WO1998050530A2 - Enzymatic nucleic acids: synthesis, selection and use - Google Patents

Abstract

Nucleic acid catalysts which modulate the expression of Raf gene; method of delivery, screening, identification, synthesis, deprotection, purification, of nucleic acid catalysts and processes for identification of nucleic acid molecules is described.

Description

DESCRIPTION

ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF EXPRESSION OF C-RA-F

Background Of The Invention

This invention relates to methods and reagents for the treatment of diseases or conditions relating to the levels of expression of raf genes.

The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.

The Raf family of serine/threonine kinases function as cytoplasmic signaling proteins that transduce mitogenic signals in response to activation of various growth factor receptors (for reviews, see Daum, 1994 Trends in Biochem. Sci. 19, 474; Katz,

1997, Curr. Opin. Genet. Devel. 7, 75; Marais, 1996, Cancer Surveys 27; Naumann,

1997, Cancer Res. 143, 237). c-Raf is the cellular homolog of v-Raf, the transforming element of the murine sarcoma virus 3611. The Raf family consists of three highly conserved isozymes in vertebrates: c-Raf-1, which is constitutively expressed in all tissues, A-Raf, which is expressed in urogenital tissue and B-Raf which is expressed in and cerebrum and testes (Storm, 1990, Oncogene 5, 345). Inappropriate expression of these key genes involved in cell growth and differentiation can result in uncontrolled cell proliferation and/or propagation of damaged DNA, leading to hyperproliferative disorders such as cancer, restenosis, psoriasis and rheumatoid arthritis.

Raf is one of the major downstream effectors of Ras, a member of the class of small GDP/GTP-binding proteins involved in cellular signal transduction pathways (figure 35; Marshall, 1995, Molec. Reprod. Devel, 42, 493). Appropriate mitogenic signals cause an increase in levels of the GTP -bound Ras. In its GTP-bound active state, Ras binds Raf and localizes it to the plasma membrane. This results in activation of the Raf kinase activity. Activated Raf in turn phosphorylates MEK, thereby activating the MAP kinase signaling cascade leading to cell cycle progression. Amino terminal truncation of Raf leads to constitutively active protein. Expression of either constitutively active Raf or constitutively active MEK is sufficient for oncogenic transformation of fibroblasts (Cowley, 1994, Cell 77, 81; Mansour, 1994, Science 265, 966; Kolch, 1991, Nature 349, 426). In normal cells, the expression level of Raf is limiting in cellular transformation (Cuadrado, 1993, Oncogene 8, 2443). The pivotal position that the Ras and Raf family of proteins occupy in cellular signal transduction pathways emphasizes their importance in the control of normal cellular growth.

Activation of Raf in mammalian cells is triggered by a variety of growth factors and cytokines. Raf activation has been observed in cardiac myocyte cultures stimulated by fibroblast growth factor (FGF), endothelin or phorbol ester (Bogoyevitch, 1995, J. Biol. Chem. 270, 1). Activation has also been seen in Swiss 3T3 cells treated with bombesin and platelet derived growth factor (Mitchell, 1995, J. Biol. Chem. 270, 8623) or with colony stimulating factor or lipopolysacchride (Reimann, 1994, J. Immun. 153, 398), in L6 myoblasts stimulated with insulin-like growth factor (Cross, 1994, Biochem J. 303, 21), as well as in B cells stimulated via the immunoglobulin receptor (Kumar, 1995, Biochem J. 307, 215).

There is growing evidence from a number of laboratories that suggests that the Ras/Raf pathway may also be involved in cell motility (Bar-Sagi and Feramisco, 1986 Science 233, 1061; Partin et al, 1988 Cancer Res. 48-6050; Fox et al, 1994 Oncogene 9, 3519). These studies show that cell lines transfected with activated Ras show an increase in ruffling, pseudopod extension and chemotactic response, all of which are cell-motility- related processes. Uncontrolled cell motility has been implicated in several pathological processes such as restenosis, angiogenesis and wound healing.

Raf activation leads to induction of several immediate early transcription factors including NF-kB and AP-1 (Bruder, 1992, Genes Devel. 6, 545; Finco, 1993, J. Biol Chem. 268, 17676). AP-1 regulates expression of a variety of proteases (Sato, 1994 Oncogene 8, 395; Gaire, 1994, J Biol Chem 269, 2032; Lauricell-Lefebvre, 1993, Invasion Metastasis 13, 289; Troen, 1991, Cell Growth Differ 2, 23). A cascade of MMP and serine proteinase expression is implicated in the acquisition of an invasive phenotype as well as in angiogenesis in tumors (MacDougall, 1995, Cancer and Metastasis Reviews 14, 351). Thus, Raf signaling is expected to contribute to increased invasiveness in tumor cells, leading to metastasis.

Coexpression studies of Raf- 1 and Bcl-2 have shown that these proteins bind and interact to synergistically suppress apoptosis (Wang, 1994, Oncogene 9, 2751). Thus, overexpression of Raf- 1 in tumor cells is likely to contribute to malignant transformation and increased resistance to chemotherapeutic agents. Overexpression of c-Raf-1 is observed in squamous cell carcinomas of the head and neck taken from patients resistant to radiation therapy (Riva, 1995, Oral Oncol, Eur. J. Cancer 31B, 384) and in lung carcinomas (Rapp, 1988, The Oncogene Handbook, 213). Activated (truncated) Raf has been detected in a number of human cancers including small-cell lung, stomach, renal, breast and laryngeal cancer (Rapp, 1988, The Oncogene Handbook, 213).

Therapeutic intervention in down-regulating Raf expression have focused on antisense oligonucleotide approaches:

Antisense oligonucleo tides targeting c-Raf-1 were used to demonstrate that IL-2 stimulated growth of T cells requires c-raf (Riedel, 1993, EMr. J. Immunol. 23, 3146). Antisense oligonucleotides targeting c-Raf-1 in SQ-20B cells showed reduced Raf expression and increased radiation sensitivity (Soldatenkov, 1997, The Cancer J. from Scientific American 3, 13). Rapp et al. have disclosed a method for inhibiting c-Raf-1 gene expression using a vector expressing the gene in the antisense orientation (International PCT Publication No. WO 93/04170). Antisense oligonucleotides targeting c-Raf-1 in SQ-20B cells showed reduced DNA synthesis in response to insulin stimulation in rat hepatoma cells (Tomkvist, 1994, J. Biol. Chem. 269, 13919). Monia et al. have disclosed a method for inhibiting Raf expression using antisense oligonucleotides (U.S. Patent No. 5,563,255) and shown that antisense oligonucleotides targeting c-Raf-1 can inhibit Raf mRNA expression in cell culture, and inhibit growth of a variety of tumor types in human tumor xenograft models (Monia et al, 1996, Proc. Natl. Acad. Sci.93, 15481; Monia et al, 1996, Nature Med. 2, 668). No toxicity was observed in these studies following systemic administration of c-Raf antisense oligonucleotides, suggesting that at least partial down regulation of Raf in normal tissues is not overtly toxic.

It has been proposed that synthetic ribozymes can be delivered to target cells exogenously in the presence or absence of lipid delivery vehicles (Thompson et al, International PCT Publication No. WO 93/23057; Sullivan et al, International PCT Publication No. WO 94/02595).

Recently Sandberg et al, 1996, Abstract, IBC USA Conferences on Angiogenesis Inhibitors and other novel therapeutics for Ocular Diseases of Neovascularization, reported pharmacokinetics of a chemically modifies hammerhead ribozyme targeted against a vascular endothelial growth factor (VEGF) receptor RNA in normal and tumor bearing mice after daily bolus or continuous infusion.

Desjardins et al, 1996, J. Pharmacol. Exptl. Therapeutic, 27, 8, 1419, reported pharmacokinetics of a synthetic, chemically modified hammerhead ribozyme against the rat cytochrome P-450 3A2 mRNA after single intravenous injection.

The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes to cleave Raf RNA.

Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate normal Raf gene expression in mammalian cells and/or whole animal.

Summary Of The Invention

This invention relates to identification, synthesis and use of nucleic acid catalysts to cleave RNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by c-raf gene. Such ribozymes may be used to inhibit the hyper-proliferation of tumor cells in one or more cancers, restenosis, psoriasis, fibrosis and rheumatoid arthritis.

In the present invention, ribozymes that cleave c-raf RNA are described. Moreover, applicant shows that these ribozymes are able to inhibit gene expression and cell proliferation in vitro and in vivo, and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described herein, that other ribozymes that cleave target RNAs required for cell proliferation may be readily designed and are within the invention.

By "inhibit" is meant that the activity of c-raf or level of RNAs encoded by c-raf is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes is preferably below that level observed in the presence of an inactive

RNA molecule able to bind to the same site on the mRNA, but unable to cleave that

RNA.

By "nucleic acid catalyst" is meant a nucleic acid molecule capable of catalyzing (altering the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific manner. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur.

100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific nucleic acid catalysts described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in a nucleic acid catalyst of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.

By "substrate binding arm" or "substrate binding domain" is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figure 1 and 3. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions. The ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).

In one of the preferred embodiments of the inventions herein, the nucleic acid catalyst is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis d virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al, 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al, 1990 Nucleic Acids Res. 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al, 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al, 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al, International PCT Publication No. WO 96/22689; 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 a nucleic acid catalyst 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 have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

By "equivalent" RNA to c-raf is meant to include those naturally occurring RNA molecules associated with cancer in various animals, including human, rodent, primate, rabbit and pig. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like.

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

In a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The nucleic acid catalyst is preferably targeted to a highly conserved sequence region of a target mRNAs encoding c-raf proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such nucleic acid catalysts can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.

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 levels of c- Raf activity in a cell or tissue.

By "related" is meant that the inhibition of c-raf RNAs and thus reduction in the level of respective protein activity will relieve to some extent the symptoms of the disease or condition.

In preferred embodiments, the ribozymes have binding arms which are complementary to the target sequences in TablesXII-XIX. Examples of such ribozymes are also shown in Tables XII-XIX. 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 or core 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.

Thus, in a first aspect, the invention features ribozymes that inhibit gene expression and/or cell proliferation. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited. In a preferred embodiment, ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In another preferred embodiment, the ribozyme is administered to the site of c-raf expression (e.g., tumor cells) in an appropriate liposomal vehicle.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit c-raf activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510). In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.

By "patient" is meant an organism which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which nucleic acid -. „

catalysts can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.

By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

These ribozymes, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with c-raf levels, the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.

In a further embodiment, the described ribozymes can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described ribozymes could be used in combination with one or more known therapeutic agents to treat cancer.

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 1-501, 1078-1152, 1461-1768, 1841-1912, 2354-2794 and 2846-2956. Examples of such ribozymes are shown as Seq. I.D. Nos. 502-1002, 1003-1077, 1153-1460, 1769-1840, 1913-2353 and

2795-2845. Other sequences may be present which do not interfere with such cleavage.

Ribozymes that cleave the specified sites in Raf mRNAs represent a novel therapeutic approach to treat tumor angiogenesis, ocular diseases, rhuematoid arthritis, psoriasis and others. Applicant indicates that ribozymes are able to inhibit the activity of Raf 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 Raf mRNAs may be readily designed and are within the invention.

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 shows the secondary structure model for seven different classes of nucleic acid catalysts. Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273). RNase P (M1RNA): EGS represents external guide sequence (Forster et al, 1990, Science, 249, 783; Pace et al, 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS means 3 '-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al, 1994, Biochemistry, 33, 2716). VS RNA: I- VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577). HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047). Hammerhead Ribozyme: : I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527). Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a 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 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 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.

(Burke et al, 1996, Nucleic Acids & Mol. Biol, 10, 129; Chowrira et al, US Patent No. 5,631,359).

Figure 2 shows a general approach to accessible site and target discovery using nucleic acid catalysts.

Figure 3 is a diagram of a hammerhead ribozyme. The consensus hammerhead cleavage site in a target RNA is a "U" followed by "H" (anything but "G"). The hammerhead ribozyme cleaves after the "H." This simple di-nucleotide sequence occurs, on average, every 5 nt in a target RNA. Thus, there are approximately 400 potential hammerhead cleavage sites in a 2-Kb mRNA. Stems I and III are formed by hybridization of the hammerhead binding arms with the complementary sequence in target RNA; it is these binding arms that confer specificity to the hammerhead ribozyme for its target. The binding arms of the hammerhead are interrupted by the catalytic domain that forms part of the structure responsible for cleavage.

Figure 4 shows a scheme for the design and synthesis of a Defined Library: simultaneous screen of 400 different ICAM-targeted ribozymes is used as an example. DNA oligonucleotides encoding each ICAM-targeted ribozyme are synthesized individually (A), pooled (B), then cloned and converted to retroviral vectors as a pool. The resulting retroviral vector particles are used to transduce a target cell line that expresses ICAM (B). Cells expressing ribozymes that inhibit ICAM expression (ICAM- low) are sorted from cells expressing ineffective ribozymes by FACS sorting (C), effective ribozymes enriched in the ICAM-low population of cells are identified by filter hybridization (D).

Figure 5 A) shows randomization of the binding arms of a hammerhead ribozyme to produce a Random Library. The binding arms can be of any length and any symmetry, i.e., symmetrical or assymmetrical. B) shows complexities of hammerhead Random Ribozyme Libraries comprising a 6-nt or a 7-nt long binding arms.

Figure 6 is a schematic overview of Target Discovery strategy. An oligonucleotide is prepared in a single reaction vessel in which all 4 standard nucleotides are incorporated in a random fashion in the target binding arm(s) of the ribozyme to produce a pool of all possible ribozymes (A). This pool is cloned into an appropriate vector in a single tube to produce the Random Library expression vector (B) and retroviral vector particles are produced from this pool in a single tube (C). The resulting Random Ribozyme Library retroviral expression vector pool is then used to transduce a cell type of interest (D). Cells exhibiting the desired phenotype are then separated from the rest of the population using a number of possible selection strategies (E and see text). Genes that are critical for expression of the selected phenotype can then be identified by sequencing the target binding arms of ribozymes contained in the selected population (F).

Figure 7 shows an example of application of Random Ribozyme Libraries to identify genes critical for the induction of ICAM expression. Human Umbilical Vein Endothelial Cells (HUVECs) are transduced with a Random Ribozyme Library (A), ICAM expression is induced using TNF-alpha (B), and cells expressing ribozymes that inhibit ICAM induction are selected from cells expressing ineffective ribozymes by sorting ICAM-low cells (C). Genes critical for ICAM induction are identified by sequencing the binding arms of the ribozymes that inhibit ICAM expression in the ICAM- low cells.

Figure 8 is an example of an efficient cloning strategy for producing a Defined or Random Ribozyme Libraries. DNA oligos encoding ribozyme coding regions and restriction sites for cloning are designed to also contain a stem-loop structure on the 3' ends (1). This stem loop forms an intramolecular primer site for extension to form a double-stranded molecule by DNA polymerase (2). The double-stranded fragment is cleaved with appropriate restriction endonucleases to produce suitable ends for subsequent cloning (3).

Figure 9 shows molecular analysis of the PNP -targeted Defined Ribozyme

Library: sequence analysis. Plasmid DNA from the PNP-targeted Defined Ribozyme

Library was prepared and sequenced as a pool. The sequencing primer used reads the non-coding strand of the region encoding the ribozymes. Note that the sequence diverges at the binding arm, converges at the catalytic domain (5'

TTTCGGCCTAACGGCCTCATCAG-3'), and then diverges at the other binding arm. These results are consistent with those expected for a sequence of a heterogeneous pool of clones containing different sequences at the ribozyme binding arms.

Figure 10 shows molecular analysis of the PNP-targeted Defined Ribozyme Library: sequence analysis after propagation in Sup TI human T cells and selection in 10 mmol 6-thioguanosine. Sup TI cells were transduced with retroviral vector-based

Defined Ribozyme Library comprised of 40 different PNP-targeted ribozyme oligos cloned into the U6+27 transcription unit (Figure 11D). The cells were propagated for 2 weeks following transduction, then subjected to 16 days of selection in 10 mmol 6- thioguanosine. Surviving cells were harvested, and ribozyme sequences present in the selected population of cells were amplified and sequenced. Note that, relative to the original Library where sequences of the binding arms were ambiguous due to the presence of 40 different ribozymes (Figure 9), the sequence of the binding arms in the selected population corresponded to only 1 of the 40 ribozymes included in the Library. These results suggest that this ribozyme was the most-potent ribozyme of 40 ribozymes tested.

Figure 11 is a schematic representation of transcription units suitable for expression ribozyme library of the instant invention. A) is a diagrammatic representation of some RNA polymerase (Pol) II and III ribozyme (RZ) transcription units. CMV Promoter Driven is a Pol II transcript driven by a cytomegalovirus promoter; the transcript can designed such that the ribozyme is at the 5'- region, 3 '-region or some where in between and the transcript optionally comprises an intron. tRNA-DC is a Pol III transcript driven by a transfer RNA (tRNA) promoter, wherein the ribozyme is at the 3'- end of the transcript; the transcript optionally comprises a stem-loop structure 3' of the ribozyme. U6+27 is a Pol III transcript driven by a U6 small nuclear (snRNA) promoter; ribozyme is 3' of a sequence that is homologous to 27 nucleotides at the 5 '-end of a U6 snRNA; the transcript optionally comprise a stem-loop structure at the 3 '-end of the ribozyme. VAI-90 is a Pol III transcript driven by an adenovirus VA promoter; ribozyme is 3' of a sequence homologous to 90 nucleotides at the 5 '-end of a VAI RNA; the transcript optionally comprises a stem-loop structure at the 3 '-end of the ribozyme. VAC is a Pol III transcript driven by an adenovirus VAI promoter; the ribozyme is inserted towards the 3'- region of the VA RNA and into a S35 motif, which is a stable greater than or equal to 8 bp long intramolecular stem formed by base-paired inteaction between sequences in the 5 '-region and the 3 '-region flanking the ribozyme (see Beigelman et al, International PCT Application No. WO 96/18736); the S35 domain positions the ribozyme away from the main transcript as an independent domain. TRZ is a Pol III transcript diven by a tRNA promoter; ribozyme is inserted in the S35 domain and is positioned away from the main transcript (see Beigelman et al, International PCT Application No. WO 96/18736). B) shows various transcription units based on the UI small nuclear RNA (snRNA) system. C) is a schematic representation of a retroviral vectors encoding ribozyme genes. NGFR, nerve growth factor receptor is used as a selectable marker, LTR, long terminal repeat of a retrovirus, UTR, untranslated region. D) shows a U6+27 hammerhead ribozyme transcription unit based on the U6 snRNA. The ribozyme transcript comprises the first 27 nt from the U6 snRNA which is reported to be necessary for the stability of the transcript. The transcript terminates with a stretch of uridine residues. The hammerhead ribozyme shown in the figure has random (N) binding arm sequence. Figure 12 is a schematic representation of a combinatorial approach to the screening of ribozyme variants.

Figure 13 shows the sequence of a Starting Ribozyme to be used in the screening approach described in Figure 12. The Starting Ribozyme is a hammerhead (HH) ribozyme designed to cleave target RNA A (HH-A). Position 7 in HH-A is also referred to in this application as position 24 to indicate that U24 is . the 24th nucleotide incorporated into the HH-A ribozyme during chemical synthesis. Similarly, positions 4 and 3 are also referred to as positions 27 and 28, respectively. s indicates phosphorothioate substitution. Lower case alphabets in the HH-A sequence indicate 2'- O-methyl nucleotides; uppercase alphabets in the sequence of HH-A at positions 5, 6, 8, 12 and 15J indicate ribonucleotides. Positions 3, 4 and 7 are shown as uppercase, large alphabets to indicate the positions selected for screening using the method shown in Figure 12. • indicates base-paired interaction. iB represents abasic inverted deoxy ribose moiety.

Figure 14 shows a scheme for screening variants of HH-A ribozyme. Positions

24, 27 and 28 are selected for analysis in this scheme.

Figure 15 shows non-limiting examples of some of the nucleotide analogs that can be used to construct ribozyme libraries. 2'-O-MTM-U represents 2'-O-methylthiomethyl uridine; 2'-O-MTM-C represents 2'-O-methylthiomethyl cytidine; 6-Me-U represents 6- methyl uridine (Beigelman et al, International PCT Publication No. WO 96/18736 which is incorporated by reference herein).

Figure 16 shows activity of HH-A variant ribozymes as determined in a cell-based assay. * indicates the substitution that provided the most desirable attribute in a ribozyme.

Figure 17A shows the sequence and chemical composition of ribozymes that showed the most desirable attribute in a cell. -. _

Figure 17B shows formulae for four different novel ribozyme motifs.

Figure 18 shows the formula foe a novel ribozyme motif.

Figure 19 shows the sequence of a Starting Ribozyme to be used in the screening approach described in Figure 14. A HH ribozyme targeted against RNA B (HH-B) was chosen for analysis of the loop II sequence variants.

Figure 20 shows a scheme for screening loop-II sequence variants of HH-B ribozyme.

Figure 21 shows the relative catalytic rates (k^) for RNA cleavage reactions catalyzed by HH-B loop-II variant ribozymes.

Figure 22 is a schematic representation of HH-B ribozyme-substrate complex and the activity of HH-B ribozyme with either the 5'-GAAA-3' or the 5'-GUUA-3' loop-II sequence.

Figure 23 shows a scheme for using a combinatorial approach to identify potential ribozyme targets by varying the binding arms.

Figure 24 shows a scheme for using a combinatorial approach to identify novel ribozymes by the varying putative catalytic domain sequence.

Figure 25 shows a table of accessible sites within a Bcl-2 transcript ((975 nucleotides) which were found using the combinatorial in vitro screening process.

Figure 26 shows a table of accessible sites with a Kras transcript (796 nucleotides) which were found using the combinatorial in vitro screening process as well as a graphic depiction of relative activity of ribozymes to those sites.

Figure 27 shows a table of accessible sites with a UP A transcript (400 nucleotides) which were found using the combinatorial in vitro screening process as well as a graphic depiction of relative activity of ribozymes to those sites. i o

Figure 28 shows a graph displaying data from a ribonuclease protection assay

(RPA) after treatment of MCF-7 cells with ribozymes to targeted to site 549 of the transcript (Seq. ID #9). The Bcl-2 mRNA isolated from MCF-7 cells is normalized to

GAPDH which was also probed in the RPA. The graph includes an untreated control and an irrelevant ribozyme (no complementarity with Bcl-2 mRNA).

Figure 29 displays a schematic representation of NTP synthesis using nucleoside substrates.

Figure 30 depicts a scheme for the synthesis of a xylo ribonucleoside phosphoramidite.

Figure 31 is a diagrammatic representation of hammerhead (HH) ribozyme targeted against stromelysin RNA (site 617) with various modifications.

Figure 32 is a is a schematic representation of a one pot deprotection of RNA synthesized using RNA phosphoramidite chemistry.

Figure 33 is a comparison of a one-pot and a two-pot process for deprotection of RNA.

Figure 34 shows the results of a one-pot deprotection with different polar organic reagents.

Figure 35 is a diagrammatic represention of ras signal transduction pathway.

Figure 36 is a diagrammatic representation of hammerhead ribozymes targeted against c-raf RNA.

Figure 37 is a graphical representation of c-raf 2' -C- allyl 1120 hammerhead (HH) ribozyme-mediated inhibition of cell proliferation.

Figure 38 is a graphical representation of inhibition of cell proliferation mediated by c-raf 2'-C-allyl 1120 and 1251 hammerhead (HH) ribozymes. Figure 39 shows the effects of flt-1 ribozymes (active/inactive) on LLC-HM primary tumor growth in mice.

Figure 40 shows the effects of flt-1 ribozymes on LLC-HM primary tumor volume immediately following the cessation of treatment.

Figure 41 shows the effects of flt-1 ribozymes on lung metastatic indices (number of metastases and lung mass).

Figure 42 shows the effects of flk-1 ribozymes (active/inactive) on LLC-HM primary tumor growth in mice.

Figure 43 shows the effects of flk-1 ribozymes on LLC-HM primary tumor volume immediately following the cessation of treatment.

Figure 44 shows the effects of flk-1 ribozymes on lung metastatic indices (number of metastases and lung mass).

Nucleic Acid Catalysts:

Catalytic nucleic acid molecules (ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such nucleic acid catalysts can be used, for example, to target cleavage of virtually any RNA transcript (Zaug et al, 324, Nature 429 1986 ; Cech, 260 JAMA 3030, 1988; and Jefferies et al, 17 Nucleic Acids Research 1371, 1989). Catalytic nucleic acid molecules mean any nucleotide base-comprising molecule having the ability to repeatedly act on one or more types of molecules, including but not limited to nucleic acid catalysts. By way of example but not limitation, such molecules include those that are able to repeatedly cleave nucleic acid molecules, peptides, or other polymers, and those that are able to cause the polymerization of such nucleic acids and other polymers. Specifically, such molecules include ribozymes, DNAzymes, external guide sequences and the like. It is expected that such molecules will also include modified nucleotides compared to standard nucleotides found in DNA and RNA.

Because of their sequence-specificity, trans-cleaving nucleic acid catalysts show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Nucleic acid catalysts can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non- functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited. In addition, nucleic acid catalysts can be used to validate a therapeutic gene target and/or to determine the function of a gene in a biological system (Christoffersen, 1997, Nature Biotech. 15, 483).

There are at least seven basic varieties of enzymatic RNA molecules derived from naturally occurring self-cleaving RNAs (see Table I). Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a substrate/target RNA. Such binding occurs through the substrate/target binding portion of an nucleic acid catalyst which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic and selective cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and thus can repeatedly bind and cleave new targets.

In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R.

Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al, 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Breaker, 1997, Nature Biotech. 15, 427).

There are several reports that describe the use of a variety of in vitro and in vivo selection strategies to study structure and function of catalytic nucleic acid molecules (Campbell et al, 1995, RNA 1, 598; Joyce 1989, Gene, 82,83; Lieber et al, 1995, Mol Cell Biol. 15, 540; Lieber et al, International PCT Publication No. WO 96/01314; Szostak 1988, in Redesigning the Molecules of Life, Ed. S. A. Benner, pp 87, Springer- Verlag, Germany; Kramer et al, U.S. Patent No. 5,616,459; Draper et al, US Patent No. 5,496,698; Joyce, U.S. Patent No. 5,595,873; Szostak et al, U.S. Patent No. 5,631,146).

The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme sufficient to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme (enzymatic nucleic acid) molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the 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.

The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (k_CΑJ) of ~1 min^-1 in the presence of saturating (10 mM) concentrations of Mg²⁺ cofactor. However, the rate for this ribozyme in Mg²⁺ concentrations that are closer to those found inside cells (0.5 - 2 mM) can be 10- to 100- fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a ^cat °f ~30 min^-1 under optimal assay conditions. An artificial 'RNA ligase' ribozyme (Bartel et al, supra) has been shown to catalyze the corresponding self-modification reaction with a rate of -100 min^-1. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min^-1. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may not be optimized to give maximal catalytic activity, or that entirely new RNA motifs could be made that display significantly faster rates for RNA phosphoester cleavage.

By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a sugar moiety. Nucleotide generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other ; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art and has recently been summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g., 6-methyluridine) and others (Burgin et al, 1996, Biochemistry, 35, 14090). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core of the enzyme and/or in the substrate-binding regions.

In another preferred embodiment, catalytic activity of the molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al, 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al, International Publication No. WO 93/15187; and Rossi et al, International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et al, supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All of these publications are hereby incorporated by reference herein).

There are several examples in the art describing sugar and phosphate modifications that can be introduced into nucleic acid catalysts without significantly effecting catalysis and with significant enhancement in their nuclease stability and efficacy. Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'- flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 35, 14090). Sugar modification of nucleic acid catalysts has been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Peπault et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al, 1995 J. Biol Chem. 270, 25702; all of the references are hereby incorporated in their totality by reference herein).

Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid catalysts of the instant invention.

In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such a nucleic acid is also, generally, more resistant to nucleases than the corresponding unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such modifications herein are said to "maintain" the enzymatic activity on all RNA ribozyme.

In a preferred embodiment, the nucleic acid catalysts of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. Using the methods described herein, other nucleic acid catalysts that cleave target nucleic acid may be derived and used as described above. Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures. Sullivan, et al, WO 94/02595, describes the general methods for delivery of nucleic acid catalysts. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al, supra and Draper et al, WO 93/23569 which have been incorporated by reference herein.

Such nucleic acid catalysts can be delivered exogenously to specific cells as required. In the preferred hammerhead motif the small size (less than 60 nucleotides, preferably between 30-40 nucleotides in length) of the molecule allows the cost of treatment to be reduced.

Therapeutic ribozymes delivered exogenously must remain stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al, 1995 Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes by introducing nucleotide modifications to enhance their nuclease stability as described above. o

Synthesis. Deprotection. and Purification of Nucleic Acid Catalysts:

Generally, RNA molecules are chemically synthesized and purified by methodologies based on the use of tetrazole to activate the RNA phosphoramidite, ethanolic-NH4OH to remove the exocyclic amino protecting groups, tetra-n- butylammonium fluoride (TBAF) to remove the -OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA. Examples of chemical synthesis, deprotection, purification and analysis procedures for RNA are provided by Usman et al., 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al. Nucleic Acids Res. 1990, 18, 5433- 5341; Perreault et al. Biochemistry 1991, 30 4020-4025; Slim and Gait Nucleic Acids Res. 1991, 19, 1183-1188. All the above noted references are all hereby incorporated by reference herein.

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 nucleic acid motifs (e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes) are used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure. However, these nucleic acid molecules can also be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci.USA 83, 399; SullengerScanlon et al, 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992 J. Virol, 66, 1432-41; Weerasinghe et al, 1991 J. Virol, 65, 5531-4; Ojwang et al, 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al, 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al, 1990 Science 247, 1222-1225; Thompson et al, 1995 Nucleic Acids Res. 23, 2259). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO93/23569, and Sullivan et al, PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994 J. Biol Chem. 269, 25856).

The ribozymes were 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; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; and Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2'-O- methylated nucleotides. Table II outlines the amounts, and the contact times, of the reagents used in the synthesis cycle. A 6.5-fold excess (163 μL of 0J M = 16.3 μmol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 μL of 0.25 M = 59.5 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, were 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer: detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF (Millipore). B & j Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc.

Deprotection of the RΝA was performed as follows. The polymer-bound oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4mL glass screw top vial and suspended in a solution of methylamine (MA) at 65 °C for 10 min.

After cooling to -20 °C, the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCΝ:H2θ/3:l :l, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder.

The base-deprotected oligoribonucleotide was resuspended in anhydrous

TEA^»HF/NMP solution (250 μL of a solution of 1.5mL N-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA^»3HF to provide a 1.4M HF concentration) and heated to 65°C for

1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.

For anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the RΝA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.

Deprotection of RΝA:

For high throughput chemical synthesis of oligoribonucleotides, it is important that the two main steps involved in the deprotection of oligoribonucleotides (i.e., aqueous basic treatment to remove exocyclic amino protecting groups and phosphate protecting groups and fluoride treatment to remove the 2' -OH alkylsilyl protecting groups such as the tButylDiMethylSilyl) are condensed.

Stinchcomb et al, supra describe a time-efficient (~ 2 hrs) one-pot deprotection protocol based on anhydrous methylamine and triethylamine trihydrogen fluoride. Since it has recently been reported that water contamination during fluoride treatment may be detrimental to the efficiency of the desilylation reaction (Hogrefe et al, Nucleic Acids Res. (1993), 21 4739-4741), it is necessary to use an anhydrous solution of base such as a 33% methylamine in absolute ethanol followed by neat triethylamine trihydro fluoride to effectively deprotect oligoribonucleotides in a one-pot fashion. However it may be cumbersome to apply such a protocol to plate format deprotection where the solid- support is preferentially separated from the partially deprotected oligoribonucleotides prior to the 2 '-hydroxyl deprotection. Indeed, because the methylamine solution used is anhydrous, it may not be suitable to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. Therefore, applicant investigated a 1 : 1 mixture of the ethanolic methylamine solution and different polar additives such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), l-methyl-2-pyrrolidinone (NMP) or 2- methoxyethyl ether (glyme). Of all these additives, dimethylsufoxide is capable of efficiently solubilizing partially deprotected oligoribonucleotides (figure 34). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in figure 33.

The deprotection process commonly involves the deprotection of the exocyclic amino protecting groups by NH4OH, which is time consuming (6-24 h) and inefficient. This step is then followed by treatment with TBAF to facilitate the removal of alkylsilyl protecting groups, which again is time consuming and not very effective in achieving efficient deprotection.

A recent modification of this two-step strategy for oligoribonucleotide deprotection has been reported by Wincott et al, (Nucleic Acids Res., 1995, 23, 2677- 2784) and by Vinayak et al, (Nucleic Acids Symposium series, 1995. 33, 123-125). The optimized conditions make use of aqueous methylamine at 65°C for 15 minutes in place of the ammonium hydroxide cocktail to remove exocyclic amino protecting groups while the desilylation treatment needed to remove the 2'-OH alkylsilyl protecting groups utilizes a mixture of triethylamine trihydrogen fluoride (TEA.3ΗF), N-methyl- pyrrolidinone and triethylamine at 65°C for 90 minutes, thereby replacing tetrabutyl ammonium fluoride.

Stinchcomb et al, International PCT Publication No. WO 95/23225 describe a process for one pot deprotection of RNA. On page 73, it states that:

"In an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol... According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 minutes and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 minutes in a TEA^«3HF reagent. The reaction is quenched with 16 mM TEAB solution."

This invention concerns a one-pot process for the deprotection of RNA molecules. This invention features a novel method for the removal of protecting groups from the nucleic acid base and 2'-OH groups, which accelerates the process for generating synthetic RNA in a high throughput manner (e.g., in a 96 well format).

Chemical synthesis of RNA is generally accomplished using a traditional column format on a RNA synthesizer where only one oligoribonucleotide is synthesized at a time. Simultaneous synthesis of more than one RNA molecule in a time efficient manner requires alternate methods to the traditional column format, such as synthesis in a 96 well plate format where up to 96 RNA molecules can be synthesized at the same time. To expedite this process of simultaneous synthesis of multiple RNA molecules, it is important to accelerate some of the time consuming processes such as the deprotection of RNA following synthesis (i.e., removal of base protecting group, such as the exocyclic amino protecting group and the phosphate protecting groups and the removal of 2' -OH protecting groups, such as the tButylDiMethylSilyl). In a preferred embodiment, the invention features a one-pot process for rapid deprotection of RNA.

