C~~.~a~~C',~Ar~ r1'~CUCffC,~WA
3 0 nC~~CGCGG C'~C"~'"C~:-.~a~'~Gr.~"C"~1 ' 31 C'"~L'LJG:~CGC'U C'LT"~=~L1G~C--:n.C~G.~.."CC-.~?. ?F~GGG..'ZJUUQG

Table 31: RSV (1B) HH Target Sequence at. HH Tarpet Sequenceat. fib Target Sequence positioa positioa "EAU A AaUC:.aU 276 "'~,2.U A CQG~,UA

14 AALRAAU C A:~WC~,,2 83 ACL,'G~U ,~ C?.AC
aCA

18 ~UC.AAU U C:,O~~~.~A295 U A L'GC~:.~,C'J

ADCn:~~LJ C AGCCnAC303 DG:,G~C~T U UCC~,~

54 C.'-~ADGAU A aLmCACC304 C.:~:~C.W U CCLUAL~

57 UC,AU'.~U A C~CCAG13OS GC_ACUW C CC:,hL'GC

77 UC~U C ACJ~GACA 309 UUUC'JCU A LJGC,;
~1U

94 A~.CC~ U 6JCACW 317 L'GCCArIU A UUG~UC1 97 CCuQLIC~iT C AC~~"AG319 C~.:~I1AU U G~L'G"~7 101 ~ U G:~GACCA 320 C:~:~1UU C :,UG~?.UC

,1 p AA 323 UAUQC?,U C

1,13 CCA~ A AG1DCAC 327 CAUCAAU C aDG?,L'GG

~ 8 AImACAU C ACDAACC 337 G,L'G",~U U

122 CAUCACD A 1CC~GAG 338 :~L'Gw"W C ULTAGaAU

13 4 GAGAGAU C ADAACAC 3 4 0 GCWJt'CU U AG:~L'iGC

137 AC'~L'CAU A nC~UC'~341 Cv"~CCW A GnAUG~..A

14 8 CHAD U UA~C _ 3 5 ~GGG~,U U G:~T~UG~

149 AG.AAUD U 356 DCGC"~IJ U AAGC,C~A

150 C.~.hADW A L?A~CUQ357 DGC~.:~L'LJ A AGCCVAC

152 A ~.C~A 363 t~G;.Cp p, CAp.AC.;~

154 ~U A CGUG:.UA 372 ~nG~.U A CUCCC1L7 157 AIJAIJACL7 U G~AU 375 G:.AL~CV C CChU,~I7 161 ACCIDGaU A AF~DCAL7G380 C'JCCG~U A AUAUACA

165 Cy,~AU C ADGAF.L1G383 CGUAiaU A ~IC~IGtT

17 6 U A G~JG:~GAA 3 8 5 AUA:,DF,U A CAAGLTAU

1 B 8 C~AAA~CU U G:~GAAF,3 91 L'.~C~GU A UGaUCGC

2 08 GCC3~AD U UACADUC 3 9 6 GJAL'G?,U C UC~.AUCC

209 CCAC~ U AGAUC'CC 398 AUGaUCLI C AAUCCAU

210 CACAUW A CAUBCCQ 402 UCUCnAU C CFaImAAU

214 OUDACAU U CCBG.~'UC406 :,At7CCAU A Ap,WU~

215 L~JAC~.W C 410 C_~L?~AAU U UC~.ACAC

221 "LJ C AACLIF.I7G 411 ALTn~,:,W U CAACACA

226 C,aCAACU A BGn:~F~UG4?.2 UFmUULT C pp,~~

239 L7~AACQ A ULTACACA421 AC.~C.'.AU A UUCACAC

241 AF~C~U U ACACAAA 423 AG.AUAU U CACACAA

242 AACQAUU A C3~C~AG 424 C:~F.BAL'~J C ACACAAU

251 ACJJJyC~ A ~' ~ 432 ACAG,AU C LZp.AAACA

261 AA~~GU A AAtZA~A 434 AC:,?.UCU A AAACAaC

2 65 ACUAAAU A AAA 4 4 6 AF,~,~7 C U

2 67 ~F.TJAU A AA:~alsA~14 4 8 C:,r~ CGCV A UGCAL'r~.A

274 AAAAAAU A LIACL7GAP,454 U'nL'C~,U A AC'~",UAC

4 c~~,AC~ A ~A
s a 9 tTnACL~U A ~~

4 CUAIJACQ C CAt~lC

4 ~U A GJC..aGa 470 CC G~GAUG~

4 t G~aFv~U U :~I1AG~1AA

490 GAAAAUU a ~G~,AU

492 :LV;UffAU A GRAI7UU

495 UL1F~ A ADQL~AA

Tsble 32: RSV (1B) HH Ribozyme Sequence at. 88 Ribozyms Saguenc~
Poait3on iaUDGAW CUGAGG.'aGG~.~C"~AG~'GAA ADULTx_"C

14 C'JG~?.W CL~CGAAaG:~..~CGaA
AL'U~W

18 UQG:r.~u~G C'9G1~CCAAAGGC"CGAA
AUL'GAW

1g GQCK~~0 CUGCCG?~A. AG~"CGlr1 AAWG1U

54 G~U. CUG~I~aAGGC'C~~1'~G sCC~.aaA
AUGWG

57 u~G.~"CG CUG~ -LT~G:~'"~
'~AAG~.'~A AWAUGA

77 UGJC0G0 CL1GALG~~C w AUC?.UCA
, 94 A~G%~ ~L~ ~1 ACG.~~UCU

9? C'0 'C.~e~GR CL'GADGAGGCAC'~AC'"..G

1Ol UG.~"QCL'C ~CG~A AGGCCGAA AGUGaCA

110 ADGLTDAU CDG~LJGF,GGCCGAAAGG.."C'G~A
AiJG.~"UGV

1? GL'GAUCa'U C~L~GC,~.~CGaAA~;CCGr~A AD~I1GG

118 G.~"UQAGU COGAI"C~~~ aA ABU

122 C'OCCflGGU COGr'~ ~"CGaA A~.1GAUG

134 G~AU C~CC~'~A~CCG~
AUGJCUC

137 UGUQ3GQ C'DG:~BG.~~GGCC'GAAAG: ~"'C"~AA
AUGaL7GJ

148 GL~UA ~an~I~ IGGCCGAP. AWUGC1G

149 ABU ~ .'~ADG~a T

150 nAL~LU1 COGAL7GAGGCCGaAAG~CC"~rlA
AAAL7C10Ci 152 Q~ ' ' " ALU~AI7U

154 L~L1C'4AG C~UGAGaCCGrl3'~AG.~~.~C~ AL~r~IAA

157 AUUOAUC CUGF~ ~ .~CCGrI~r AG'L1A
?I7A~J

161 CAD"'~FaLJQ CVGF.UGnC,~,~.CG?aFaA~.~vGaA
AIJCAAC~T

165 caWC~u cvc;~uc~CC~AAAG;~~cGaA
A~mvC

17s Wcvcac cvc~~-.~, cG~-r~ A~
~c:A~ ~
AVv .. , .

188 UUUG'~DC CVGAZ~AGG.- ACL7QWC

208 GnFaUGffA '" ADGflGw 2 09 " AADG'JGG

210 A~~AUG CL?GAUG:,6G.,.AAADGUG

214 G~CAGG " " AUG'JAAA

215 ~~G CUG~ GwCGAA AAL'GLTAA

221 CAUF.GQtJ CUGnLK~G:~CC'G~AA~CG?.?a ACCAG
art 226 CAWUC~ CQGALTGAGGCCGAAAAGJUG~IC

239 L~AA CUG~UG~GCCG?~T~AGGCCGAA
AGUUUCA

2 41 ~ " ~ CGaA ALU1GLTUU

242 WWGL~G CVGT,IIGTaG:~CCGnA~~s~~C'CGAA
AA~GW

251 UGCUOCC G~GAL1G~G AG~~CCGaA ACUUUGU
GCCGAA

261 ~ ULRIIAUU.CUGAUG~CCGAAAGGCCGAA AGUGCUU

265 Wtn7UUA CUGADGaGGCC ~ _ ADUUAGU

267 QAUUUUO C~G~ ~GaA AUAL1WA

274 WG'~ CUG1U"~GGGCGAA AGGCCGAA AUUUUUU

276 UAUCCAG CUG~UGAGG.~.CGAA?1~CGAA
AU'fsUVW

2 83 -uLT~ ~ ~ ~ '"C"~,74A A(JC~'1~l 2 9 ~ C?. C~~GG.~C""'uMAGC-~~C
G.~a nDLJU~T

3 03 ~C~.'~' .~'.,~ G~C".~AA aGUGC:Ca 304 G.UAC~ ' " G,,lr'~Fs ~C'CGAA AAC,'L~'a~,."C

3 05 G: ~.I~.GG C'C's~'C'GAA~CCGa.A
' ~ C

3 09 ~UG~CA C'L~'G~ . , .