Stinchcomb et al, supra described a one-pot protocol for RNA deprotection using anhydrous methylamine and triethylamine trihydrogen fluoride. This procedure involves the use of an anhydrous solution of base such as a 33% methylamine in absolute ethanol followed by neat triethylamine trihydrofluoride to effectively deprotect oligoribonucleotides in a one-pot fashion. However such a protocol may be cumbersome for deprotection of RNA synthesized on a plate format, such as a 96 well plate, because it may be necessary to separate the solid-support from the partially deprotected RNA prior to the 2 '-hydroxyl deprotection. Also, since the methylamine solution used is anhydrous, -, ,

31

it may be difficult to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. So, in a first aspect the invention features the use of a 1 J mixture of the ethanolic methylamine solution and a polar additive, such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), 1- methyl-2-pyrrolidinone (NMP), 2-methoxyethyl ether (glyme) or the like. More specifically, dimethylsufoxide is used to partially deprotect oligoribonucleotides (Figure 32). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in Figure 33.

This invention also concerns a rapid (high through-put) deprotection of RNA in a 96-well plate format. More specifically rapid deprotection of enzymatic RNA molecules in greater than microgram quantities with high biological activity is featured. It has been determined that the recovery of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its deprotection.

In a preferred embodiment, the invention features a process for one-pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, e.g., methylamine), trialkylamine (where alkyl can be branched or unbranched, methyl, propyl or butyl and is preferably ethyl, e.g., ethylamine) and dimethylsulfoxide, preferably in a 10:3:13, or 1:0.3:1 proportion at temperature 20-30 °C for about 30-100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group (e.g., 2-cyanoethyl) (vs 4-20 h at 55-65 °C using

NH₄OH/EtOH or NH₃/EtOH, or 10-15 min at 65 °C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylamine»hydrogen fluoride (3HF^»TEA) and heating at about 50-70 °C, preferably at 65 °C, for about 5-30 min, preferably 15 min to remove the 2 '-hydroxyl protecting group (vs 8 - 24 h using TBAF, 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) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 M). Although some other buffers can be used to quench the desilylation reaction (i.e., triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5'-O-dimethoxytrityl group at the 5 '-end of the oligoribonucleotide thereby facilitating a reverse phase-based solid-phase extraction purification protocol.

By "one-pot" deprotection is meant that the process of deprotection RNA is carried out in one container instead of multiple containers as in two-pot deprotection.

In another preferred embodiment, the invention features a process for one pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, e.g., methylamine), and dimethylsulfoxide, preferably in a 1:1 proportion at 20-30 °C temperature for about 30-100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group (e.g., 2-cyanoethyl) (vs 4-20 h at 55-65 °C using NH₄OH/EtOH or NH₃/EtOH, or 10-15 min at 65°C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylamine'hydrogen fluoride (3HF»TEA) and heating at about 50-70 °C, preferably at 65 °C, for about 5-30 min, preferably 15 min to remove the 2'-hydroxyl protecting group (Other alkylamine*HF complexes may also be used, e.g., trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 M). Although some other buffers can be used to quench the desilylation reaction (i.e., triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5'-O-dimethoxytrityl group at the 5 '-end of the oligoribonucleotide thereby facilitating a reverse phase-based solid-phase extraction purification protocol.

In another aspect the invention features a process for RNA deprotection where the exocyclic amino and phosphate deprotection reaction is performed with the ethanolic methylamine solution at room temperature for about 90 min or at 65°C for 15 min or at 45 °C for 30 min or at 35°C for 60 min.

In a prefeπed embodiment, the process for deprotection of RNA of the present invention is used to deprotect a ribozyme synthesized using a column format as described in (Scaringe et al, supra; Wicott et al, supra).

Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res., 20, 3252).

The average stepwise coupling yields were >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).

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 Uhlenbeck, 1989, Methods Enzymol 180, 51).

Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren,

1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al,

1996 Biochemistry 6, 14090).

Ribozymes were purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al, International PCT Publication No. WO 95/23225, the totality of which is hereby incorporated herein by reference) and are resuspended in water.

The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Tables XII-XIX. 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 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, can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. Preferably, no more than 200 bases are inserted at these locations. The sequences listed in Tables XII-XIX may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables.

Nucleotide Triphosphates:

The use of modified nucleotide triphosphates would greatly assist in the combinatorial chemistry. The synthesis of nucleoside triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research. The polymerase enzyme utilizes nucleoside triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3 ' hydroxyl group of the oligonucleotide' s last nucleotide onto the 5' triphosphate of the next nucleotide. Nucleotides are incorporated one at a time into the oligonucleotide in a 5' to 3' direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.

Most natural polymerase enzymes incorporate standard nucleoside triphosphates into nucleic acid. For example, a DNA polymerase incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerases that are capable of incorporating non-standard nucleoside triphosphates into nucleic acids (Joyce, 1997, PNAS 94, 1619- 1622, Huang et al., Biochemistry 36, 8231-8242).

Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes they must first be converted into nucleoside triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl₃ and trialkyi phosphates was shown to yield nucleoside 5'- phosphorodichloridates (Yoshikawa et al, 1969, Bull. Chem. Soc. (Japan) 42, 3505). Adenosine or 2'-deoxyadenosine 5 '-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981, Ada Biochim. et Biophys. Acad. Sci. Hung. 16, 131-133).

Non-standard nucleoside triphosphates are not readily incorporated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate incorporation of deoxyribonucleotides into RNA (Sousa & Padilla, 1995, EMBO J. 14,4609-4621 , Bonner et al, 1992, EMBO J. 11, 3767-3775, Bonner et al., 1994, J. Biol. Chem. 42, 25120-25128, Aurup et al, 1992, Biochemistry 31, 9636-9641).

McGee et al, International PCT Publication No. WO 95/35102, describes the incorporation of 2'-NH₂-NTP's, 2'-F-NTP's, and 2'-deoxy-2'-benzyloxyamino UTP into RNA using bacteriophage T7 polymerase.

Wieczorek et al, 1994, Bioorganic & Medicinal Chemistry Letters 4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA transcript using bacteriophage T7 RNA polymerase.

Lin et al, 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2'-NH₂-CTP and 2'-NH₂-UTP into RNA using bacteriophage T7 RNA polymerase and ,, -.

36

polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).

The invention features NTP's having the formula triphosphate-OR, for example the following formula I:

where R is any nucleoside; specifically the nucleosides 2'-O-methyl-2,6-diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine riboside; 2'-(N-alanyl) amino-2'-deoxy- uridine; 2'-(N-phenylalanyl)amino-2'-deoxy-uridine; 2'-deoxy -2'-(N-β-alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-amino-uridine; 2'-O- methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-O-methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-Deoxy-2'-(N-histidyl) amino uridine; 2'- deoxy-2'-amino-5-methyl cytidine; 2'-(N-β-carboxamidine-β-alanyl)amino-2'-deoxy- uridine; 2'-deoxy-2'-(Ν-β-alanyl)-guanosine; and 2'-O-amino-adenosine.

In a second aspect, the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2'-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tris- triazolides, phospho-tris-triimidazolides and the like), trialkyi phosphate (such as triethylphosphate or trimethylphosphate or the like) and dimethylaminopyridine (DMAP) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates. By "pyrimidines" is meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.

By "nucleotide triphosphate" or "NTP" is meant a nucleoside bound to three inorganic phosphate groups at the 5' hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1 ' position of the sugar may comprise a nucleic acid base or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group (i.e., allow incorporation into an RNA molecule).

In another preferred embodiment, nucleoside triphosphates (NTP's) of the instant invention are incorporated into an oligonucleotide using an RNA polymerase enzyme. RNA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases.

In yet another preferred embodiment, the invention features a process for incorporating modified NTP's into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified

NTP incorporation under conditions suitable for the incorporation of the modified NTP into the oligonucleotide.

By "enhancer of modified NTP incorporation" is meant a reagent which facilitates the incorporation of modified nucleotides into a nucleic acid transcript by an RNA polymerase. Such reagents include but are not limited to methanol; LiCl; polyethylene glycol (PEG); diethyl ether; propanol; methyl amine; ethanol and the like.

In another preferred embodiment, the modified nucleoside triphosphates can be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull et al, 1995 Pharmaceutical Res. 12, 465). 3o

By "antisense" it is meant a non-nucleic acid catalyst that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al,

1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004; Agrawal et al, U.S. Patent No. 5,591,721; Agrawal, U.S. Patent No. 5,652,356).

By "2-5A antisense chimera" it is meant, an antisense oligonucleotide containing a 5' phosphorylated 2'-5'-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al, 1993 Proc. Natl. Acad. Sci. USA 90, 1300).

By "triplex forming oligonucleotides (TFO)" it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al, 1992 Proc. Natl. Acad. Sci. USA 89, 504).

By "oligonucleotide" as used herein is meant a molecule having two or more nucleotides. The polynucleotide can be single, double or multiple stranded and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

In yet another preferred embodiment, the modified nucleoside triphosphates of the instant invention can be used for combinatorial chemistry or in vitro selection of nucleic acid molecules with novel function. Modified oligonucleotides can be enzymatically synthesized to generate libraries for screening.

Nucleoside modifications of bases and sugars, have been discovered in a variety of naturally occurring RNA (e.g., tRNA, mRNA, rRNA; reviewed by Hall, 1971 77*e Modified Nucleosides in Nucleic Acids, Columbia University Press, New York; Limbach et al, 1994 Nucleic Acids Res. 22, 2183). In an attempt to understand the biological significance, structural and thermodynamic properties, and nuclease resistance of these nucleoside modifications in nucleic acids, several investigators have chemically -

synthesized nucleosides, nucleotides and phosphoramidites with various base and sugar modifications and incorporated them into oligonucleotides.

Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the use of certain nucleoside modifications to stabilize antisense oligonucleotides.

Usman et al, International PCT Publication Nos. WO/93/15187; and WO

95/13378; describe the use of sugar, base and backbone modifications to enhance the nuclease stability of nucleic acid catalysts.

Eckstein et al, International PCT Publication No. WO 92/07065 describe the use of sugar, base and backbone modifications to enhance the nuclease stability of nucleic acid catalysts.

Grasby et al, 1994, Proc. Indian Acad. Sci., 106, 1003, review the "applications of synthetic oligoribonucleotide analogues in studies of RNA structure and function".

Eaton and Pieken, 1995, Annu. Rev. Biochem., 64, 837, review sugar, base and backbone modifications that enhance the nuclease stability of RNA molecules.

Rosemeyer et al, 1991, Helvetica Chem. Ada, 74, 748, describe the synthesis of l-(2'-deoxy-β-D-xylofuranosyl) thymine-containing oligodeoxynucleotides.

Seela et al, 1994, Helvetica Chem. Ada, 77, 883, describe the synthesis of l-(2'- deoxy-β-D-xylofuranosyl) cytosine-containing oligodeoxynucleotides.

Seela et al, 1996, Helvetica Chem. Ada, 79, 1451, describe the synthesis xylose- DNA containing the four natural bases.

In another preferred embodiment, catalytic activity of the molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nαtwre 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al, International Publication No. WO 93/15187; and Rossi et al, International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et al, supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein.).

By "enhanced enzymatic activity" is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties is increased or not significantly (less that 10 fold) decreased in vivo compared to an all RNA ribozyme.

In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme.

In a most preferred embodiment the invention features a method of synthesizing ribozyme libraries of various sizes. This invention describes methods to chemically synthesize ribozyme libraries of various sizes from suitable nucleoside analogs.

Considerations for the selection of nucleotide building blocks and determination of coupling efficiency: In addition to structural considerations (hydrogen bond donors and acceptors, stacking properties, pucker orientation of sugars, hydrophobicity or hydrophilicity of some subgroups constitutive of the nucleotides) that may lead to the selection of a specific nucleotide to be included in the design of a ribozyme library, one of the important features that needs to be considered when selecting nucleotide building blocks is the chemical compatibility of such building blocks with ribozyme synthesis. A "nucleotide building block" is a nucleoside or nucleoside analog that possess a suitably protected phosphorus atom at the oxidation state V reacting readily, upon activation, to give a P^v-containing intemucleoside linkage. A suitable nucleoside building block may also contain a phosphorus atom at the oxidation state III reacting readily, upon activation, to give a P^πι-containing intemucleoside linkage that can be oxidized to the desired P^v- containing intemucleoside linkage. Applicant has found that the phosphoramidite chemistry (P^ιπ) is a preferred coupling method for ribozyme library synthesis. There are several other considerations while designing and synthesizing certain ribozyme libraries, such as: a) the coupling efficiencies of the nucleotide building blocks considered for a ribozyme library should not fall below 90% to provide a majority of full-length ribozyme; b) the nucleotide building blocks should be chemically stable to the selected synthesis and deprotection conditions of the particular ribozyme library; c) the deprotection schemes for the nucleotide building blocks incorporated into a ribozyme library, should be relatively similar and be fully compatible with ribozyme deprotection protocols. In particular, nucleoside building blocks requiring extended deprotection or that cannot sustain harsh treatment should be avoided in the synthesis of a ribozyme library. Typically, the reactivity of the nucleotide building blocks should be optimum when diluted to 100 mM to 200 mM in non-protic and relatively polar solvent. Also the deprotection condition using 3:1 mixture of ethanol and concentrated aqueous ammonia at 65 degrees C. for 4 hours followed by a fluoride treatment as exemplified in Wincott et al. supra, is particularly useful for ribozyme synthesis and is a preferred deprotection pathway for such nucleotide building blocks.

In one preferred embodiment, a "nucleotide building block mixing" approach to generate ribozyme libraries is described. This method involves mixing various nucleotide building blocks together in proportions necessary to ensure equal representation of each of the nucleotide building blocks in the mixture. This mixture is incorporated into the ribozyme at position(s) selected for randomization.

The nucleotide building blocks selected for incorporation into a ribozyme library, are typically mixed together in appropriate concentrations, in reagents, such as anhydrous acetonitrile, to form a mixture with a desired phosphoramidite concentration. This approach for combinatorial synthesis of a ribozyme library with one or more random positions within the ribozyme (X as described above) is particularly useful since a standard DNA synthesizer can handle a building block mixture similar to a building block solution containing a single building block. Such a nucleotide building block mixture is coupled to a solid support or to a growing ribozyme sequence attached to a solid-support. To ensure that the ribozyme library synthesized achieves the desired complexity, the scale of the synthesis is increased substantially above that of the total complexity of the library. For example, a 2.5 μmole ribozyme synthesis provides ~ 3x10¹⁷ ribozyme molecules corresponding to sub-nanomolar amounts of each member of a billion compounds ribozyme library.

Divinylbenzene highly cross-linked polystyrene solid-support constitutes the preferred stationary phase for ribozyme library synthesis. However, other solid-support systems utilized in DNA or RNA synthesis can also be used for ribozyme library synthesis. This includes silica-based solid-supports such as controlled-pore glass (CPG) or polymeric solid-supports such as all types of derivatized polystyrene resins, grafted polymers of chloromethylated polystyrene crosslinked with ethyiene glycol, oligoethylene glycol.

Because of different coupling kinetics of the nucleotide building blocks present in a mixture, it is necessary to evaluate the relative incorporation of each of the members of the mixture and to adjust, if needed, the relative concentration of the building blocks in the mixture to get equimolar representation, compensating thereby the kinetic parameter.

Typically a building block that presents a slow coupling kinetic will be over-represented in the mixture and vice versa for a building block that presents a fast coupling kinetic. When equimolar incorporation is sought, acceptable limits for unequal incorporation may generally be +/-10%.

Synthesis of a random ribozyme library can be performed either with the mixture of desired nucleotide building blocks, or with a combination of certain random positions (obtained by using one or more building block mixtures) and one or more fixed positions that can be introduced through the incorporation of a single nucleotide building block reagent. For instance, in the oligonucleotide model 5'-TT XXXX TTB-3' used in example 2 infra, the positions from 3 '-end 1 is fixed as 2'-deoxy-inverted abasic ribose (B), positions 2, 3, 8 and 9 have been fixed as 2'-deoxy-thymidine (T) while the X positions 4-7 correspond to an approximately equimolar distribution of all the nucleotide building blocks that make up the X mixture.

In another preferred embodiment, a "mix and split" approach to generate ribozyme libraries is described. This method is particularly useful when the number of selected nucleotide building blocks to be included in the library is large and diverse (greater than 5 nucleotide building blocks) and/or when the coupling kinetics of the selected nucleotide building blocks do not allow competitive coupling even after relative concentration adjustments and optimization. This method involves a multi-step process wherein the solid support used for ribozyme library synthesis is "split" (divided) into equal portions, (the number of portions is equal to the number of different nucleotide building blocks (n) chosen for incorporation at one or more random positions within the ribozyme). For example, if there are 10 different nucleotide building blocks chosen for incorporation at one or more positions in the ribozyme library, then the solid support is divided into 10 different portions. Each portion is independently coupled to one of the selected nucleotide building blocks followed by mixing of all the portions of solid support. The ribozyme synthesis is then resumed as before the division of the building blocks. This enables the synthesis of a ribozyme library wherein one or more positions within the ribozyme is random. The number of "splitting" and "mixing" steps is dependent on the number of positions that are random within the ribozyme. For example if three positions are desired to be random then three different splitting and mixing steps are necessary to synthesize the ribozyme library.

Random ribozyme libraries are synthesized using a non-competitive coupling procedure where each of the selected nucleotide analogs "n" separately couple to an inverse "n" (1/n) number of aliquots of solid-support or of a growing ribozyme chain on the solid-support. A very convenient way to verify completeness of the coupling reaction is the use of a standard spectrophotometric DMT assay (Oligonucleotide Synthesis, A Practical Approach, ed. M. Gait, pp 48, IRC Press, Oxford, UK; incorporated by reference herein). These aliquots may be subsequently combined, mixed and split into one new aliquot. A similar approach to making oligonucleotide libraries has recently been described by Cook et al, (US Patent No. 5,587,471) and is incorporated by reference herein.

Nucleotide Synthesis Addition of dimethylaminopyridine (DMAP) to the phosphorylation protocols known in the art can greatly increase the yield of nucleoside monophosphates while decreasing the reaction time (Fig. 29). Synthesis of the nucleosides of the invention have been described in several publications and Applicants previous applications (Beigelman et al, International PCT publication No. WO 96/18736; Dudzcy et al, Int. PCT Pub. No. WO 95/11910; Usman et al, Int. PCT Pub. No. WO 95/13378; Matulic-Adamic et al, 1997, Tetrahedron Lett. 38, 203; Matulic-Adamic et al, 1997, Tetrahedron Lett. 38, 1669; all of which are incorporated herein by reference). These nucleosides are dissolved in triethyl phosphate and chilled in an ice bath. Phosphorus oxychloride (POCl₃) is then added followed by the introduction of DMAP. The reaction is then warmed to room temperature and allowed to proceed for 5 hours. This reaction allows the formation of nucleoside monophosphates which can then be used in the formation of nucleoside triphosphates. Tributylamine is added followed by the addition of anhydrous acetonitrile and tributylammonium pyrophosphate. The reaction is then quenched with TEAB and stirred overnight at room temperature (about 20C). The triphosphate is purified using column purification and HPLC and the chemical structure is confirmed using NMR analysis. Those skilled in the art will recognize that the reagents, temperatures of the reaction, and purification methods can easily be alternated with substitutes and equivalents and still obtain the desired product.

The invention provides nucleoside triphosphates which can be used for a number of different functions. The nucleoside triphosphates formed from nucleosides found in table III are unique and distinct from other nucleoside triphosphates known in the art. Incorporation of modified nucleotides into DNA or RNA oligonucleotides can alter the properties of the molecule. For example, modified nucleotides can hinder binding of nucleases, thus increasing the chemical half-life of the molecule. This is especially important if the molecule is to be used for cell culture or in vivo. It is known in the art that the introduction of modified nucleotides into these molecules can greatly increase the stability and thereby the effectiveness of the molecules (Burgin et al, 1996, Biochemistry 35, 14090-14097; Usman et al, 1996, Curr. Opin. Struct. Biol. 6, 527-533).

Modified nucleotides are incorporated using either wild type and mutant polymerases. For example, mutant T7 polymerase is used in the presence of modified nucleotide triphosphate(s), DNA template and suitable buffers. Those skilled in the art will recognize that other polymerases and their respective mutant versions can also be utilized for the incorporation of NTP's of the invention. Nucleic acid transcripts were detected by incorporating radiolabelled nucleotides (α-³²P NTP). The radiolabeled NTP contained the same base as the modified triphosphate being tested. The effects of methanol, PEG and LiCl were tested by adding these compounds independently or in combination. Detection and quantitation of the nucleic acid transcripts was performed using a Molecular Dynamics Phosphorlmager. Efficiency of transcription was assessed by comparing modified nucleotide triphosphate incorporation with all-ribonucleotide incorporation control. Wild type polymerase was used to incorporate NTP's using the manufacturers buffers and instructions (Boehringer Mannheim). 4o

Transcription Conditions

Incorporation rates of modified nucleoside triphosphates into oligonucleotides can be increased by adding to traditional buffer conditions, several different enhancers of modified NTP incorporation. Applicant has utilized methanol and LiCl in an attempt to increase incorporation rates of dNTP using RNA polymerase. These enhancers of modified NTP incorporation can be used in different combinations and ratios to optimize transcription. Optimal reaction conditions differ between nucleoside triphosphates and can readily be determined by standard experimentation. Overall however, inclusion of enhancers of modified NTP incorporation such as methanol or inorganic compound such as lithium chloride, have been shown by the applicant to increase the mean transcription rates.

Administration of Nucleoside mono, di or triphosphates

The nucleotide monophosphates, diphosphates, or triphosphates can be used as a therapeutic agent either independently or in combination with other pharmaceutical components. These molecules of the inventions can be administered to patients using the methods of Sullivan et al, PCT WO 94/02595. Molecules of the invention may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the modified nucleotide triphosphate, diphosphate or monophosphate/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and or intrathecal delivery. More detailed descriptions of delivery and administration are provided in Sullivan et al, supra and Draper et al, PCT WO93/23569 which have been incorporated by reference herein. This invention further relates to a compound having the Formula II:

wherein, R, is OH, O-R3, where R3 is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester; C-R3, where R3 is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester; halo, NHR4 (R4=alkyl (Cl-22), acyl (Cl-22), substituted or unsubstituted aryl), or OCH2SCH3 (methylthiomethyl), ONHR₅ where R₅ is independently H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester, ON=R₆, where R₆ is independently pyridoxal residue, pyridoxal-5 -phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl , alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl; B is independently a nucleotide base or its analog or hydrogen; X is independently a phosphoms-containing group; and R₂ is independently blocking group or a phosphoms-containing group.

Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxy, cyano, alkoxy, NO2 or N(CH₃)₂, amino, or SH.

The term "alkenyl" group refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO₂, halogen, N(CH₃)2, amino, or SH.

The term "alkynyl" refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂ or N(CH₃)₂, amino or SH.

An "aryl" group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) on aryl groups are halogen, trihalomethyl, hydroxyl, SH, 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, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl 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, or alkylaryl.

A "blocking group" is a group which is able to be removed after polynucleotide synthesis and/or which is compatible with solid phase polynucleotide synthesis.

A "phosphorus containing group" can include phosphorus in forms such as dithioates, phosphoramidites and/or as part of an oligonucleotide.

In a preferred embodiment, the invention features a process for synthesis of the compounds of formula II.

In a preferred embodiment the invention features a process for the synthesis of a xylofuranosyl nucleoside phosphoramidite comprising the steps of: a) oxidation of a 2' and 5 '-protected ribonucleoside with a an oxidant such as chromium oxide/pyridine/aceticanhydride, dimethylsulfoxide/aceticanhydride, or Dess-Martin reagent (periodinane) followed by reduction with a reducing agent such as, triacetoxy sodium borohydride, sodium borohydride, or lithium borohydride, under conditions suitable for the formation of 2' and 5'-protected xylofuranosyl nucleoside; b) phosphitylation under conditions suitable for the formation of xylofuranosyl nucleoside phosphoramidite.

In yet another preferred embodiment, the invention features the incorporation of the compounds of Formula II into polynucleotides. These compounds can be incorporated into polynucleotides enzymatically. For example by using bacteriophage T7 RNA polymerase, these novel nucleotide analogs can be incorporated into RNA at one or more positions (Milligan et al, 1989, Methods Enzymol, 180, 51). Alternatively, novel nucleoside analogs can be incorporated into polynucleotides using solid phase synthesis (Brown and Brown, 1991, in Oligonucleotides and Analogues: A Practical Approach, p. 1, ed. F. Eckstein, Oxford University Press, New York; Wincott et al, 1995, Nucleic Acids Res., 23, 2677; Beaucage & Camthers, 1996, in Bioorganic Chemistry: Nucleic Acids, p 36, ed. S. M. Hecht, Oxford University Press, New York). The compounds of Formula II can be used for chemical synthesis of nucleotide- tri-phosphates and/or phosphoramidites as building blocks for selective incorporation into oligonucleotides. These oligonucleotides can be used as an antisense molecule, 2-5A antisense chimera, triplex forming oligonucleotides (TFO) or as an nucleic acid catalyst. The oligonucleotides can also be used as probes or primers for synthesis and/or sequencing of RNA or DNA.

The compounds of the instant invention can be readily converted into nucleotide diphosphate and nucleotide triphosphates using standard protocols (for a review see Hutchinson, 1991, in Chemistty of Nucleosides and Nucleotides, v.2, pp 81-160, Ed. L. B. Townsend, Plenum Press, New York, USA; incorporated by reference herein).

The compounds of Formula II can also be independently or in combination used as an antiviral, anticancer or an antitumor agent. These compounds can also be independently or in combination used with other antiviral, anticancer or an antitumor agents.

In one of the preferred embodiments of the inventions herein, the nucleic acid catalyst is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis d vims, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al, 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al, 1990 Nucleic Acids Res. 18, 299; of the hepatitis d vims motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al, 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II _ -.

introns are described by Griffin et al, 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al, International PCT Publication No. WO 96/22689; 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 a nucleic acid catalyst 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 have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

In a preferred embodiment, a polynucleotide of the invention would bear one or more 2'-hydroxylamino functionalities attached directly to the monomeric unit or through the use of an appropriate spacer. Since oligonucleotides have neither aldehyde nor hydroxylamino groups, the formation of an oxime would occur selectively using oligo as a polymeric template. This approach would facilitate the attachment of practically any molecule of interest (peptides, polyamines, coenzymes, oligosaccharides, lipids, etc.) directly to the oligonucleotide using either aldehyde or carboxylic function in the molecule of interest.

Scheme 1. Post synthetic Oxime Bond Formation

H

OLIGO ^■ONH₂ + H- iCf I -R OLIGO N=C— R

Scheme 2. Chemical Ligation of Oligonucleotides

Oligol — ONH H — fl C — (CH ₂)_n fl C - H ,NO Oligo2

Oligol — N =C 1 "(CH ₂)ή — C =N— O - Oligo2

Advantages of oxime bond formation:

• The oximation reaction proceeds in water

• Quantitative yields

• Hydrolytic stability in a wide pH range (5 - 8)

• The amphoteric nature of oximes allows them to act either as weak acids or weak bases.

• Oximes exhibit a great tendency to complex with metal ions

In yet another preferred embodiment, the aminooxy "tether" in oligonucleotides, such as a ribozyme, is reacted with different compounds bearing carboxylic groups (e.g., aminoacids, peptides, "cap" stmctures ,etc.) resulting in the formation of oxyamides as shown below. Scheme 3. Post synthetic oxyamide bond formation

OLIGO ^■ONH + HO - ^<CS— R OLIGO ^■ - VN— fl C— R

Target Discovery: Applicant has developed an efficient and rapid method for screening libraries of catalytic nucleic acid molecules capable of performing a desired function in a cell. The invention also features the use of a catalytic nucleic acid library to modulate certain attributes or processes in a biological system, such as a mammalian cell, and to identify and isolate a) nucleic acid catalysts from the library involved in modulating the cellular process/attribute of interest; and b) modulators of the desired cellular process/attribute using the sequence of the nucleic acid catalyst.

More specifically, the method of the instant invention involves designing and constmcting a catalytic nucleic acid library, where the catalytic nucleic acid includes a catalytic and a substrate binding domain, and the substrate binding domain (arms) are randomized. This library of catalytic nucleic acid molecules with randomized binding arm(s) are used to modulate certain processes/attributes in a biological system. The method described in this application involves simultaneous screening of a library or pool of catalytic nucleic acid molecules with various substitutions at one or more positions and selecting for ribozymes with desired function or characteristics or attributes. This invention also features a method for constmcting and selecting for catalytic nucleic acid molecules for their ability to cleave a given target nucleic acid molecule or an unknown target nucleic acid molecule (e.g., RNA), and to inhibit the biological function of that target molecule or any protein encoded by it.

It is not necessary to know either the sequence or the stmcture of the target nucleic acid molecule in order to select for catalytic nucleic acid molecules capable of cleaving the target in this cellular system. The cell-based screening protocol described in the instant invention (i.e., one which takes place inside a cell) offers many advantages over extracellular systems, because the synthesis of large quantities of RNA by enzymatic or chemical methods prior to assessing the efficacy of the catalytic nucleic acid molecules is not necessary. The invention further describes a rapid method of using catalytic nucleic acid molecule libraries to identify the biological function of a gene sequence inside a cell. Applicant describes a method of using catalytic nucleic acid molecule libraries to identify a nucleic acid molecule, such as a gene, involved in a biological process; this nucleic acid molecule may be a known molecule with a known function, or a known molecule with a previously undefined function or an entirely novel molecule. This is a rapid means for identifying, for example, genes involved in a cellular pathway, such as cell proliferation, cell migration, cell death, and others. This method of gene discovery is not only a novel approach to studying a desired biological process but also a means to identify active reagents that can modulate this cellular process in a precise manner.

Applicant describes herein, a general approach for simultaneously assaying the ability of one or more members of a catalytic nucleic acid molecule library to modulate certain attributes/process(es) in a biological system, such as plants, animals or bacteria, involving introduction of the library into a desired cell and assaying for changes in a specific "attribute," "characteristic" or "process." The specific attributes may include cell proliferation, cell survival, cell death, cell migration, angiogenesis, tumor volume, tumor metastasis, levels of a specific mRNA(s) in a cell, levels of a specific protein(s) in a cell, levels of a specific protein secreted, cell surface markers, cell morphology, cell differentiation pattern, cartilage degradation, transplantation, restenosis, viral replication, viral load, and the like. By modulating a specific biological pathway using a catalytic nucleic acid molecule library, it is possible to identify the gene(s) involved in that pathway, which may lead to the discovery of novel genes, or genes with novel function. This method provides a powerful tool to study gene function inside a cell. This approach also offers the potential for designing novel catalytic oligonucleotides, identifying ribozyme accessible sites within a target, and for identifying new nucleic acid targets for ribozyme-mediated modulation of gene expression. In another aspect the invention involves synthesizing a Random Binding Arm Nucleic Acid Catalyst Library (Random Library) and simultaneously testing all members of the Random Library in cells. This library has ribozymes with random substrate binding arm(s) and a defined catalytic domain. Cells with an altered attribute (such as inhibition of cell proliferation) as a result of interaction with the members of the Random Library are selected and the sequences of the ribozymes from these cells are determined. Sequence information from the binding arm(s) of these ribozymes can be used to isolate nucleic acid molecules that are likely to be involved in the pathway responsible for the desired cellular attribute using standard technology known in the art, e.g., nucleic acid amplification using techniques such as polymerase chain reaction (PCR). This method is a powerful means to isolate new genes or genes with new function.

By "Random Library" as used herein is meant ribozyme libraries comprising all possible variants in the binding arm (s) of a given ribozyme motif. Here the complexity and the content of the library is not defined. The Random Library is expected to comprise sequences complementary to every potential target sequence, for the ribozyme motif chosen, in the genome of an organism. This Random Library can be used to screen for ribozyme cleavage sites in a known target sequence or in a unknown target. In the first instance, the Random Library is introduced into the cell of choice and the expression of the known target gene is assayed. Cells with an altered expression of the target will yield the most effective ribozyme against the known target. In the second instance, the Random Library is introduced into the cell of choice and the cells are assayed for a specific attribute, for example, survival of cells. Cells that survive the interaction with the Random Library are isolated and the ribozyme sequence from these cells is determined. The sequence of the binding arm of the ribozyme can then be used as probes to isolate the gene(s) involved in cell death. Because, the ribozyme(s) from the Random Library is able to modulate (e.g., down regulate) the expression of the gene(s) involved in cell death, the cells are able to survive under conditions where they would have otherwise died. This is a novel method of gene discovery. This approach not only provides the information about mediators of certain cellular processes, but also provides a means to modulate the JO

expression of these modulators. This method can be used to identify modulators of any cell process in any organism, including but not limited to mammals, plants and bacteria.

The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the nucleic acid sequence of a desired target. The nucleic acid catalyst is preferably targeted to a highly conserved sequence region of a target such that specific diagnosis and/or treatment of a disease or condition can be provided with a single enzymatic nucleic acid.

In a first aspect the invention features a method for identifying one or more nucleic acid molecules, such as gene(s), involved in a process (such as, cell growth, proliferation, apoptosis, moφhology, angiogenesis, differentiation, migration, viral multiplication, dmg resistance, signal transduction, cell cycle regulation, temperature sensitivity, chemical sensitivity and others) in a biological system, such as a cell. The method involves the steps of: a) providing a random library of nucleic acid catalysts, with a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, to the biological system under conditions suitable for the process to be altered; b) identifying any nucleic acid catalyst present in that biological system where the process has been altered by any nucleic acid catalyst; and c) determining the nucleotide sequence of at least a portion of the binding arm of such a nucleic acid catalyst to allow identification of the nucleic acid molecule involved in the process in that biological system.

In a related aspect the invention features a method for identification of a nucleic acid molecule capable of modulating a process in a biological system. The method includes: a) introducing a library of nucleic acid catalysts with a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, into the biological system under conditions suitable for modulating the process; and b) determining the nucleotide sequence of at least a portion of the substrate binding domain of any nucleic acid catalyst from a biological system where the process has been modulated to allow said identification of the nucleic acid molecule capable of modulating said process in that biological system.

In a second aspect, the invention the invention further concerns a method for identification of a nucleic acid catalyst capable of modulating a process in a biological system. This involves: a) introducing a library of nucleic acid catalysts with a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, into the biological system under conditions suitable for modulating the process; and b) identifying any nucleic acid catalyst from a biological system where the process has been modulated.

By "enzymatic portion" or "catalytic domain" is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 3).

By "nucleic acid molecule" as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. An example of a nucleic acid molecule according to the invention is a gene which encodes for macromolecule such as a protein.

The "biological system" as used herein may be a eukaryotic system or a prokaryotic system, may be a bacterial cell, plant cell or a mammalian cell, or may be of plant origin, mammalian origin, yeast origin, Drosophila origin, or archebacterial origin.

This invention further relates to novel nucleic acid molecules with catalytic activity, which are particularly useful for cleavage of RNA or DNA. The nucleic acid catalysts of the instant invention are distinct from other nucleic acid catalysts known in the art. This invention also relates to a method of screening variants of nucleic acid catalysts using standard nucleotides or modified nucleotides. Applicant has determined an efficient method for screening libraries of catalytic nucleic acid molecules, particularly those with chemical modifications at one or more positions. The method described in this application involves systematic screening of a library or pool of ribozymes with various substitutions at one or more positions and selecting for ribozymes with desired function or characteristic or attribute.

In one preferred embodiment, a method for identifying a nucleic acid molecule involved in a process in a cell is described, including the steps of: a) synthesizing a library of nucleic acid catalysts, having a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence; b) testing the library in the cell under conditions suitable to cause the process in the cell to be altered (such as: inhibition of cell proliferation, inhibition of angiogenesis, modulation of growth and /or differentiation, and others); c) isolating and enriching the cell with the altered process; d) identifying and isolating the nucleic acid catalyst in the altered cell; e) using an oligonucleotide, having the sequence homologous to the sequence of the substrate binding domain of the nucleic acid catalyst isolated from the altered cell, as a probe to isolate the nucleic acid molecule from the cell or the altered cell. Those nucleic acid molecules identified using the selection/screening method described above are likely to be involved in the process that was being assayed for alteration by the member(s) of the ribozyme library. These nucleic acid molecules may be new gene sequences, or known gene sequences, with a novel function. One of the advantages of this method is that nucleic acid sequences, such as genes, involved in a biological process, such as differentiation, cell growth, disease processes including cancer, tumor angiogenesis, arthritis, cardiovascular disease, inflammation, restenosis, vascular disease and the like, can be readily identified using the Random Library approach. Thus theoretically, one Random Library for a given ribozyme motif can be used to assay any process in any biological system.

In another preferred embodiment the invention involves synthesizing a Defined Arm Nucleic Acid Catalyst Library (Defined Library) and simultaneously testing it against known targets in a cell. The library includes ribozymes with binding arm(s) of known complexity (Defined) and a defined catalytic domain. Modulation of expression of the target gene by ribozymes in the library will cause the cells to have an altered phenotype. Such cells are isolated and the ribozymes in these cells are the ones most suited for modulating the expression of the desired gene in the cell.

By "Defined Library" as used herein is meant a library of nucleic acid catalysts, wherein each member nucleic acid catalyst is designed and produced independently, then added to the library. Thus, the content, complexity (number of different ribozymes contained in the library) and ratios of library members are defined at the outset. Defined Library comprises > 2 ribozymes. The process involves screening the sequence of the known target RNA for all possible sites that can be cleaved by a given ribozyme motif and as described, for example in McSwiggen, US Patent No. 5,525,468, incoφorated by reference herein. Synthesizing a representative number of different ribozymes aginst the target sequence. Combining the ribozymes and introducing the pooled ribozymes into a biological system comprising the target RNA under conditions suitable to facilitate modulation of the expression of the target RNA in said biological system.

Screening of Nucleic Acid Catalysts

Applicant describes herein, a general combinatorial approach for assaying ribozyme variants based on ribozyme activity and/or a specific "attribute" of a ribozyme, such as the cleavage rate, cellular efficacy, stability, delivery, localization and the like. Variations of this approach also offer the potential for designing novel catalytic oligonucleotides, identifying ribozyme accessible sites within a target, and for identifying new nucleic acid targets for ribozyme-mediated modulation of gene expression.

In one preferred embodiment, the method relies upon testing mixtures (libraries) of ribozymes with various nucleotides, nucleotide analogs, or other analog substitutions, rather than individual ribozymes, to rapidly identify the nucleotide, nucleotide analog, or other analog that is variable at one or more positions within a ribozyme. In the first step (step 1, Figure 2), a desired number of positions (for example, 3 positions as shown in Figure 2) are chosen for variation in a first ribozyme motif (Starting Ribozyme); there is no requirement on the number of positions that can be varied and these positions may or may not be phylogenetically conserved for the ribozyme. In addition, these position may reside within the catalytic core, binding arms, or accessory domains. The number of positions that are chosen to be varied defines the number of "Classes" of ribozyme libraries that will be synthesized. In the example illustrated in Figure 2, three positions (designated positions 1, 2 and 3) are varied, so three different Classes of ribozyme pool are synthesized. In the next step (step 2), ribozyme pools are synthesized containing a random mixture of different nucleotides, nucleotide analogs, or other analogs at all of the desired positions (designated "X") to be varied except one, which is the "fixed" position (designated "F"). The fixed position contains a specific nucleotide, nucleotide analog or other analog. There is no requirement for the number of nucleotides, or analogs be used. The number of nucleotides or analogs defmes the number of pools (designated n) in each Class. For example if ten different nucleotides or analogs are chosen, ten different pools (n=10) will be synthesized for each Class; each of the pools will contain a specific modification at one fixed position (designated F) but will contain an equal mixture of all ten modifications at the other positions (designated X). In a subsequent step (step 3), the different pools of ribozymes are tested for desired activity, phenotype, characteristic or attribute. For example, the testing may be determining in vitro rates of target nucleic acid cleavage for each pool, testing ribozyme-substrate binding affinities, testing nuclease resistance, determining pharmacodynamic properties, or determining which pool is most efficacious in a cellular or animal model system. Following testing, a particular pool is identified as a desired variant (designated " Desired Variant- 1") and the nucleotide or the analog present at the fixed position within the Desired Variant- 1 is made constant (designated "Z") for all subsequent experiments; a single position within a ribozyme is therefore varied, i.e., the variable nucleotide or analog at a single position, when all other X positions are random, is identified within a ribozyme motif. Subsequently, new ribozyme pools (Classes 2, 3 etc.) are synthesized containing an equal mixture of all nucleotides or analogs at the remaining positions to be optimized except one fixed position and one or more constant positions. Again, a specific nucleotide or analog is "fixed" at a single position that is not randomized and the pools are assayed for a -,

61

particular phenotype or attribute (step 4). This process is repeated until all desired positions have been varied and screened. For example if three positions are chosen for optimization, the synthesis and testing will need to be repeated three times (3 Classes). In the first two Classes, pools will be synthesized; in the final Class, specific ribozymes will be synthesized and tested. When the final position is analyzed (step 5), no random positions will remain and therefore only individual ribozymes are synthesized and tested. The resulting ribozyme or ribozymes (designated "second ribozyme motif) will have a defined chemical composition which will likely be distinct from the Starting Ribozyme motif (first ribozyme motif). This is a rapid method of screening for variability of one or more positions within a ribozyme motif.

In another preferred embodiment, the invention involves screening of chemical modifications at one or more positions within a hammerhead ribozyme motif. More specifically, the invention involves variability in the catalytic core sequence of a hammerhead ribozyme. Particularly, the invention describes screening for variability of positions 3, 4 and 7 within a hammerhead ribozyme. The invention also features screening for optimal loop II sequence in a hammerhead ribozyme.

In yet another preferred embodiment, the invention features a rapid method for screening accessible ribozyme cleavage sites within a target sequence. This method involves screening of all possible sequences in the binding arm of a ribozyme. The sequence of the binding arms determines the site of action of certain ribozymes. The combinatorial approach can be used to identify desirable and/or accessible sites within a target sequence by essentially testing all possible arm sequences. The difficulty with this approach is that ribozymes require a certain number of base pairs (for example, for hammerhead ribozymes the binding arm length is approximately 12-16 nucleotides) in order to bind functionally and sequence-specifically. This would require, for example 12- 16 different groups of hammerhead ribozyme pools; 12-16 positions would have to be optimized which would require 12-16 different groups being synthesized and tested. Each pool would contain the four different nucleotides (A, C, U and G) or nucleotide -

analogs (p = 4 for nucleotides). It would be very time consuming to test each group, identify the best pool, synthesize another group of ribozyme pools with one additional position constant, and then repeat the procedure until all 12-16 groups had been tested. However it is possible to decrease the number of Classes by testing multiple positions within a single Class. In this case, the number of pools within a Class equals the number of nucleotides or analogs in the random mixture (i.e., n) to the w power, where w equals the number of positions fixed in each Class. The number of Classes that need to be synthesized to optimize the final ribozyme equals the total number of positions to be optimized divided by the number of positions (w) tested within each Class. The number of pools in each Class = n . The number of Class = total number of positions Λv.

In another preferred embodiment, the invention features a rapid method of screening for new catalytic nucleic acid motifs by keeping the binding arms constant and varying one or more positions in a putative catalytic domain. Applicant describes a method to vary positions within the catalytic domain, without changing positions within the binding arms, in order to identify new catalytic motifs. An example is illustrated in Figure 24. It is unclear how many positions are required to obtain a functional catalytic domain in a nucleic acid molecule, however it is reasonable to presume that if a large number of functionally diverse nucleotide analogs can be used to constmct the pools, a relatively small number of positions could constitute a functional catalytic domain. This may especially be tme if analogs are chosen that one would expect to participate in catalysis (e.g., acid/base catalysts, metal binding, etc.). In the example illustrated, four positions (designated 1, 2, 3 and 4) are chosen. In the first step, ribozyme libraries (Class 1) are constmcted: position 1 is fixed (F,) and positions 2, 3 and 4 are random (X₂, X₃ and X₄, respectively). In step 2, the pools (the number of pools tested depends on the number of analogs used; n) are assayed for activity. This testing may be performed in vitro or in a cellular or animal model. Whatever assay that is used, the pool with the desired characteristic is identified and libraries (class 2) are again synthesized with position 1 now constant (Z,) with the analog that was identified in class 1. In class 2, position 2 is fixed (F₂) and positions 3 and 4 are random (X₃ and X₄). This process is repeated until oi

every position has been made constant and the chemical composition of the catalytic domain is determined. If the number of positions in the catalytic domain to be varied are large, then it is possible to decrease the number of Classes by testing multiple positions within a single Class. The number of pools within a Class equals the number of nucleotides or analogs in the random mixture (i.e., n) to the w power, where w equals the number of positions fixed in each Class. The number of Classes that need to be synthesized to optimize the final ribozyme equals the total number of positions to be optimized divided by the number of positions (w) tested within each Class. The number of pools in each Class= n^w. The number of Classes- total number of positions /w.

In a preferred embodiment a method for identifying variants of a nucleic acid catalyst is described comprising the steps of: a) selecting at least three (3) positions, preferably 3-12, specifically 4-10, within said nucleic acid catalyst to be varied with a predetermined group of different nucleotides, these nucleotides are modified or unmodified (non-limiting examples of nucleotides that can used in this method are shown in Figure 15); b) synthesizing a first class of different pools of said nucleic acid catalyst, wherein the number of pools synthesized is equal to the number of nucleotides in the predetermined group of different nucleotides (for example if 10 different nucleotides are selected to be in the group of predetermined nucleotides then 10 different pools of nucleic acid catalysts have to be synthesized), wherein at least one of the positions to be varied in each pool comprises a defined nucleotide (fixed position; F) selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides (X positions) selected from the predetermined group of different nucleotides; c) testing the different pools of said nucleic acid catalyst under conditions suitable for said pools to show a desired attribute (including but not limited to improved cleavage rate, cellular and animal efficacy, nuclease stability, enhanced delivery, desirable localization) and identifying the pool with said desired attribute and wherein the position with the defined nucleotide (F) in the pool with the desired attribute is made constant (Z position) in subsequent steps; d) synthesizing a second class of different pools of nucleic acid catalyst, wherein at least one of the positions to be varied in each of the second class of different pools comprises a defined nucleotide (F) selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture (X) of nucleotides selected from the predetermined group of different nucleotides (this second class of pools therefore has F, X and Z positions); e) testing the second class of different pools of said nucleic acid catalyst under conditions suitable for showing desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant (Z) in subsequent steps; and f) this process is repeated until every position selected in said nucleic acid catalyst to be varied is made constant.

In yet another preferred embodiment, a method for identifying novel nucleic acid molecules in a biological system is described, comprising the steps of: a) synthesizing a pool of nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence; b) testing the pools of nucleic acid catalyst under conditions suitable for showing a desired effect (such as inhibition of cell proliferation, inhibition of angiogenesis, modulation of growth and /or differentiation, and others) and identifying the catalyst with said desired attribute; c) using an oligonucleotide, comprising the sequence of the substrate binding domain of the nucleic acid catalyst showing said desired effect, as a probe, screening said biological system for nucleic acid molecules complementary to said probe ; and d) isolating and sequencing said complementary nucleic acid molecules.

These nucleic acid molecules identified using a nucleic acid screening method described above may be new gene sequences, or known gene sequences. The advantage of this method is that nucleic acid sequences, such as genes, involved in a biological process, such as differentiation, cell growth, disease processes including cancer, tumor angiogenesis, arthritis, cardiovascular disease, inflammation, restenosis, vascular disease and the like, can be readily identified. In a preferred embodiment, the invention features a nucleic acid molecule with catalytic activity having one of the formulae III- VII:

Formula III

(N) o — C— G — A — A — A M— 3'

L

(N) G — A — G— NT" — G — C4— U3— Q— 5'

Formula IV

Z4

A G

A A

G C^ Z7

A^G c • Z3= 2'-0-MTM-U

G

ZA= 2'-C-Allyl-U

(N)o ( )n Zl= 6-Methyl-U

\ / L

Formula V

^3' M_χ / Q ^5'

A Z3

Λ ^{Z4 A} G

A A

G _ΛG ^Z7

A

C # G _Z3=

= 2'-0-MTM-U

(N)o (N)n _Z4= ^: 2'-0-MTM-C ^{x /} Z7= = 6-Methyl-U

Formula VI

3' M

\ / Q

A Z3

Z4 A G A A G r^ Z7 A^G Z3= 2'-0-MTM-U

C , G Z4= 2'-0-MTM-C (N)o (N)n Z7= 2'-C-Allyl-U

\ / Formula VII

3' M / Q 5' A Z3

Z4

A G

A A

G C Z7

A^G c • G Z3= 2'-0-MTM-U

(N)o (N)n Z4= 2'-0-MTM-C

\ / L Z7= Pyridin-4-One

In each of the above formulae, N represents independently a nucleotide or a non- nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact (e.g., by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA mixed polymers); preferably the length of Q is greater than or equal to 3 nucleotides and the length of M is preferably greater than or equal to 5 nucleotides; o and n are integers greater than or equal to 1 and preferably less than about 100, wherein if (N)₀ and (N)_n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent (i.e., the molecule is assembled from two separate molecules), but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; and _ represents a chemical linkage (e.g., a phosphate ester linkage, amide linkage or others known in the art). 2'-O-MTM-U and 2'-O-MTM-C refers to 2'-O-methylthiomethyl uridine and 2'-O-methylthiomethyl-cytidine, respectively. A, C, U and G represent adenosine, cytidine, uridine and guanosine nucleotides, respectively. The nucleotides in the formulae are unmodified or modified at the sugar, base, and/or phosphate portions as known in the art. oo

In yet another embodiment, the nucleotide linker (L) is a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al, 1995, Ann . Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp 511, CSH Laboratory Press). A "nucleic acid aptamer" as used herein is meant to indicate nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, dmgs, toxins, transition state analogs, peptides, lipids, proteins, aminoacids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

In yet another embodiment, the non-nucleotide linker (L) is as defined herein.

In particular, the invention features modified ribozymes having a base substitution selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl.

In yet another embodiment, the non-nucleotide linker (L) is as defined herein.

The term "non-nucleotide" as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 75:6353 and Nucleic Acids Res. 1987, 75:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 773:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 773:5109; Ma et al., Nucleic Acids Res. 1993, 27:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 75:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 70:287; Jschke et al, Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al, International Publication No. WO 89/02439; Usman et al, International Publication No. WO 95/06731; Dudycz et al, International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 773:4000, all hereby incoφorated by reference herein. Thus, in a preferred embodiment, the invention features a nucleic acid catalyst having one or more non-nucleotide moieties, and having enzymatic -._

69

activity to cleave an RNA or DNA molecule. By the term "non-nucleotide" is meant any group or compound which can be incoφorated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. The terms "abasic" or "abasic nucleotide" as used herein encompass sugar moieties lacking a base or having other chemical groups in place of base at the 1' position.

In preferred embodiments, the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety. The necessary RNA components are known in the art, see, e.g.,

Usman, supra. By RNA is meant a molecule comprising at least one ribonucleotide residue.

As the term is used in this application, non-nucleotide-containing enzymatic nucleic acid means a nucleic acid molecule that contains at least one non-nucleotide component which replaces a portion of a ribozyme, e.g., but not limited to, a double- stranded stem, a single-stranded "catalytic core" sequence, a single-stranded loop or a single-stranded recognition sequence. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such molecules can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

The specific nucleic acid catalysts described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in a nucleic acid catalyst of this invention is that it has a specific substrate binding site (e.g., M and/or Q of Formulae III- VII above) which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule. Vector Expression of Enzymatic Nucleic Acid

The nucleic acid catalysts of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci.USA 83, 399; Scanlon et al, 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992 J. Virol, 66, 1432-41; Weerasinghe et al, 1991 J. Virol, 65, 5531-4; Ojwang et al, 1992 Proc Natl Acad. Sci. USA 89, 10802-6; Chen et al, 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al, 1990 Science 247, 1222-1225; Thompson et al, 1995 Nucleic Acids Res. 23, 2259; Good et al, 1997, Gene Therapy, 4, 45; all of the references are hereby incoφorated in their totality by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994 J. Biol Chem. 269, 25856; all of the references are hereby incoφorated in their totality by reference herein).

In another aspect of the invention, nucleic acid catalysts that cleave target molecules are expressed from transcription units (see for example Figure 11) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constmcted based on, but not limited to, adeno-associated vims, retrovims, adenovims, or alphavims. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. The active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510).

In a preferred embodiment, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid catalyst of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.

In one embodiment, the expression vector comprises: a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a gene encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'- side of the gene encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).

Transcription of the ribozyme sequences are driven from a promoter for eukaryotic

RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).

Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl.

Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72;

Lieber et al., 1993 Methods Enzymol, 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol, 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g., Kashani-Sabet et al, 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992 Proc. Natl Acad. Sci. USA, 89, 10802-6; Chen et al, 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al, 1992 EMBO J. 11, 4411-8; Lisziewicz et al, 1993 Proc. Natl. Acad. Sci. U. S. A., 90, 8000-4; Thompson et al, 1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovims VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, US Patent No. 5,624,803; Good et al, 1997, Gene Ther. 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736; all of these publications are incoφorated by reference herein. Examples of transcription units suitable for expression of ribozymes of the instant invention are shown in Figure 11. The above ribozyme transcription units can be incoφorated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovims or adeno-associated vims vectors), or viral RNA vectors (such as retroviral or alphavims vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In a preferred embodiment an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecule of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another preferred embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

Delivery of Nucleic Acid Catalysts:

In a preferred embodiment, the nucleic acid catalysts are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incoφoration in biopolymers. Using the methods described herein, other nucleic acid catalysts that cleave target nucleic acid may be derived and used as described above. Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures.

Sullivan, et al, supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incoφoration into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al, supra and Draper et al, supra which have been incoφorated by reference herein.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., ammonium, sodium, calcium, magnesium, lithium, and potassium salts.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.

By "systemic administration" is meant in vivo systemic absoφtion or accumulation of dmgs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absoφtion include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., NTP's, to an accessible diseased tissue. The rate of entry of a dmg into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other dmg carrier comprising the compounds of the instant invention can potentially localize the dmg, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of dmg with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the dmg to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells.

The invention also features the use of the a composition comprising surface- modified liposomes containing poly (ethyiene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes). These formulations offer an method for increasing the accumulation of dmgs in target tissues. This class of dmg carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated dmg (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al, Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Ada, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of dmgs, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol Chem. 1995, 42, 24864-24870; Choi et al, International PCT Publication No. WO 96/10391; Ansell et al, International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392; all of these are incoφorated by reference herein). Long-circulating liposomes are also likely to protect dmgs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incoφorated by reference herein.

The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incoφorated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These include sodium benzoate, sorbic acid and esters of /?-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between OJ mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. In a one aspect, the invention provides nucleic acid catalysts that can be delivered exogenously to specific cells as required.

Local ribozyme administration offers the advantages of achieving high tissue concentrations of ribozymes and limiting their exposure to catabolic and excretory mechanisms. Although local routes of administration provide access to pathologies involving a number of organ systems, systemic administration would make ribozyme treatment of several other major human diseases feasible.

It has been demonstrated that certain tissues accumulate oligonucleotides and/or oligonucleotide formulations following systemic administration. These tissues include sites of inflammation (Wu et al. 1993, Cancer Res. 53: 3765-3767), solid tumors (Yuan et al. 1994, Cancer Res. 54: 3352-3356), kidney (Cossum et al. 1993, J. Pharmaco. and Exp. Ther. 267: 1181-1190), brain (Wu et al. 1996, J. Pharmacol. Exp. Ther. 276: 206- 1 ) and those rich in reticulo-endothelial cells (liver, spleen, lymphatics; Litzinger et al. 1994, Biochim. Biophys. Ada 1190: 99-107; Agrawal et al. 1991, Proc. Natl. Acad. Sci. USA 88: 7595-7599; Agrawal et al. 1995, Clin. Pharmacology 28: 7-16; Sands et al. 1994, Molecular Pharmacol. 45: 932-943; Saijo et alA994, Oncology Research 6: 243- 249).

The kidney, as well as organs of the reticulo-endothelial system (RES), are mainly responsible for clearance of ribozymes following intravenous (i.v.) administration. Diseases involving these tissues are good candidates for systemic ribozyme therapy by virtue of their tendency to accumulate ribozymes.

In one prefeπed embodiment, the invention features method of treating inflammation using ribozymes. Inflammatory processes underlie the pathology of a large number of human diseases. Many of these processes can be blocked by inhibiting the expression of inflammatory mediators and or their receptors (Cohen et al. 1995, Am. J. Med. 99: 45 S- 52S). Systemic administration of monoclonal antibodies specific to these mediators have been shown to be efficacious in animal models of rheumatoid arthritis, inflammatory bowel disease, and acute respiratory distress syndrome (Arend et al. 1990, Arthritis and Rheumatism 33: 305-315). One potential way for systemic administration of ribozymes to impact systemic inflammatory disease is through inhibition of TNF-a production by macrophages. TNF-a has been shown participate in a variety of inflammatory processes and is produced mainly by macrophages which are known to accumulate cationic lipid- formulated ribozymes (Masahiro et al. 1990, J. Immunology. 144: 1425-1431). Anti- mouse TNF- a ribozymes were effective in cell culture, thus, it may be possible that systemic delivery of ribozymes by a liposome formulation could be an effective therapeutic in the above mentioned inflammatory disease states.

In another preferred embodiment, the invention features methods of treating diseases involving RES using ribozymes. A number of studies have shown that systemically administered oligonucleotides distribute to RES tissues (liver, spleen and lymphatics).

Several studies with cationic lipid complexed oligonucleotides have also shown specific biodistribution to these. Pathology involving the RES includes a number of infectious /o

diseases of major importance, such as human immunodeficiency vims (HIV), mycobacterium infections including tuberculosis (TB), avium, and leprae (leprosy). These diseases are all associated with, for example, oveφroduction of interleukin- 10 (IL- 10), a potent immunosuppressive cytokine (Barnes et al. 1993, Infect. Immun. 61: 3482- 9). Some of these infections can potentially be ameliorated by administration of neutralizing antibodies to IL-10.

In yet another preferred embodiment, the invention features method of treating cancer using ribozymes. As evidence of the potential use of systemic oligonucleotides as anticancer agents, antisense phosphorothioates have been have been reported to exhibit antitumor efficacy in a murine model of Burkitt's lymphoma (Huang et al. 1995, Mol. Med. 1: 647-658). The molecular targets of systemic antineoplastic ribozymes could include oncogenes, protooncogenes, or angiogenic factors and receptors. Although the link between oncogenes and tumorigenesis is now well established, the specific mutations that lead to activation of a proto-oncogene can be widely diverse. Upregulation of protooncogene products is also common in human cancer. Reducing the levels of these gene products may be beneficial in treatment of cancer. In addition, since many tumors are highly vascularized, angiogenic factors or receptors may provide good alternate or adjunct targets to oncogenes for the therapy of solid tumors and their metastases. Applicant, in a non-limiting example infra, show ribozymes targeting angiogenic mediators.

The potential number of molecular targets in cancer is quite large. Among these targets are oncogenes, protooncogenes, metalloproteinases, growth factors, and angiogenic factors. However, a common denominator in many forms of metastatic solid tumors is extensive vascularization of the tumor. As tumors exceed about 1 mm in diameter, they require neovascularization for continued growth (Gimbrone et al, 1972, J. Exp. Med., 136, 261). In addition, the appearance of new blood vessels within a tumor correlates with the initiation of the process of metastasis (Martiny-Baron and Marme, 1995). It is possible that by using a systemically administered ribozyme targeting a key player in the process of angiogenesis would reduce both primary tumor growth, tumor progression and tumor metastasis.

"Angiogenesis" refers to formation of new blood vessels from existing blood vessels which is an essential process in reproduction, development and wound repair.

"Tumor angiogenesis" refers to the induction of the growth of blood vessels from surrounding tissue into a solid tumor. Tumor growth and tumor metastasis are dependent on angiogenesis (for a review see Folkman, 1985, Nature Med. 1: 27-31; Folkman 1990 J. Natl. Cancer Inst, 82, 4; Folkman and Shing, 1992 J. Biol. Chem. 267, 10931).

"Tumor metastasis" refers to the transfer and/or migration of tumor cells, originating from a primary tumor, from one part of the body or organ to another. Most malignant tumors have the capacity to metastasize.

"Tumor" refers to a new growth of tissue wherein the cells multiply, divide and grow uncontrolled.

In a preferred embodiment, the invention features a method of treating non-hepatic ascites using ribozymes. Nonhepatic ascites or peritoneal fluid accumulation resulting from abdominal cancer and ovarian hyperstimulation syndrome (OHSS) can result in significant fluid loss from the intravascular space and hypovolemia. If ascites volumes are large, abdominal pain, hypovolemic hypotension, electrolyte abnormalities and respiratory difficulties can ensue. Thus, if ascites is left untreated, it can be life threatening. Evidence is now accumulating that nonhepatic ascites may be induced, at least in part, by vascular endothelial growth factor (VEGF). For this reason, nonhepatic ascites may be a potential therapeutic indication for ribozymes directed against vascular endothelial growth factor (VEGF) receptors delivered either systemically or regionally to the peritoneum.

Ovaries can be overstimulated by hormonal therapy during fertility treatment. As a result, women can experience ovarian hyperstimulation syndrome which is associated with grossly enlarged ovaries and extreme ascites fluid accumulation. This fluid accumulation is thought to be induced by the release of a vascular permeability agent which may interact with vessels of the peritoneal cavity leading to plasma extravasation. Abramov and co-workers (1997, Fertil Steril 67: 261) have shown that plasma VEGF levels are elevated in OHSS and return to normal upon resolution of the syndrome. An earlier study has shown that VEGF is elevated in the semm and follicular fluid of OHSS patients and that the source of this VEGF may be the luteinizing granulosa cells of the ovary (Krasnow et al., 1996, Fertil. Steril. 65: 552). McClure et al. (1994, Lancet 344, 235) concluded that VEGF is the key mediator of OHSS ascites production since rhVEGF increases OHSS ascites but not liver ascites and that this increase is reversible by rhVEGF antisemm. Thus, reducing the expression of VEGF receptors in the vasculature of the peritoneum may have a therapeutic benefit in OHSS by substantially reducing OHSS- stimulated ascites production. Since VEGF can interact with VEGF receptors on vessels throughout the peritoneum from ovarian release of VEGF into systemic circulation, systemic treatment may represent the best option for treating this syndrome.

Malignant ascites: Another form of ascites can be induced by malignancies of the peritoneum including breast, pancreatic, uterine and colorectal cancers. It is thought that certain cancers produce factors which influence peritoneal vascular permeability leading to plasma extravasation (Garrison et al., 1986; Ann. Surg. 203: 644; Garrison et al., 1987, J. Surg. Res. 42: 126; Nagy et al, 1993, Cancer Res. 53: 2631). Several solid tumors including some colorectal and breast carcinomas are known to secrete VEGF to recmit blood vessels for sustained growth and metastasis. This secreted VEGF may also serve to increase local vasculature permeability. In support of this hypothesis, Nagy et al. (supra) showed in mice that peritoneal fluid resulting from MOT and TTA3/St carcinomas contained elevated levels of VEGF whose concentration correlated directly with fluid accumulation and development of hypeφermeable microvessels. Therefore, ribozymes directed against VEGF receptors administered systemically may impact both the tumor growth and metastases of VEGF secreting tumors as well as ascites induced by VEGF interacting with the vasculature of the peritoneum. o l

Strategies for Systemic Delivery

Methods to enhance tissue accumulation

Tissue accumulation of ribozymes can be improved by formulation, conjugation, or further chemical stabilization of the ribozyme. Elimination due to glomeralar filtration can be slowed by increasing the apparent molecular weight of the ribozyme, e.g., by liposome encapsulation or bioconjugation to PEG. Applicant has observed that the rate of catabolism can be slowed by a factor of 100 and lung accumulation increased 500 fold by formulation with DMRIE/DOPE reagents. Liposomal encapsulation is likely to have a similar effect on the rate of catabolism. The rate of clearance into non-target tissues could also be reduced by encapsulation into liposomes, provided that the liposomes were surface modified with PEG such that RES clearance were avoided. Increasing the rate of uptake by target tissues can also be enhanced, for example, by conjugation of cholesterol to the ribozymes. Applicant has also observed that in tissues of the RES, accumulation has been increased several hundred fold by complexation with a cationic lipid carrier.

Sustained release as a means to increase exposure

Sustained or continuous delivery devices, such as ALZET^® osmotic mini-pumps, may also enhance accumulation in target tissues by increasing exposure relative to bolus i.v. administration. Sustained delivery from ALZET^® pumps has been shown to be an effective way of administering a phosphorothioate antisense molecule for inhibition of tumor growth in mice (Huang et al. 1995, supra). Applicant has observed that the rate of ribozyme catabolism in and rate of clearance from the circulation is concentration dependent and may relate to the equilibrium plasma protein binding of the ribozyme. Phosphorothioate DNA is rapidly cleared from circulation when its concentration exceeds the plasma protein binding constant, as is the case after i.v. bolus administration. Osmotic pumps administer oligonucleotides at a slower and constant rate, and therefore may maintain plasma levels near the equilibrium binding capacity. This would result in less of the administered dose being lost to glomerular filtration (elimination) and hepatic extraction (catabolism); more of the administered dose may be available for uptake into target tissues.

Animal Models

Use of murine models For a typical systemic study involving 10 mice (20 g each) per dose group, 5 doses

(1, 3, 10, 30 and 100 mg/kg daily over 14 days continuous administration), approximately 400 mg of ribozyme, formulated in saline would be used. A similar study in young adult rats (200 g) would require over 4 g. Parallel pharmacokinetic studies may involve the use of similar quantities of ribozymes further justifying the use of murine models.

Ribozymes and Lewis lung carcinoma and B-16 melanoma murine models

Identifying a common animal model for systemic efficacy testing of ribozymes is an efficient way of screening ribozymes for systemic efficacy.

The Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 10⁶ tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter). Metastasis also may be modeled by injecting the tumor cells directly i.v.. In the Lewis lung model, microscopic metastases can be observed approximately 14 days following implantation with quantifiable macroscopic metastatic tumors developing within 21-25 days. The B-16 melanoma exhibits a similar time course with tumor neovascularization beginning 4 days following implantation. Since both primary and metastatic tumors exist in these models after 21-25 days in the same animal, multiple measurements can be taken as indices of efficacy. Primary tumor volume and growth latency as well as the number of micro- and macroscopic metastatic lung foci or number of animals exhibiting metastases can be quantitated. The percent increase in lifespan can also be measured. Thus, these models would provide suitable primary efficacy assays for screening systemically administered ribozymes/ribozyme formulations.

In the Lewis lung and B-16 melanoma models, systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation inoculation with either continuous or multiple administration regimens. Concurrent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of ribozymes can be achieved for pharmacodynamic effect to be expected. Furthermore, primary tumors and secondary lung metastases can be removed and subjected to a variety of in vitro studies (i.e., target mRNA reduction).

Anti-VEGF receptor ribozymes

Sustained tumor growth and metastasis depend upon angiogenesis. In fact, the appearance of vessels in a growing tumor is correlated with the beginning of metastatic potential. Several studies have shown that antiangiogenic agents alone or in combination with cytotoxic agents reduce lung metastases and/or primary tumor volume in the Lewis lung and B-16 melanoma models (Borgstrom et al. 1995, Anticancer Res. 15: 719-728; Kato et al. 1994, Cancer Res. 54: 5143-5147; O'Reilly et al. 1994, Cell 79: 315-328; Sato et al. 1995, Jpn. J. Cancer Res. 86: 374-382).

A major factor implicated in the induction of solid tumor angiogenesis is vascular endothelial growth factor (VEGF; Folkman, 1995, supra). Several human tumors have been shown to synthesize and secrete. With regard to treating lung metastasis, VEGF and VEGF receptors of both subtypes and their expression are upregulated in the lung under conditions of hypoxia (Tuder et al. 1994, J. Clin. Invest. 95: 1798-1807). This may lead to neovascularization which provides the means by which tumor cells gain access to circulation (Mariny-Baron and Marme, 1995). Thus, VEGF and its receptors may be important targets in the treatment of metastatic disease.

Applicant has shown that a catalytically active ribozyme targeting flt-1 RNA inhibits VEGF-induced neovascularization in a dose-dependent manner in a rat comeal model of angiogenesis. Testing with cytotoxic agents in combination with antiangiogenic ribozymes may also prove useful.

Anti-K- and H-ras ribozymes

Mutations involving ras underlie a number of human cancers. Ras also plays a role in metastatic potential (Shekhar and Miller, 1994, Invasion Metastasis 14: 27-37) and may do so, in part, by influencing endothelial cell migration (Fox et al. 1994, Oncogene 9: 3519-26). With regard to lung cancer, ras has been shown to induce abnormal mitoses in lung fibroblasts (Lyubuski et al. 1994, Cytobios 80: 161-178) and is a clinical marker in non-small cell lung tumors (Niklinski and Furman, 1995, Eur. J. Cancer Prev. 4: 129- 138). Studies in cells cultured from human small cell lung tumor xeno grafts demonstrated overexpression of K-ras (Arvelo et al. 1994, Anticancer Res. 14: 1893- 1901). This evidence provides ample support for the systemic testing of ribozymes directed against H- and K-ras in the murine cancer models (primary and secondary metastasis) discussed above.