317 UC~~.A C~J".~"C~AA G:~.."~AA AUOG"~1 319 ~WG:~ITG CUG:~DGT~G:~CC3aAA~"C~.~1r1 AI~L~', .2O G.~D~JG.~D CJC,~LtC~'~G~..."C~.~.,A~~CG?~A
~1ADAD~.~

323 CAL.K~UUQ ~,G...~C "~ '.a~A AIT,~

327 CC~UUU CDG?.DGAG~~.,~"CGaAr~~G'~?a ADCGrIDG

337 UUC~G CCGADG~CCG1~.~ G'~.~C~~lA
ACS

3 3 ~DL7CL1AA CL'G~~~ C~.~A

3 4 NCO " . ~A: ~Gw~C".-~A
0 ~G~,CCC

3 41 UG~.~UC CGGAUG~.. AAGaACC
.

350 LAADG..,.,C az7GCADQ

3 5 '"W CQG:~G?.G~"CGAAA ~.~CG~ ADG~"G1A

5 7 ~G~U C'UGa ' .~~CC".~.1A

3 63 CC,C~UffG AGG'pCO~

372 :~L'GG:~.G CUGr.DG ~.."C~A A1~0 ~

375 CQG ~CGA<1 Ac~ADGC

3 8O L~U C'UGaIKa~'~a.~'Cl~hAAG~CCGr.A GAG

3 83 F~CUU~ CDGnGC'~75C',:,w"CGAAA~aCC"~A ADS

3 85 nL~.CQUG CUG:.BGAG.~""C:GnAAGG..'"C".~iA
~aUADC~

z of C-~UCA ' w ACQD'~I~

3 9 G,:~~UOG?, ' ~ ~ AtJ~UAC

3 9 .~L'G~ADQ CQG~GAGG'"CG~AAG'~CC'".~AA
8 .~DCAD

402 :~UUL~.UG CZJG.UG~GG.."CG'~'~.A ADUGAGA

406 UG~nAW CUG.'. ' ~ ~"CGAA A~AUp 410 G~G~ CGGAaGAG: CC CGAA ADUD~,L'G

4?'_ UG,~1GG C'UGaL~AG~"Ct=.AP.AGCaCCGAA AAIJtIUAU

sit ~ro c~:~~cAG;o~c,~A AGu,.-~ccaa A~o~, t21 G~AA CL1GA ~ ADD

423 UQ~lGat"a C~G~DC,AG~~~CG7~Lf~ALr~CCGaA AImOUC'Os 42 F U~GO '~

4 3 UGJUC1~ ' App 434 G~7GUW CoC~DGnGGCC ~ aGADDW

4 4 :-.2~L~. CCJGAL1C~J~GGCCGAF~"CGAA AGQLIGpQ

4 4 LCDGF.DGAC',:,CC AC,~

454 Gff~L~GU CL~UGaGG. ,Ap, Ap~At~

458 UGH, CL1GALIG~AG~CCGAAA~CCGaA ApL~:pG

4 60 L~t~c~G A p~p~

4 63 G~aC'~tJG CUGhBGF,CCCGT~;i4I~~,CCGAA
F~P,UAG

4 67 UCQG~C C'LTGr',L1GAC~GA,~,~~

470 C~,..F~DCUG C'UGAt~G~"CCrCGAA ACBADGG

4 8 L'L:AC~D CCIGnL~a 9 ~' 490 AUCACUA Cl?GAL7C'~G~~CGhAF~:".~CCA~, F~AU~JpC

492 AAATjDAC CUGr~.U~ AG;~CCGnA AL~aUUQ

499 L'LZJ?,AF,U CUG:,L1G~GGCCGAAAG.~,CCG?l.~
AC'UP.LIAp~

Table 33 : RSV (1C) HH taiget Sequence at. Tn.=yet Se~sence at Target SeQueace Positioa Fositioa l o U A ac~L~ ~ 6=_ ~ a ar~c~

36 UFaaGnAD U UGr~.LTr~IG169 UQ~C,~ A AC'".n:uL'Q

17 AaGr.:~U U G?~ _ 5 ~U U

n ;~coGav A Ac~c:a 17 s aac:~.--t~-Q tt ~",~.-,~~

25 GaUhAGO A CGsCuGA 181 ULJt,C-C,.,.~J
A ,s~,GJ

31 U ?.AAUP '_92 C~GVT,~,U A Cf,LaC~1 32 ACCA~ A :~.UQ~ 196 GAUAG,U A C'~UCaA

36 C~~,F,AQ U ~COCC 201 ?,tJ~C~V C A

37 UDAAAIJQ U AACOCCC206 AU~,~U ~

38. L~AAUQU A r~CQCCCU216 AUC,~U U ~-G

42 UUUAACL1 C CC~.~"'U221 AU~,~ U U

46 ACUCCCIT U G.~~ 222 Ups 'U ~~UG

50 CCUQGGJ U AGaG~aLJG23i UCaG'LT,J U i1L1L7P.G',e~

51 CLJCG~'ZJU A GAGT.LTG.;2.2 G~,'G;,'p A U~

67 CT~,AU U CADflG~G 234 .~~U U AC~AGCTA

68 aGCA~L'tJ C AL'f3G~GJ~ 235 y,W ,~ C~

71 AAUUCAU U GAG 241 ~p A

76 ALZ7GAGU' A UGAL~AA247 ~,~,U ,~ U~~.y 81 6,TAUGa,U A :.i~Aplm249 G"'Gp,U~,U U U~-C

87 L~AGLJ U AGDDAC 250 U~,~W U ~"

88 AAAAG'vJU A G~LTQAC~,256 UG~~ ,~ ,~V,~~

92 GL~GAU U ACAAaAU 259 CCCL~,U :, avAavAU

93 L~GaDD A 'C.~,AAW 2 62 ~,~,~,U A p 100 ACAF~AAU U UGLtOUGA2 65 U A UUGUAGp 1 Ol CF,AAAUQ U GU9~C 2 67 ;,Up"~U Q G.~

104 AAUDUC~ U UG:~CAAD270 aUaD~,'J ?, GNU

?05 AUUUGUU U GaCAAUG 273 UUG~G~J A AAADCCA

12 0 AUGr.AG'J A GCAUC1GU2 7 8 G'JAA~.AU C CAAUL~1C

125 GUAG~U U GUQAAAA 283 AUCCAAU U UCAC~AC

8 G.=:~WGJ U ' ' 2 84 UCCArIL'U U CACAACA

~9 CAzJUGUtJ A AA3~k~A285 CCAAUW C AG'~A~G~A

335 ~'~AAAU A ACAUG.'U:00 LGCG,G'J A CZ'Je~G'~A

143 ACAUGCU A LIAC'UGAU3 03 C~CU .A CT.p.AAilG

145 AUG."L~U A CDGAUAA316 UG~~G"~u U AaAtTAUG

151 LTACUGAZJ A ~aF.Ut~,AU317 ~,~ A ~~~

155 GAUAAAU U AAUACAU 319 AG.~~OUAI7 A UAL

15 6 A~W A AUACAUV 3 21 GJUAUAU A Uc~;~"AAA

15 9 AAUUAAV A CAUULTAA3 3 8 A 'UC~:,AU U ;,:,CaCAL7 163 ~ ?.AUACAU U UAACUAA339 L'G~~AW A ACACAW.

164 AUACAUIT U nACUAAC3 4 6 i.ACACr,U U Ct'Z1CUCA

350 C~UD~p C UCnACCCI

3 52 UU~~DCp C AACC~A

.58 UC:,rICCQ A ADG~C~

3 64 L~,AD~ C

3 66 AUG.~"~ A CVAG?LT

3 69 GQC'UT,CQ A W

379 t~ACAAD U G~AAD

3 8 ' ' A AT,UOD~

392 AWAF~AU Q C'UCCaAA

3 93 L'DAAF,Dp C UC'CAAApr 3 95 :~,AUOC~ C G~.AAAA~.