Four of the current synthetic ribozymes directed against human K- ras will cleave homologous mouse K-ras targets at four sites and inhibit cultured rat aortic smooth muscle cell proliferation.

Anti-c-fos ribozymes

The protein product of the proto-oncogene c-fos is a nuclear transcription factor which is involved in tumorigenesis. In support of the possible use of systemically administered ribozymes directed against c-fos, null mouse mutations of c-fos have been shown to result in viable mice. Using this mouse model, it has been shown that c-fos is important in malignant conversion of papillomas. Additionally, c-fos has been shown to up-regulate tumor metalloproteinases (Schonthal et al. 1988, Cell 54: 325-334). ■ It is possible that c-fos may play a role in tumor angiogenesis as evidenced by VEGF mRNA levels being significantly reduced in c-fos deficient tumors. It has also been shown that c- os is highly expressed in some B-16 cell and human melanoma cell lines (Kroumpouzos et al. 1994, Pigment Cell Res. 7: 348-353; Nakayama et al. 1995, J. Dermatol 22: 549- 559; Peris et al. 1991, Arch. Dermatol. Res. 283: 500-505). The expression of c-fos may be directly proportional to metastatic potential in B-16 melanoma cell lines. With this evidence, it is reasonable to conclude that c-fos represents a suitable systemic ribozyme target in either the Lewis lung, B-16 melanoma, or human melanoma models.

Delivery of ribozymes and ribozyme formulations in the Lewis lung model

Several ribozyme formulations, including cationic lipid complexes which may be useful for inflammatory diseases (e.g., DIMRIE/DOPE, etc.) and RES evading liposomes which may be used to enhance vascular exposure of the ribozymes, are of interest in cancer models due to their presumed biodistribution to the lung. Thus, liposome formulations can be used for delivering ribozymes to sites of pathology linked to an angiogenic response.

The sequences of the ribozymes that are chemically synthesized, useful in this study, are non-limiting examples. 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 can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem stmcture can form. Similarly, stem-loop IV sequence of haiφin ribozymes listed in Tables IV (5'-CACGUUGUG-3^?) can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem stmcture can form. Preferably, no more than 200 bases are inserted at these locations. The sequences listed in Tables III and IV may be formed of ribonucleotides or other nucleotides or non- 06

nucleotides. Such ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables.

Target sites

Targets for useful ribozymes can be determined as disclosed in Draper et al, WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al, WO 94/02595; Draper et al, WO 95/04818; McSwiggen et al, US Patent No. 5,525,468 and hereby incoφorated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.

The sequence of human c-raf mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or haiφin ribozyme cleavage sites were identified. These sites are shown in Tables XII-XIX (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 nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

Because Raf RNAs are highly homologous in certain regions, some ribozyme target sites are also homologous (see Table XVIII and XIX). In this case, a single ribozyme will target different classes of Raf RNA. The advantage of one ribozyme that targets several classes of Raf RNA is clear, especially in cases where one or more of these RNAs may contribute to the disease state.

Hammerhead or haiφin ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et al, 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary stmcture. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding o /

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. Ribozymes of the hammerhead or haiφin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above.

Examples

The following are non-limiting examples showing the selection, isolation, synthesis and activity of enzymatic nucleic acids of the instant invention.

The following examples demonstrate the selection of ribozymes that cleave c-raf RNA. The methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA targets required for cell division. Also provided is a description of how such ribozymes may be delivered to cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture and modulate gene expression in vivo. Moreover, significantly reduced inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes.

Example 1 : Identification of Potential Ribozyme Cleavage Sites in Human c-raf RNA

The sequence of human c-raf RNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding stmctures and contained potential hammerhead and/or haiφin ribozyme cleavage sites were identified. The sequences of these cleavage sites are shown in tables XII-XIX.

Example 2: Selection of Ribozyme Cleavage Sites in Human c-raf RNA

To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in c-raf RNA, 20 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of human c-raf (GenBank Accession No. X03484; Bonner et al, 1986, Nucleic Acids Research, 14, oo

1009-1015) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 1) and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary stmcture. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozyme target sites within A-Raf were chosen by analyzing genomic sequences of human A-raf-1 (GenBank Accession No. X04790; Beck et al, 1987, Nucleic Acids Research, 115, 595-609). Ribozyme target sites within B-Raf were chosen by analyzing genomic sequences of human B-raf-1 (GenBank Accession No. M95712 M95720 X54072; Sitanandam et al, 1990, Oncogene, 5, 1775- 1780).

Example 3: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of c-rαfRNA

Ribozymes of the hammerhead or haiφin motif were designed to anneal to various sites in the RNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'- end. The average stepwise coupling yields were >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). Haiφin ribozymes were synthesized in two parts and annealed to reconstmct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized o9

from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were modified 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 were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incoφorated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table XII-XIX.

Example 4: Ribozyme Cleavage of c-raf RNA Target in vitro

Ribozymes targeted to the human c-raf RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example using the following procedure. The target sequences and the nucleotide location within the c-raf mRNA are given in Table XII.

Cleavage Reactions: Full-length or partially full-length, internally-labeled target

RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-³²p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32p-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCtø) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at o

37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., πbozyme excess. The reaction is quenched by the addition of an equal volume of 95% formamide,

20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the o sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.

Example 5: Ability of c-raf Ribozymes to Inhibit Smooth Muscle Cell Proliferation.

Ribozymes targeting sites in c-Raf mRNA were synthesized using modifications that confer nuclease resistance (Beigelman, 1995, J. Biol. Chem. 270, 25702). The ribozymes were screened for their ability to inhibit cell proliferation in serum-starved primary rat aortic smooth muscle cells as described by Jarvis et al. (1996, 7?NJ 2, 419; incoφorated by reference herein). The ribozyme targeting site represented by Seq ID Νos 175 and 198 showed particularly high activity in inhibiting cell proliferation. An inactive control ribozyme was synthesized which had identical substrate binding arms but contained mutations in the catalytic core that eliminate cleavage activity. Inhibition of cell proliferation by active versus inactive c-Raf ribozymes is shown in Figures 37 and 38. The data are presented as proliferation relative to the semm-stimulated untreated control cells. Clearly the active ribozyme is showing substantial inhibition relative to both the untreated control and its corresponding inactive control, thus indicating that the inhibition of proliferation is mediated by ribozyme-mediated cleavage of c-Raf.

In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, C. F., et al., 1992, Mol. Pharmacology, 41, 1023-1033). In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) formulation of DOSPA (2,3-dioleyloxy-Ν-[2(sperminecarboxamido)ethyl]-Ν,Ν- dimethyl-1-propanaminium trifluoroacetate) and dioleoyl phosphatidyl ethanolamine (DOPE)), was purchased from Life Technologies, Inc. DMRIE (N-[l-(2,3- ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide) was obtained from VICAL. DMRIE was resuspended in CHCI3 and mixed at a 1:1 molar ratio with dioleoyl phosphatidyl ethanolamine (DOPE). The CHCI3 was evaporated, the lipid was resuspended in water, vortexed for 1 minute and bath sonicated for 5 minutes. Ribozyme and cationic lipid mixtures were prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives was warmed to room temperature (about 20- 25 °C), cationic lipid was added to the final desired concentration and the solution was vortexed briefly. RNA oligonucleotides were added to the final desired concentration and the solution was again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex was serially diluted into DMEM following the 10 minute incubation.

Serum-starved smooth muscle cells were washed twice with PBS, and the RNA/lipid complex was added. The plates were incubated for 4 hours at 37°C. The medium was then removed and DMEM containing 10% FBS, additives and 10 μM bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation.

The plates were incubated at 37°C for 20-24 hours, fixed with 0.3% H₂O₂ in 100% methanol, and stained for BrdU incoφoration by standard methods. In this procedure, cells that have proliferated and incoφorated BrdU stain brown; nonproliferating cells are counter-stained a light puφle. Both BrdU positive and BrdU negative cells were counted under the microscope. 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incoφorated BrdU (% cell proliferation) is presented. Errors represent the range of duplicate wells. Percent inhibition then is calculated from the % cell proliferation values as follows: % inhibition = 100 - 100((Ribozyme - 0% semm)/(Control - 0% serum)).

From this initial screen, hammerhead ribozyme targeted against c-raf site 1120

(Figure 36) was further tested. The active ribozyme was able to inhibit proliferation of smooth muscle cell, whereas, the control inactive ribozyme, that cannot cleave c-raf RNA due to alterations in their catalytic core sequence, fails to inhibit smooth muscle cell proliferation (Figure 37). Thus, inhibition of cell proliferation by these hammerhead sequences is due to their ability to cleave c-raf RNA, and not because of any non- ribozyme activity.

Example 6: Oligonucleotide design and preparation for cloning Defined and Random Libraries

The DNA oligonucleotides used in this study to constmct Defined and Random

Ribozyme Libraries were purchased from Life Technologies (BRL). A schematic of the oligonucleotide design used to constmct said Defined or Comprehensive Ribozyme Libraries is shown in Figure 8. This example is meant to illustrate one possible means to constmct such libraries. The methods described herein are not meant to be inclusive of all possible methods for constmcting such libraries. The oligonucleotides used to construct the hammerhead ribozyme libraries were designed as follows:

5 '-CGAAATCAATTG-(N1)_X- {CatalyticCore} -(N2)_x-CGTACGACACGAAAGTATCG-3 '

Where Nl = the Stem I target-specific binding arm of length x, Catalytic Core = the hammerhead catalytic domain 5'-CTGATGAGGCCGUUAGGCCGAAA-3', and N2 = the Stem III target specific binding arm of length x. The oligonucleotides were designed to self-prime via formation of a stem-loop stmcture encoded at the 3' ends of the oligos (Figure 8A). This intramolecular interaction favored an unbiased extension of complex pools of ribozyme-encoding oligonucleotides. In the case of Defined Ribozyme Library described below (Figures 9-10), Nl and N2 were 8 nt each and were designed to be complimentary to the RNA encoded by the purine nucleoside phosphorylase (PNP) gene. In the case of Random Ribozyme Libraries, Nl and N2 were randomized during synthesis to produce a single pool of all possible hammerhead ribozymes.

In the example shown (Figures 9-10), oligonucleotides encoding 40 different PNP-specific hammerhead ribozymes (greater than 40 ribozymes can be used) were pooled to a final concentration of 1 μM total oligonucleotides (2.5 nM each individual oligo). Oligos were heated to 68°C for 30 min and then cooled to ambient temperature to promote formation of the 3' stem-loop for self-priming (Figure 8A). The 3' stem loop was extended (Figure 8B) using Klenow DNA polymerase (1 μM total oligonucleotides in 1 ml of 50 mM Tris pH 7.5, lOmM MgC12, 100 μg/ml BSA. 25 μg M dNTP mix, and 200 U Klenow) by incubating for 30 min at 37°C. The reaction mixtures were then heated to 65°C for 15 min to inactivate the polymerase. The double-stranded oligos (approximately 30 μg) were digested with the 100 U of the 5' restriction endonuclease Mfe I (NEB) as described by the manufacturer, then similarly digested with the 3' restriction endonuclease BsiWI (Figure 8C). To reduce the incidence of multiple ribozyme inserts during the cloning steps, the cleaved products were treated with Calf Intestinal Phosphatase (CIP, Boehringer Mannheim) as described by the manufacturer to remove the phosphate groups at the 5' ends. This step inhibits intra- and intermolecular ligation of the ribozyme-encoding fragments. Full-length product corresponding to the double-stranded, restriction digested and phosphatase-treated products was gel-purified following electrophoresis through 10% non-denaturing acrylamide gels prior to cloning to enrich for full-length material.

Example 7: Cloning of Defined and Random Libraries

The cloning vectors used contained the following cloning sites: 5'- Mfel - Cla I - BsiWI -3'. Vectors were digested with Mfe I and BsiWI prior to use. Thus, vectors cleaved with both enzymes should lack the Cla I site present between the sites, while vectors cleaved with only one of the enzymes should still retain the Cla I site. Pooled oligos were ligated to vector using a 2:1 or 5:1 molar ratio of double-stranded oligo to vector in 50-mL reactions containing 500 ng vector and 5 U ligase in lx ligase buffer (Boehringer Mannheim). Ligation reactions were incubated over night at 16°C, then heated to 65°C 10 min to inactivate the ligase enzyme. The desired products contain a single ribozyme insert and lack the original Cla I site included between the Mfe I and BsiWI cloning sites. Any unwanted, background vector lacking ribozyme inserts and thus still containing the Cla I sites were inactivated by cleaving the product with 5 U of the restriction endonuclease Cla I for 1 h at 37°C. Approximately 150 ng of ligated vector was used to transform 100 μl XL-2 Blue competent bacteria as described by the supplier (Stratagene).

Example 8: Simultaneous screening of 40 different ribozymes targeting PNP using Defined Ribozyme Libraries.

A Defined Ribozyme Library containing 40 different hammerhead ribozymes targeting PNP was constmcted as described above (Figures 8-10). PNP is an enzyme that plays a critical role in the purine metabolic/salvage pathways. PNP was chosen as a target because cells with reduced PNP activity can be readily selected from cells with wild-type activity levels using the dmg 6-thioguanosine. This agent is not toxic to cells until it is converted to 6-thioguanine by PNP. Thus, cells with reduced PNP activity are more resistant to this dmg and can be selectively grown in concentrations of 6-thioguanosine that are toxic to cells with wild-type activity levels.

The PNP-targeted Defined Ribozyme Library expression vectors were converted into retroviral vector particles, and the resulting particles were used to transduce the Sup TI human T cell line. A T-cell line was chosen for study because T lymphocytes are more dependent on the purine salvage pathway and thus are highly susceptible to 6-thioguanosine killing. Two weeks after transduction, the cells were challenged with 10 mmol 6-thioguanosine. Resistant cells began to emerge two weeks after initiation of selection. 6-Thioguanosine-resistant cells were harvested, and the ribozyme-encoding region of the expression vector was amplified using PCR and sequenced. The sequence pattern of the ribozyme region in the selected cells was significantly different from that produced from the starting library shown in Figure 9. In the original library, sequences of the binding arms were ambiguous due to the presence of all 40 PNP-targeted ribozymes (Figure 9). However, the sequence of the ribozyme- encoding regions from the 6-thioguanosine selected cells was clearly weighted towards one of the ribozymes contained in the original pool - the ribozyme designed to cleave at nucleotide #32 of PNP mRNA. These data suggests that the ribozyme targeting position 32 of the PNP mRNA appears to be more active than the other 39 PNP-targeted ribozymes included in the pool.

Example 9: Optimizing Loop II sequence of a Hammerhead Ribozyme (HH-B) for Enhanced Catalytic Rates

To test the feasibility of the combinatorial approach described in Figure 12 approach, Applicant chose to optimize the sequence of loop-II of a hammerhead ribozyme (HH-B) (see Figure 22). Previous studies had demonstrated that a variety of chemical modifications and different sequences within loop-II may have significant effects on the rate of cleavage in vitro, despite the fact that this sequence is not phylogenetically conserved and can in fact be deleted completely. According to the standard numbering system for the hammerhead ribozyme, the four positions within loop II are numbered 12 J, 12.2, 12.3, and 12.4. The Starting Ribozyme (HH-B) contained the sequence G ₁₂ , A ₁₂₂ A _]23 A ₁₂₄. For simplicity, the four positions will be numbered 5 ' to 3 ' : G _I2 ,= 1 ; A ₁₂₂= 2; A ,_{2 3}=3; A ₁₂₄= 4. The remainder of the hammerhead ribozyme "template" remained constant and is based on a previously described hammerhead motif (Draper et al, International PCT Publication No. WO 95/13380, incoφorated by reference herein).

A strategy for optimizing the four (number of Classes = 4) loop-II positions is illustrated in Figure 180. The four standard ribose nucleotides (A, C, U and G) were chosen to constmct the ribozyme pools (n = 4). In the first step, four different pools were synthesized by the nucleotide building block mixing approach described herein. Applicant first chose to "fix" (designated F) position 3 because preliminary experiments indicated that the identity of the base at this position had the most profound effects on activity; positions 1, 2 and 4 are random. The four pools were assayed under stoichiometric conditions (lμM ribozyme; lμM substrate), to help ensure that the entire population of ribozymes in each pool was assayed. Substrate and ribozyme were pre- annealed and the reactions were initiated with the addition of lOmM MgCl₂. The rate of cleavage for each library was derived from plots of fraction of substrate cleaved as a function of time. Reactions were also performed simultaneously with the starting . ..

96

ribozyme (i.e., homogenous, loop-II = GAAA). The relative rate of cleavage for each library (k^) was calculated by dividing the observed rate of the library by the rate of the control/starting ribozyme and is plotted in Figure 21. The error bars indicate the standard error derived from the curve fits. The results show that all four pools had similar rates (K_e\). however, the library possessing "U" at position 3 was slightly faster.

Ribozyme pools were again synthesized (Class 2) with position 3 being made constant (U₃), position 4 was fixed (F₄) and positions 1 and 2 were random (X). The four pools were assayed as before; the pool containing "A" at position 4 was identified as the most desirable pool. Therefore, during the synthesis of the next pool (Class 3), positions 3 and 4 were constant with U₃ and A₄, position 2 was fixed (F₂) and position 1 was random (X). The four pools were again assayed; all four pools showed very similar, but substantially elevated rates of cleavage. The pool containing U at position 2 was identified as the fastest. Therefore, during the synthesis of the final four ribozymes (Class 4), position 3, 4 and 2 were made constant with U₃, A₄ and U₂; position 1 was fixed with A, U, C or G. The final ribozyme containing G at position 4 was clearly identified as the fastest ribozyme, allowing the identification of G₁₂ , U₁₂₂ U₁₂₃ A₁₂₄ as the optimized ribozyme motif.

To confirm that the final ribozyme (G₁₂ , U₁₂₂ U₁₂₃ A₁₂₄) was indeed faster that the starting ribozyme (G₁₂ , A₁₂₂ A₁₂₃ A₁₂₄), we compared the two ribozymes (illustrated in Figure 22) under single-turnover conditions at saturating ribozyme concentrations. The observed rates should therefore measure the rate of the chemical step, k₂. The fraction of substrate remaining uncleaved as a function of time is shown in Figure 22 (lower panel), and the derived rate contents are shown. The results show that the optimized ribozyme cleaves >10 times faster (3.7 min^"1 vs. 0.35 min^"1) than the starting ribozyme.

Example 10: Optimizing Core Chemistry of a Hammerhead Ribozyme (HH-A)

To further test the feasibility of the approach described in Figure 12, we chose to optimize the three pyrimidine residues within the core of a hammerhead ribozyme (HH- A). These three positions (shown in Figure 13 as U7, U4 and C3) were chosen because previous studies indicated that these positions are critical for both stability (Beigelman et al, 1995, supra) and activity (Ruffner et al,, 1990, supra; Burgin et al, 1996, supra) of the ribozyme. According to the standard numbering system for the hammerhead ribozyme, the three pyrimidine positions are 7, 4 and 3. For constmction of the libraries, the ribozyme positions are numbered 3' to 5': position 24 = 7, position 27 = 4, and position 28 = 3 (see Figure 13). The remainder of the hammerhead ribozyme "template" remained constant and is based on a previously described hammerhead motif (Thompson et al, US Patent No. 5,610,052, incoφorated by reference herein). The starting ribozyme template is targeted against nucleotide position 823 of k-ras mRNA (Site A). Down regulation of this message, as a result of ribozyme action, results in the inability of the cells to proliferate. Therefore in order to optimize a ribozyme, we chose to identify "variants" which were successful in inhibiting cell proliferation.

Cell Culture Assay: Ribozyme: lipid complex formation

Ribozymes and LipofectAMINE were combined DMEM at final concentrations of 100 nM and 3.6 μM, respectively. Complexes were allowed to form for 15 min at 37 C in the absence of semm and antibiotics.

Proliferation Assay Primary rat aortic smooth muscle cells (RASMC) were seeded at a density of 2500 cells/well in 48 well plates. Cells were incubated overnight in DMEM, supplemented with 20% fetal bovine semm (FBS), Na-pyravate, penicillin (50 U/ml), and streptomycin (50 μg/ml). Subsequently cells were rendered quiescent by a 48 h incubation in DMEM with 0.5% FBS.

Cells were incubated for 1.5 h with semm- free DMEM ribozyme:lipid complexes.

The medium was replaced and cells were incubated for 24 h in DMEM with 0.25% FCS. Cells were then stimulated with 10% FBS for 24 h. ³H-thymidine (0.3 μCi//well) was present for the last 12 h of semm stimulation.

At the end of the stimulation period the medium was aspirated and cells were fixed in icecold TCA (10%) for 15 min. The TCA solution was removed and wells were washed once with water. DNA was extracted by incubation with 0J N NaOH at RT for 15 min. Solubilized DNA was quantitatively transferred to minivials. Plates were washed once with water. Finally, ³H-thymidine incoφoration was determined by liquid scintillation counting.

A strategy for optimizing the three (number of Class = 3) pyrimidine residues is illustrated in Figure 20. Ten different nucleotide analogs (illustrated in Figure 15) were chosen to constmct the ribozyme library (n = 10). In the first step, ten different pools (Class 1) were synthesized by the mix and split approach described herein. Positions 24 and 27 were random and position 28 was fixed with each of the ten different analogs. The ten different pools were formulated with a cationic lipid (Jarvis et al, 1996, RNA, 2,419; incoφorated by reference herein), delivered to cells in vitro, and cell proliferation was subsequently assayed (see Figure 16). A positive control (active ribozyme) inhibited cell proliferation by -50% and an inactive control (inactive) resulted in a less than 25% reduction in cell proliferation. The ten ribozyme pools resulted in intermediate levels of reduction. However, the best pool could be identified as X₂₄ X₂₇ 2'-MTM-U₂₈ (positions 24 and 27 random; 2'-O-MTM-U at position 28). Therefore, a second ribozyme library (Class 2) was synthesized with position 28 constant (2'-O-MTM-U); position 24 was random (X₂₄) and position 27 was fixed with each of the ten different analogs (F₂₇). Again, the ten pools were assayed for their ability to inhibit cell proliferation. Among Class 2, two pools inhibited proliferation equally well: X₂₄ 2'-C-allyl-U₂₇ 2'-O-MTM-U₂₈ and X₂₄ 2'-O-MTM-C₂₇ 2'-O-MTM-U₂₈. Because a single "winner" could not be identified in Class 2, position 27 was made constant with either 2'-C-allyl-U or with 2'- O-MTM-C and the ten analogs were placed individually at position 24 (Class 3). Therefore in Class 3, twenty different ribozymes were assayed for their ability to inhibit cell proliferation. Because both positions 27 and 28 are constant, the final twenty ribozymes contain no random positions. Thus in the final group (Class 3), pure ribozymes and not pools were assayed. Among the final groups four ribozymes inhibited cell proliferation to a greater extent than the control ribozyme (Figure 22). These four winners are illustrated in Figure 23A. Figure 23B shows general formula for four different motifs. A formula for a novel ribozyme motif is shown in Figure 18.

Example 11 : Identifying Accessible Sites for Ribozyme Action in a target

In the previous two examples (9 and 10), positions within the catalytic domain of the hammerhead ribozyme were optimized. The number of groups that needed to be tested equals = the total number of positions within the ribozyme that were chosen to be tested. A similar procedure can be used on the binding arms of the ribozyme. The sequence of the binding arms determines the site of action of the ribozyme. The combinatorial approach can be used to identify those sites by essentially testing all possible arm sequences. The difficulty with this approach is that ribozymes require a certain number of base pairs (12-16) in order bind tightly and specifically. According to the procedure outlined above, this would require 12-16 different groups of ribozyme pools; 12-16 positions would have to be optimized which would require 12-16 different groups being synthesized and tested. Each pool would contain the four different nucleotides (A, C, U and G) or nucleotide analogs (n = 4). It would be very time consuming to test each group, identify the best pool, synthesize another group of ribozyme pools with one additional position constant, and then repeat the procedure until all 12-16 groups had been tested. However it is possible to decrease the number of groups by testing multiple positions within a single group. In this case, the number of pools within a group equals the number of nucleotides or analogs in the random mixture (i.e., n) to the w power, where w equals the number of positions fixed in each group. The number of groups that need to be synthesized to optimize the final ribozyme equals the total number of positions to be optimized divided by the number of positions (w) tested within each group. The number of pools in each group = n . The number of groups = total number of positions / w.

For example, Figure 23 illustrates this concept on a hammerhead ribozyme containing 12 base pair binding arms. Each of the two binding arms form 6 base pairs with it's corresponding RNA target. It is important to note that for the hammerhead ribozyme one residue (A15J) must remain constant; A15J forms a base pair with a substrate nucleotide (U16J) but is also absolutely required for ribozyme activity. It is the only residue within the hammerhead ribozyme that is part of both the catalytic domain, and the binding domain (arms). In the example this position is not optimized. In the first Group, three positions are fixed (designated F) with the four different 2'-O-methyl nucleotides (A, C, U and G). The 2 '-O-methyl modification stabilizes the ribozyme against nuclease degradation and increases the binding affinity with it's substrate. The total number of pools in each group does not equal n, as in the previous examples. The number of pools in each group equals 4³ = (four analogs)^Λ(number of positions fixed; 3) = 64. In all 64 pools, all other positions in the arm are made random (designated X) by the nucleotide mixing building block approach. The catalytic domain is not considered in this example and therefore remains part of the ribozyme template (i.e., constant).

In the first step, all 64 ribozyme pools are tested. This test may be cleavage in vitro (see Example 9), or efficacy in a cellular (see Example 10) or animal model, or any other assayable end-point. This end-point however, should be specific to a particular RNA target. For example, if one wishes to identify accessible sites within the mRNA of GeneB, a suitable end-point would be to look for decreased levels of GeneB mRNA after ribozyme treatment. After a winning pool is identified, since each pool specifies the identity of three positions (w), three positions can be made constant for the next group (Class 2). Class 2 is synthesized containing 64 different pools; three positions that were fixed in Class 1 are now constant (designated TJ), three more positions are fixed (F), and the remaining positions (X) are a random mix of the four nucleotides. The 64 pools are assayed as before, a winning pool is identified, allowing three more positions to be constant in the next Class of ribozyme pools (Class 3) and the process is repeated again. In the final Class of ribozymes (Class 4), only two positions are fixed, all other positions have been previously fixed. The total number of ribozymes is therefore n^w = 4² = 16; these ribozymes also contain no random positions. In the final step (step 4), the 16 ribozymes are tested; the winning ribozyme defmes the sequence of the binding arms for a particular target.

Fixing multiple positions within a single group it is possible to decrease the overall number of groups that need to be tested. As mentioned, this is particularly useful when a large number of different positions need to be optimized. A second advantage to this approach is that it decreases the complexity of molecules in each pool. If one would expect that many combinations within a given pool will be inactive, by decreasing the number of different ribozymes in each pool, it will be easier to identify the "winning" pool. In this approach, a larger number of pools have to be tested in each group, however, the number of groups is smaller and the complexity of each ribozyme pool is smaller. Finally, it should be emphasized there is not a restriction on the number of positions or analogs that can be tested. There is also no restriction on how many positions are tested in each group.

Example 12: Identifying new RNA targets for Ribozymes

As described above for identifying ribozyme-accessible sites, the assayed used to identify the "winning" pool of ribozymes is not defined and may be cleavage in vitro (see Example 8), or efficacy in a cellular (see Example 9) or animal model, or any other assayable end-point. For identifying accessible sites, this end-point should be specific to a particular RNA target (e.g., mRNA levels). However, the . end-point could also be nonspecific. For example, one could choose a disease model and simply identify the winning ribozyme pool based on the ability to provide a desired effect. In this case, it is not even necessary to know what the cellular target that is being acted upon by the ribozyme is. One can simply identify a ribozyme that has a desired effect. The advantage to this approach is that the sequence of the binding arms will be complementary to the RNA target. It is therefore possible to identify gene products that are involved in a disease process or any other assayable phenotype. One does not have to know what the target is prior to starting the study. The process of identifying an optimized ribozyme (arm combinatorial) identifies both the dmg (ribozyme) and the RNA target, which may be a known RNA sequence or a novel sequence leading to the discovery of new genes.

Example 13: Identifying New Ribozyme Catalytic Domains

In the previous two examples, positions within the binding domain of the hammerhead ribozyme were varied and positions within the catalytic domain were not changed. Conversely, it is possible to vary positions within the catalytic domain, without changing positions within the binding arms, in order to identify new catalytic motifs. An example is illustrated in Figure 24. The hammerhead ribozyme, for example comprises about 23 residues within the catalytic domain. It is unclear how many of these 23 positions are required to obtain a functional catalytic domain, however it is reasonable to presume that if a large number of functionally diverse nucleotide analogs can be used to constmct the pools, a relatively small number of positions could constitute a functional catalytic domain. This may especially be tme if analogs are chosen that one would expect to participate in catalysis (e.g., acid/base catalysts, metal binding, etc.). In the example illustrated in Figure 24, four positions (designated 1, 2, 3 and 4) are chosen. In the first step, ribozyme libraries (Class 1) are constmcted: position 1 is fixed (FJ) and positions 2, 3 and 4 are random (X₂, X₃ and X₄, respectively). In step 2, the pools (the number of pools tested depends on the number of analogs used; n) are assayed for activity. This testing may be performed in vitro or in a cellular or animal model. Whatever assay that is used, the pool with the most activity is identified and libraries (class 2) are again synthesized with position 1 now constant (Z,) with the analog that was identified in class 1. In class 2, position 2 is fixed (F₂) and positions 3 and 4 are random (X₃ and X₄). This process is repeated until every position has been made constant, thus identifying the catalytic domain or a new motif. EXAMPLE 14: Determination of Coupling Efficiency of the phosphoramidite derivatives of 2'-C-allyl-uridine, 1; 4'-thio-cytidine. 2; 2'-methylthiomethyl-uridine. 3; 2'- methylthiomethyl-cytidine. 4; 2'-amino-uridine. 5; N3-methyl-uridine, 6; 1-b-D- (ribofuranosylVpyridin-4-one, 7; l-b-D-(riboftιranosyr)-pyridin-2-one-, 8; 1-b-D- (ribofuranosyl)-phenyl, 9; 6-methyl-uridine, 10 to be used in a split and mix approach.

The determination of the coupling efficiency of amidites 1 to 10 was assessed using ten model sequences agacXGAuGa (where upper case represents ribonucleotide residues, lower case represents 2'-O-methyl ribonucleotide residues and X is amidites 1 to 10, to be used in the constmction of a hammerhead ribozyme library wherein the modified amidites 1 to 10 would be incoφorated. Ten model sequences were synthesized using ten 0J 12 g aliquots of 5'-O-DMT-2'-O-Me- Adenosine Polystyrene (PS) solid- support loaded at 22.3 μmol/g and equivalent to a 2.5 μmol scale synthesis. Synthesis of these ten decamers were performed on ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites and phosphoramidites 1, 2, 3, 4, 6, 7, 8, 9, 10, 12.5 min coupling time for the 2'-amino-uridine phosphoramidite, amidite 5 and 2.5 min coupling time for the 2'- O-methyl nucleoside phosphoramidites.

Oligomers were cleaved from the solid support by treatment with a 3 : 1 mixture of ammonium hydroxide:absolute ethanol at 65 degree C for 4 hrs followed by a desilylation treatment and butanol precipitation as described in Wincott et al. (Wincott et al, Nucleic Acids Res, 1995, 23, 2677-2684; incoφorated by reference herein). Oligonucleotides were analyzed directly on an anion-exchange HPLC column (Dionex, Nucleopac, PA- 100, 4x250 mm) using a gradient of 50% to 80% of B over 12 minutes (A = 10 mM sodium perchlorate, 1 mM Tris, pH 9.43; B = 300 mM sodium perchlorate, 1 mM Tris, pH 9.36) and a Hewlett-Packard 1090 HPLC system.

The average stepwise yield (ASWY), indicating the coupling efficiency of phosphoramidites, 1 to 10, were calculated from peak-area percentages according to the equation ASWY = (FLP%)^{1 n} where FLP% is the percentage full-length product in the cmde chromatogram and n the number of synthesis cycles. ASWY ranging from of 96.5%o to 97.5% were obtained for phosphoramidites, 1 to 10. The experimental coupling efficiencies of the phosphoramidites 1 to 10, as determined using a standard spectrophotometric dimethoxytrityl assay were in complete agreement with the ASWY and were judged satisfactory to proceed with the X24, X27, X28 ribozyme library synthesis.

EXAMPLE 15: Determination of optimal relative concentration of a mixture of 2'-O- methyl-guanosine, cytidine, uridine and adenosine providing equal representation of the four nucleotides.

A mixture N, composed of an equimolar mixture of the four 2'-O-Me- nucleoside phosphoramidites (mG=2'-O-methyl guanosine; mA=2'-O-methyl adenosine; mC=2'-O- methyl cytidine; mU=2'-O-methyl uridine) was used in the synthesis of a model sequence TTXXXXTTB, where T is 2'-deoxy-thymidine and B is a 2'-deoxy-inverted abasic polystyrene solid-support as described in Example 14. After standard deprotection (Wincott et al, supra), the cmde nonamer was analyzed on an anion-exchange HPLC column (see example 6). From the HPLC analysis, an averaged stepwise yield (ASWY) of 99.3% was calculated (see example 14) indicating that the overall coupling efficiency of the mixture N was comparable to that of 2'-deoxythymidine. To further assess the relative incoφoration of each of the components within the mixture, N, the full-length product TTXXXXTTB (over 94.3% at the cmde stage) was further purified and subjected to base composition analysis as described herein. Purification of the FLP from the failures is desired to get accurate base composition.

Base composition analysis summary: A standard digestion/HPLC analysis was performed: To a dried sample containing

0.5 A.sub.260 units of TTXXXXTTB, 50 μl mixture, containing 1 mg of nuclease PI (550 units/mg), 2.85 ml of 30 mM sodium acetate and 0.3 ml of 20 mM aqueous zinc chloride, was added. The reaction mixture was incubated at 50 degrees C overnight. Next, 50 μl of a mixture comprising 500 μl of alkaline phosphatase (1 units/μl), 312 μl of 500 mM Tris pH 7.5 and 2316 μl water was added to the reaction mixture and incubated at 37 degrees C for 4 hours. After incubation, the samples were centrifuged to remove sediments and the supernatant was analyzed by HPLC on a reversed-phase C18 column equilibrated with 25 mM KH2PO4. Samples were analyzed with a 5% acetonitrile isocratic gradient for 8 min followed by a 5% to 70% acetonitrile gradient over 8 min.

The HPLC percentage areas of the different nucleoside peaks, once corrected for the extinction coefficient of the individual nucleosides, are directly proportional to their molar ratios.

The results of these couplings are shown in Table IV.

As can be seen in Table IV, the use of an equimolar mixture of the four 2'-O- methyl phosphoramidites does not provide an equal incoφoration of all four amidites, but favors 2'-O-methyl-U and G and incoφorates 2'-O-methyl-A and C to a lower efficiency. To alleviate this, the relative concentrations of 2 '-O-methyl- A, G, U and C amidite were adjusted using the inverse of the relative incoφoration as a guide line. After several iterations, the optimized mixture providing nearly identical incoφoration of all four amidites was obtained as shown in Table V below. The relative representation do not exceed 12% difference between the most and least incoφorated residue corresponding to a +/- 6% deviation from equimolar incoφoration.

EXAMPLE 16: A Non-competitive coupling method for the preparation of the X24, X27 and N28 ribozyme library 5'- a_sc_sa_sa_;ag aFX GAY Gag gcg aaa gcc Gaa Age ecu cB -3' wherein 2'-C-allyl-uridine. 1; 4'-thio-cytidine. 2; 2 '-methylthiomethyl -uridine. 3; 2'- methylthiomethyl-cytidine. 4; 2'-amino-uridine. 5; N3-methyl-uridine, 6: 1-b-D-

(ribofuranosyl)-pyrimidine-4-one. 7; l-b-D-(ribofuranosyl)-pyrimidine-2-one. 8; 1-b-D- (ribofuranosyl)-phenyl. 9; and/or 6-methyl-uridine. 10 are incoφorated at the X24, X27 and F28 positions through the mix and split approach.

The synthesis often different batches of 2.5 μmol scale Gag gcg aaa gcc Gaa Age ecu cB sequence was performed on 2'-deoxy inverted abasic polystyrene solid support B on a 394 ABI DNA synthesizer (Applied Biosystems, Foster City, CA). These ten aliquots were then separately reacted with phosphoramidite building blocks 1 to 10 according to the conditions described in example 11. After completion of the individual incoφoration of amidites 1 to 10, their coupling efficiencies were determined to be above 95 % as judged by trityl monitoring. The 10 different aliquots bearing the ten different sequences were mixed thoroughly and divided into ten equal subsets. Each of these aliquots were then successively reacted with ribo-A, ribo-G amidites and one of the amidites 1 to 10. The ten aliquots were combined, mixed and split again in 10 subsets.

At that point, the 10 different polystyrene aliquots, exhibiting the following sequence: X GA Gag gcg aaa gcc Gaa Age ecu cB, were reacted again with amidites 1 to 10 separately. The aliquots were not mixed, but kept separate to obtain a unique residue at the 28th position of each of the ten pools. The ribozyme synthesis was then finished independently to yield ten random ribozymes pools. Each pool comprises a 3'-terminal inverted abasic residue B, followed by the sequence Gag gcg aaa gcc Gaa Age ecu c, followed with one random position X in the 24th position corresponding to a mixture of amidites 1 to 10, followed by the sequence GA, followed one random position X in the 27th position corresponding to a mixture of amidites 1 to 10, followed by a fixed monomer F (one of the amidites 1 to 10) in the 28th position and finally the 5'-terminal sequence a_sc_sa_sa_sa g a. This is represented by the sequence notation 5'- a_sc_sa_sa_sag aFX

GAX- Gag gcg aaa gcc Gaa Age ecu cB-3', in which X are random positions and F is a

₃ unique fixed position. The total complexity of such a ribozyme library was 10 or 1,000 members separated in 10 pools of 100 different ribozyme sequences each.

EXAMPLE 17: Competitive coupling method (monomer mixing approach) for the preparation of the x₂ ^ and X_? _ ς "binding arms" ribozyme library

Synthesis of 5'-x_sx_sx xFF cwG AM G Agg ccg uua ggc cGA AAF xxx xB-3' is described, with F being a defined 2'-O-methyl-ribonucleoside chosen among 2'-Ο- methyl-ribo-adenosine (mA), -guanosine (mG), -cytidine (mC), -uridine (mU) and x being an equal mixture of 2'-O-methyl-ribo-adenosine, -guanosine, -cytidine, -uridine.

The syntheses of this ribozyme library was performed with an ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites (upper case) and 2'-amino-uridine phosphoramidite, u, (2.5 min) coupling time for the 2'-O-methyl-ribonucleoside phosphoramidites (lower case) and the 2'-O-methyl-ribonucleoside phosphoramidites mixture, n.

Sixty four (64) batches of 0.086 g aliquots of 3'-O-DMT-2'-deoxy-inverted abasic-Polystyrene (B) solid-support loaded at 29 μmol/g and equivalent to a 2.5 μmol -

scale synthesis were individually reacted with a 27:32:19:22 / v:v:v:v mixture, x, of mA:mC:mG:mU diluted in dry acetonitrile to OJ M as described in example 7. This synthesis cycle was repeated for a total of four times. The 64 aliquots were then grouped into four subsets of sixteen aliquots (Class 1) that were reacted with either mA, mG, mC, mU to synthesize the n6 position. This accomplished, the sequence: 5'- cwG AH G Agg ccg uua ggc cGA AA was added onto the 6 position of the 64 aliquots constituting Class 1. Each subset of Class 1 was then divided into four subsets of four aliquots (Class 2) that were reacted with either mA, mG, mC, mU to synthesize the F30 position. Each subset of Class 2 was then divided into four subsets of one aliquot (Class 3) that were reacted with either mA, mG, mC, mU to synthesize the F31 position. Finally, the random sequence 5'- x_sx_sx x was added onto each of the 64 aliquots.

The ribozyme library yielded sixty four random ribozymes pools each having an equal mixture of the four 2'-O-methyl-nucleoside at the position x2 to 6 and x30 to 35, and a defined 2'-O-methyl-nucleoside chosen among mA, mC, mG, mU at the positions F6, F30 and F31. The total complexity of such a "binding arms" ribozyme library was

4 or 4,194,304 members separated in 64 pools of 65,536 different ribozyme sequences each.

EXAMPLE 18: Competitive coupling method (monomer mixing approach) for the preparation of the position 15 to 18 "loop II" ribozyme library

Synthesis of 5' UCU CCA UCU GAU GAG GCC XXF XGG CCG AAA AUC

CCU 3' is described, with F being a defined ribonucleoside chosen among adenosine (A), guanosine (G), cytidine (C), uridine (U) and X being an equal mixture of adenosine (A), guanosine (G), cytidine (C), uridine (U).

The syntheses of this ribozyme library was performed with an ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites (A, G, C, U) and the ribonucleoside phosphoramidite mixture, X.

Four batches (4) of 2.5 μmol scale of GG CCG AAA AUC CCU sequence were synthesized on 0.085 g samples of 5'-O-DMT-2'-O-TBDMS-3'-succinyl-uridine- Polystyrene (U) solid-support loaded at 29.8 μmol/g. To synthesize the position XI 5, the four aliquots of solid-supports were individually reacted with a 30:26:24:20 / v:v:v:v mixture, X, of A:C:G:U diluted in dry acetonitrile to 0J M according to the optimized conditions for the DNA phosphoramidites mixed-base coupling as described in the DNA Synthesis Course Manual published by Perkin-Elmer-Applied Biosystem Division. (DNA Synthesis Course Manual : Evaluating and isolating synthetic oligonucleotides, the complete guide, p. 2-4, Alex Andrus, August 1995). The four aliquots of solid-supports were then individually reacted with either of the four ribonucleoside phosphoramidites (A, G, C, U) to create the F16 position. The position XI 7 and XI 8 were then added onto the F16 (either A, G, C or U) of the four aliquots of solid-supports by repeating twice the same procedure used for the position XI 5.

The synthesis of the ribozyme library was then ended by adding the sequence 5'- UCU CCA UCU GAU GAG GCC on the position XI 8 of each of the four subsets of the ribozyme library. The ribozyme library yielded four random ribozymes pools that each have an equal mixture of the four ribonucleoside (A, G, C and U) at the position XI 5, XI 7 and XI 8, and a discrete ribonucleoside chosen among A, C, G or U at the positions F16. The total complexity of such a loop II ribozyme library was 256 members separated in 4 pools of 64 different ribozyme sequences.

Example 19: Arm-Combinatorial Library Screening For Bcl-2, K-ras and Urokinase plasminogen Activator (UP A) Substrate synthesis through in vitro transcription: Run-off transcripts for Bcl-2 and Kras were prepared using linearized plasmids (975 and 796 nucleotides respectively). Transcripts for UPA were produced from a PCR generated DNA fragment containing a T7 promoter (400 nucleotides). Transcription was performed using the T7 Megascript transcription kit (Ambion, Inc.) with the following conditions: a 50ul reaction volume containing 7.5mM each of ATP, CTP, UTP, and GTP, 2mM guanosine, 5ul lOx T7 reaction buffer, 5ul T7 enzyme mix, and 0.5ug of linearized plasmid or PCR'd DNA template. The mixture was incubated at 37°C for 4 hours (6 hours for transcripts < 500 bases). Guanosine was added to the transcription reactions so that the final transcript could be efficiently 5 '-end labeled without prior phosphatase treatment. Transcription volume was then increased to 200ul with buffer containing 50mM TRIS pH 7.5, lOOmM KC1, and 2mM MgCl₂ and spin column purified over Bio-Gel P-60 (BioRad) equilibrated in the same buffer. lOOul of transcript was then applied to 750ul of packed resin. Spin column flow- through was used directly in a 5 '-end labeling reaction as follows (lOOul final volume): 82ul of P-60 spin column purified transcript, lOul lOx polynucleotide kinase buffer, 4ul lOU/ul Polynucleotide Kinase (Boehringer/Mannheim) and 4ul 150uCi/ul Gamma-32P-ATP (NEN) were incubated together at 37°C for one hour. The reaction volume was increased to 200ul with buffer containing 50mM TRIS pH 7.5, lOOmM KC1 and 2mM MgCl₂ and the sample was then purified over Bio-Gel P-60 packed spin column as described above. Approximate specific activities of the 5 '-end labeled transcripts were determined via BioScan and stored frozen at -20°C. Synthesis of Ribozyme pools: In vitro ribozyme-transcript cleavage reactions: Cleavage reactions were carried out as follows: 5 '-end labeled transcript (~2-4 x 10⁴ dpm ul final) was incubated with lOuM ribozyme pool in 50mM TRIS pH 7.5, 50mM NaCl, 2mM MgCl₂ and 0.01% SDS for 24- 48 hours at room temperature (~22°C). An equal volume of gel loading dye (95% formamide, 0.01M EDTA, 0.0375% bromophenol blue, and 0.0375% xylene cyanol) was added to stop the reaction and the samples are heated to 95°C. Reactions (1-2 x 10⁵ dpm per lane) were mn on a 5% denaturing polyacrylamide gel containing 7M urea and lx TBE. Gels are dried and imaged using the Phosphorlmager system (Molecular Dynamics). Ambion, Inc. RNA Century Marker Plus RNA standards body labeled in a T7 Megascript reaction as described above using 3ul of lOmCi/ml Alpha-³²P-ATP (BioRad) and 0.5ug Century RNA template and subsequently spin column purified over Bio-Gel P-6 (Bio-Rad) were used as a size reference on the gel. Cleavage product sizes were determined using the RNA standards which provided an approximate site of cleavage (est. Size in Figure). Because each of the ribozyme pools has three positions within the binding arms fixed, it is possible to identify all of the potential ribozyme sites that can potentially be cleaved by that pool. The estimated size of the cleavage product is therefore compared with the potential sites to identify the exact site of cleavage. This protocol has been completed on three different transcripts: Bcl-2 (figure 25), K-ras (figure 26), and UPA (figure 27). The data is summarized in the figures. All potential hammerhead ribozyme cleavage sites are indicated in the graph with a short vertical line. The actual sites identified are indicated in the graph. The size of the bar reflects the intensity of the cleavage product from the cleavage reaction. The combinatorial pool used to identify each site, the actual sequence of each site, the position of cleavage within the transcript (based on the known sequence), and the estimated size of the cleavage product (based on gel analysis) are listed. Example 20: Reduction of Bcl-2 mRNA using Optimized Ribozymes

Two ribozymes targeted against the same site in the bcl-2 transcript (SeqJD#9, figure 25) were synthesized, but the two ribozymes were stabilized using two different chemistries (U4/U7 amino and U4 c-allyl). Ribozymes (200 nM) were delivered using lipofectamine (7.2 mM) for 3 hours into MCF-7 cells (50% confluency). Cellular RNA was harvested 24 hours after delivery, analyzed by RNase protectection analysis (RPA) and normalized to GAPDH mRNA in triplicate samples. Both ribozymes gave a reduction in bcl-2 mRNA (see Figure 28). A ribozyme targeted against an irrelevant mRNA (c-myb) had no effect on the ratio of bcl-2 mRNA to GAPDH mRNA. All reduction of bcl-2 RNA was statistically significant with respect to untreated samples and samples treated with the irrelevant ribozyme.

Example 21: Synthesis of purine nucleoside triphosphates: 2'-O-methyl-guanosine-5'- triphosphate

2'-O-methyl guanosine nucleoside (0.25 grams, 0.84 mmol) was dissolved in triethyl phosphate (5.0) ml by heating to 100 C for 5 minutes. The resulting clear, , , „

112

colorless solution was cooled to 0 C using an ice bath under an argon atmosphere. Phosphorous oxychloride (1.8 eq., 0J41 ml) was then added to the reaction mixture with vigorous stirring. The reaction was monitored by HPLC, using a sodium perchlorate gradient. After 5 hours at 0 C, tributylamine (0.65 ml) was added followed by the addition of anhydrous acetonitrile (10.0 ml), and after 5 minutes (reequilibration to 0 C) tributylammonium pyrophosphate (4.0 eq., 1.53 g) was added. The reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 0 C (HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then stirred ovemight at room temperature, the mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in 50 ml 0.05M TEAB. DEAE sephadex purification was used with a gradient of 0.05 to 0.6 M TEAB to obtain pure triphosphate (0.52 g, 66.0% yield) (elutes around 0.3M TEAB); the purity was confirmed by HPLC and NMR analysis. Example 22: Synthesis of Pyrimdine nucleoside triphosphates: 2'-O-methylthiomethyl- uridine-5 ' -triphosphate 2'-O-methylthiomethyl uridine nucleoside (0.27 grams, 1.0 mmol) was dissolved in triethyl phosphate (5.0 ml). The resulting clear, colorless solution was cooled to 0 C with an ice bath under an argon atmosphere. Phosphoms oxychloride (2.0 eq., 0J90 ml) was then added to the reaction mixture with vigorous stirring. Dimethylaminopyridine (DMAP, 0.2eq., 25 mg) was added, the solution warmed to room temperature and the reaction was monitored by HPLC, using a sodium perchlorate gradient. After 5 hours at 20 C, tributylamine (1.0 ml) was added followed by anhydrous acetonitrile (10.0 ml), and after 5 minutes tributylammonium pyrophosphate (4.0 eq., 1.8 g) was added. The reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 20 C (HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then stirred ovemight at room temperature. The mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in 50 ml 0.05M TEAB. DEAE fast flow Sepharose purification with a gradient of 0.05 to 1.0 M TEAB was used to obtain pure triphosphate (0.40 g, 44% yield) (elutes around 0.3M TEAB) as determined by HPLC and NMR analysis. - -

Example 23: Utilization of DMAP in Uridine 5 '-Triphosphate Synthesis

The reactions were performed on 20 mg aliquots of nucleoside dissolved in 1 ml of triethyl phosphate and 19 ul of phosphoms oxychloride. The reactions were monitored at 40 minute intervals automatically by HPLC to generate yield-of-product curves at times up to 18 hours. A reverse phase column and ammonium acetate/ sodium acetate buffer system (50mM & lOOmM respectively at pH 4.2) was used to separate the 5', 3', 2' monophosphates (the monophosphates elute in that order) from the 5'- triphosphate and the starting nucleoside. The data is shown in table VI. These conditions doubled the product yield and resulted in a 10-fold improvement in the reaction time to maximum yield (1200 minutes down to 120 minutes for a 90% yield). Selectivity for 5'- monophosphorylation was observed for all reactions. Subsequent triphosphorylation occurred in nearly quantitative yield.

Materials Used in Bacteriophage T7 RNA Polymerase Reactions BUFFER 1: Reagents are mixed together to form a 10X stock solution of buffer 1 (400 mM Tris-Cl (pH 8J), 200 mM MgCl₂, 100 mM DTT, 50 mM spermidine, and 0.1% triton X-100. Prior to initiation of the polymerase reaction methanol, LiCl is added and the buffer is diluted such that the final reaction conditions for condition 1 consisted of : 40mM tris pH (8.1), 20mM MgCl₂, 10 mM DTT, 5 mM spermidine, 0.01% triton X-100, 10% methanol, and 1 mM LiCl. BUFFER 2: Reagents are mixed together to form a 10X stock solution of buffer 2(400 mM Tris-Cl (pH 8J), 200 mM MgCl₂, 100 mM DTT, 50 mM spermidine, and 0.1% triton X-100. Prior to initiation of the polymerase reaction PEG, LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 2 consisted of : 40mM tris pH (8.1), 20mM MgCl₂, 10 mM DTT, 5 mM spermidine, 0.01% triton X-100, 4% PEG, and 1 mM LiCl.

BUFFER 3: Reagents are mixed together to form a 10X stock solution of buffer 3 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl₂, 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG is added and the buffer is , , ,

114

diluted such that the final reaction conditions for buffer 3 consisted of : 40mM tris pH (8.0), 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, and 4% PEG. BUFFER 4: Reagents are mixed together to form a 10X stock solution of buffer 4 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl₂, 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 4 consisted of : 40mM tris pH (8.0), 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, 10% methanol, and 4% PEG. BUFFER 5: Reagents are mixed together to form a 10X stock solution of buffer 5 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl₂, 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 5 consisted of : 40mM tris pH (8.0), 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, 1 mM LiCl and 4% PEG. BUFFER 6: Reagents are mixed together to form a 10X stock solution of buffer 6 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl₂, 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 6 consisted of : 40mM tris pH (8.0), 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, 10% methanol, and 4% PEG.

Example 24: Screening of Modified Nucleoside triphosphates with Mutant T7 RNA Polymerase

Each modified nucleotide triphosphate was individully tested in buffers 1 through 6 at two different temperatures (25 and 37°C). Buffers 1-6 tested at 25°C were designated conditions 1-6 and buffers 1-6 test at 37°C were designated conditions 7-12 (table VII). In each condition, Y639F mutant T7 polymerase (Sousa and Padilla, Supra) (0.3-2 mg/20 ml reaction), NTP's (2 mM each), DNA template (10 pmol), inorganic pyrophosphatase (5U/ml) and α-³²P NTP(0.8 mCi/pmol template) were combined and heated at the designated temperatures for 1-2 hours. The radiolabeled NTP used was different from the modified triphosphate being testing. The samples were resolved by polyacrylamide gel electrophoresis. Using a phosphorlmager (Molecular Dynamics, Sunnyvale, CA), the amount of full-length transcript was quantified and compared with an all-RNA control reaction. The data is presented in Table VIII; results in each reaction is expressed as a percent compared to the all-ribonucleotide triphosphate (rNTP) control. The control was ran with the mutant T7 polymerase using commercially available polymerase buffer (Boehringer Mannheim, Indianapolis, IN). Example 25: Incoφoration of Modified NTP's using Wild-type T7 RNA polymerase

Bacteriophage T7 RNA polymerase was purchased from Boehringer Mannheim at 0.4 U/μL concentration. Applicant used the commercial buffer supplied with the enzyme and 0.2 μCi alpha-³²P NTP in a 50 μL reaction with nucleotides triphosphates at 2 mM each. The template was double-stranded PCR fragment, which was used in previous screens. Reactions were carried out at 37°C for 1 hour. 10 μL of the sample was mn on a 7.5% analytical PAGE and bands were quantitated using a Phosphorlmager. Results are calculated as a comparison to an "all ribo" control (non-modified nucleoside triphosphates) and the results are in Table IX.

Example 26: Incoφoration of Multiple Modified Nucleoside triphosphates Into Oligonucleotides

Combinations of modified nucleoside triphosphates were tested with the transcription protocol described in example 9, to determine the rates of incoφoration of two or more of these triphosphates. Incoφoration 2'-Deoxy-2'-(L-histidine) amino uridine (2'-his-NH₂-UTP) was tested with unmodified cytidine nucleoside triphosphates, rATP and rGTP in reaction condition number 9. The data is presented as a percentage of incoφoration of modified NTP's compared to the all rNTP control and is shown in Table Xa.

Two modified cyti dines (2'-NH₂-CTP or 2'dCTP) were incoφorated along with 2'-his-NH₂-UTP with identical efficiencies. 2'-his-NH₂-UTP and 2'-NH₂-CTP were then tested with various unmodified and modified adenosine triphosphates in the same buffer (Table Xb). The best modified adenosine triphosphate for incoφoration with both 2'-his- NH₂-UTP and 2'-NH₂-CTP was 2'-NH₂-DAPTP.

EXAMPLE 27: Optimization of Reaction conditions for Incoφoration of Modified Nucleotide Triphosphate The combination of 2'-his-NH₂-UTP, 2'-NH₂-CTP, 2'-NH₂-DAP, and rGTP was tested in several reaction conditions (Table XI) using the incoφoration protocol described in example 14. The results demonstrate that of the buffer conditions tested, incoφoration of these modified nucleoside triphosphates occur in the presence of both methanol and LiCl. Example 28: Deprotection of Ribozyme in a 96 Well Plate

A ribozyme sequence (200nmole) was synthesized as described herein on a polystyrene solid support in a well of a 96 well plate. A 10:3:13 mixture (800 μL) of anhydrous methylamine (308μL), triethylamine (92μL) and dimethylsulfoxide (DMSO) (400 μL) was prepared of which half (400 μL) was added to the well and incubated at room temperature for 45 minutes. Following the reaction the solution was replaced with the remaining 400 μL and incubated as before. At the end of the reaction, the solid support was filtered off, all 800 μL of MA/TEA/DMSO solution was collected together and 100 μL of TEA.3HF was added. The reaction was then heated at 65°C for 15 minutes and then cooled to room temperature. The solution was then quenched with aqueous NΗ HCO3^" (ImL) (see Figure 30). HPLC chromatography of the reaction mixture afforded 32 O. D.u_{260 nm} of which 46% was full length ribozyme. Example 29: Column Deprotection of Ribozyme

A ribozyme was synthesized using the column format as described herein. The polystyrene solid-support with protected oligoribonucleotide or modified oligoribonucleotide (200 nmole) was transferred into a glass vial equipped with a screw cap. A 10:3:13 mixture of anhydrous methylamine ( 308 μL), triethylamine (92 μL) and dimethylsulfoxide (DMSO) (400 μL) was added followed by vortexing of the glass vial. After allowing the reaction for 1.5 hours, the solid support was filtered off. 100 μL of TEA.3HF was added at room temperature to the vial and the reaction was mixed causing the solution to gel. The reaction was then heated at 65 °C for 15 minutes and then cooled to room temperature. The solution was then quenched with 1.5 M aqueous NH₄ΗCO₃ ^"

(ImL). HPLC chromatography of the reaction mixture afforded 32 O. D.u_{260 nm} of which 46% was full length ribozyme.

Example 30: Column Deprotection of Ribozyme with anhydrous ethanolic methylamine

A ribozyme was synthesized using the column format as described herein. The polystyrene solid-support with protected oligoribonucleotide or modified oligoribonucleotide (200 nmole) was transferred into a glass vial equipped with a screw cap. A 1:1 mixture of anhydrous ethanolic methylamine ( 400 μL) and dimethylsulfoxide (DMSO) (400 μL) was added followed by vortexing of the glass vial. After allowing the reaction for 1.5 hours, the solid support was filtered off. 100 μL of TEA.3HF was added at room temperature to the vial and the reaction was mixed causing the solution to gel. The reaction was then heated at 65 °C for 15 minutes and then cooled to room temperature. The solution was then quenched with 1.5 M aqueous NH₄ΗCO₃ ^" (ImL). HPLC chromatography of the reaction mixture afforded 32 O. D.u_{260 nm} of which 46% was full length ribozyme.

Example 31. Large-scale One-Pot Deprotection of Ribozyme

A ribozyme was synthesized at the 0.5 mmol scale using the column format as described herein. The polystyrene solid-support (24 grs) with protected oligoribonucleotide or modified oligoribonucleotide (500 μmole) was transferred into a IL Schott bottle equipped with a screw cap. A 1:1.3 mixture of anhydrous ethanolic methylamine ( 150 mL) and dimethylsulfoxide (DMSO) (200 mL) was added followed by vortexing (200 φm) of the glass bottle for 1.5 hours. The reaction mixture was then frozen at -70 °C for 30 minutes. 50 mL of neat TEA.3HF was then added at room temperature to the reaction mixture and the reaction was placed in a shaking oven (200 φm) where it was heated at 65 °C for 60 minutes and subsequently frozen at -70 °C for 30 minutes. The solution was then quenched with 1.5 M aqueous NH₄ΗCO₃ ^" (200 mL). 1 l o

The reaction mixture was separated from the polystyrene solid-support by filtration on a sintered glass funnel (10-20 μm porosity). UN. spectrophotometric quantification and HPLC chromatography of the reaction mixture afforded 160,000 O.D.u_{260 nm} of which 46.4%) was full length ribozyme. After allowing the reaction for 1.5 hours, the solid support was filtered off

Example 32: Antitumor and antimetastatic efficacy of ribozymes directed against the mRΝA encoding the two VEGF receptor subtypes, flt-1 and flk-1 in the mouse Lewis lung-HM carcinoma model of primary tumor growth and metastasis

The Lewis lung carcinoma (LLC) model is a syngeneic mouse model of metastatic cancer commonly used for antitumor agent efficacy screening. According to Folkman (1995, supra), primary tumor growth and metastasis in this model is dependent upon VEGF-induced angiogenesis. Two variants of the LLC model exist. The low metastatic form involves the implantation of a tumor, usually subcutaneous, which sends micrometastases to the lungs whose growth is suppressed by the presence of the primary tumor. The highly metastatic (HM) form differs from the low metastatic variant in that the growth of metastases is not suppressed by the presence of the primary tumor. Thus, the HM form is a model in which it is possible to measure pharmacologic efficacy on both primary tumor growth and metastasis in the same mouse without excision of the primary tumor.

Applicant selected the highly metastatic variant of the Lewis lung model for antitumor/metastatic screening of ribozymes directed against VEGF receptor (flt-1 and flk-1) mRΝA. These ribozymes have been shown to reduce VEGF binding and VEGF- stimulated proliferation in cultured MVEC's as well as VEGF-induced neovascularization of the rat cornea (Cushman et al., 1996, Angiogenesis Inhibitors and Other Novel Therapeutic Strategies for Ocular Diseases of Neovascularization, IBC Conference Abstract). Pharmacokinetically, Applicant has found that ribozymes distribute systemically following continuous i.v. infusion (via Alzet osmotic minipumps) at significant concentrations within most tissues including subcutaneously implanted tumors. This study examines the antitumor/antimetastatic efficacy of flt-1 and flk-1 ribozymes continuously infused i.v. in the LLC-HM mouse model.

Methods

Ribozymes The ribozymes used in this study were hammerhead ribozymes comprising a 4 base pair stem II, four phosphorothioate linkages at the 5 '-end, a 2'-C-allyl substitution at position 4 (see Figure 1), and an inverted abasic nucleotide substitution at the 3 '-end. The catalytically active and inactive ribozymes were RPI.4610/4611 (active/inactive) and RPI.4733/4734 directed against ^7t- and flk-1 messages, respectively. Ribozymes solutions were prepared in normal saline (USP).

Test solutions (ribozymes or saline control) were dispensed into Alzet^® osmotic minipumps (Model # 1002-total volume capacity including excess = 200 μl) which dispense 0.5 μl/h at 37 °C when exposed to interstitial water. Pumps were either filled with normal saline (USP) or 167.0, 50.0, 16.7, 5.0, or 1.7 mg/ml ribozyme solutions. Prior to animal implantation, osmotic minipumps were placed in 37 °C sterile water for at least four hours to activate pumping.

Tumor inoculation

All animal procedures in this study were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (1996), USDA regulations, and the policies and procedures of the RPI Institutional Animal Care and Use Committee. A total of 210 female C57BL/6J mice weighing between 20-25 g were used in this study. All animals were housed under 12 h on 12 h off light cycles and received ad libitum food and water.

Highly metastatic variant Lewis lung carcinoma (LLC-HM) tumors were propogated in vivo from an LLC-HM cell line. These tumors needed to be propogated in vivo because they can revert to the low metastatic phenotype in culture. LLC-HM cells were initially cultured in DMEM + 10% FCS + 1 % GPS. For in vivo propogation, 5 X

10 cells were injected subcutaneously in mice. Tumors were allowed to grow for 25 days at which time animals were euthanized by CO₂ inhalation and lung macrometastases were counted. Animals with the most macrometastases (approximately 15-20) were selected for preparation of tumor breis and propogation. When tumors in animals selected for propogation reached a volume of approximately 1500 mm , animals were euthanized by CO₂ inhalation and tumors were excised. Tumors were seived through a 100 μm pore size sterile nylon mesh. LLC-HM cells were resuspended in normal saline to a final concentration of 5 x 10 viable cells/ml (via hemocytometer). Three days prior to ribozyme dosing, all animals were subcutaneously inoculated on the right flank with 5 x

10 cells (in a volume of 100 μl).

Ribozyme or saline dosing

Each ribozyme solution was prepared to deliver 100, 30, 10, 3, or 1 mg/kg/day in a volume of 12 ml. A total of 10 animals per dose or saline control group were surgically implanted on the left flank with osmotic minipumps pre-filled with the respective test solution three days following tumor inoculation. Pumps were attached to indwelling jugular vein catheters. The specifications for the model #1002 Alzet osmotic minipump show that they accurately deliver aqueous solutions at 0.5 μl/h for 14 days. Table III summarizes the experimental groups.

Tumor volume and metastatic index quantitation

Beginning four days and ending 24 days days following tumor inoculation, the length and width of all primary tumors were measured every other day using microcalipers. Tumor volumes were calculated using the standard formula for an elipsoid volume, (L^'W )/2. Tumor volumes were calculated in triplicate for each animal. A mean tumor volume was calculated for each animal. Group means and standard error of the group means were calculated from individual animal mean tumor volumes. Twenty-five days following tumor inoculation, all animals were euthanized by CO₂ inhalation and lungs and primary tumors harvested. Lung macrometastases were counted under a dissecting microscope (2.5 X magnification). Lungs and primary tumors were also weighed on an analytical balance. Lung weights served as an index of total lung metastatic burden.

Statistical analysis

For all treatment groups, group tumor volume means on day 18 (end of treatment) as well as means of primary tumor and lung weight and numbers of lung metastases were evaluated for normality and subjected to analysis of variance. Statistical differences between group means were evaluated using the Tukey-Kramer post-hoc test (alpha = 0.05). Comparisons with the control group (saline control) were made using the Dunnett's test (alpha = 0.05).

Results

Flt-1 The effects of several doses of active and inactive E/t-7 ribozymes (RPI.4610/4611, respectively) on primary LLC-HM tumor growth are summarized in Figure 39 (A-E). At the lowest dose (Figure 39A), both active and inactive reduce primary tumor growth similarly throughout the entire time course compared to saline controls. However, with increasing dose, active ribozyme reduces primary tumor growth to a greater extent than the inactive ribozyme, with the largest difference observed at 30 mg/kg/day (Figure 39D). The magnitude of the maximal reduction compared to saline was approximately four fold with the active ribozyme RPI.4610 at 30 mg/kg/day. It should be noted that this observed four fold reduction is still present at day 24 even though treatment ended 7 days earlier.

The growth curve data was subjected to exponential regression. The curve fits show that the tumor growth data fits an exponential curve with a high correlation coefficient

(R>0.95). Thus, there appears to be no long lasting toxic effect on tumor growth. Since the calculated slope of the exponential curve at any point indicates the rate of tumor growth, it should be possible to compare rates of growth between treatments. Since the curve fits do not assume that the tumor growth starts from the same point (which is a correct assumption since the all tumors start with the same tumor cell inoculum concentration), an accurate calculation of the slope of the exponential curve is not possible since the curve fitting algorithm extrapolates a t = 0 tumor size which is then used to calculate the slope. In the analysis, the saline tumor size at t = 0 is much greater than the other treatment groups, thus comparisons with saline are not necessarily accurate. If the curve fit algorithm is restricted to the same tumor size, a dose-dependent reduction in the rate of tumor growth is observed with the active ribozyme. However, the curve fits show lower correlation coefficients in some cases.

In order to see whether a the ribozyme treatments statistically reduce primary tumor growth, primary tumor volume measurements at each dose immediately following treatment (day 18) were compared (Figure 40). Active ribozyme RPI.4610 produced a statistically significant (p < 0.05) and dose-dependent reduction in primary tumor volume. Although the inactive ribozyme (RPI.4611) showed some reduction in primary tumor volume at the lowest and highest doses, there was no dose-dependent reduction observed. At doses between 3 and 30 mg/kg/day, the inactive ribozyme showed no significant reduction in primary tumor volume. There was a significant difference (p < 0.05) between active and inactive ribozymes (Tukey-Kramer test) at doses of 10 and 30 mg/kg/day.

Applicant has also observed that the active ribozyme RPI.4610 produced a significant reduction in primary tumor mass at all doses tested (1-100 mg/kg/day) 25 days following inoculation.

Figures 41 A and B illustrate that the active ribozyme reduced both the number of lung metastases and lung mass in a dose-dependent manner. The active flt-1 ribozyme showed a significant reduction (p < 0.05 by Dunnetts) in the number of lung metastases at the 30 and 100 mg/kg/day doses compared to saline. There was also a significant difference between active and inactive ribozymes at these doses (p , 0.05 by Student's t). RPI.4610 reduced the lung weight to almost normal levels at the highest dose (100 mg/kg/day). There was no observable dose-related effect of the inactive ribozyme on either the number of lung metastases or lung weight. A significant reduction (p < 0.05, Student's t) in lung mass, an index of metastatic burden, was observed between saline and the active ribozyme. The lack of significance using more stringent statistical tests (Dunnet's or Tukey-Kramer), which take into account the variance within all groups, was due to high variability, especially in the inactive ribozyme group. However, since five doses were tested, it is possible to say that there is a dose-dependent trend in the reduction of lung metastases/lung weight.

Example 33: Effects of flk-1 ribozymes (active/inactive) on LLC-HM primary tumor growth in mice.

The dose-related effects of active and inactive flk-1 directed ribozymes (RPI.4733/4734, respectively) on primary LLCare shown in Figure 38 A-E.