412 J~GLT~.D U CnACAAD

413 AGUGADL7 C AACAAL~G

428 ACC~IADp A U

430 CAADI~ A L~A~

436 ~AAD C AADUADC

440 AADCAAD U ADCL7C',?,A

441 AIJC3,F~IJQ p, 443 CAAZTUAQ C UCv,A~

449 UCUGAAU U ACL7~A

4 5 C~GF,ADp A C~~.;AD

453 AADt'~CU ~ G;~7pp~

458 C~ p~,~pp 459 UGGG~Dp O GADC~L7F, 4 63 AUQt7GAQ C pL~ADCC

4 s U~cv tr AA~ccau s 4 6 L3GADC~ A ADL~A~

477 c~,I~AAD Q ADAAI70~

478 ALg,AAUp A

480 AAAIJtmD A ADLJAALp, 483 UDA~,A~ U AAffADCA

484 ~AI7L7 A AAA

487 AADL~AD A UCAACQA

4 89 UUAAI~U C AACL~GC

494 AIICA.ACU A "Cu,AADC

07 t~ C A~AA~

511 L~1CACQ A ACACCAD

519 ACACCAU D AGUL~AD

520 ~p A ~7~,A~

523 ~ ~ A~,~

524 ABL~,G~ A l~t~AA

Table 34: RSV (1C) $H Ribozyme Sequence 8H Ribc~ym~ Se~ueac~
~oaitioa 1 'r~n:~~Ct7 C~~aAGCCWnAA~G~..~C~'.y,A
Q a~DGCG~"C

.6 C~Fai3C.~ COGAL~:~~CG,'h?~AG:~"'~.1 ~

1'7 ?.C'JLRIJC C?G:~DG~IAGa.."'CGr~
~1ADGC~Q

c. L'G.~IaCZ1 CG'G?.Lr~.'~.G:,C ~~C:,C~.~?, ?S~C~.~aD

25 Q'~G: CLT~Li~t-:~..~CGAaF.~1"C~~
1LZ.~t7C

31 tT'.~F~?.LW CGGat~?~~C~.~.AAC~"'C'..:a.1 .~G,'C~

32 L'IT.iu\W CL'GAL~G-',C,~."C~"C.~sarl 3 G;~CCtm CGGL~G:~.,.~CG~AC~'~CG~1 37 G;~~GUU " ~ r~rlD~tlAe1 38 :T CffG'r.~'C~G~."CGr~I ~1AU~7C~a 42 :~CC~AG: C~C'~~~C~ ~lA

4 L'CL~CC CCC,A~~C'Ga~~1 ACT

50 C,DC'CCZ7 CZG~G:~G:~c~G~.."C~.~.1 aCG'~AG~

~l CG~UCUC CVC~GC1C"~ ~,aG

CL'G~:,TIG CQGW ~~CG.~G.~'~ .1L'Ll'..~CG

68 nC~-iA~ -CflGni~:aG:,~"CC,~AG~.."CC,~r rl AAL'D~.~~7 71 C.'-~CGC ~' '~~"'CC~AG".~..~C~~?.r1 F~ADQ

6 WQABC~ C9~ ~ ~ ~ . . -.~ ~..~'1~AD

81 L~:.C'CW CCG?I~G:~G:~"CGAV~CCGM
~L~C

87 6JiaAL2TJ CLJGaI7Cv:G~WG:sw"'C"aTv's aCZJDGC1A

88 L'GJAALlC CQGADG~G~CC"CGaA .~UU

g2 nLTv'OCGLJ CLG:~~C?.C:~C~~ e~.UC~1AC

03 ~.~1UI~'UG CUGT~~"C'".~'~AG:r.~CGA~
aADC'~1A

oo vc~wcJi cvr~;~:~:~a:~aAA~~ccr~.~A
~

o muc~AAC cvc~~c~c~aa~,.-,c~aA A~nw~

104 n ,~C?~ C~CG?.AAGC,w~C~.a:a?, ACaAAW

l ~C ' AA~A~

?ZO nC'-~?.BGC CQGFa~r'aG.~~'AG~"CC~A
aCLiJGQ

125 L~AC CDGAL'G~~'C:G~:~CCG :A ?.LT~'~AC

?2 LnUL7LIW CUG?.L7G?.G.~a~~t ''~~C~.~:~A
8 AC?.r'~C

129 L'L~DUOQ CU~~C".~AAG:~CG:~1 aACAAUG

13 F.G:.ADGU CDGF.LT~G~CCGAF~AGGCC.GAA

143 nL'C'~ Ctl~. -UCn'C,~~CCGaF~AG~.."CGAFa ?~G~"'~IJfa~

145 UL7AtiCAG C~1C,T~ ' ~ C'GJ'~AA nVAGC'rIUU

131 '~L~F~u'O COGA ?aQCAfa'C~l ~ .'~.~QtT CUG?.D~' ~

r~

5 :~UG~AU ~-~CGAA AADU~rO

139 LAG CflL~~C'CAAA~CCGAA nD~AUO

183 UC~G~JL~ CC7C,?~DGAG~CC~~AG~CCG.'~A
aL7GJAxJi1 1 GG~.GIJU CtJGAUGF~~C'~AAG:~CCGAA
~ :~.DC~,U

03 CGJIJ?.GU CLiG~.LKar':GCZCG a~aF.C,GCCG.~, ?t. :L'C~Ja 169 A~-'~ ~~~~'~~~ AGC~.~

175 ~C~ C'CG~.~' ~".yJ~G:~CC"'.~.AA
~"~7LTA

176 CVCAG-~C C~.~G:~CC"~,C'T;yCCGAA
aAGC'G7D

181 ACL1GCL~J C'GG~L~G~~C"~euaG;~~C.~,,AA
~.G~"~?u~

192 DRUG COC~.'~.GG..~CCGAA ~CQG

1 g U~UL1G CG~~,G~ C~,-? A AUG<IALC

201 UCAABOU C~'"CGAAAC'~~.~.."CGAA AVOU~U
.

206 G~~CAWC CCTGA ' GV.~CGaA ABWGaU

216 GAACAC CVGhDGF,G:aC ~ ulAC"'~,~CCG~A
AUG~~C'~U

221 :~D""~ CUGADGnGaCCGAAAG~CGA<1 AC~G'-,P.U

231 UL7G~AU ~ '~'CG~nF.G,CCCa?~A .~G~UG~

232 C~G'~A C~A~.-"C~~AAG~~C~.~A AaC~LJC-C

234 ''L~CIJCTGtJ C'JCA~GGCCCGAA AUAAC?.U

235 CZ.mCCUG CUGAL ~ ~CGaA AALmACa 241 AUAUCAC CbGAL3G~.GGCCGAAAGGCCGaA
ACOL'GUA

247 G;~.AAA CVGaL~CGAAAGGCCGaA ?.LG'~C',7A

249 BAGwCA COGAC'"~1A~.C~CCGAA

250 DL~GGGC ~ A~.QCA

256 ~ADL~t1 C'CK~"C'GA7?,3~f~C'CGAA
AG~Ar~

2 5 A~nUC~U ~~...~C'~AAAG;~CCGaA AL1L~G~

2 62 AC ;ALmU CUGr~'~C1G<".GG~."CC~?~AAG~CC~.~, aUCTAUUr~.

2 65 ACUACAA ~GF~~CCG.~A ADCIAUG~a 2 67 UC~CL~C ' " - AAC'~.~CCGAA A~DCmU

270 AUtJL7CAC C'L~"CGCGAA AG~AL~U
.

273 ~CGaA ACL~CA?r U~%~ CHUG ~

278 GAFsAUUG CLCC'GAAAGGCC'".~A AU~C

283 GAGA CflGhUGAGuCCC~Ae,GGCCGaA AWG:~1U

2 84 wG CLTGF.DC'snG'vC s~.AUtJG~

285 ~'~1UGU CDGAL7GhGu'CGAA~aG~CGAA
AAaLTGGG

300 UWG'~.G C'CTC~AGC~CCG:~AGGCCC~A
ACL'C,:~C?~

303 CJ~JUG C " ~ uGGCCGAA AGVALUG

316 G',L~U CUGA . CGAA ACCUCCA

31? CCA~'~L~ COGF,OGAG~C'G74AAGGCCGAA
AACCUCC

319 CCC'".~.~ C'O~I7GacHCCGA3i,~IG~CCCuIA
ALmACCCT

321 UtmCCCA C~GAiIG:~"CGAAAGGCCGAA aI7AL~C

338 AB~.JGUtJ CQGALKAG~~'C:c:~,AG;,CCGaA
ADUCCAU

339 AAL1GUGU CUGAI~FSG:~~CGAAAG~GCCGAA
AAIJUCC~

3 4 BGAGAGC CUGAUGnGG~~'C'GAAAG:~. C~.~A
6 ALT~GQL7 350 AG"~GA CL1GAUGAGG~."'C~"~AC~CC"..AA
AG~~A~JG

3 52 UCAGu''TJU CDGAL~C'~G:~CCGAA~'~GGCCGAA
ACAGCAA

358 AGACCAU Ct7GAL~t~:~C~GCCGAAGGCCG~.A
AG,rJQGa 364 UC~GOA CQGF~GGCCGAAAC~:~CCGAA ACCAL1QA

366 CAUCUAG NGAL~G:~~'C:GAAAGGCCGaA
AC~ACCAU

369 UG'JCAUC C'CGnI7GAG~CC'GAAA~CGAA
p~p,C

379 AUUUCAC C'UGAUG'~~,GCC''"AA AUUGUCA

3 s7 A~:"~uuo cQC~uc~.G~cccvw.GuccG:~
avwc~c 3sa G:~Atnr cvcAUGaGvCcGaAACc,ccG.~
AavwC~, 392 QCIUG:zAG CDGAUGAGG:.C~.~Af~AGGCCG~
AUWAAU