The dose-related effects of active and inactive flk-1 directed ribozymes

(RPI.4733/4734, respectively) on primary LLCare shown in Figure 42 A-E. At the lowest dose, there was no observable effect on primary tumor growth with the active flk-1 ribozyme (Figure 42 A). The inactive ribozyme showed a modest reduction in primary tumor growth. At higher doses (3-100 mg/kg/day, Figure 42B-E), the active flk-1 ribozyme reduced primary tumor growth while the inactive ribozyme showed little, if any, antitumor efficacy over the dose range between 10 and 100 mg/kg/day (Figures 42C-E). The antitumor efficacy of both active and inactive flk-1 ribozymes are similar at 3 mg/kg/day (Figure 42B).

As in the case of the flt-1 ribozymes, tumor growth followed exponential growth kinetics. Again, since the t = 0 tumor size could not accurately be estimated by the curve fit program, it is not possible to calculate the slope of the exponential curve fits for theflk- 1 ribozymes. Immediately following the cessation of treatment (day 18), the active flk-1 ribozyme showed a significant reduction in primary tumor volume from 3 to 100 mg/kg/day (Figure 43). The magnitude of the reduction is approximately four fold and appeared to be maximal at 3 mg/kg/day. The lowest dose had no significant effect on primary tumor volume. The inactive flk-1 ribozyme had a significant antitumor effect at doses of 1 and 3 mg/kg/day; however, this effect disappeared between 10 and 100 mg/kg/day.

The antimetastatic effects of the flk-1 ribozymes are illustrated in Figure 44 A and B. Although neither ribozyme showed a statistically significant effect on the number of lung metastases at any dose, it appears that the active flk-1 ribozyme showed a significant reduction in lung mass over the dose range between 3 and 100 mg/kg/day.

Applicant has further observed that the lung mass was reduced to normal over the entire dose range. The inactive ribozyme reduced lung mass at 1 and 3 mg/kg/day (Figure 41C); however, this trend was not observed at higher doses (3-100 mg/kg/day).

Example 34: Ribozyme-mediated decrease in vascularitv of tumor

Three tumors from each of three treatment groups (saline controls, inactive

RPI.4611 and active RPI.4610, 30 mg/kg/day dose only) were analyzed for vascularity using an immunohistochemical assay which stains endothelial cells for CD31 (PECAM). The vascularity was quantitated in a blinded fashion. From the raw data the average number of vessels per high magnification field (400X) were calculated. They are as follows: SALINE CONTROL = 24.1; RPI.4611 (Inactive) - 27.6; RPI.4610 (Active) - 16.0.

It is suggestive that ribozyme-specific antiangiogenic effect is exhibited by the active Flt-1 ribozyme in Lewis lung tumors. Thus, the mechanism of action for the observed reduction in the primary tumor volumes may be due to an antiangiogenic effect. Similar delivery strategies can be used to deliver c-raf ribozymes to treat a variety of diseases. Use of Ribozymes Targeting c-raf

Overexpression of the c-raf oncogene has been reported in a number of cancers (see above). Thus, inhibition of c-raf expression (for example using ribozymes) can reduce cell proliferation of a number of cancers, in vitro and in vivo and can reduce their proliferative potential. A cascade of MMP and serine proteinase expression is implicated in the acquisition of an invasive phenotype as well as in angiogenesis in tumors (MacDougall & Matrisian, 1995, Cancer & Metastasis Reviews 14, 351;Ritchlin & Winchester, 1989, Springer Semin Immunopathol, 11, 219).

A number of human diseases are characterized by the inappropriate proliferation of cells at sites of injury or damage to the normal tissue architecture. These diseases include restenosis, caused by the local proliferation of medial smooth muscle cells at sites of arterial wall dismption by surgery; psoriasis, caused by proliferation of keratinocytes at regions of endothelial cell damage in the skin, and various fibrosis, caused by the inappropriate replication of cells during wound healing processes. In certain inflammatory processes, cell proliferation may not be causative, yet it exacerbates the disease pathology. For example, in rheumatoid arthritis, synovial hypeφlasia leads to accelerated cartilage damage due to secretion of proteases by the expanding population of synovial fibroblasts. Any number of these diseases and others which involve cellular proliferation or the loss of proliferative control, such as cancer, could be treated using ribozymes which inhibit the expression of the cellular Raf gene products. Alternatively, ribozyme inhibition of the cellular growth factor receptors could be used to inhibit downstream signalling pathways. The specific growth factors involved would depend upon the cell type indicated in the proliferative event.

Ribozymes, with their catalytic activity and increased site specificity (see above), are likely to represent a potent and safe therapeutic molecule for the treatment of cancer.

In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation.

From those practiced in the art, it is clear from the examples described, that the same ribozymes may be delivered in a similar fashion to cancer cells to block their proliferation.

Diagnostic uses

Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of c-raf RNA in a cell. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base- pairing and three-dimensional stmcture of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA stmcture 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.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNAs associated with c-raf 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., c-raf) 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.

Additional Uses

Potential usefulness of sequence-specific nucleic acid catalysts of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al, 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence.

The use of NTP's described in this invention have several research and commercial applications. These modified nucleoside triphosphates can be used for in vitro selection (evolution) of oligonucleotides with novel functions. Examples of in vitro selection protocols are incoφorated herein by reference (Joyce, 1989, Gene, 82, 83-87 Beaudry et al, 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97 Breaker et al, 1994, TIBTECH 12, 268; Bartel et α/.J993, Science 261 :1411-1418 Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB , 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442). Additionally, these modified nucleoside triphosphates can be employed to generate modified oligonucleotide combinatorial chemistry libraries. Several references for this technology exist (Brenner et al, 1992, PNAS 89, 5381-5383, Eaton, 1997, Curr. Opin. Chem. Biol. 1, 10-16) which are all incoφorated herein by reference. Nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al, 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence. Nucleic acid molecules (e.g., ribozymes) of the invention can be used, for example, to target cleavage of virtually any RNA transcript (Zaug et al, 324, Nature 429 1986 ; Cech, 260 JAMA 3030, 1988; and Jefferies et al, 17 Nucleic Acids Research 1371, 1989). Such nucleic acids can be used as a therapeutic or to validate a therapeutic gene target and/or to determine the function of a gene in a biological system (Christoffersen, 1997, Nature Biotech. 15, 483).

Various ligands can be attached to oligonucleotides using the componds containing zylo modification for the puφoses of cellular delivery, nuclease resistance, cellular trafficking and localization, chemical ligation of oligonucleotide fragments. Incoφoration of one or more compounds of Formula II into a ribozyme may increase its effectiveness. Compounds of Formula II can be used as potential antiviral agents.

Other embodiments are within the following claims. TABLE I

Characteristics of naturally occurring ribozymes Group I Introns

• Size: -150 to > 1000 nucleotides.

• Requires a U in the target sequence immediately 5' of the cleavage site. Binds 4-6 nucleotides at the 5'-side of the cleavage site.

• Reaction mechanism: attack by the 3' -OH of guanosine to generate cleavage products with 3' -OH and 5 '-guanosine. • Additional protein cofactors required in some cases to help folding and maintainance of the active structure.

Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others. • Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [',²].

• Complete kinetic framework established for one ribozyme [³,⁴,⁵,⁶].

• Studies of ribozyme folding and substrate docking underway [⁷,⁸,⁹].

• Chemical modification investigation of important residues well established [ , ].

¹ . Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1 (1 ), 5-7.

² . Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17.

³ . Herschlag, Daniel; Cech, Thomas R„ Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71.

⁴ . Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80.

⁵ . Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70.

⁶ . Bevilacqua, Philip C; Sugimoto, Naoki; Turner, Douglas H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.

⁷ . Li, Yi; Bevilacqua, Philip C; Mathews, David; Turner, Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9.

⁸ . Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.

⁹ . Zarrinkar, Patrick P.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.

¹⁰ . Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995), 267(5198), The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" β- galactosidase message by the ligation of new β-galactosidase sequences onto the defective message [¹²].

RNAse P RNA (M1 RNA)

Size: -290 to 400 nucleotides.

• RNA portion of a ubiquitous ribonucleoprotein enzyme. • Cleaves tRNA precursors to form mature tRNA [¹³].

2+

• Reaction mechanism: possible attack by M -OH to generate cleavage products with 3'- OH and 5 '-phosphate.

RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates. • Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA [¹⁴,¹⁵]

• Important phosphate and 2' OH contacts recently identified [¹⁶,¹⁷]

Group II Introns

• Size: > 1000 nucleotides.

• Trans cleavage of target RNAs recently demonstrated [¹⁸,¹⁹].

• Sequence requirements not fully determined.

• Reaction mechanism: 2'-OH of an internal adenosine generates cleavage products with 3'- OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.

675-9.

¹¹ . Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and

Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.

¹². Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371 (6498), 619-22.

". Robertson, H.D.; Altman, S.; Smith, J.D. J. Biol. Chem., 247, 5243-5251 (1972).

¹⁴. Forster, Anthony C; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C, 1883-) (1990), 249(4970), 783-6.

¹⁵. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.

¹⁶ . Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1 (2), 210-18.

¹⁷ . Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2'-hydroxyl- base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(26), 12510-14.

¹⁸ . Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25.

¹⁹ . Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77. • Only natural ribozyme with demonstrated participation in DNA cleavage [²⁰,²¹] in addition to RNA cleavage and ligation.

• Major structural features largely established through phylogenetic comparisons [²²].

• Important 2' OH contacts beginning to be identified [²³] • Kinetic framework under development [²⁴]

Neurospora VS RNA

Size: -144 nucleotides. • Trans cleavage of hairpin target RNAs recently demonstrated [²⁵].

• Sequence requirements not fully determined.

• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2', 3 '-cyclic phosphate and 5'-OH ends.

• Binding sites and structural requirements not fully determined. • Only 1 known member of this class. Found in Neurospora NS RΝA.

Hammerhead Ribozyme

(see text for references)

• 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.

• Reaction mechanism: attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2', 3 '-cyclic phosphate and 5'-OH ends.

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

• Essential structural features largely defined, including 2 crystal structures [²⁶,²⁷]

• Minimal ligation activity demonstrated (for engineering through in vitro selection) [²⁸]

²⁰ . Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RΝA is a catalytic component of a DΝA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38. ²¹ . Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DΝA and RΝA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem. Biol. (1995), 2(11 ), 761-70. ² . Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61.

²³ . Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic roie of 2'-hydroxyl groups within a group II intron active site. Science (Washington, D. C.) (1996), 271(5254), 1410- 13. ⁴ . Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1 ), 31-49.

²⁵ . Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-loop RΝA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76.

²⁶ . Scott, W.G., Finch, J.T., Aaron, K. The crystal structure of an all RNA hammerhead ribozyme-Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81 , 991-1002.

²⁷ . McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403.

²⁸ . Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. US Patent No. • Complete kinetic framework established for two or more ribozymes [²⁹].

• Chemical modification investigation of important residues well established [³⁰] .

Hairpin Ribozyme

• Size: -50 nucleotides.

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

• Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable number to the 3 '-side of the cleavage site. • Reaction mechanism: attack by 2'-OH 5 ' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.

• 3 known members 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. • Essential structural features largely defined [³¹,³²,³³,³⁴]

• Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [³⁵]

Complete kinetic framework established for one ribozyme [³⁶].

• Chemical modification investigation of important residues begun [³⁷,³⁸].

5,633,133.

²⁹ . Hertel, K.J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374-3385. Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

³⁰ . Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

³¹ . Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299-304.

³² . Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991 ), 354(6351 ), 320-2.

³³ . Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel E.; Burke, John M.. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73.

³⁴ . Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel E.. Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1 ), 130-8.

³⁵ . Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1 ), 129-34.

³⁶ . Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.

³⁷ . Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068-76.

³⁸ . Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik S.; Gait, Michael J.. Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81. . ..

Hepatitis Delta Virus (HDV) Ribozyme

Size: -60 nucleotides.

• Trans cleavage of target RNAs demonstrated [³⁹]. • Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [⁴⁰]. Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3 '-cyclic phosphate and 5 '-OH ends.

• Only 2 known members of this class. Found in human HDV. • Circular form of HDV is active and shows increased nuclease stability [⁴¹]

³⁹ . Perrotta, Anne T.; Been, Michael D.. Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta, virus RNA sequence. Biochemistry (1992), 31 (1 ), 16-21.

⁴⁰ . Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structure required for efficient self- cleavage of hepatitis delta virus RNA. Nature (London) (1991 ), 350(6317), 434-6.

⁴¹ . Puttaraju, M.; Perrotta, Anne T.; Been, Michael D.. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21 (18), 4253-8.

Table II: 2.5 μmol RNA Synthesis Cycle

Reagent Equivalents Amount Wait

Time*

Phosphoramidites 6.5 163 μL 2.5

S-Ethyl Tetrazole 23.8 238 μL 2.5

Acetic Anhydride 100 233 μL 5 sec

N-Methyl Imidazole 186 233 μL 5 sec

TCA 83.2 1.73 mL 21 sec

Iodine 8.0 1.18 mL 45 sec

Acetonitrile ΝA 6.67 mL ΝA

Wait time does not include contact time during delivery.

TABLE III. NUCLEOSIDES USED FOR CHEMICAL SYNTHESIS OF MODIFIED NUCLEOTIDE TRIPHOSPHATES

Table VI. PHOSPHORYLATION OF URIDINE IN THE PRESENCE OF DMAP

Table VII Detailed Description of the NTP Incoφoration Reaction Conditions

Table VIII. INCORPORATION OF MODIFIED NUCLEOTIDE TRIPHOSPHATES

Table IX: INCORPORATION OF MODIFIED

NUCLEOTIDE TRIPHOSPHATES USING WILD TYPE

BACTERIOPHAGE T7 POLYMERASE

Table Xa: Incorporation of 2'-his-UTP and Modified CTP's

Table Xb: Incorporation of 2'-his-UTP, 2-amino CTP, and Modified ATP's

Numbers shown are a percentage of incorporation compared to the all-RNA control

* -Bold number indicates best observed rate of modified nucleotide triphosphate incorporation

Table XI. INCORPORATION OF 2'-his-UTP, 2'-NH₂-CTP, 2'-NH₂-DAP, and rGTP USING VARIOUS REACTION CONDITIONS

Numbers shown are a percentage of incorporation compared to the all-RNA control

* Two highest levels of incoφoration contained both methanol and LiCl

Table XII: Human C-rø/Hammerhead Ribozyme and Target Sequences nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

17 GACCGCCUC CCGCUCCC 1 GGGAGCGG CUGAUGAG X CGAA AGGCGGUC 502

23 CUCCCGCUC CCUCACCC 2 GGGUGAGG CUGAUGAG X CGAA AGCGGGAG 503

27 CGCUCCCUC ACCCGCCG 3 CGGCGGGU CUGAUGAG X CGAA AGGGAGCG 504

82 CAGGACGUU GGGGCGGC 4 GCCGCCCC CUGAUGAG X CGAA ACGUCCUG 505

97 GCCUGGCUC CCUCAGGU 5 ACCUGAGG CUGAUGAG X CGAA AGCCAGGC 506

101 GGCUCCCUC AGGUUUAA 6 UUAAACCU CUGAUGAG X CGAA AGGGAGCC 507

106 CCUCAGGUU UAAGAAUU 7 AAUUCUUA CUGAUGAG X CGAA ACCUGAGG 508

107 CUCAGGUUU AAGAAUUG 8 CAAUUCUU CUGAUGAG X CGAA AACCUGAG 509

108 UCAGGUUUA AGAAUUGU 9 ACAAUUCU CUGAUGAG X CGAA AAACCUGA 510

114 UUAAGAAUU GUUUAAGC 10 GCUUAAAC CUGAUGAG X CGAA AUUCUUAA 511

117 AGAAUUGUU UAAGCUGC 11 GCAGCUUA CUGAUGAG X CGAA ACAAUUCU 512

118 GAAUUGUUU AAGCUGCA 12 UGCAGCUU CUGAUGAG X CGAA AACAAUUC 513

119 AAUUGUUUA AGCUGCAU 13 AUGCAGCU CUGAUGAG X CGAA AAACAAUU 514

128 AGCUGCAUC AAUGGAGC 14 GCUCCAUU CUGAUGAG X CGAA AUGCAGCU 515

141 GAGCACAUA CAGGGAGC 15 GCUCCCUG CUGAUGAG X CGAA AUGUGCUC 516

151 AGGGAGCUU GGAAGACG 16 CGUCUUCC CUGAUGAG X CGAA AGCUCCCU 517

162 AAGACGAUC AGCAAUGG 17 CCAUUGCU CUGAUGAG X CGAA AUCGUCUU 518

172 GCAAUGGUU UUGGAUUC 18 GAAUCCAA CUGAUGAG X CGAA ACCAUUGC 519

173 CAAUGGUUU UGGAUUCA 19 UGAAUCCA CUGAUGAG X CGAA AACCAUUG 520

174 AAUGGUUUU GGAUUCAA 20 UUGAAUCC CUGAUGAG X CGAA AAACCAUU 521

179 UUUUGGAUU CAAAGAUG 21 CAUCUUUG CUGAUGAG X CGAA AUCCAAAA 522

180 UUUGGAUUC AAAGAUGC 22 GCAUCUUU CUGAUGAG X CGAA AAUCCAAA 523

194 UGCCGUGUU UGAUGGCU 23 AGCCAUCA CUGAUGAG X CGAA ACACGGCA 524

195 GCCGUGUUU GAUGGCUC 24 GAGCCAUC CUGAUGAG X CGAA AACACGGC 525

203 UGAUGGCUC CAGCUGCA 25 UGCAGCUG CUGAUGAG X CGAA AGCCAUCA 526

213 AGCUGCAUC UCUCCUAC 26 GUAGGAGA CUGAUGAG X CGAA AUGCAGCU 527

215 CUGCAUCUC UCCUACAA 27 UUGUAGGA CUGAUGAG X CGAA AGAUGCAG 528

217 GCAUCUCUC CUACAAUA 28 UAUUGUAG CUGAUGAG X CGAA AGAGAUGC 529

220 UCUCUCCUA CAAUAGUU 29 AACUAUUG CUGAUGAG X CGAA AGGAGAGA 530

225 CCUACAAUA GUUCAGCA 30 UGCUGAAC CUGAUGAG X CGAA AUUGUAGG 531

228 ACAAUAGUU CAGCAGUU 31 AACUGCUG CUGAUGAG X CGAA ACUAUUGU 532

229 CAAUAGUUC AGCAGUUU 32 AAACUGCU CUGAUGAG X CGAA AACUAUUG 533

236 UCAGCAGUU UGGCUAUC 33 GAUAGCCA CUGAUGAG X CGAA ACUGCUGA 534

237 CAGCAGUUU GGCUAUCA 34 UGAUAGCC CUGAUGAG X CGAA AACUGCUG 535

242 GUUUGGCUA UCAGCGCC 35 GGCGCUGA CUGAUGAG X CGAA AGCCAAAC 536

244 UUGGCUAUC AGCGCCGG 36 CCGGCGCU CUGAUGAG X CGAA AUAGCCAA 537

257 CCGGGCAUC AGAUGAUG 37 CAUCAUCU CUGAUGAG X CGAA AUGCCCGG 538

273 GGCAAACUC ACAGAUCC 38 GGAUCUGU CUGAUGAG X CGAA AGUUUGCC 539

280 UCACAGAUC CUUCUAAG 39 CUUAGAAG CUGAUGAG X CGAA AUCUGUGA 540

283 CAGAUCCUU CUAAGACA 40 UGUCUUAG CUGAUGAG X CGAA AGGAUCUG 541

284 AGAUCCUUC UAAGACAA 41 UUGUCUUA CUGAUGAG X CGAA AAGGAUCU 542

286 AUCCUUCUA AGACAAGC 42 GCUUGUCU CUGAUGAG X CGAA AGAAGGAU 543

301 GCAACACUA UCCGUGUU 43 AACACGGA CUGAUGAG X CGAA AGUGUUGC 544

303 AACACUAUC CGUGUUUU 44 AAAACACG CUGAUGAG X CGAA AUAGUGUU 545

309 AUCCGUGUU UUCUUGCC 45 GGCAAGAA CUGAUGAG X CGAA ACACGGAU 546

310 UCCGUGUUU UCUUGCCG 46 CGGCAAGA CUGAUGAG X CGAA AACACGGA 547

311 CCGUGUUUU CUUGCCGA 47 UCGGCAAG CUGAUGAG X CGAA AAACACGG 548

312 CGUGUUUUC UUGCCGAA 48 UUCGGCAA CUGAUGAG X CGAA AAAACACG 549

314 UGUUUUCUU GCCGAACA 49 UGUUCGGC CUGAUGAG X CGAA AGAAAACA 550

339 ACAGUGGUC AAUGUGCG 50 CGCACAUU CUGAUGAG X CGAA ACCACUGU 551

362 AAUGAGCUU GCAUGACU 51 AGUCAUGC CUGAUGAG X CGAA AGCUCAUU 552

375 GACUGCCUU AUGAAAGC 52 GCUUUCAU CUGAUGAG X CGAA AGGCAGUC 553

376 ACUGCCUUA UGAAAGCA 53 UGCUUUCA CUGAUGAG X CGAA AAGGCAGU 554

387 AAAGCACUC AAGGUGAG 54 CUCACCUU CUGAUGAG X CGAA AGUGCUUU 555

425 UGCAGUGUU CAGACUUC 55 GAAGUCUG CUGAUGAG X CGAA ACACUGCA 556

426 GCAGUGUUC AGACUUCU 56 AGAAGUCU CUGAUGAG X CGAA AACACUGC 557

432 UUCAGACUU CUCCACGA 57 UCGUGGAG CUGAUGAG X CGAA AGUCUGAA 558 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

433 UCAGACUUC UCCACGAA 58 UUCGUGGA CUGAUGAG X CGAA AAGUCUGA 559

435 AGACUUCUC CACGAACA 59 UGUUCGUG CUGAUGAG X CGAA AGAAGUCU 560

451 ACAAAGGUA AAAAAGCA 60 UGCUUUUU CUGAUGAG X CGAA ACCUUUGU 561

464 AGCACGCUU AGAUUGGA 61 UCCAAUCU CUGAUGAG X CGAA AGCGUGCU 562

465 GCACGCUUA GAUUGGAA 62 UUCCAAUC CUGAUGAG X CGAA AAGCGUGC 563

469 GCUUAGAUU GGAAUACU 63 AGUAUUCC CUGAUGAG X CGAA AUCUAAGC 564

475 AUUGGAAUA CUGAUGCU 64 AGCAUCAG CUGAUGAG X CGAA AUUCCAAU 565

488 UGCUGCGUC UUUGAUUG 65 CAAUCAAA CUGAUGAG X CGAA ACGCAGCA 566

490 CUGCGUCUU UGAUUGGA 66 UCCAAUCA CUGAUGAG X CGAA AGACGCAG 567

491 UGCGUCUUU GAUUGGAG 67 CUCCAAUC CUGAUGAG X CGAA AAGACGCA 568

495 UCUUUGAUU GGAGAAGA 68 UCUUCUCC CUGAUGAG X CGAA AUCAAAGA 569

507 GAAGAACUU CAAGUAGA 69 UCUACUUG CUGAUGAG X CGAA AGUUCUUC 570

508 AAGAACUUC AAGUAGAU 70 AUCUACUU CUGAUGAG X CGAA AAGUUCUU 571

513 CUUCAAGUA GAUUUCCU 71 AGGAAAUC CUGAUGAG X CGAA ACUUGAAG 572

517 AAGUAGAUU UCCUGGAU 72 AUCCAGGA CUGAUGAG X CGAA AUCUACUU 573

518 AGUAGAUUU CCUGGAUC 73 GAUCCAGG CUGAUGAG X CGAA AAUCUACU 574

519 GUAGAUUUC CUGGAUCA 74 UGAUCCAG CUGAUGAG X CGAA AAAUCUAC 575

526 UCCUGGAUC AUGUUCCC 75 GGGAACAU CUGAUGAG X CGAA AUCCAGGA 576

531 GAUCAUGUU CCCCUCAC 76 GUGAGGGG CUGAUGAG X CGAA ACAUGAUC 577

532 AUCAUGUUC CCCUCACA 77 UGUGAGGG CUGAUGAG X CGAA AACAUGAU 578

537 GUUCCCCUC ACAACACA 78 UGUGUUGU CUGAUGAG X CGAA AGGGGAAC 579

551 ACACAACUU UGCUCGGA 79 UCCGAGCA CUGAUGAG X CGAA AGUUGUGU 580

552 CACAACUUU GCUCGGAA 80 UUCCGAGC CUGAUGAG X CGAA AAGUUGUG 581

556 ACUUUGCUC GGAAGACG 81 CGUCUUCC CUGAUGAG X CGAA AGCAAAGU 582

566 GAAGACGUU CCUGAAGC 82 GCUUCAGG CUGAUGAG X CGAA ACGUCUUC 583

567 AAGACGUUC CUGAAGCU 83 AGCUUCAG CUGAUGAG X CGAA AACGUCUU 584

576 CUGAAGCUU GCCUUCUG 84 CAGAAGGC CUGAUGAG X CGAA AGCUUCAG 585

581 GCUUGCCUU CUGUGACA 85 UGUCACAG CUGAUGAG X CGAA AGGCAAGC 586

582 CUUGCCUUC UGUGACAU 86 AUGUCACA CUGAUGAG X CGAA AAGGCAAG 587

591 UGUGACAUC UGUCAGAA 87 UUCUGACA CUGAUGAG X CGAA AUGUCACA 588

595 ACAUCUGUC AGAAAUUC 88 GAAUUUCU CUGAUGAG X CGAA ACAGAUGU 589

602 UCAGAAAUU CCUGCUCA 89 UGAGCAGG CUGAUGAG X CGAA AUUUCUGA 590

603 CAGAAAUUC CUGCUCAA 90 UUGAGCAG CUGAUGAG X CGAA AAUUUCUG 591

609 UUCCUGCUC AAUGGAUU 91 AAUCCAUU CUGAUGAG X CGAA AGCAGGAA 592

617 CAAUGGAUU UCGAUGUC 92 GACAUCGA CUGAUGAG X CGAA AUCCAUUG 593

618 AAUGGAUUU CGAUGUCA 93 UGACAUCG CUGAUGAG X CGAA AAUCCAUU 594

619 AUGGAUUUC GAUGUCAG 94 CUGACAUC CUGAUGAG X CGAA AAAUCCAU 595

625 UUCGAUGUC AGACUUGU 95 ACAAGUCU CUGAUGAG X CGAA ACAUCGAA 596

631 GUCAGACUU GUGGCUAC 96 GUAGCCAC CUGAUGAG X CGAA AGUCUGAC 597

638 UUGUGGCUA CAAAUUUC 97 GAAAUUUG CUGAUGAG X CGAA AGCCACAA 598

644 CUACAAAUU UCAUGAGC 98 GCUCAUGA CUGAUGAG X CGAA AUUUGUAG 599

645 UACAAAUUU CAUGAGCA 99 UGCUCAUG CUGAUGAG X CGAA AAUUUGUA 600

646 ACAAAUUUC AUGAGCAC 100 GUGCUCAU CUGAUGAG X CGAA AAAUUUGU 601

658 AGCACUGUA GCACCAAA 101 UUUGGUGC CUGAUGAG X CGAA ACAGUGCU 602

669 ACCAAAGUA CCUACUAU 102 AUAGUAGG CUGAUGAG X CGAA ACUUUGGU 603

673 AAGUACCUA CUAUGUGU 103 ACACAUAG CUGAUGAG X CGAA AGGUACUU 604

676 UACCUACUA UGUGUGUG 104 CACACACA CUGAUGAG X CGAA AGUAGGUA 605

694 ACUGGAGUA ACAUCAGA 105 UCUGAUGU CUGAUGAG X CGAA ACUCCAGU 606

699 AGUAACAUC AGACAACU 106 AGUUGUCU CUGAUGAG X CGAA AUGUUACU 607

708 AGACAACUC UUAUUGUU 107 AACAAUAA CUGAUGAG X CGAA AGUUGUCU 608

710 ACAACUCUU AUUGUUUC 108 GAAACAAU CUGAUGAG X CGAA AGAGUUGU 609

711 CAACUCUUA UUGUUUCC 109 GGAAACAA CUGAUGAG X CGAA AAGAGUUG 610

713 ACUCUUAUU GUUUCCAA 110 UUGGAAAC CUGAUGAG X CGAA AUAAGAGU 611

716 CUUAUUGUU UCCAAAUU 111 AAUUUGGA CUGAUGAG X CGAA ACAAUAAG 612

717 UUAUUGUUU CCAAAUUC 112 GAAUUUGG CUGAUGAG X CGAA AACAAUAA 613

718 UAUUGUUUC CAAAUUCC 113 GGAAUUUG CUGAUGAG X CGAA AAACAAUA 614

724 UUCCAAAUU CCACUAUU 114 AAUAGUGG CUGAUGAG X CGAA AUUUGGAA 615

725 UCCAAAUUC CACUAUUG 115 CAAUAGUG CUGAUGAG X CGAA AAUUUGGA 616

730 AUUCCACUA UUGGUGAU 116 AUCACCAA CUGAUGAG X CGAA AGUGGAAU 617 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

732 UCCACUAUU GGUGAUAG 117 CUAUCACC CUGAUGAG X CGAA AUAGUGGA 618

739 UUGGUGAUA GUGGAGUC 118 GACUCCAC CUGAUGAG X CGAA AUCACCAA 619

747 AGUGGAGUC CCAGCACU 119 AGUGCUGG CUGAUGAG X CGAA ACUCCACU 620

756 CCAGCACUA CCUUCUUU 120 AAAGAAGG CUGAUGAG X CGAA AGUGCUGG 621

760 CACUACCUU CUUUGACU 121 AGUCAAAG CUGAUGAG X CGAA AGGUAGUG 622

761 ACUACCUUC UUUGACUA 122 UAGUCAAA CUGAUGAG X CGAA AAGGUAGU 623

763 UACCUUCUU UGACUAUG 123 CAUAGUCA CUGAUGAG X CGAA AGAAGGUA 624

764 ACCUUCUUU GACUAUGC 124 GCAUAGUC CUGAUGAG X CGAA AAGAAGGU 625

769 CUUUGACUA UGCGUCGU 125 ACGACGCA CUGAUGAG X CGAA AGUCAAAG 626

775 CUAUGCGUC GUAUGCGA 126 UCGCAUAC CUGAUGAG X CGAA ACGCAUAG 627

778 UGCGUCGUA UGCGAGAG 127 CUCUCGCA CUGAUGAG X CGAA ACGACGCA 628

788 GCGAGAGUC UGUUUCCA 128 UGGAAACA CUGAUGAG X CGAA ACUCUCGC 629

792 GAGUCUGUU UCCAGGAU 129 AUCCUGGA CUGAUGAG X CGAA ACAGACUC 630

793 AGUCUGUUU CCAGGAUG 130 CAUCCUGG CUGAUGAG X CGAA AACAGACU 631

794 GUCUGUUUC CAGGAUGC 131 GCAUCCUG CUGAUGAG X CGAA AAACAGAC 632

807 AUGCCUGUU AGUUCUCA 132 UGAGAACU CUGAUGAG X CGAA ACAGGCAU 633

808 UGCCUGUUA GUUCUCAG 133 CUGAGAAC CUGAUGAG X CGAA AACAGGCA 634

811 CUGUUAGUU CUCAGCAC 134 GUGCUGAG CUGAUGAG X CGAA ACUAACAG 635

812 UGUUAGUUC UCAGCACA 135 UGUGCUGA CUGAUGAG X CGAA AACUAACA 636

814 UUAGUUCUC AGCACAGA 136 UCUGUGCU CUGAUGAG X CGAA AGAACUAA 637

824 GCACAGAUA UUCUACAC 137 GUGUAGAA CUGAUGAG X CGAA AUCUGUGC 638

826 ACAGAUAUU CUACACCU 138 AGGUGUAG CUGAUGAG X CGAA AUAUCUGU 639

827 CAGAUAUUC UACACCUC 139 GAGGUGUA CUGAUGAG X CGAA AAUAUCUG 640

829 GAUAUUCUA CACCUCAC 140 GUGAGGUG CUGAUGAG X CGAA AGAAUAUC 641

835 CUACACCUC ACGCCUUC 141 GAAGGCGU CUGAUGAG X CGAA AGGUGUAG 642

842 UCACGCCUU CACCUUUA 142 UAAAGGUG CUGAUGAG X CGAA AGGCGUGA 643

843 CACGCCUUC ACCUUUAA 143 UUAAAGGU CUGAUGAG X CGAA AAGGCGUG 644

848 CUUCACCUU UAACACCU 144 AGGUGUUA CUGAUGAG X CGAA AGGUGAAG 645

849 UUCACCUUU AACACCUC 145 GAGGUGUU CUGAUGAG X CGAA AAGGUGAA 646

850 UCACCUUUA ACACCUCC 146 GGAGGUGU CUGAUGAG X CGAA AAAGGUGA 647

857 UAACACCUC CAGUCCCU 147 AGGGACUG CUGAUGAG X CGAA AGGUGUUA 648

862 CCUCCAGUC CCUCAUCU 148 AGAUGAGG CUGAUGAG X CGAA ACUGGAGG 649

866 CAGUCCCUC AUCUGAAG 149 CUUCAGAU CUGAUGAG X CGAA AGGGACUG 650

869 UCCCUCAUC UGAAGGUU 150 AACCUUCA CUGAUGAG X CGAA AUGAGGGA 651

877 CUGAAGGUU CCCUCUCC 151 GGAGAGGG CUGAUGAG X CGAA ACCUUCAG 652

878 UGAAGGUUC CCUCUCCC 152 GGGAGAGG CUGAUGAG X CGAA AACCUUCA 653

882 GGUUCCCUC UCCCAGAG 153 CUCUGGGA CUGAUGAG X CGAA AGGGAACC 654

884 UUCCCUCUC CCAGAGGC 154 GCCUCUGG CUGAUGAG X CGAA AGAGGGAA 655

899 GCAGAGGUC GACAUCCA 155 UGGAUGUC CUGAUGAG X CGAA ACCUCUGC 656

905 GUCGACAUC CACACCUA 156 UAGGUGUG CUGAUGAG X CGAA AUGUCGAC 657

913 CCACACCUA AUGUCCAC 157 GUGGACAU CUGAUGAG X CGAA AGGUGUGG 658

918 CCUAAUGUC CACAUGGU 158 ACCAUGUG CUGAUGAG X CGAA ACAUUAGG 659

927 CACAUGGUC AGCACCAC 159 GUGGUGCU CUGAUGAG X CGAA ACCAUGUG 660

960 AGGAUGAUU GAGGAUGC 160 GCAUCCUC CUGAUGAG X CGAA AUCAUCCU 661

972 GAUGCAAUU CGAAGUCA 161 UGACUUCG CUGAUGAG X CGAA AUUGCAUC 662

973 AUGCAAUUC GAAGUCAC 162 GUGACUUC CUGAUGAG X CGAA AAUUGCAU 663

979 UUCGAAGUC ACAGCGAA 163 UUCGCUGU CUGAUGAG X CGAA ACUUCGAA 664

989 CAGCGAAUC AGCCUCAC 164 GUGAGGCU CUGAUGAG X CGAA AUUCGCUG 665

995 AUCAGCCUC ACCUUCAG 165 CUGAAGGU CUGAUGAG X CGAA AGGCUGAU 666

1000 CCUCACCUU CAGCCCUG 166 CAGGGCUG CUGAUGAG X CGAA AGGUGAGG 667

1001 CUCACCUUC AGCCCUGU 167 ACAGGGCU CUGAUGAG X CGAA AAGGUGAG 668

1010 AGCCCUGUC CAGUAGCC 168 GGCUACUG CUGAUGAG X CGAA ACAGGGCU 669

1015 UGUCCAGUA GCCCCAAC 169 GUUGGGGC CUGAUGAG X CGAA ACUGGACA 670

1027 CCAACAAUC UGAGCCCA 170 UGGGCUCA CUGAUGAG X CGAA AUUGUUGG 671

1046 AGGCUGGUC ACAGCCGA 171 UCGGCUGU CUGAUGAG X CGAA ACCAGCCU 672

1092 GCACCAGUA UCUGGGAC 172 GUCCCAGA CUGAUGAG X CGAA ACUGGUGC 673

1094 ACCAGUAUC UGGGACCC 173 GGGUCCCA CUGAUGAG X CGAA AUACUGGU 674

1119 AACAAAAUU AGGCCUCG 174 CGAGGCCU CUGAUGAG X CGAA AUUUUGUU 675

1120 ACAAAAUUA GGCCUCGU 175 ACGAGGCC CUGAUGAG X CGAA AAUUUUGU 676 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1126 UUAGGCCUC GUGGACAG 176 CUGUCCAC CUGAUGAG X CGAA AGGCCUAA 677

1141 AGAGAGAUU CAAGCUAU 177 AUAGCUUG CUGAUGAG X CGAA AUCUCUCU 678

1142 GAGAGAUUC AAGCUAUU 178 AAUAGCUU CUGAUGAG X CGAA AAUCUCUC 679

1148 UUCAAGCUA UUAUUGGG 179 CCCAAUAA CUGAUGAG X CGAA AGCUUGAA 680

1150 CAAGCUAUU AUUGGGAA 180 UUCCCAAU CUGAUGAG X CGAA AUAGCUUG 681

1151 AAGCUAUUA UUGGGAAA 181 UUUCCCAA CUGAUGAG X CGAA AAUAGCUU 682

1153 GCUAUUAUU GGGAAAUA 182 UAUUUCCC CUGAUGAG X CGAA AUAAUAGC 683

1161 UGGGAAAUA GAAGCCAG 183 CUGGCUUC CUGAUGAG X CGAA AUUUCCCA 684

1184 GAUGCUGUC CACUCGGA 184 UCCGAGUG CUGAUGAG X CGAA ACAGCAUC 685

1189 UGUCCACUC GGAUUGGG 185 CCCAAUCC CUGAUGAG X CGAA AGUGGACA 686

1194 ACUCGGAUU GGGUCAGG 186 CCUGACCC CUGAUGAG X CGAA AUCCGAGU 687

1199 GAUUGGGUC AGGCUCUU 187 AAGAGCCU CUGAUGAG X CGAA ACCCAAUC 688

1205 GUCAGGCUC UUUUGGAA 188 UUCCAAAA CUGAUGAG X CGAA AGCCUGAC 689

1207 CAGGCUCUU UUGGAACU 189 AGUUCCAA CUGAUGAG X CGAA AGAGCCUG 690

1208 AGGCUCUUU UGGAACUG 190 CAGUUCCA CUGAUGAG X CGAA AAGAGCCU 691

1209 GGCUCUUUU GGAACUGU 191 ACAGUUCC CUGAUGAG X CGAA AAAGAGCC 692

1218 GGAACUGUU UAUAAGGG 192 CCCUUAUA CUGAUGAG X CGAA ACAGUUCC 693

1219 GAACUGUUU AUAAGGGU 193 ACCCUUAU CUGAUGAG X CGAA AACAGUUC 694

1220 AACUGUUUA UAAGGGUA 194 UACCCUUA CUGAUGAG X CGAA AAACAGUU 695

1222 CUGUUUAUA AGGGUAAA 195 UUUACCCU CUGAUGAG X CGAA AUAAACAG 696

1228 AUAAGGGUA AAUGGCAC 196 GUGCCAUU CUGAUGAG X CGAA ACCCUUAU 697

1245 GGAGAUGUU GCAGUAAA 197 UUUACUGC CUGAUGAG X CGAA ACAUCUCC 698

1251 GUUGCAGUA AAGAUCCU 198 AGGAUCUU CUGAUGAG X CGAA ACUGCAAC 699

1257 GUAAAGAUC CUAAAGGU 199 ACCUUUAG CUGAUGAG X CGAA AUCUUUAC 700

1260 AAGAUCCUA AAGGUUGU 200 ACAACCUU CUGAUGAG X CGAA AGGAUCUU 701

1266 CUAAAGGUU GUCGACCC 201 GGGUCGAC CUGAUGAG X CGAA ACCUUUAG 702

1269 AAGGUUGUC GACCCAAC 202 GUUGGGUC CUGAUGAG X CGAA ACAACCUU 703

1289 AGAGCAAUU CCAGGCCU 203 AGGCCUGG CUGAUGAG X CGAA AUUGCUCU 704

1290 GAGCAAUUC CAGGCCUU 204 AAGGCCUG CUGAUGAG X CGAA AAUUGCUC 705

1298 CCAGGCCUU CAGGAAUG 205 CAUUCCUG CUGAUGAG X CGAA AGGCCUGG 706

1299 CAGGCCUUC AGGAAUGA 206 UCAUUCCU CUGAUGAG X CGAA AAGGCCUG 707

1317 GUGGCUGUU CUGCGCAA 207 UUGCGCAG CUGAUGAG X CGAA ACAGCCAC 708

1318 UGGCUGUUC UGCGCAAA 208 UUUGCGCA CUGAUGAG X CGAA AACAGCCA 709

1344 GUGAACAUU CUGCUUUU 209 AAAAGCAG CUGAUGAG X CGAA AUGUUCAC 710

1345 UGAACAUUC UGCUUUUC 210 GAAAAGCA CUGAUGAG X CGAA AAUGUUCA 711

1350 AUUCUGCUU UUCAUGGG 211 CCCAUGAA CUGAUGAG X CGAA AGCAGAAU 712

1351 UUCUGCUUU UCAUGGGG 212 CCCCAUGA CUGAUGAG X CGAA AAGCAGAA 713

1352 UCUGCUUUU CAUGGGGU 213 ACCCCAUG CUGAUGAG X CGAA AAAGCAGA 714

1353 CUGCUUUUC AUGGGGUA 214 UACCCCAU CUGAUGAG X CGAA AAAAGCAG 715

1361 CAUGGGGUA CAUGACAA 215 UUGUCAUG CUGAUGAG X CGAA ACCCCAUG 716

1386 CUGGCAAUU GUGACCCA 216 UGGGUCAC CUGAUGAG X CGAA AUUGCCAG 717

1416 AGCAGCCUC UACAAACA 217 UGUUUGUA CUGAUGAG X CGAA AGGCUGCU 718

1418 CAGCCUCUA CAAACACC 218 GGUGUUUG CUGAUGAG X CGAA AGAGGCUG 719

1434 CUGCAUGUC CAGGAGAC 219 GUCUCCUG CUGAUGAG X CGAA ACAUGCAG 720

1448 GACCAAGUU UCAGAUGU 220 ACAUCUGA CUGAUGAG X CGAA ACUUGGUC 721

1449 ACCAAGUUU CAGAUGUU 221 AACAUCUG CUGAUGAG X CGAA AACUUGGU 722

1450 CCAAGUUUC AGAUGUUC 222 GAACAUCU CUGAUGAG X CGAA AAACUUGG 723

1457 UCAGAUGUU CCAGCUAA 223 UUAGCUGG CUGAUGAG X CGAA ACAUCUGA 724

1458 CAGAUGUUC CAGCUAAU 224 AUUAGCUG CUGAUGAG X CGAA AACAUCUG 725

1464 UUCCAGCUA AUUGACAU 225 AUGUCAAU CUGAUGAG X CGAA AGCUGGAA 726

1467 CAGCUAAUU GACAUUGC 226 GCAAUGUC CUGAUGAG X CGAA AUUAGCUG 727

1473 AUUGACAUU GCCCGGCA 227 UGCCGGGC CUGAUGAG X CGAA AUGUCAAU 728

1489 AGACGGCUC AGGGAAUG 228 CAUUCCCU CUGAUGAG X CGAA AGCCGUCU 729

1502 AAUGGACUA UUUGCAUG 229 CAUGCAAA CUGAUGAG X CGAA AGUCCAUU 730

1504 UGGACUAUU UGCAUGCA 230 UGCAUGCA CUGAUGAG X CGAA AUAGUCCA 731

1505 GGACUAUUU GCAUGCAA 231 UUGCAUGC CUGAUGAG X CGAA AAUAGUCC 732

1521 AAGAACAUC AUCCAUAG 232 CUAUGGAU CUGAUGAG X CGAA AUGUUCUU 733

1524 AACAUCAUC CAUAGAGA 233 UCUCUAUG CUGAUGAG X CGAA AUGAUGUU 734

1528 UCAUCCAUA GAGACAUG 234 CAUGUCUC CUGAUGAG X CGAA AUGGAUGA 735 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1541 CAUGAAAUC CAACAAUA 235 UAUUGUUG CUGAUGAG X CGAA AUUUCAUG 736

1549 CCAACAAUA UAUUUCUC 236 GAGAAAUA CUGAUGAG X CGAA AUUGUUGG 737

1551 AACAAUAUA UUUCUCCA 237 UGGAGAAA CUGAUGAG X CGAA AUAUUGUU 738

1553 CAAUAUAUU UCUCCAUG 238 CAUGGAGA CUGAUGAG X CGAA AUAUAUUG 739

1554 AAUAUAUUU CUCCAUGA 239 UCAUGGAG CUGAUGAG X CGAA AAUAUAUU 740

1555 AUAUAUUUC UCCAUGAA 240 UUCAUGGA CUGAUGAG X CGAA AAAUAUAU 741

1557 AUAUUUCUC CAUGAAGG 241 CCUUCAUG CUGAUGAG X CGAA AGAAAUAU 742

1568 UGAAGGCUU AACAGUGA 242 UCACUGUU CUGAUGAG X CGAA AGCCUUCA 743

1569 GAAGGCUUA ACAGUGAA 243 UUCACUGU CUGAUGAG X CGAA AAGCCUUC 744

1581 GUGAAAAUU GGAGAUUU 244 AAAUCUCC CUGAUGAG X CGAA AUUUUCAC 745

1588 UUGGAGAUU UUGGUUUG 245 CAAACCAA CUGAUGAG X CGAA AUCUCCAA 746

1589 UGGAGAUUU UGGUUUGG 246 CCAAACCA CUGAUGAG X CGAA AAUCUCCA 747

1590 GGAGAUUUU GGUUUGGC 247 GCCAAACC CUGAUGAG X CGAA AAAUCUCC 748

1594 AUUUUGGUU UGGCAACA 248 UGUUGCCA CUGAUGAG X CGAA ACCAAAAU 749

1595 UUUUGGUUU GGCAACAG 249 CUGUUGCC CUGAUGAG X CGAA AACCAAAA 750

1605 GCAACAGUA AAGUCACG 250 CGUGACUU CUGAUGAG X CGAA ACUGUUGC 751

1610 AGUAAAGUC ACGCUGGA 251 UCCAGCGU CUGAUGAG X CGAA ACUUUACU 752

1624 GGAGUGGUU CUCAGCAG 252 CUGCUGAG CUGAUGAG X CGAA ACCACUCC 753

1625 GAGUGGUUC UCAGCAGG 253 CCUGCUGA CUGAUGAG X CGAA AACCACUC 754

1627 GUGGUUCUC AGCAGGUU 254 AACCUGCU CUGAUGAG X CGAA AGAACCAC 755

1635 CAGCAGGUU GAACAACC 255 GGUUGUUC CUGAUGAG X CGAA ACCUGCUG 756

1645 AACAACCUA CUGGCUCU 256 AGAGCCAG CUGAUGAG X CGAA AGGUUGUU 757

1652 UACUGGCUC UGUCCUCU 257 AGAGGACA CUGAUGAG X CGAA AGCCAGUA 758

1656 GGCUCUGUC CUCUGGAU 258 AUCCAGAG CUGAUGAG X CGAA ACAGAGCC 759

1659 UCUGUCCUC UGGAUGGC 259 GCCAUCCA CUGAUGAG X CGAA AGGACAGA 760

1680 GAGGUGAUC CGAAUGCA 260 UGCAUUCG CUGAUGAG X CGAA AUCACCUC 761

1693 UGCAGGAUA ACAACCCA 261 UGGGUUGU CUGAUGAG X CGAA AUCCUGCA 762

1703 CAACCCAUU CAGUUUCC 262 GGAAACUG CUGAUGAG X CGAA AUGGGUUG 763

1704 AACCCAUUC AGUUUCCA 263 UGGAAACU CUGAUGAG X CGAA AAUGGGUU 764

1708 CAUUCAGUU UCCAGUCG 264 CGACUGGA CUGAUGAG X CGAA ACUGAAUG 765

1709 AUUCAGUUU CCAGUCGG 265 CCGACUGG CUGAUGAG X CGAA AACUGAAU 766

1710 UUCAGUUUC CAGUCGGA 266 UCCGACUG CUGAUGAG X CGAA AAACUGAA 767

1715 UUUCCAGUC GGAUGUCU 267 AGACAUCC CUGAUGAG X CGAA ACUGGAAA 768

1722 UCGGAUGUC UACUCCUA 268 UAGGAGUA CUGAUGAG X CGAA ACAUCCGA 769

1724 GGAUGUCUA CUCCUAUG 269 CAUAGGAG CUGAUGAG X CGAA AGACAUCC 770

1727 UGUCUACUC CUAUGGCA 270 UGCCAUAG CUGAUGAG X CGAA AGUAGACA 771

1730 CUACUCCUA UGGCAUCG 271 CGAUGCCA CUGAUGAG X CGAA AGGAGUAG 772

1737 UAUGGCAUC GUAUUGUA 272 UACAAUAC CUGAUGAG X CGAA AUGCCAUA 773

1740 GGCAUCGUA UUGUAUGA 273 UCAUACAA CUGAUGAG X CGAA ACGAUGCC 774

1742 CAUCGUAUU GUAUGAAC 274 GUUCAUAC CUGAUGAG X CGAA AUACGAUG 775

1745 CGUAUUGUA UGAACUGA 275 UCAGUUCA CUGAUGAG X CGAA ACAAUACG 776

1767 GGGGAGCUU CCUUAUUC 276 GAAUAAGG CUGAUGAG X CGAA AGCUCCCC 777

1768 GGGAGCUUC CUUAUUCU 277 AGAAUAAG CUGAUGAG X CGAA AAGCUCCC 778

1771 AGCUUCCUU AUUCUCAC 278 GUGAGAAU CUGAUGAG X CGAA AGGAAGCU 779

1772 GCUUCCUUA UUCUCACA 279 UGUGAGAA CUGAUGAG X CGAA AAGGAAGC 780

1774 UUCCUUAUU CUCACAUC 280 GAUGUGAG CUGAUGAG X CGAA AUAAGGAA 781

1775 UCCUUAUUC UCACAUCA 281 UGAUGUGA CUGAUGAG X CGAA AAUAAGGA 782

1777 CUUAUUCUC ACAUCAAC 282 GUUGAUGU CUGAUGAG X CGAA AGAAUAAG 783

1782 UCUCACAUC AACAACCG 283 CGGUUGUU CUGAUGAG X CGAA AUGUGAGA 784

1795 ACCGAGAUC AGAUCAUC 284 GAUGAUCU CUGAUGAG X CGAA AUCUCGGU 785

1800 GAUCAGAUC AUCUUCAU 285 AUGAAGAU CUGAUGAG X CGAA AUCUGAUC 786

1803 CAGAUCAUC UUCAUGGU 286 ACCAUGAA CUGAUGAG X CGAA AUGAUCUG 787

1805 GAUCAUCUU CAUGGUGG 287 CCACCAUG CUGAUGAG X CGAA AGAUGAUC 788

1806 AUCAUCUUC AUGGUGGG 288 CCCACCAU CUGAUGAG X CGAA AAGAUGAU 789

1823 CCGAGGAUA UGCCUCCC 289 GGGAGGCA CUGAUGAG X CGAA AUCCUCGG 790

1829 AUAUGCCUC CCCAGAUC 290 GAUCUGGG CUGAUGAG X CGAA AGGCAUAU 791

1837 CCCCAGAUC UUAGUAAG 291 CUUACUAA CUGAUGAG X CGAA AUCUGGGG 792

1839 CCAGAUCUU AGUAAGCU 292 AGCUUACU CUGAUGAG X CGAA AGAUCUGG 793

1840 CAGAUCUUA GUAAGCUA 293 UAGCUUAC CUGAUGAG X CGAA AAGAUCUG 794 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1843 AUCUUAGUA AGCUAUAU 294 AUAUAGCU CUGAUGAG X CGAA ACUAAGAU 795

1848 AGUAAGCUA UAUAAGAA 295 UUCUUAUA CUGAUGAG X CGAA AGCUUACU 796

1850 UAAGCUAUA UAAGAACU 296 AGUUCUUA CUGAUGAG X CGAA AUAGCUUA 797

1852 AGCUAUAUA AGAACUGC 297 GCAGUUCU CUGAUGAG X CGAA AUAUAGCU 798

1884 AGGCUGGUA GCUGACUG 298 CAGUCAGC CUGAUGAG X CGAA ACCAGCCU 799

1905 AAGAAAGUA AAGGAAGA 299 UCUUCCUU CUGAUGAG X CGAA ACUUUCUU 800

1921 AGAGGCCUC UUUUUCCC 300 GGGAAAAA CUGAUGAG X CGAA AGGCCUCU 801

1923 AGGCCUCUU UUUCCCCA 301 UGGGGAAA CUGAUGAG X CGAA AGAGGCCU 802

1924 GGCCUCUUU UUCCCCAG 302 CUGGGGAA CUGAUGAG X CGAA AAGAGGCC 803

1925 GCCUCUUUU UCCCCAGA 303 UCUGGGGA CUGAUGAG X CGAA AAAGAGGC 804

1926 CCUCUUUUU CCCCAGAU 304 AUCUGGGG CUGAUGAG X CGAA AAAAGAGG 805

1927 cucuuuuuc CCCAGAUC 305 GAUCUGGG CUGAUGAG X CGAA AAAAAGAG 806

1935 CCCCAGAUC CUGUCUUC 306 GAAGACAG CUGAUGAG X CGAA AUCUGGGG 807

1940 GAUCCUGUC UUCCAUUG 307 CAAUGGAA CUGAUGAG X CGAA ACAGGAUC 808

1942 UCCUGUCUU CCAUUGAG 308 CUCAAUGG CUGAUGAG X CGAA AGACAGGA 809

1943 CCUGUCUUC CAUUGAGC 309 GCUCAAUG CUGAUGAG X CGAA AAGACAGG 810

1947 UCUUCCAUU GAGCUGCU 310 AGCAGCUC CUGAUGAG X CGAA AUGGAAGA 811

1956 GAGCUGCUC CAACACUC 311 GAGUGUUG CUGAUGAG X CGAA AGCAGCUC 812

1964 CCAACACUC UCUACCGA 312 UCGGUAGA CUGAUGAG X CGAA AGUGUUGG 813

1966 AACACUCUC UACCGAAG 313 CUUCGGUA CUGAUGAG X CGAA AGAGUGUU 814

1968 CACUCUCUA CCGAAGAU 314 AUCUUCGG CUGAUGAG X CGAA AGAGAGUG 815

1977 CCGAAGAUC AACCGGAG 315 CUCCGGUU CUGAUGAG X CGAA AUCUUCGG 816

1990 GGAGCGCUU CCGAGCCA 316 UGGCUCGG CUGAUGAG X CGAA AGCGCUCC 817

1991 GAGCGCUUC CGAGCCAU 317 AUGGCUCG CUGAUGAG X CGAA AAGCGCUC 818

2000 CGAGCCAUC CUUGCAUC 318 GAUGCAAG CUGAUGAG X CGAA AUGGCUCG 819

2003 GCCAUCCUU GCAUCGGG 319 CCCGAUGC CUGAUGAG X CGAA AGGAUGGC 820

2008 CCUUGCAUC GGGCAGCC 320 GGCUGCCC CUGAUGAG X CGAA AUGCAAGG 821

2029 CUGAGGAUA UCAAUGCU 321 AGCAUUGA CUGAUGAG X CGAA AUCCUCAG 822

2031 GAGGAUAUC AAUGCUUG 322 CAAGCAUU CUGAUGAG X CGAA AUAUCCUC 823

2038 UCAAUGCUU GCACGCUG 323 CAGCGUGC CUGAUGAG X CGAA AGCAUUGA 824

2054 GACCACGUC CCCGAGGC 324 GCCUCGGG CUGAUGAG X CGAA ACGUGGUC 825

2070 CUGCCUGUC UUCUAGUU 325 AACUAGAA CUGAUGAG X CGAA ACAGGCAG 826

2072 GCCUGUCUU CUAGUUGA 326 UCAACUAG CUGAUGAG X CGAA AGACAGGC 827

2073 CCUGUCUUC UAGUUGAC 327 GUCAACUA CUGAUGAG X CGAA AAGACAGG 828

2075 UGUCUUCUA GUUGACUU 328 AAGUCAAC CUGAUGAG X CGAA AGAAGACA 829

2078 CUUCUAGUU GACUUUGC 329 GCAAAGUC CUGAUGAG X CGAA ACUAGAAG 830

2083 AGUUGACUU UGCACCUG 330 CAGGUGCA CUGAUGAG X CGAA AGUCAACU 831

2084 GUUGACUUU GCACCUGU 331 ACAGGUGC CUGAUGAG X CGAA AAGUCAAC 832

2093 GCACCUGUC UUCAGGCU 332 AGCCUGAA CUGAUGAG X CGAA ACAGGUGC 833

2095 ACCUGUCUU CAGGCUGC 333 GCAGCCUG CUGAUGAG X CGAA AGACAGGU 834

2096 CCUGUCUUC AGGCUGCC 334 GGCAGCCU CUGAUGAG X CGAA AAGACAGG 835

2136 GCACCACUU UUCUGCUC 335 GAGCAGAA CUGAUGAG X CGAA AGUGGUGC 836

2137 CACCACUUU UCUGCUCC 336 GGAGCAGA CUGAUGAG X CGAA AAGUGGUG 837

2138 ACCACUUUU CUGCUCCC 337 GGGAGCAG CUGAUGAG X CGAA AAAGUGGU 838

2139 CCACUUUUC UGCUCCCU 338 AGGGAGCA CUGAUGAG X CGAA AAAAGUGG 839

2144 UUUCUGCUC CCUUUCUC 339 GAGAAAGG CUGAUGAG X CGAA AGCAGAAA 840

2148 UGCUCCCUU UCUCCAGA 340 UCUGGAGA CUGAUGAG X CGAA AGGGAGCA 841

2149 GCUCCCUUU CUCCAGAG 341 CUCUGGAG CUGAUGAG X CGAA AAGGGAGC 842

2150 cucccuuuc UCCAGAGG 342 CCUCUGGA CUGAUGAG X CGAA AAAGGGAG 843

2152 cccuuucuc CAGAGGCA 343 UGCCUCUG CUGAUGAG X CGAA AGAAAGGG 844

2171 ACACAUGUU UUCAGAGA 344 UCUCUGAA CUGAUGAG X CGAA ACAUGUGU 845

2172 CACAUGUUU UCAGAGAA 345 UUCUCUGA CUGAUGAG X CGAA AACAUGUG 846

2173 ACAUGUUUU CAGAGAAG 346 CUUCUCUG CUGAUGAG X CGAA AAACAUGU 847

2174 CAUGUUUUC AGAGAAGC 347 GCUUCUCU CUGAUGAG X CGAA AAAACAUG 848

2184 GAGAAGCUC UGCUAAGG 348 CCUUAGCA CUGAUGAG X CGAA AGCUUCUC 849

2189 GCUCUGCUA AGGACCUU 349 AAGGUCCU CUGAUGAG X CGAA AGCAGAGC 850

2197 AAGGACCUU CUAGACUG 350 CAGUCUAG CUGAUGAG X CGAA AGGUCCUU 851

2198 AGGACCUUC UAGACUGC 351 GCAGUCUA CUGAUGAG X CGAA AAGGUCCU 852

2200 GACCUUCUA GACUGCUC 352 GAGCAGUC CUGAUGAG X CGAA AGAAGGUC 853 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2208 AGACUGCUC ACAGGGCC 353 GGCCCUGU CUGAUGAG X CGAA AGCAGUCU 854

2218 CAGGGCCUU AACUUCAU 354 AUGAAGUU CUGAUGAG X CGAA AGGCCCUG 855

2219 AGGGCCUUA ACUUCAUG 355 CAUGAAGU CUGAUGAG X CGAA AAGGCCCU 856

2223 CCUUAACUU CAUGUUGC 356 GCAACAUG CUGAUGAG X CGAA AGUUAAGG 857

2224 CUUAACUUC AUGUUGCC 357 GGCAACAU CUGAUGAG X CGAA AAGUUAAG 858

2229 CUUCAUGUU GCCUUCUU 358 AAGAAGGC CUGAUGAG X CGAA ACAUGAAG 859

2234 UGUUGCCUU CUUUUCUA 359 UAGAAAAG CUGAUGAG X CGAA AGGCAACA 860

2235 GUUGCCUUC UUUUCUAU 360 AUAGAAAA CUGAUGAG X CGAA AAGGCAAC 861

2237 UGCCUUCUU UUCUAUCC 361 GGAUAGAA CUGAUGAG X CGAA AGAAGGCA 862

2238 GCCUUCUUU UCUAUCCC 362 GGGAUAGA CUGAUGAG X CGAA AAGAAGGC 863

2239 ccuucuuuu CUAUCCCU 363 AGGGAUAG CUGAUGAG X CGAA AAAGAAGG 864

2240 cuucuuuuc UAUCCCUU 364 AAGGGAUA CUGAUGAG X CGAA AAAAGAAG 865

2242 UCUUUUCUA UCCCUUUG 365 CAAAGGGA CUGAUGAG X CGAA AGAAAAGA 866

2244 UUUUCUAUC CCUUUGGG 366 CCCAAAGG CUGAUGAG X CGAA AUAGAAAA 867

2248 CUAUCCCUU UGGGCCCU 367 AGGGCCCA CUGAUGAG X CGAA AGGGAUAG 868

2249 UAUCCCUUU GGGCCCUG 368 CAGGGCCC CUGAUGAG X CGAA AAGGGAUA 869

2273 GAAGCCAUU UGCAGUGC 369 GCACUGCA CUGAUGAG X CGAA AUGGCUUC 870

2274 AAGCCAUUU GCAGUGCU 370 AGCACUGC CUGAUGAG X CGAA AAUGGCUU 871

2290 UGGUGUGUC CUGCUCCC 371 GGGAGCAG CUGAUGAG X CGAA ACACACCA 872

2296 GUCCUGCUC CCUCCCCA 372 UGGGGAGG CUGAUGAG X CGAA AGCAGGAC 873

2300 UGCUCCCUC CCCACAUU 373 AAUGUGGG CUGAUGAG X CGAA AGGGAGCA 874

2308 CCCCACAUU CCCCAUGC 374 GCAUGGGG CUGAUGAG X CGAA AUGUGGGG 875

2309 CCCACAUUC CCCAUGCU 375 AGCAUGGG CUGAUGAG X CGAA AAUGUGGG 876

2318 CCCAUGCUC AAGGCCCA 376 UGGGCCUU CUGAUGAG X CGAA AGCAUGGG 877

2331 CCCAGCCUU CUGUAGAU 377 AUCUACAG CUGAUGAG X CGAA AGGCUGGG 878

2332 CCAGCCUUC UGUAGAUG 378 CAUCUACA CUGAUGAG X CGAA AAGGCUGG 879

2336 CCUUCUGUA GAUGCGCA 379 UGCGCAUC CUGAUGAG X CGAA ACAGAAGG 880

2354 GUGGAUGUU GAUGGUAG 380 CUACCAUC CUGAUGAG X CGAA ACAUCCAC 881

2361 UUGAUGGUA GUACAAAA 381 UUUUGUAC CUGAUGAG X CGAA ACCAUCAA 882

2364 AUGGUAGUA CAAAAAGC 382 GCUUUUUG CUGAUGAG X CGAA ACUACCAU 883

2393 CCAGCUGUU GGCUACAU 383 AUGUAGCC CUGAUGAG X CGAA ACAGCUGG 884

2398 UGUUGGCUA CAUGAGUA 384 UACUCAUG CUGAUGAG X CGAA AGCCAACA 885

2406 ACAUGAGUA UUUAGAGG 385 CCUCUAAA CUGAUGAG X CGAA ACUCAUGU 886

2408 AUGAGUAUU UAGAGGAA 386 UUCCUCUA CUGAUGAG X CGAA AUACUCAU 887

2409 UGAGUAUUU AGAGGAAG 387 CUUCCUCU CUGAUGAG X CGAA AAUACUCA 888

2410 GAGUAUUUA GAGGAAGU 388 ACUUCCUC CUGAUGAG X CGAA AAAUACUC 889

2419 GAGGAAGUA AGGUAGCA 389 UGCUACCU CUGAUGAG X CGAA ACUUCCUC 890

2424 AGUAAGGUA GCAGGCAG 390 CUGCCUGC CUGAUGAG X CGAA ACCUUACU 891

2434 CAGGCAGUC CAGCCCUG 391 CAGGGCUG CUGAUGAG X CGAA ACUGCCUG 892

2462 CAUGGGAUU UUGGAAAU 392 AUUUCCAA CUGAUGAG X CGAA AUCCCAUG 893

2463 AUGGGAUUU UGGAAAUC 393 GAUUUCCA CUGAUGAG X CGAA AAUCCCAU 894

2464 UGGGAUUUU GGAAAUCA 394 UGAUUUCC CUGAUGAG X CGAA AAAUCCCA 895

2471 UUGGAAAUC AGCUUCUG 395 CAGAAGCU CUGAUGAG X CGAA AUUUCCAA 896

2476 AAUCAGCUU CUGGAGGA 396 UCCUCCAG CUGAUGAG X CGAA AGCUGAUU 897

2477 AUCAGCUUC UGGAGGAA 397 UUCCUCCA CUGAUGAG X CGAA AAGCUGAU 898

2493 AUGCAUGUC ACAGGCGG 398 CCGCCUGU CUGAUGAG X CGAA ACAUGCAU 899

2506 GCGGGACUU UCUUCAGA 399 UCUGAAGA CUGAUGAG X CGAA AGUCCCGC 900

2507 CGGGACUUU CUUCAGAG 400 CUCUGAAG CUGAUGAG X CGAA AAGUCCCG 901

2508 GGGACUUUC UUCAGAGA 401 UCUCUGAA CUGAUGAG X CGAA AAAGUCCC 902

2510 GACUUUCUU CAGAGAGU 402 ACUCUCUG CUGAUGAG X CGAA AGAAAGUC 903

2511 ACUUUCUUC AGAGAGUG 403 CACUCUCU CUGAUGAG X CGAA AAGAAAGU 904

2536 CCAGACAUU UUGCACAU 404 AUGUGCAA CUGAUGAG X CGAA AUGUCUGG 905

2537 CAGACAUUU UGCACAUA 405 UAUGUGCA CUGAUGAG X CGAA AAUGUCUG 906

2538 AGACAUUUU GCACAUAA 406 UUAUGUGC CUGAUGAG X CGAA AAAUGUCU 907

2545 UUGCACAUA AGGCACCA 407 UGGUGCCU CUGAUGAG X CGAA AUGUGCAA 908

2577 CCGAGACUC UGGCCGCC 408 GGCGGCCA CUGAUGAG X CGAA AGUCUCGG 909

2600 AGCCUGCUU UGGUACUA 409 UAGUACCA CUGAUGAG X CGAA AGCAGGCU 910

2601 GCCUGCUUU GGUACUAU 410 AUAGUACC CUGAUGAG X CGAA AAGCAGGC 911

2605 GCUUUGGUA CUAUGGAA 411 UUCCAUAG CUGAUGAG X CGAA ACCAAAGC 912 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2608 UUGGUACUA UGGAACUU 412 AAGUUCCA CUGAUGAG X CGAA AGUACCAA 913