393 UOWG,~~A Cu'CC:GTaAAG~~~GA~ AAUOQAA

Up~pG ~~CC~1',AAG.'~~~~~.A AG:~u~.D0t7 405 AAUCACD CL7"~~~C'GAAA~CCGaA AGJOO~t7 412 AU~G ~~C~A~.~C".~A ADi~CO~T

413 ~ ~'"~ AAUCACD

427 UflCnIRU COGF ' ' "C'GaA ADL7C,GUC

428 ~ CDGF~GG~ ~ .~AA AADOGGD

430 UGaDOCA CRGF.L~GG.. .~A ADAADDG

436 GraUFaAULI CUGAi~G~~CGAAA~."vGaA AOQCa' Im 440 U ~~'"'~~ ALnTvIW

443 A~A~ ~~C~ AADtKAI7 443 LTAFaUOU C'GC~G~' ~aAA~"C'".~1A ~1D~

450 C~U'"~A AADOCAG

453 G'~AAUCC C'LtC~GAC~C'GAAA~.."CGAA A6.1AAW

458 AAGAUCA CRGA~AC.GCCGAAAGG~GAA AUC~IAG

459 I~1GAUC CGGAL~G~..~C"'WvlA s~.,C'C'.~A
AAUC''.Ae1 4 63 G~ADLmA CLTGAL7GAG~,COGAAA~.."CGAA
AI7CAAaL7 465 ACC~~~AA~C~A AGaDCAA

4 66 ~aDGGaU C~GF.~G:~CCC~aAG.~~~CaA AAGaDCA

4 69 %.DQOT~I~ -CUGUGA~CGAAAG~.""'C".~r1 A~AGA

473 ~'~~:~C ~ A AUG~raL7CT

477 UAAL'tg,U CoG<,'UG~G~~'CCaAhGG..TGaA
ADUC~

478 UUAAULm CUGaDG?,GG.."CGaAA~CCGAA AADQC~4D

480 Ln'~UDAAU CUCCGAAAGGCC~A AUAADOl7 4 83 UGT.~.UtJ CD'Gr.BG.'1G~;~CCG?~A:~"CCAA
AUtmi~, 484 UOGAImU C'.~.~AG~CG?~AAGGCCG1D, Ap.DOAUA

487 L~Ga CQGACGAAA~.."CGaA :,UC~ApO

4 89 G~."OAGW CUGr.L~'~~C~~AAF~CC".~AA ADADQAA

494 G:,D~7~ CUGAi7G~C,C~~'~GAAAGG."CGAF, AG~T(3GAIJ

501 DG?~CALU CL'GaLK~raC~~~~~C.'Gi~AAG~CCC~A
AUWGCp 507 UGUQ7iGU CCTGA~GGCC'GAAAG~CtGAA ACAT;70GA

511 A~ ~ ~ CGAA AGJGAC~

519 AUOAAC~T CC.'G~,UGAGGCCC,AAW."CGAA
AL~L~

320 WiDDAAC CnGaDGAG.~~.~CGAAA~C.'~Fa AAD~,~pG

523 UtU~DU C~LCC~CGAA AC"~,ADG

324 UUUAI~J CL~G~sUGAG~,.~DGnAAG~sCCGr~A
AAC~T.~AU

Table 33: RSV (I~ HH Target Sequence nt . &8 Ta.r Qet 9equeaceat. H8 Tappet SeCuenca Pcsition Positioa 9 '-CPU A C'~AAGAII 217 G,~'?AUGU U ,~U?,UC~~

21 GLTC.:,.."ZJ C 218 G'v~ A
UGAGCa?.

23 ~ JCJ Q AG:aAaG 2 2 0 AUC,'OL~J A L'GC".~G

24 G:,~'~ .~ GC,'~AaGLT229 G'.~,I~ C v..~
.

32 G''~CL7 C : '~nG~GA231 GnU~CU A Cz'~~~, 37 ~~C~GU U C-~:AL~?.U235 LTCU'.,~G.~"U U
' 45 G~GAQ .~ C?.CLJC~r236 CQAG;JU A G;~"~"~, 0 AUACACU C AACAAAG 2 5 4 .~Cr,C~,.1U ,~

60 C-J~GaU C AACUQCU 2 60 UAA?~1?JJ A C','G'-~G

65 AU 'C.~D U CUGDCAU2 53 AAA~,Cp C

66 UG~ALVU C UGDCADC 277 G~'~U ,'~, LCD

70 C~UC~GU C AUCG'uC-C279 G.~~U C A~pA

73 cJGUCAU C CAC~CAAA2 84 ~ AUCADGLT ,~, A

82 ~G-;~,AU A CACC~L'C299 r.LT::.=,f,'U ,~
G~AA

89 ACAC;.?U C C.'-.AC~.~305 UAGALGLJ ,'~, ACC

108 AG~G~.U a G~TAUUGA315 AACAG~Q C C;rG~

111 GU A UC,~',~ . 318 AC~.L'C~ C A:~.C~1C~.U

113 AIR .~r U U Gr'~~L'UC3 2 6 naG~G~,U U ~..~.LT~?, 117 LV,BUGAU A CpC~ 32? AG?GWJLJ A AUG

1.2 0 L~LRCLT C CI,RAUUA3 4 6 ?.UGAAa,U U GG~t~,G

123 ZIAC'JCCLJ .~, 347 L'GnA~.UU U -C
AUZ?AUGA ' v T

12 6 UC CL~U U ADG:,UG'J? 5 5 G:~AG~1GV U A?
C.~UUG

12 7 ccz~u a 3 5 s :~,-a a ac~.L~Gc 14 6 :~ACKCAU C Aria 3 61 UUAACAU U

150 G~UCJ-.AU A .~GZ~~IJG370 G:J,AG'~p U 1"~,G~CU

154 ~AAGD U AUG~C 371 G~,AG'~W A p,Ca,~,-,G

155 A UGUC~vCA 383 C~JG~,r~U U C~UC.~

'! 6 6 C~F~BGU U AU~ADC 3 8 4 L'G:Js~.UU C AAAUCAA

16'l G~.~BGJU A ULIF,AIfiA3 89 UL'C'.~.AAU C AAC?,UCG

16 9 A ~ ~ U U AAI1CAC~3 9 5 UCA?.C~U U GaG~G

17 0 UGau.~W A AUCACAG 4 O 1 UL'G?L? U A GnAUCUA

17 3 L~UUAAU C AC:~F~AG4 0 6 ALTAGAAB C L~CA
A~

18 6 :~G:,DG~"G A AUCAUAA4 0 8 AG:~.UC'J A ' ' ' UC

1 8 9 v~~~U C AUA~W 415 AG~U C CQAC~.AA

192 L~UG~U A AAUUCAC 418 AAAUCCU A G~AA,p.AA

19 6 CA~r '~U U Cf~CLIG4 31 AAAUGCU A AA.AGAAA
;G

197 AL;AF~AUtJ C ACUG~~.~LJ449 GAGAG,"'U A C,CL'CCaG

205 ACCIG:~.~~IJ U 453 G"~L1AG.~p C
AAITAGGQ

2 0 6 C~JG;N'~T A AUF 4 6 0 C ~U A C-~G"~,U
G.,'ZTA

209 G:w~~~.T'n:~U A 4?2 C~L"G~CU C UCCL'Ga.U
G.~~JAIJGU

213 AAU'nGa.-U A UGUL'AUA4 7 4 UG.CZTCU C CUG,ULTG

4 8 ~U. U GUGC.:~1 E 9 6 UUUDG.3U A UAGCAC.~

4 91 ~GAQ A Ai7?~UL~D E 9 8 UQG.~~JAU ?~ GCAG~AU

494 UG~AD A U~ 706 G~~G'~U C WCCACC

496 A~IJ U AL~Im 708 ACAADCQ U CQACC~G

497 ~~W A UG~.IIAG 709 CAAUC'OU C UACC.~

501 A ~GC 711 AUCULJCU A CG~GaGV, 503 ~~U A GNU 726 UGGCAGU A G~Gpr 511 "G~C~ U AGJAaUA 731 G~G?.V'U U GAAGG
art 'S~ G'1GCAUl7 A G~F.:,L~A740 ~~ U U WUGC3~G

515 C~ A AL~ACa~1 741 AG~~UO U UUGCAGa 518 A ACLU.A.~O 742 G:rC~DL'U U L~uG~1 522 AnBAACO A AADUAGC 743 G:a~U~TUU U G~GaarU

52 AC~AU U A 7 51 GC.~U U G'JCUAUG

527 C~:,DU A GGC~G 754 G:~WGU U UAUGA?rU

544 G%~AG~ C ~'~ 755 GaUUGDU U AUGAAUG

549 AUC~ C UOACAGC 756 AUUGaUO A UGAAUGC

551 C~JCQ U AC<.GCCG 766 AAL'GCCU A UG.~~JG~A

552 A G~GQ 787 C~GFsL~ U ACG:~GG

563 CC~U U AG:~,GAG 788 UGat~LJU A CGG'LJG
a6 564 CVs A G~1CAGC 800 G~GU C UCU1GCAA

573 "Q A A~ 802 G~.GUCD U AGC.~AAA

576 AG.."DA~aU A ALG'UCCU803 GhGQCW A GC~.AAaU

581 A~AUW C CaJ,F~T,AA 811 GCAAAAU C AGJUAAA

584 ADGDC'CD A A~A~G 815 aAUC~.GiJ U AAAAAUA

603 G~AACGa U AC~AGG 816 AtICAGW A aAAA~U

604 AAAC.;UU A CAC ~ 822 LJAAAA.rIU A U~JC~

613 AAAGGCU U ACUACCC 824 AAAAL?~U U AUGUUAG

614 AT~COQ A C~CCCA 825 AAAUAUU A UG~Ga 629 AC~GAU A ~CAACA 830 ULD~UGW A GGACAL'G

640 AAC~G.."'Q U CQAL~,AA840 ACr.UGCU A GuGUG.~~, 641 AG~GC~ C D~.BGAA6 866 AACAAGU U GWGaGa 643 AG~."~C9 A G 869 aAC~GLT U GAG""UnLT

652 G:~AG~ U UGJ~AAA 875 UQG~~U U UAUGAAU

E53 A~ U G'~.:~AC 876 UGAGV~UU U AL'GAAVA

663 AAAACAD C CCCiCDO 877 Ga,C~'~JLJLT A
UGaAVAU

670 CCCCACL7 U ~r~GAU 883 UFsUGaAU A UGCCCAA

671 CC~.:,C~ U AUAG~G 895 CJ,?.AAAU U G:>GLJGa~U

672 ~C.'L70U A UAGhDC~ 913 G:~1C:,~.U U CUACCAU

674 ACUDLIAU A GABGUW 914 CAGGhUU C UACCAUA

680 ~D"' ALT U ~70C 916 G:~:.UUCU A CCA~VA

681 AGAUGW U UUG'WCA 921 CUACCAU A UAUUGAA

682 GAUGDW U DGaUCAU 923 ACCAUAU A UUGAACA

683 AUGUQW U GWCJ,W 925 CAVAUAU U GAACAAC

686 UUQUDGU U CAUWDG 943 AAAG~J,U C AULmULTA

687 UUUamU C ADUCT~ 946 GCAUCAU U AUL~1UCQ

E90 LIGLnJCAU U UUG~D 947 CAUCAUU A UUAUCW

691 GLIffCAUU U UG.~''i~UA949 UCAUVAU U AUCUUE1G

692 WC~.~JU U G~'~nDAG 950 Cr.UUAW A UCUWG?r 52 UDAUL~SU C UL'UGACO

954 :~UI~,iJCV U
UGaLZJCA

055 ~3'aDCULJ U GAL"DC1~

960 WUGACU C AF~DQDCC

964 ACffCAAU U UCC~C

965 CUCAAUU U CC9C:~1CQ

966 C Cep 969 ~BOQCCU C AC~CGC

973 C~21G~CQ U CUCCAGO

974 C'JC~.CL'U C
UC'C~GL'G

9 e3 c~~ a caA~~aG

986 GLG~GU A Ut~,G,CA

988 C~AC~U U AG:~CAAU

1007 CQG:,~."'CQ A
CC.~1LJA~, ol mmc~ a auGGVa~c 102 "G~G~L;LT A CAGAG;G'U

.032 C:~~ A CAL~CGAG

1044 G~G~ C CQ

'_ 050 UC :~GaU C

:052 :~T~~ A ~GAL'G

.054 G-'~C~',U A UGa,UGC~a 1072 aACr,CAU A ~~

085 :~.ACAACp C AAAGAAA

1103 ~U U AAC~1CA

.104 BGUGAUU A AL~ACAG

.108 ?.ULmACn A CAGE

i'_15 AC?.GvJGU A CGAGAC'p .? 18 GJGJACC A GaGZIpGa, '-123 U C-~AGCA

1 '-3 1,AGAACL1 A GAGGCLm 1146 "U A

1 14 AG~C'UAQ C AA.ACADC

1_55 CAAACAU C AGCD~, 160 AUCA.GCp U AaDCCAp, 11 61 L1CAC,C~ A Aat:CAp,A

4 t'ZTUAF,U C CAhAAGA
C

7 3 AAP FLU A F.UC~DGU

'_'_ ALIG:yUGU A GAGCUL1I7 =a7 -v U UGac~

8 g vp -- U G:~GOLIAA

--g3 U A.AUAAAA

'-'--94WGAG~p A ADAAAAA

Table 36: RSV (I~ HH Ribozpme Sequence at. EH Ribozyme Sequence positioa g AL'CUOUCa CVGA~C~CC~.1 ADUG'GCC

21 UCG..~JAA CL'C,AiI~GGCC~"'C".~r1 AG..'"C~aL'C

23 C~TppG,,"U CDC~A~C~'~G:~: C".~ AG?~GCCA

24 ACIItJL'GC CJG?JD~GGCCGAAAC~."C"'~?.ar M

32 UGaCVII CUC~UG'aG aCCG aAAGVCC:~r1 AC'JUUGC

37 AUCAUUC CUG~GAG: ~."C'G1~~SCC".~1?l ACGt'GaC

45 UQGAGUG C'CIt~JGAG~.~CG~AGGCCG1~
AUG1UG~C

50 CUtIiIGLIU CUGA ' "'~ :,GJG'JAU

60 aG:.AG'UU CUGaUC,~GGCC~~:~AAGGCC~.~1 65 AUGACAG CDGP.UGDGS,'CCGC".-. AA AG70GAU

66 GC~7GALJGAG ;CCG?~AAGG.~C'.~e1 ~'~

70 Gt'OGGp.U " .. -.~A aCAG?.?~G

73 UWGCUG CUGAUGAG a~"CCCG~ A~GACAG

82 C~UG~"'~7G CZJG~GGCC~.CCG~1 ALUG'G.'V

89 UCCGiIUG CQGAUGAG~~ "~CGAA ~~T,U
~

108 UG'~AUAC CUG?U
~ ~ ~ CGnA AQCUCCO

111 GUAUCAA CUGAUGAGGCV"~A:~AG:~CC".~
a ACUAUC'J

1?3 C~AUC CUGAUGAG~CUGAAAGr.~CGaA ADACDAU

117 UUAGGAG CUGADGAGGCCGaAAG:~."CGaA

120 UAAUUAG CW:.L7GT~C,GCCGnAACs:~C'".~1 AGUAUCA

123 UCAUAAU CQGAUGAGGCCG.~AACsav."C~,?.A
AGG1G"UA

12 AGUCAZJ CUGA "~CGAA .'~rL~

127 CACADCA CUC,?aL~G.ovC'CGr'~r~' ~'1r~

146 ACOUALJQ CQGAUG~ ' ALGA

.50 CAUAACO CDGADGAGG~~C~C~.~A ADWFaLTG

154 G~"CACAU CUGr.UGAGGCCG ~ ., ACUCTAUU

'~5 DG'' CACA CZIG~DGA~GGC ~"CG?.A AAC~UAU

166 GAUUAAD CCICiA~GCCGTa~arlC~~~.CGaA
AG'aDGv.~C

1 s~ Uc~umA cvcAC~,~ ~ AACaUCc 169 U~JGAW CUGAUG:~G:~CCGA?~CG1A ADAACAU
~

170 CQG'JGAU CCiGAUGnG~C'G~aAAGGCC".yaA
?i.~T~?aCA

173 CWCUGU CiJ~GADGAG;~C'C:GAT~AGG~~C:GnA
AD07iAUA

186 Ut~UGAU CUGAL7GAGGCCG AAC,GCCGaA
AC,~DCU

189 AAUUUAU CUGAUG CGAP. AU~.GC~1 192 GUGAAUU CUGF~I1GAGGCCGAAAG sCCG~
ALG?.WA

196 CC".~,GUG CtJGAL7GAGGCCG.'~F~AG:,cC~a?.A
AIJWAUG

197 ACCCAGU CUGT~I~aGGCC:GaAACGCC:GaPa AAL'OQAU

205 ACr'JAW CUGAT1GAGGC~CGaA ACCCAGU

206 UACCUAU CUGALJGaGGCCGAAAGGCC~.~,A
AnCCCAG

209 AGUACC CUGAUGAGG..~~AC~.~.C~~ AL'UAACC
~

213 UAUAACA ~ CUGhL~;C;~CGT~'~GGCCC~, ACCUAUU

217 C~~ ' ~ '~~ ACF.uACC

218 UC"~ cJC~ G: w "~""C GAA AAGUAC

22o c~cc~a ~x~: C~AAGwCc~A ago 229 cn,~,cc~A c ' ~~cGt.Ar~cccaA
Ac ~ucc~C

231 CCL1FACC CUG'r~GG.~'~AnG:~CG?.A AC,'~.G'iUC

235 CC~C~1 C~B" ~-~C~~C~.3AA AC~CA

236 CL'CUQCC CUGr'.~AG:~C'""..~P.AC~.."CGAA
AACC'~1G

2 54 G~L'~U CUGZ.UGhGGCCG~3!~~CNCC GA,A
AB~Gp 2 6 C'9C'CG.'~ AGGCC"vAA,ALvCt GAA
0 C ~Z~ AUWQC~Fr 263 ' v C' ~ ' AGUAUW

277 LTAC~DGA COGAL~G~s~~AACCCGAA AUCCCGC

279 ~CkU CL1GF~U GAGGC.:.~~AAGuCCGaA

284 ULiC~'~ CL1GAI~GG..~~"CGAA ACAL1GA'U

299 ABC CLTG:~ ~C~~CGaA AL'VC".~U

305 ~ CL~ GGCCGaAA~C'~A ACABCLm 315 UDC CGG~ GC,~,~C'C~AAA~CCGAA
ADG~JU

318 F~DC~JC C~IGA~AG~.Cu%~AG:~-"CC~A
AL'GA~V

326 UBCC3~D0 G~A~ AG~~CGAAAGw'tGAA A~~

327 C'~R ~"~AAC"~"CGaA AADGaCQ

3 4 ~ " AUDC~4D

347 ACAC~C CLJC~ GGCCGAAA~CCGAA AAULNCA

355 ~O CUGAD GAGGCC'GAAAG:~CCGAA
AC~CQtaC

356 CG~J CL7G:~ AGGCCGT,AA~CC".~AA
AACACC)t1 ~

3 61 G~."'.1BG,."C ~ AUC"~A

370 AGCY CDG.',I ~~,GGC~~CGAA AGCWGC

371 CAC~QG'J ~ AAGC'L7C1G

3 83 BG~G "CG?,A AU~C.1~

384 QL~AU00 AAUC70C~1 3 89 C: ~.UG~ CDC~UGAG~
AD(JUG7~pr 395 CQAUCUC CIJGT~DG:,G.~~CCGAAAGu.."CGAA
AUGaL7GA

401 UACnBOC C~G:,UG3~GGCCGAAAGGCCGAA
AUCDCAA

406 L'QQUCOA CC~ AG~~'~AAAG~,CCGAA
ADnCQAD

4 08 GAUQLJOC COGT~""~'CGAA AGT,DUC"0 415 UU~G COGZ.L ~C~;t~CCGAAA~~CGAA
ALTppOC~

418 U~ COGAD G?~GGCCC',aAT~C~A
AC~AVC7U

431 ~ AGCADOp 449 CL~C ACCCJCQC

453 AC,C~CC

460 AUGC'CUG CDG;,~ 'AA~CCGaA ADUC'OGG

472 AUC~ C~L 3GnGGw~CGAAAGC",CCGAA
A~

474 G,:SUCAG ~ A~

480 AUCCCAC C'L1G~~I1GAGGCCt'~7~.~~lA~CCGAp, AUCAGGA

491 ~.tIF~BAU C~D ~G~F,GC~CCGAAAG~~CGAA
ADCADCC

4 9 LmCADAA CL~' ~AC~C ~ ADUAL7CA

4 9 UAIB~CAg AQAU~

497 C~ffACA Ct~ 7GAGGC'0.'C~F.AA~CCGAA
AF.L~UtJA

501 G~.'Z1G.."~ ~AGG..~C'C~.AAGGCCGAA
CL1G:~L ACALmAx1' 503 nUC~' .~GC
CD~VGAG~:CGa,AAGGCCGAA
ALmCF,.LTA

511 LT"nLTJ'i,CLJ 3GAGGiC:CG.~AAG~~CGAA
CUGAL AUGC'UGC

512 ~t~C CIJGADG' ~ ,, _ , . ...C,..~A
AADG.."L1G

53 ~~ CUGr~~CGiirIAWCC'". ',r~,1 ACG~1r1L7G

518 ADOCAGV C~~ w;AAG~CCGaA ADC~CC~

522 G.~J~W ~ . ....~CGaA

526 ~~0 CC~"CJAAAG~CC~.sAA ADI~GfJ ' 527 Cv~~a~ c~C~'~:~cCCaA AF.ov~

544 'UCCA CO~GAL7C~G~CC'~AA~CGAA AOCDGCC

549 G.~'D~A . CQ~v'r~~"v,:~G~..~C"'.~AAG~CCGAA
ACCAGaLI

551 C~~:COGO C ~CG~1 AGACG'~G

_552 AC~.~;~~OG CG~..~~AAG~..'~CGaA AT,GACCA

63 C;1CL1CC0 . .. A~CA~CG~

564 G~"OC~CC CUGaCCG%~'~J~I. ~CCGaW
?~AUCACG

573 aC:~DL~U COG:~~CC~~G:~."CG~1A AG~'UCL:C

57 .aG:~Ca~ C'~aDC~G~.."C'" ~ AD~GCZJ

581 TJtnJUflA~ COGAT~G~~C"~ArJaG:~CC".~A
ACliLIt~D

584 C:JJOflW CCG?WG~CC~C'".~A AG~~AG~U

603 C~ C~C~A~CCG?~A ACCVOLIC

604 G."N09G CDG~L~?~AAO~."CGAA AACGi7G0 613 ~ ' AG~~CZJCtT

614 ~'~ ' AAGCCW

617 CC~OG:~G C~D"~AG~CCCAAA~CCG?~1 AGLmAGC

629 ~~CCC~~C~ A~CC'J

640 WCALnG CC1~UG " ' CGAA AG~.'"I7~Cr'LT

641 CC~Lm C~G:~GGC'CG~AA~~'CG~A AAGCO~

643 CACWCA CG~~G~CCGAAAG~~CG?~A AGaAGCU

652 UUQWCA C'"'CG?,h~~C~CCGaA ACJ~CDCC

653 GQUL1WC CGGF~L~ a:,CA~

6 63 iIAGO~ CL'G%~~C~..GGCCa,.~CGAA A'CC~7U~7tJ

670 AL7Ct~.tm CQG:~DGAGGCCG.~r'..'~CGAA
AGLA~:~G s 671 C~C"CFU C~GaG~CC~-' .~CC,Ar1 AAGLIC, ;~;

672 A~~ CL~CCGaAAGr.CGAA AAAGiIG

674 F~F.ACAUC CC~ ~ CGAA AtTAAAGL7 680 G~ACAAA Cue. " CGAA ACAUCQp, 681 CG:~AC~Fa CUG'~r 'nG~CCGAAAG:yCCGAA
AACADCU

682 ACA CflC,F~L~GA ~ "~C~.aAA AAACAUC
.

683 AAI~AC C
" ~ CGAA AAAACAQ

6 8 CP.AAJ~ItG ACAAAAA

687 C~~AU ' ~ a~CGAA AACAAAA

6 9 ALmC'CAA CDG:.DG?~G~CC'C~.AACuCCG.~.F, 691 UF~JACCA CUGr'.L~t'GAAAG~CCG?.A
AA~GAAC

692 CLRDACC C' ~ ~ AAAL7GaA

696 L'GLJG~"tD~ CDGALJCv.GC.CCG;AAG~CCGAA
ACCAAAA

698 AL1L1GUGC CUGAL1GAG~CCGAAFaGGCCGaA
AITACCAA

706 G.~"UF.GAA CUGF~ ' ""C~C'GaA Am~C

708 ~ CUC-~"L~G CDG:,IJGAGGCC'GAAA~G~CCGAA
ACADLJGG

7 09 UCL1G~~JA C'DGF.LJ~C'~:~CCGAAAG:~CGAA
AAGAIJG'C;

711 CC~CUGG CLT.J. ' ~ C~CGAA AGAAG?.Q

726 LG'~ACZ7C.Ci3GA~GAG~CCGAAAwCCGAA
AC~GCCA

731 vcccvuc cvcavcacNccc"~,~c;~CCr,:~
AcUCCn~C

7 4 C~v.=riAAC~C'~'aG:~C CC~lGw'"C
0 -GiA AUCCC~U

741 ~JG:~A C'~Cv,L~AG~CC""C".~1 hAIICCC:J

742 CC~G:~ CGGntIGnC'~CC"~,~',A~xGaA
.~AADCCC

743 :~BCC'JGCC'"'wAL~aGvCCC'~inr?duCCG?~
AAAAUCC

7 51 G~AC CCGi~IR'~~CG~C,~~CG~A
ADC~GC

754 ~WCAUA C'JGnI~G:~CCG~A:,GGCCGAA
ACAAUCC

7 55 G.DQG~U ' AACF.ADC

7 5 C.~.~BL'G~CL'G;~L~A~.."'CG~.ArI~"C~.~A
6 AAACAaU

766 i~:~:CC~ CLTC,~L~C,:,~ GaA~G~."CGA?r AC,~Up 787 CC~CC"~.JCUGAL~C,hG;~~C".,Ar~C~..-.?~A
AG~L'CAC

7 8 CC G~CCG ' ' aAC~~C?, 8 00 ,p, C J - . .

8 02 L'~.."Q CL'~~"Ci'~A:,C,"v.'"CG:~A

803 :~L~GC CL'G<1UG:~GGCC~~CG1~ AAGAC':C

e11 Lc~AC~ c~G.:.tx~G~CCG.~aa.~,.~ccaa A~ov~;.

~uw c~G~. ' a 816 AL~DUW CCC'G~AA~CC~~ ;?, AaC~lU

c22 ~ Ci7~"'CGAA.A~~'C'GaA AUppp0~1 824 C~CAU ,~,BpUp 825 CC~ACP. CLlc"CGAa,F~t~LCGAA AA~Q

E 2 ADGUCCQ CL~~GG..~C~~..~;1 ACA~AU

8 3 CADGL1CC C'CGht~G:~CCG?.~~AG~CCGr'L~

0 4 DC,C.~CACCL ~a"C ' ' AG~aUG'J

8 6 '~CT~AC C'~ -G.BGAGGC'CGAAAC'~..."CGAA
6 AC~LT

869 AAACCOC CC~C~"CGAAAGGCCG?~ AC~ACW

875 :~UDCF~tIAgyp, 876 BAUOCAU C~ AACCCG

877 AUAL1UCA C'JG~,G:,CC".,Fu~."CGA?, A:,ACCVC

8 8 LUGw:A ~ - p~p~, c 9 AC",r=.CCCC'JG:~ ~ ~C'"AA AUI7pDUG

913 :~'G:~,~GC~?GhL~CG~,G:~..~CG~aA
AQCCQGC

914 ~L7~~ C~UC"~AGGCCGAAA~.~CGAA

916 G CCGA2~,Cr,CC",,,AF,AGu.."~A
AGAp 921 BQGJ~A ' ,w.~CGA1 AUG~"~G

923 UC~CAA

9 2 GuLIC;JOCCDG? 'CGAA ,'~~L~T, 943 U~AU ~-~pUp 9 4 AC~AU CQGF.LIGnGGCCGAA~,G~;~CCGaA
6 AaGALTGC

947 AAGA~A CUGAL~F~G:".."CG.~T,A~CCGP.A
~AL1G

9 4 CAA~GF~IJCLJG:-.IJC~G;~~G?..'~
9 A~AI3GF, o c LT w~.,~,~C'CC"C'CGAAAGGV."CGAA

952 A~ CDG~I7GAGS,~C~~' AnGGCCGap.
ALRAL~A

5 4 L'GAGi7C~C~JG:~L7G~Cr,CC'~'~,AG',CCGAA
AGA~AU

55 L'L'GAGUCC'C1C~AC~CCGAAA~~CCGA?~
AnGAUAA

960 GC~AAW CL'GA w ' CC.CGAA ~GaCAAp, 9 64 ~ CUGAI~."CGAAAGGCCGAa, AUL~7 965 nGCGi~GG CDGF.UGF~c:~CCCGAAF~CCGAA
AALJG'GAG

966 ~JC~,G CUG;~L'GAGG,.~r'C~A~,T,G:,CCGA?, ,~Ap.L~3Gp~

969 C,~GnAGU CUG:~L'Gr~C,GCCGAF~AGGCCG?.A
?.C,G~AU

573 ACOG:v~ CUGhLTv,AGGCCGAA~~G:,CC".~.
~.G""'v~GG

974 C~C~~s?,r'~AGv~:C~~ ae~uara' G

976 UACACUG CQGAB'"~~C'CAAF~.."C".~
:~' 083 C.'~'UAC CLIGn,I~AAr'~CC,1A aG~

086 UC,.~.CLTAA CUC,nL~s'nC,.~~CC~aA'n?.G:~C~u~A
r~C?~

988 rUDGCCU CUGi.L'C~~GG.'W~'~r~G:~:.:f~a a~..CLUsC

c8g CAUO'GCC CQGAI~~CGAFaAG~~~ ar.DACUA

1007 C CUGaL~a~.GVaCCGr~?.AWCCv~a aC,~vW'~1G

1 pi CLICCCfIU C~"C"va."~e1 aL'GCCLJ~s 1024 ACCZJCLTG CL7GAUGAGG.."C":AAnG.IC~~
aCLCr'CC

1032 CUC"w;JG CflG,AI~G:~CC"'~SG~~:.CGaA

1044 AGnUCW CCIGnI~~z~CC~aA a~.s~.~CCsn?a nUrL.'CCrC

1050 UCaUAUA CUGAL'GaGGCC~~?.~Z ".v'v'~
:~':C:rL:GA

10_'2 C~UC.~UA CUGAL~G:,CCGAP.AGG..~A
?.G~rCW

1054 UC.:AIJCA COGAL1G~.G~.CCyIr~C~,:.CGAA
aI~C

1072 UUG~ CUG:,QG:~GGCCGaAaC~cCG:,a aL'CZCW

1085 UG'UCULJU CUC~~'C~AAG:,CCG,?~a aGvL'GL'U

" 03 UC~G7D C'C~t~aI~AGG~~CC~A1C,:,~~CG?.~
?LT~G1C

1104 cvG~~u ~ A~~

pa c,c~ ~ ~:.a aG~a~r 11 ? AL~1L~~G CUGAW' "~C~.y,a aC?.~CGGZT

cc~ACUC cuca~~;T.~ ~aAa~:~c~a~ ac-aACac 1..23 UG'-vGUC cncc~ap.A~c~ ~

1 ~ L'FaC,CCUC CUGnI:~C~CCCGAAAG:~...~'CGnFa 39 r1C",vCTC'~

,146 UC-JIJUC'~A CUGhUGAGGCCGaAAGGCC"~a aG~.'WUCQ

'! 14 GaUGUUU CL~GA~.G:~..' CC'~CC, a 8 .~~GCCfJ

v~,a.GC-u c~,~;~uc~;~;~c~ - . -ar~wG

" 60 UL~:~,W CUGALT:.:~G:~.:CGAAF.G~CC''.:A?.
.~,G.."UC,1U

1 1 UUtJGGP.U CUG:~L'~,GGCCGaAF~G;~CCG?.A
61 :.aC~."L7G?a .

1154 UCUUUUG CUGnUG~CCGr'~.r'~AG'vCC~~ar'aa aUL~G.

1.73 AG.UCAU CU"~I3GAGGCC~~CG?~?~ aLCUUW

11 E1 F.AF.G~'"QC ' ~"C.sAaF.G:~CC"'.~:~
aC?~L'GD

1187 t~CDG1 ~ ~1 aG.'Z.'CUA

11 a UUFaACUC CQGa;~CGr'J~AG,~~C~. .~.e~
8 ar~,GC~C'J

1 ~ UUUUAUU CU~~CGA7~,F~G:~CCGaa aC

1 ? UUWUAU CUGAL~GwCGnaAC~CCG~ AACJC?.a ~

m ~ 5 m ~ ~
~I

y ~

~ ~

' 5~5 m N
a O iC
r.
N $ I
~ c~
.r w a ~~5 y y o .-i c"
~..i r a, r .a ~ ~ a ~., w m c's ~
~555~
m v c m v v m c ~ a 5~~5 ~

~

o ~~ 55~5 O ~ U
U
~
U

L~l~ C ~ ~
~
<

C~
A

t WC
~ ~

O .

rC G
iC yC
~

~ 5 .. C~ C9 G ~
< C~
C
G
G
G
G

W U
C iC

z _ G ~~~

ao O

. yo wo d " O m y o ' u~
e~
o, ~

y r , w wo o, ~

G O

H

Table 39: Large-Scale Synthesis Sequence Activator Amidite Time' % Full [ AddedlFinal][AddedlFinal] Length (min) (min) Product A9T T [0.50!0.33][0.1/0.02] '15 m 85 A9T S (0.2510.17][0.1/0.02] 15 m 89 (GGU)3GGT T [0.5010.33][0.110.02] ~ 5 m 78 (GGU)3GGT S [0.2510.17][0.1!0.02] 15 m 81 C9T T [0.5010.33][0.1!0.02] 15 m 90 C9T S [0.2510.17][0.110.02] 15 m 97 U9T T [0.50/0.33][0.1/0.02] 15 m 80 U9T S [0.25/0.17][0.1/0.02] 1 ~ m 85 A (36-mer) T [0.50/0.33][0.110.02] 15/15m 21 A (36-mer) S [0.25/0.17][0.1/0.02] 15I~5 m 25 A (36-mer) S [0.5010.241[0.1!0.03] 15l'l5 m 25 A (36-mer) S [0.50/0.18]j0.1l0.05] 15115 m 38 A (36-mer) S [0.5010.18][0.110.05] 10J5 m 42 'Where two coupling times ndicated A coupling sre i the first refers to RN

and the second to 2'-O-methylcoupling. 5-S-Ethyltetrazole, S = T =

tetra.zole activator. cA UCU GAU AGG CCG
A is 5' -ucu c GAG GCC
GAA

AAA Auc ccu -3' where lewerecase represents 2'-O-methylnucleetides.

Table 40: Base Deprotection Sequence Deprotection Time T C ~ ~ Full Reagent (min) Length Product i8u(GGU)4 NH,~OHIEiOH 16 h 55 62.5 MA 10 m 65 62.7 AMA 10 m 65 74.8 MA 10 m 55 75.0 AMA 10 m 55 772 iPrP(GGU)4NH4OH/EtOH 4 h 65 44.8 MA 10 m 65 65.9 AMA 10 m 65 59.8 MA 10 m 55 61.3 AMA . 10 m 55 60.1 .

C9U NH40H/EtOH 4 h 65 , 75.2 MA 10 m 65 79.1 AMA 10 m 65 77.1 MA 10 m 55 79.8 AMA 10 m 55 75.5 A (36-mer)NH40H/EiOH 4 h 65 22.7 MA 10 m 65 28.9 .. .

Table 41: 2'-O-Alkylsilyl Deprotection Sequence Deprotection Time T % ~Futl C

Reagent (min) Length Product AgT TBAF 24 h 20 84.5 1.4 M HF 0.5 65 81.0 h (GGU)4 TBAF 24 h 20 60.9 1.4MHF 0.5h 65 67.8 TBAF 24 h 20 86.2 1.4 M HF 0.5 65 86.1 h U ~ p TBAF 24 h 20 84.8 1.4 M HF 0.5 65 84.5 h B (36-mer) iBAF 24 h 20 25.2 1.4MHF 1.5h 65 30.6 A (36-mer) THAF 24 h 20 29.7 1.4 M HF 1.5 65 30.4 h B is 5'- UCU AG GCC
CCA UCU GAU GAA
G AGG
CCG
AAA
AUC
CCU

-3'.

' 287 'N .

.

N
~ CD r1 C C

'fJ CIA L'~ GV C C
CV C~ r~

r~ 1~

~ o~

!~3 o ~ O imp O ~ ~

~

N 1! 1~
rl r-c IWl !V

~1 , '~ d~ e'" to CD cC
r ~~

~ O ~ p O O

d v~ ~n m v~
L'~ , Zp to k A N N

N ri r1 h.s O ~ :~ : ;~ ~ .c r-' ~. ~. ~" ;, ...

cV ~ -~r o c~ co o, ~a ~ N
N N ~ N

r"~.i~ C'~~'~~ ~ L' L' ~ 7 n U~

Zsa N

.N
N. , ' N ,-.

C ~ 00 CO

V

v~ m tJ N O

M N M O
yS M

k N ~ N ,-t N

'N

p' ~ ~ ~ b ca ~ ~

Ltd ~ ~ o ~ o '~ >

O L'7 .d, ""r L7 d' b~ K

1; SG
N r-t N

a~ e~ a~

.
r., : N N CV
L' w .
r , M ~ p ~

_ c''7e'~ m e'~ M
L.

H

Table ~4. Kinetics of Self=Processing In Intro SE:lf Processing Constructs k {~ 1) 1.16 ~ 0.08 ~y ~ o.5s = o.i5 o.ss = o.os Hp(G~ i 0.054 = O.G03 k represents the uaimolecular rate constant for n'bozpme self cleavage determined from a non-linear, least-squares fit (KaleidaGraph, Synergy Software, Reeling, PA) to the equatioa:
(fraction Uncleaved Transcript) _ ~ (1-e'~
The equation describes the extent of r~ozyme processing in the presense of ongoing transcription (Long & Uhlenbeck, 1°94 Proc. Natl. Acad. ~ci' LTSA 91, 697 as a function of time {t) and the unimolecular rate constant for cleavage (k). Each value of k represents the average (j range) of values determined from two experiments.

Table 45 entry Modification t (m) ty~ (m) p = tS/tA

., ActivityStability X '18 (tA) (tS) 1 U4 & U7 = U 1 0.1 1 2 U4 & U7 = 2'-G~-Me-U4 260 650 3 U4 = 2'=CHrU 6.5 120 180 4 U7 = 2'=CHZ-U 8 280 350 U4 & U7 = 2'=CH2-U 9.5 120 130 6 U4 = 2'=CF~-U 5 320 640 7 U7 = 2'=CFz-U 4 220 550 8 U4 8 U7 = 2'=CF2-U 20 320 160 9 U4 = 2'-F-U 4 320 800 U7 ~ 2'-F-U 8 400 500 11 U4 8~ U7 = 2'-F-U 4 300 750 12 U4 = 2'-C-Afly!-U 3 >500 >1700 13 U7 - 2'-C-Aily!-U 3 220 730 14 U4 ~ U7 = 2'-C-Ally!-U3 120 400 U4 = 2'-araF-U 5 > 500 > 1000 16 U7 ~ 2'-araF-U 4 350 875 17 U4 $ U7 = 2'-araF-U15 500 330 18 U4 = 2'-NH2-U 10 500 500 19 U7 = 2'-NH2-U 5 500 7 000 U4 8 U7 = 2'-NH2-U 2 300 1500 21 U4 = dU 6 100 170 22 U4 8. U7 = dU 4 ' 240 600

Claims (30)

1. A process for purifying chemically synthesized RNA
having one or more chemical modifications, comprising:
(a) loading the RNA onto reverse phase high-performance liquid chromatography (HPLC) column, wherein the RNA comprises a 5'-protecting group;
(b) eluting the RNA by passing a suitable buffer through the reverse phase column;
(c) removing the 5'-protecting group from the RNA
to obtain unprotected RNA;
(d) loading the unprotected RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(e) eluting the unprotected RNA by passing a suitable buffer through the anion exchange column to obtain an eluate; and (f) collecting the eluate from the anion exchange column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.

2. A process for purifying chemically synthesized RNA
having one or more chemical modifications, comprising:
(a) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(b) eluting the RNA by passing a suitable buffer through the column to obtain an eluate; and (c) collecting the eluate from the column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.

3. A process for purifying chemically synthesized RNA
having one or more chemical modifications, comprising:
(a) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(b) eluting the RNA by passing a suitable buffer through the column to obtain an eluate;
(c) collecting the eluate from the column and desalting the eluate; and (d) recovering the RNA from the desalted eluate under conditions which allow for purification of the RNA.

4. A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
(a) contacting the RNA with an alkylamine under conditions suitable for removing any exocyclic amine protecting groups or phosphate ester protecting groups;
(b) contacting the RNA with triethylamine-hydrogen fluoride under conditions suitable to remove any alkylsilyl protecting groups from the RNA;
(c) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(d) eluting the RNA by passing a suitable buffer through the column to obtain an eluate; and (e) collecting the eluate from the column and recovering the RNA from the eluate, under conditions which allow for purification of the RNA.

5. A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
(a) contacting the RNA with an alkylamine under conditions suitable for removing any exocyclic amine protecting groups or phosphate ester protecting groups;
(b) contacting the RNA with triethylamine-hydrogen fluoride under conditions suitable to remove any alkylsilyl protecting groups from the RNA;
(c) loading the RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;
(d) eluting the RNA by passing a suitable buffer through the column to obtain an eluate;
(e) collecting the eluate from the column and desalting the eluate; and (f) recovering the RNA from the desalted eluate under conditions which allow for purification of the RNA.

6. The process of any one of claims 1-5, wherein the anion exchange column is selected from the group consisting of Pharmacia Mono Q® column and Dionex NucleoPac® column.

7. The process of any one of claims 1-5, wherein the anion exchange column comprises resins that are either quaternary of tertiary amino derivatized stationary phases.

8. The process of claim 7, wherein the resins are either silica-based or polystyrene based.

9. The process of any one of claims 1-8, wherein the RNA is an enzymatic RNA.

10. The process of claim 9, wherein the enzymatic RNA
is in a hammerhead motif.

11. The process of any one of claims 1-10, wherein the RNA comprises a plurality of chemical modifications.

12. The process of any one of claims 1-11, wherein the chemical modifications are sugar modification.

13. The process of any one of claims 1-11, wherein the chemical modifications are base modification.

14. The process of any one of claims 1-11, wherein the chemical modifications are phosphate backbone modification.

15. The process of claim 12, wherein the sugar modification is 2'-O-methyl modification.

16. The process of claim 12, wherein the sugar modification is 2'-deoxy-2'-amino modification.

17. The process of claim 12, wherein the sugar modification is 2'-deoxy-2'-fluoro modification.

18. The process of claim 14, wherein the phosphate backbone modification is phosphorothioate modification.

19. The process of any one of claims 1-8, wherein the RNA is an antisense RNA.

20. The process of any one of claims 1-8, wherein the RNA is between 28 and 70 nucleotides long.

21. The process of claim 20, wherein the RNA is between 30 and 40 nucleotides long.

22. The process of any one of claims 1-11, wherein the RNA is chemically synthesized using solid phase synthesis.

23. The process of claim 22, wherein the solid phase synthesis utilizes nucleoside monomers having a 5'-protecting group and a 3'-coupling group.

24. The process of claim 23, wherein the 5'-protecting group is dimethoxytrityl group.

25. The process of claim 23, wherein the 3'-coupling group is phosphoramidite group.

26. The process of claim 22, wherein the solid phase synthesis of RNA is carried out on controlled pore glass (CPG) solid support.

27. The process of claim 22, wherein the solid phase synthesis of RNA is carried out on polystyrene solid support.

28. The process of claim 18, wherein the phosphorothioate modification is introduced using a sulfurizing reagent.

29. The process of claim 28, wherein the sulfurizing reagent is Beaucage reagent.

30. The process of claim 4 or claim 5, wherein the alkylamine is methylamine.