2616 AUGGAACUU UUCUUAGG 413 CCUAAGAA CUGAUGAG X CGAA AGUUCCAU 914

2617 UGGAACUUU UCUUAGGG 414 CCCUAAGA CUGAUGAG X CGAA AAGUUCCA 915

2618 GGAACUUUU CUUAGGGG 415 CCCCUAAG CUGAUGAG X CGAA AAAGUUCC 916

2619 GAACUUUUC UUAGGGGA 416 UCCCCUAA CUGAUGAG X CGAA AAAAGUUC 917

2621 ACUUUUCUU AGGGGACA 417 UGUCCCCU CUGAUGAG X CGAA AGAAAAGU 918

2622 CUUUUCUUA GGGGACAC 418 GUGUCCCC CUGAUGAG X CGAA AAGAAAAG 919

2633 GGACACGUC CUCCUUUC 419 GAAAGGAG CUGAUGAG X CGAA ACGUGUCC 920

2636 CACGUCCUC CUUUCACA 420 UGUGAAAG CUGAUGAG X CGAA AGGACGUG 921

2639 GUCCUCCUU UCACAGCU 421 AGCUGUGA CUGAUGAG X CGAA AGGAGGAC 922

2640 UCCUCCUUU CACAGCUU 422 AAGCUGUG CUGAUGAG X CGAA AAGGAGGA 923

2641 CCUCCUUUC ACAGCUUC 423 GAAGCUGU CUGAUGAG X CGAA AAAGGAGG 924

2648 UCACAGCUU CUAAGGUG 424 CACCUUAG CUGAUGAG X CGAA AGCUGUGA 925

2649 CACAGCUUC UAAGGUGU 425 ACACCUUA CUGAUGAG X CGAA AAGCUGUG 926

2651 CAGCUUCUA AGGUGUCC 426 GGACACCU CUGAUGAG X CGAA AGAAGCUG 927

2658 UAAGGUGUC CAGUGCAU 427 AUGCACUG CUGAUGAG X CGAA ACACCUUA 928

2667 CAGUGCAUU GGGAUGGU 428 ACCAUCCC CUGAUGAG X CGAA AUGCACUG 929

2676 GGGAUGGUU UUCCAGGC 429 GCCUGGAA CUGAUGAG X CGAA ACCAUCCC 930

2677 GGAUGGUUU UCCAGGCA 430 UGCCUGGA CUGAUGAG X CGAA AACCAUCC 931

2678 GAUGGUUUU CCAGGCAA 431 UUGCCUGG CUGAUGAG X CGAA AAACCAUC 932

2679 AUGGUUUUC CAGGCAAG 432 CUUGCCUG CUGAUGAG X CGAA AAAACCAU 933

2693 AAGGCACUC GGCCAAUC 433 GAUUGGCC CUGAUGAG X CGAA AGUGCCUU 934

2701 CGGCCAAUC CGCAUCUC 434 GAGAUGCG CUGAUGAG X CGAA AUUGGCCG 935

2707 AUCCGCAUC UCAGCCCU 435 AGGGCUGA CUGAUGAG X CGAA AUGCGGAU 936

2709 CCGCAUCUC AGCCCUCU 436 AGAGGGCU CUGAUGAG X CGAA AGAUGCGG 937

2716 UCAGCCCUC UCAGGAGC 437 GCUCCUGA CUGAUGAG X CGAA AGGGCUGA 938

2718 AGCCCUCUC AGGAGCAG 438 CUGCUCCU CUGAUGAG X CGAA AGAGGGCU 939

2728 GGAGCAGUC UUCCAUCA 439 UGAUGGAA CUGAUGAG X CGAA ACUGCUCC 940

2730 AGCAGUCUU CCAUCAUG 440 CAUGAUGG CUGAUGAG X CGAA AGACUGCU 941

2731 GCAGUCUUC CAUCAUGC 441 GCAUGAUG CUGAUGAG X CGAA AAGACUGC 942

2735 UCUUCCAUC AUGCUGAA 442 UUCAGCAU CUGAUGAG X CGAA AUGGAAGA 943

2745 UGCUGAAUU UUGUCUUC 443 GAAGACAA CUGAUGAG X CGAA AUUCAGCA 944

2746 GCUGAAUUU UGUCUUCC 444 GGAAGACA CUGAUGAG X CGAA AAUUCAGC 945

2747 CUGAAUUUU GUCUUCCA 445 UGGAAGAC CUGAUGAG X CGAA AAAUUCAG 946

2750 AAUUUUGUC UUCCAGGA 446 UCCUGGAA CUGAUGAG X CGAA ACAAAAUU 947

2752 UUUUGUCUU CCAGGAGC 447 GCUCCUGG CUGAUGAG X CGAA AGACAAAA 948

2753 UUUGUCUUC CAGGAGCU 448 AGCUCCUG CUGAUGAG X CGAA AAGACAAA 949

2768 CUGCCCCUA UGGGGCGG 449 CCGCCCCA CUGAUGAG X CGAA AGGGGCAG 950

2795 CAGCCUGUU UCUCUAAC 450 GUUAGAGA CUGAUGAG X CGAA ACAGGCUG 951

2796 AGCCUGUUU CUCUAACA 451 UGUUAGAG CUGAUGAG X CGAA AACAGGCU 952

2797 GCCUGUUUC UCUAACAA 452 UUGUUAGA CUGAUGAG X CGAA AAACAGGC 953

2799 CUGUUUCUC UAACAAAC 453 GUUUGUUA CUGAUGAG X CGAA AGAAACAG 954

2801 GUUUCUCUA ACAAACAA 454 UUGUUUGU CUGAUGAG X CGAA AGAGAAAC 955

2825 AACAGCCUU GUUUCUCU 455 AGAGAAAC CUGAUGAG X CGAA AGGCUGUU 956

2828 AGCCUUGUU UCUCUAGU 456 ACUAGAGA CUGAUGAG X CGAA ACAAGGCU 957

2829 GCCUUGUUU CUCUAGUC 457 GACUAGAG CUGAUGAG X CGAA AACAAGGC 958

2830 CCUUGUUUC UCUAGUCA 458 UGACUAGA CUGAUGAG X CGAA AAACAAGG 959

2832 UUGUUUCUC UAGUCACA 459 UGUGACUA CUGAUGAG X CGAA AGAAACAA 960

2834 GUUUCUCUA GUCACAUC 460 GAUGUGAC CUGAUGAG X CGAA AGAGAAAC 961

2837 UCUCUAGUC ACAUCAUG 461 CAUGAUGU CUGAUGAG X CGAA ACUAGAGA 962

2842 AGUCACAUC AUGUGUAU 462 AUACACAU CUGAUGAG X CGAA AUGUGACU 963

2849 UCAUGUGUA UACAAGGA 463 UCCUUGUA CUGAUGAG X CGAA ACACAUGA 964

2851 AUGUGUAUA CAAGGAAG 464 CUUCCUUG CUGAUGAG X CGAA AUACACAU 965

2868 CCAGGAAUA CAGGUUUU 465 AAAACCUG CUGAUGAG X CGAA AUUCCUGG 966

2874 AUACAGGUU UUCUUGAU 466 AUCAAGAA CUGAUGAG X CGAA ACCUGUAU 967

2875 UACAGGUUU UCUUGAUG 467 CAUCAAGA CUGAUGAG X CGAA AACCUGUA 968

2876 ACAGGUUUU CUUGAUGA 468 UCAUCAAG CUGAUGAG X CGAA AAACCUGU 969

2877 CAGGUUUUC UUGAUGAU 469 AUCAUCAA CUGAUGAG X CGAA AAAACCUG 970

2879 GGUUUUCUU GAUGAUUU 470 AAAUCAUC CUGAUGAG X CGAA AGAAAACC 971 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2886 UUGAUGAUU UGGGUUUU 471 AAAACCCA CUGAUGAG X CGAA AUCAUCAA 972

2887 UGAUGAUUU GGGUUUUA 472 UAAAACCC CUGAUGAG X CGAA AAUCAUCA 973

2892 AUUUGGGUU UUAAUUUU 473 AAAAUUAA CUGAUGAG X CGAA ACCCAAAU 974

2893 UUUGGGUUU UAAUUUUG 474 CAAAAUUA CUGAUGAG X CGAA AACCCAAA 975

2894 UUGGGUUUU AAUUUUGU 475 ACAAAAUU CUGAUGAG X CGAA AAACCCAA 976

2895 UGGGUUUUA AUUUUGUU 476 AACAAAAU CUGAUGAG X CGAA AAAACCCA 977

2898 GUUUUAAUU UUGUUUUU 477 AAAAACAA CUGAUGAG X CGAA AUUAAAAC 978

2899 UUUUAAUUU UGUUUUUA 478 UAAAAACA CUGAUGAG X CGAA AAUUAAAA 979

2900 UUUAAUUUU GUUUUUAU 479 AUAAAAAC CUGAUGAG X CGAA AAAUUAAA 980

2903 AAUUUUGUU UUUAUUGC 480 GCAAUAAA CUGAUGAG X CGAA ACAAAAUU 981

2904 AUUUUGUUU UUAUUGCA 481 UGCAAUAA CUGAUGAG X CGAA AACAAAAU 982

2905 UUUUGUUUU UAUUGCAC 482 GUGCAAUA CUGAUGAG X CGAA AAACAAAA 983

2906 UUUGUUUUU AUUGCACC 483 GGUGCAAU CUGAUGAG X CGAA AAAACAAA 984

2907 UUGUUUUUA UUGCACCU 484 AGGUGCAA CUGAUGAG X CGAA AAAAACAA 985

2909 GUUUUUAUU GCACCUGA 485 UCAGGUGC CUGAUGAG X CGAA AUAAAAAC 986

2924 GACAAAAUA CAGUUAUC 486 GAUAACUG CUGAUGAG X CGAA AUUUUGUC 987

2929 AAUACAGUU AUCUGAUG 487 CAUCAGAU CUGAUGAG X CGAA ACUGUAUU 988

2930 AUACAGUUA UCUGAUGG 488 CCAUCAGA CUGAUGAG X CGAA AACUGUAU 989

2932 ACAGUUAUC UGAUGGUC 489 GACCAUCA CUGAUGAG X CGAA AUAACUGU 990

2940 CUGAUGGUC CCUCAAUU 490 AAUUGAGG CUGAUGAG X CGAA ACCAUCAG 991

2944 UGGUCCCUC AAUUAUGU 491 ACAUAAUU CUGAUGAG X CGAA AGGGACCA 992

2948 CCCUCAAUU AUGUUAUU 492 AAUAACAU CUGAUGAG X CGAA AUUGAGGG 993

2949 CCUCAAUUA UGUUAUUU 493 AAAUAACA CUGAUGAG X CGAA AAUUGAGG 994

2953 AAUUAUGUU AUUUUAAU 494 AUUAAAAU CUGAUGAG X CGAA ACAUAAUU 995

2954 AUUAUGUUA UUUUAAUA 495 UAUUAAAA CUGAUGAG X CGAA AACAUAAU 996

2956 UAUGUUAUU UUAAUAAA 496 UUUAUUAA CUGAUGAG X CGAA AUAACAUA 997

2957 AUGUUAUUU UAAUAAAA 497 UUUUAUUA CUGAUGAG X CGAA AAUAACAU 998

2958 UGUUAUUUU AAUAAAAU 498 AUUUUAUU CUGAUGAG X CGAA AAAUAACA 999

2959 GUUAUUUUA AUAAAAUA 499 UAUUUUAU CUGAUGAG X CGAA AAAAUAAC 1000

2962 AUUUUAAUA AAAUAAAU 500 AUUUAUUU CUGAUGAG X CGAA AUUAAAAU 1001

2967 AAUAAAAUA AAUUAAAU 501 AUUUAAUU CUGAUGAG X CGAA AUUUUAUU 1002

Table XIII: Human C-rα/Hairpin Ribozyme and Target Sequence nt. Ribozyme SEQ ID. Target SEQ ID. Position Sequence No. Sequence No.

12 GCGGGA AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1003 UGACC GCC UCCCGC 1078

19 UGAGGG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1004 CUCCC GCU CCCUCA 1079

31 CCCCGC AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1005 CACCC GCC GCGGGG 1080

61 UUCGGC AGAA GCUU ACCAGAGAAACA X GUACAUUACCUGGUA 1006 AAGCU GCC GCCGAA 1081

64 UCGUUC AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 1007 CUGCC GCC GAACGA 1082

88 GAGCCA AGAA GCCC ACCAGAGAAACA X GUACAUUACCUGGUA 1008 GGGCG GCC UGGCUC 1083

205 AGAUGC AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1009 CUCCA GCU GCAUCU 1084

233 UAGCCA AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1010 CAGCA GUU UGGCUA 1085

258 GCCAUC AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 1011 CAUCA GAU GAUGGC 1086

276 AGAAGG AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1012 UCACA GAU CCUUCU 1087

370 UCAUAA AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1013 UGACU GCC UUAUGA 1088

427 GGAGAA AGAA GAAC ACCAGAGAAACA X GUACAUUACCUGGUA 1014 GUUCA GAC UUCUCC 1089

477 CGCAGC AGAA GUAU ACCAGAGAAACA X GUACAUUACCUGGUA 1015 AUACU GAU GCUGCG 1090

605 CCAUUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1016 UUCCU GCU CAAUGG 1091

626 CCACAA AGAA GACA ACCAGAGAAACA X GUACAUUACCUGGUA 1017 UGUCA GAC UUGUGG 1092

655 UGGUGC AGAA GUGC ACCAGAGAAACA X GUACAUUACCUGGUA 1018 GCACU GUA GCACCA 1093

789 CCUGGA AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1019 AGUCU GUU UCCAGG 1094

859 AUGAGG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1020 CUCCA GUC CCUCAU 1095

938 UCCACA AGAA GCGU ACCAGAGAAACA X GUACAUUACCUGGUA 1021 ACGCU GCC UGUGGA 1096

990 AGGUGA AGAA GAUU ACCAGAGAAACA X GUACAUUACCUGGUA 1022 AAUCA GCC UCACCU 1097

1002 GGACAG AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1023 CUUCA GCC CUGUCC 1098

1007 CUACUG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1024 GCCCU GUC CAGUAG 1099

1012 UGGGGC AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1025 GUCCA GUA GCCCCA 1100

1049 GUUUUC AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1026 UCACA GCC GAAAAC 1101

1089 CCCAGA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1027 CACCA GUA UCUGGG 1102

1181 CGAGUG AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1028 AUGCU GUC CACUCG 1103

1190 GACCCA AGAA GAGU ACCAGAGAAACA X GUACAUUACCUGGUA 1029 ACUCG GAU UGGGUC 1104

1215 CUUAUA AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1030 GAACU GUU UAUAAG 1105

1314 GCGCAG AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1031 UGGCU GUU CUGCGC 1106

1346 AUGAAA AGAA GAAU ACCAGAGAAACA X GUACAUUACCUGGUA 1032 AUUCU GCU UUUCAU 1107

1411 UGUAGA AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1033 CAGCA GCC UCUACA 1108

1451 UGGAAC AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1034 UUUCA GAU GUUCCA 1109

1481 UGAGCC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1035 CGGCA GAC GGCUCA 1110

1485 UCCCUG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 1036 AGACG GCU CAGGGA 1111

1653 CCAGAG AGAA GAGC ACCAGAGAAACA X GUACAUUACCUGGUA 1037 GCUCU GUC CUCUGG 1112

1705 ACUGGA AGAA GAAU ACCAGAGAAACA X GUACAUUACCUGGUA 1038 AUUCA GUU UCCAGU 1113

1712 ACAUCC AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1039 UUCCA GUC GGAUGU 1114

nt. Ribozyme SEQ ID. Target SEQ ID. Position Sequence No. Sequence No.

1716 GUAGAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1040 AGUCG GAU GUCUAC 1115

1751 CCCGUC AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1041 GAACU GAU GACGGG 1116

1796 AAGAUG AGAA GAUC ACCAGAGAAACA X GUACAUUACCUGGUA 1042 GAUCA GAU CAUCUU 1117

1833 ACUAAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1043 CCCCA GAU CUUAGU 1118

1858 CUUUGG AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1044 GAACU GCC CCAAAG 1119

1887 CACACA AGAA GCUA ACCAGAGAAACA X GUACAUUACCUGGUA 1045 UAGCU GAC UGUGUG 1120

1931 GACAGG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1046 CCCCA GAU CCUGUC 1121

1937 AUGGAA AGAA GGAU ACCAGAGAAACA X GUACAUUACCUGGUA 1047 AUCCU GUC UUCCAU 1122

1952 UGUUGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1048 GAGCU GCU CCAACA 1123

2013 AGUGUG AGAA GCCC ACCAGAGAAACA X GUACAUUACCUGGUA 1049 GGGCA GCC CACACU 1124

2045 GACGUG AGAA GCGU ACCAGAGAAACA X GUACAUUACCUGGUA 1050 ACGCU GAC CACGUC 1125

2063 AAGACA AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1051 AGGCU GCC UGUCUU 1126

2067 CUAGAA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1052 UGCCU GUC UUCUAG 1127

2090 CCUGAA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1053 CACCU GUC UUCAGG 1128

2140 AAAGGG AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1054 UUUCU GCU CCCUUU 1129

2204 CCUGUG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 1055 AGACU GCU CACAGG 1130

2292 GGAGGG AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1056 GUCCU GCU CCCUCC 1131

2326 ACAGAA AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1057 GCCCA GCC UUCUGU 1132

2333 CGCAUC AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1058 CUUCU GUA GAUGCG 1133

2381 AGCUGG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1059 GCCCA GCC CCAGCU 1134 CΛ

2387 GCCAAC AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1060 CCCCA GCU GUUGGC 1135

2390 GUAGCC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1061 CAGCU GUU GGCUAC 1136

2431 GGGCUG AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1062 AGGCA GUC CAGCCC 1137

2436 CAUCAG AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1063 GUCCA GCC CUGAUG 1138

2441 CUCCAC AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1064 GCCCU GAU GUGGAG 1139

2472 UCCAGA AGAA GAUU ACCAGAGAAACA X GUACAUUACCUGGUA 1065 AAUCA GCU UCUGGA 1140

2557 GUCCUG AGAA GUUU ACCAGAGAAACA X GUACAUUACCUGGUA 1066 AAACA GCC CAGGAC 1141

2567 AGUCUC AGAA GUCC ACCAGAGAAACA X GUACAUUACCUGGUA 1067 GGACU GCC GAGACU 1142

2582 CCUUCG AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1068 UGGCC GCC CGAAGG 1143

2596 UACCAA AGAA GGCU ACCAGAGAAACA X GUACAUUACCUGGUA 1069 AGCCU GCU UUGGUA 1144

2644 CUUAGA AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1070 UCACA GCU UCUAAG 1145

2710 UGAGAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1071 UCUCA GCC CUCUCA 1146

2725 AUGGAA AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1072 GAGCA GUC UUCCAU 1147

2761 CAUAGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1073 GAGCU GCC CCUAUG 1148

2788 GAAACA AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 1074 GGCCA GCC UGUUUC 1149

2792 UAGAGA AGAA GGCU ACCAGAGAAACA X GUACAUUACCUGGUA 1075 AGCCU GUU UCUCUA 1150

2820 AAACAA AGAA GUUU ACCAGAGAAACA X GUACAUUACCUGGUA 1076 AAACA GCC UUGUUU 1151

2933 GGGACC AGAA GAUA ACCAGAGAAACA X GUACAUUACCUGGUA 1077 UAUCU GAU GGUCCC 1152

Where "X" represents stem IV region of a Haiφin ribozyme. The length of stem IV may be 2 base-pairs

Table XIV. Hammerhead Ribozyme Sites for A-Raf

SEQ ID. SEQ

RZ No. Substrate ID. No.

UCCACCCU CUGAUGAG X CGAA AUUGGGUC 1153 GACCCAAUA AGGGUGGA 1461

CUCUGCGG CUGAUGAG X CGAA ACUCAGCC 1154 GGCUGAGUC CCGCAGAG 1462

ACUCUCGU CUGAUGAG X CGAA AUUGGCUC 1155 GAGCCAAUA ACGAGAGU 1463

GCCUCUCG CUGAUGAG X CGAA ACUCUCGU 1156 ACGAGAGUC CGAGAGGC 1464

UCCUCACA CUGAUGAG X CGAA AGUCCGCC 1157 GGCGGACUC UGUGAGGA 1465

CACGCCGC CUGAUGAG X CGAA ACAGCCGC 1158 GCGGCUGUA GCGGCGUG 1466

GAUGGGCU CUGAUGAG X CGAA AGGUGGGG 1159 CCCCACCUC AGCCCAUC 1467

UUUGUCAA CUGAUGAG X CGAA AUGGGCUG 1160 CAGCCCAUC UUGACAAA 1468

AUUUUGUC CUGAUGAG X CGAA AGAUGGGC 1161 GCCCAUCUU GACAAAAU 1469

GAGCCUUA CUGAUGAG X CGAA AUUUUGUC 1162 GACAAAAUC UAAGGCUC 1470

UGGAGCCU CUGAUGAG X CGAA AGAUUUUG 1163 CAAAAUCUA AGGCUCCA 1471

GCUCCAUG CUGAUGAG X CGAA AGCCUUAG 1164 CUAAGGCUC CAUGGAGC 1472

CUGCCCGG CUGAUGAG X CGAA AUGGCUCG 1165 CGAGCCAUC CCGGGCAG 1473

UAUACUUU CUGAUGAG X CGAA ACGGUGCC 1166 GGCACCGUC AAAGUAUA 1474

GGCAGGUA CUGAUGAG X CGAA ACUUUGAC 1167 GUCAAAGUA UACCUGCC 1475

UGGGCAGG CUGAUGAG X CGAA AUACUUUG 1168 CAAAGUAUA CCUGCCCA 1476

CCAUCCCG CUGAUGAG X CGAA ACAGUCAC 1169 GUGACUGUC CGGGAUGG 1477

GAGUCGUA CUGAUGAG X CGAA ACACUCAU 1170 AUGAGUGUC UACGACUC 1478

GAGAGUCG CUGAUGAG X CGAA AGACACUC 1171 GAGUGUCUA CGACUCUC 1479

UGUCUAGA CUGAUGAG X CGAA AGUCGUAG 1172 CUACGACUC UCUAGACA 1480

CUUGUCUA CUGAUGAG X CGAA AGAGUCGU 1173 ACGACUCUC UAGACAAG 1481

GCCUUGUC CUGAUGAG X CGAA AGAGAGUC 1174 GACUCUCUA GACAAGGC 1482

CUGAUUUA CUGAUGAG X CGAA ACCCCGCA 1175 UGCGGGGUC UAAAUCAG 1483

UCCUGAUU CUGAUGAG X CGAA AGACCCCG 1176 CGGGGUCUA AAUCAGGA 1484

GCAGUCCU CUGAUGAG X CGAA AUUUAGAC 1177 GUCUAAAUC AGGACUGC 1485

AGUCGGUA CUGAUGAG X CGAA ACCACACA 1178 UGUGUGGUC UACCGACU 1486

UGAGUCGG CUGAUGAG X CGAA AGACCACA 1179 UGUGGUCUA CCGACUCA 1487

CCCUUGAU CUGAUGAG X CGAA AGUCGGUA 1180 UACCGACUC AUCAAGGG 1488

CGUCCCUU CUGAUGAG X CGAA AUGAGUCG 1181 CGACUCAUC AAGGGACG 1489

CAGGCAGU CUGAUGAG X CGAA ACCGUCUU 1182 AAGACGGUC ACUGCCUG 1490

AGGGGAGC CUGAUGAG X CGAA AUGGCUGU 1183 ACAGCCAUU GCUCCCCU 1491

AUCCAGGG CUGAUGAG X CGAA AGCAAUGG 1184 CCAUUGCUC CCCUGGAU 1492

UCGACAAU CUGAUGAG X CGAA AGCUCCUC 1185 GAGGAGCUC AUUGUCGA 1493

ACCUCGAC CUGAUGAG X CGAA AUGAGCUC 1186 GAGCUCAUU GUCGAGGU 1494

AGGACCUC CUGAUGAG X CGAA ACAAUGAG 1187 CUCAUUGUC GAGGUCCU 1495

UCUUCAAG CUGAUGAG X CGAA ACCUCGAC 1188 GUCGAGGUC CUUGAAGA 1496

ACAUCUUC CUGAUGAG X CGAA AGGACCUC 1189 GAGGUCCUU GAAGAUGU 1497

GUCAGCGG CUGAUGAG X CGAA ACAUCUUC 1190 GAAGAUGUC CCGCUGAC 1498

CCGUACAA CUGAUGAG X CGAA AUUGUGCA 1191 UGCACAAUU UUGUACGG 1499

UCCGUACA CUGAUGAG X CGAA AAUUGUGC 1192 GCACAAUUU UGUACGGA 1500

UUCCGUAC CUGAUGAG X CGAA AAAUUGUG 1193 CACAAUUUU GUACGGAA 1501

GUCUUCCG CUGAUGAG X CGAA ACAAAAUU 1194 AAUUUUGUA CGGAAGAC 1502

GGCUGAAG CUGAUGAG X CGAA AGGUCUUC 1195 GAAGACCUU CUUCAGCC 1503

AGGCUGAA CUGAUGAG X CGAA AAGGUCUU 1196 AAGACCUUC UUCAGCCU 1504

CCAGGCUG CUGAUGAG X CGAA AGAAGGUC 1197 GACCUUCUU CAGCCUGG 1505

GCCAGGCU CUGAUGAG X CGAA AAGAAGGU 1198 ACCUUCUUC AGCCUGGC 1506

AGUCACAG CUGAUGAG X CGAA ACGCCAGG 1199 CCUGGCGUU CUGUGACU 1507

AAGUCACA CUGAUGAG X CGAA AACGCCAG 1200 CUGGCGUUC UGUGACUU 1508

UAAGGCAG CUGAUGAG X CGAA AGUCACAG 1201 CUGUGACUU CUGCCUUA 1509

UUAAGGCA CUGAUGAG X CGAA AAGUCACA 1202 UGUGACUUC UGCCUUAA 1510

AGAAACUU CUGAUGAG X CGAA AGGCAGAA 1203 UUCUGCCUU AAGUUUCU 1511

CAGAAACU CUGAUGAG X CGAA AAGGCAGA 1204 UCUGCCUUA AGUUUCUG 1512 SEQ ID. SEQ

Pos RZ No. Substrate ID. No.

547 GGAACAGA CUGAUGAG X CGAA ACUUAAGG 1205 CCUUAAGUU UCUGUUCC 1513

548 UGGAACAG CUGAUGAG X CGAA AACUUAAG 1206 CUUAAGUUU CUGUUCCA 1514

549 AUGGAACA CUGAUGAG X CGAA AAACUUAA 1207 UUAAGUUUC UGUUCCAU 1515

553 AGCCAUGG CUGAUGAG X CGAA ACAGAAAC 1208 GUUUCUGUU CCAUGGCU 1516

554 AAGCCAUG CUGAUGAG X CGAA AACAGAAA 1209 UUUCUGUUC CAUGGCUU 1517

562 GGCAACGG CUGAUGAG X CGAA AGCCAUGG 1210 CCAUGGCUU CCGUUGCC 1518

563 UGGCAACG CUGAUGAG X CGAA AAGCCAUG 1211 CAUGGCUUC CGUUGCCA 1519

567 GGUUUGGC CUGAUGAG X CGAA ACGGAAGC 1212 GCUUCCGUU GCCAAACC 1520

583 GGAACUUG CUGAUGAG X CGAA AGCCACAG 1213 CUGUGGCUA CAAGUUCC 1521

589 GCUGGUGG CUGAUGAG X CGAA ACUUGUAG 1214 CUACAAGUU CCACCAGC 1522

590 UGCUGGUG CUGAUGAG X CGAA AACUUGUA 1215 UACAAGUUC CACCAGCA 1523

600 GGAGGAAC CUGAUGAG X CGAA AUGCUGGU 1216 ACCAGCAUU GUUCCUCC 1524

603 CUUGGAGG CUGAUGAG X CGAA ACAAUGCU 1217 AGCAUUGUU CCUCCAAG 1525

604 CCUUGGAG CUGAUGAG X CGAA AACAAUGC 1218 GCAUUGUUC CUCCAAGG 1526

607 GGACCUUG CUGAUGAG X CGAA AGGAACAA 1219 UUGUUCCUC CAAGGUCC 1527

614 ACUGUGGG CUGAUGAG X CGAA ACCUUGGA 1220 UCCAAGGUC CCCACAGU 1528

623 UCAACACA CUGAUGAG X CGAA ACUGUGGG 1221 CCCACAGUC UGUGUUGA 1529

629 CUCAUGUC CUGAUGAG X CGAA ACACAGAC 1222 GUCUGUGUU GACAUGAG 1530

639 GCGGUUGG CUGAUGAG X CGAA ACUCAUGU 1223 ACAUGAGUA CCAACCGC 1531

655 UGUGGUAG CUGAUGAG X CGAA ACUGUUGG 1224 CCAACAGUU CUACCACA 1532

656 CUGUGGUA CUGAUGAG X CGAA AACUGUUG 1225 CAACAGUUC UACCACAG 1533

658 CACUGUGG CUGAUGAG X CGAA AGAACUGU 1226 ACAGUUCUA CCACAGUG 1534

668 AAAUCCUG CUGAUGAG X CGAA ACACUGUG 1227 CACAGUGUC CAGGAUUU 1535

675 UCCGGACA CUGAUGAG X CGAA AUCCUGGA 1228 UCCAGGAUU UGUCCGGA 1536

676 CUCCGGAC CUGAUGAG X CGAA AAUCCUGG 1229 CCAGGAUUU GUCCGGAG 1537

679 AGCCUCCG CUGAUGAG X CGAA ACAAAUCC 1230 GGAUUUGUC CGGAGGCU 1538

688 GCUGUCUG CUGAUGAG X CGAA AGCCUCCG 1231 CGGAGGCUC CAGACAGC 1539

705 GUUCGAGG CUGAUGAG X CGAA AGCCUCAU 1232 AUGAGGCUC CCUCGAAC 1540

709 GGCGGUUC CUGAUGAG X CGAA AGGGAGCC 1233 GGCUCCCUC GAACCGCC 1541

730 GGGUUAGC CUGAUGAG X CGAA ACUCAUUC 1234 GAAUGAGUU GCUAACCC 1542

734 UGGGGGGU CUGAUGAG X CGAA AGCAACUC 1235 GAGUUGCUA ACCCCCCA 1543

747 GGGGCUGG CUGAUGAG X CGAA ACCCUGGG 1236 CCCAGGGUC CCAGCCCC 1544

784 GGAAGGGG CUGAUGAG X CGAA AGUGCUCC 1237 GGAGCACUU CCCCUUCC 1545

785 GGGAAGGG CUGAUGAG X CGAA AAGUGCUC 1238 GAGCACUUC CCCUUCCC 1546

790 GGGCAGGG CUGAUGAG X CGAA AGGGGAAG 1239 CUUCCCCUU CCCUGCCC 1547

791 GGGGCAGG CUGAUGAG X CGAA AAGGGGAA 1240 UUCCCCUUC CCUGCCCC 1548

815 AUGCGCUG CUGAUGAG X CGAA AGGGGGGC 1241 GCCCCCCUA CAGCGCAU 1549

824 GUGGAGCG CUGAUGAG X CGAA AUGCGCUG 1242 CAGCGCAUC CGCUCCAC 1550

829 UGGACGUG CUGAUGAG X CGAA AGCGGAUG 1243 CAUCCGCUC CACGUCCA 1551

835 UGGGAGUG CUGAUGAG X CGAA ACGUGGAG 1244 CUCCACGUC CACUCCCA 1552

840 GACGUUGG CUGAUGAG X CGAA AGUGGACG 1245 CGUCCACUC CCAACGUC 1553

848 ACCAUAUG CUGAUGAG X CGAA ACGUUGGG 1246 CCCAACGUC CAUAUGGU 1554

852 GCUGACCA CUGAUGAG X CGAA AUGGACGU 1247 ACGUCCAUA UGGUCAGC 1555

857 GUGGUGCU CUGAUGAG X CGAA ACCAUAUG 1248 CAUAUGGUC AGCACCAC 1556

880 UGAGGUUG CUGAUGAG X CGAA AGUCCAUG 1249 CAUGGACUC CAACCUCA 1557

887 AGCUGGAU CUGAUGAG X CGAA AGGUUGGA 1250 UCCAACCUC AUCCAGCU 1558

890 GUGAGCUG CUGAUGAG X CGAA AUGAGGUU 1251 AACCUCAUC CAGCUCAC 1559

896 UGGCCAGU CUGAUGAG X CGAA AGCUGGAU 1252 AUCCAGCUC ACUGGCCA 1560

909 AGUGCUGA CUGAUGAG X CGAA ACUCUGGC 1253 GCCAGAGUU UCAGCACU 1561

910 CAGUGCUG CUGAUGAG X CGAA AACUCUGG 1254 CCAGAGUUU CAGCACUG 1562

911 UCAGUGCU CUGAUGAG X CGAA AAACUCUG 1255 CAGAGUUUC AGCACUGA 1563

930 UCCUCUAC CUGAUGAG X CGAA ACCGGCAG 1256 CUGCCGGUA GUAGAGGA 1564

933 ACCUCCUC CUGAUGAG X CGAA ACUACCGG 1257 CCGGUAGUA GAGGAGGU 1565

942 UCCAUCAC CUGAUGAG X CGAA ACCUCCUC 1258 GAGGAGGUA GUGAUGGA 1566 SEQ ID. SEQ

Pos RZ No. Substrate ID. No.

985 UCCCCGAG CUGAUGAG X CGAA ACACGCUG 1259 CAGCGUGUC CUCGGGGA 1567

988 UCCUCCCC CUGAUGAG X CGAA AGGACACG 1260 CGUGUCCUC GGGGAGGA 1568

1000 AAUGUGGG CUGAUGAG X CGAA ACUUCCUC 1261 GAGGAAGUC CCCACAUU 1569

1008 UGACUUGG CUGAUGAG X CGAA AUGUGGGG 1262 CCCCACAUU CCAAGUCA 1570

1009 GUGACUUG CUGAUGAG X CGAA AAUGUGGG 1263 CCCACAUUC CAAGUCAC 1571

1015 CUGCUGGU CUGAUGAG X CGAA ACUUGGAA 1264 UUCCAAGUC ACCAGCAG 1572

1042 CGGCCAAG CUGAUGAG X CGAA ACUUCCGC 1265 GCGGAAGUC CUUGGCCG 1573

1045 CAUCGGCC CUGAUGAG X CGAA AGGACUUC 1266 GAAGUCCUU GGCCGAUG 1574

1081 ANUCCCGG CUGAUGAG X CGAA ACCCCAGG 1267 CCUGGGGUA CCGGGANU 1575

1090 AAUAGCCU CUGAUGAG X CGAA ANUCCCGG 1268 CCGGGANUC AGGCUAUU 1576

1096 CCCAGUAA CUGAUGAG X CGAA AGCCUGAN 1269 NUCAGGCUA UUACUGGG 1577

1098 CUCCCAGU CUGAUGAG X CGAA AUAGCCUG 1270 CAGGCUAUU ACUGGGAG 1578

1099 CCUCCCAG CUGAUGAG X CGAA AAUAGCCU 1271 AGGCUAUUA CUGGGAGG 1579

1109 CUGGGUGG CUGAUGAG X CGAA ACCUCCCA 1272 UGGGAGGUA CCACCCAG 1580

1142 CCCGUCCC CUGAUGAG X CGAA AUCCUCUU 1273 AAGAGGAUC GGGACGGG 1581

1153 UGCCAAAC CUGAUGAG X CGAA AGCCCGUC 1274 GACGGGCUC GUUUGGCA 1582

1156 CGGUGCCA CUGAUGAG X CGAA ACGAGCCC 1275 GGGCUCGUU UGGCACCG 1583

1157 ACGGUGCC CUGAUGAG X CGAA AACGAGCC 1276 GGCUCGUUU GGCACCGU 1584

1168 GCCCUCGA CUGAUGAG X CGAA ACACGGUG 1277 CACCGUGUU UCGAGGGC 1585

1169 CGCCCUCG CUGAUGAG X CGAA AACACGGU 1278 ACCGUGUUU CGAGGGCG 1586

1170 CCGCCCUC CUGAUGAG X CGAA AAACACGG 1279 CCGUGUUUC GAGGGCGG 1587

1208 GACACCUU CUGAUGAG X CGAA AGCACCUU 1280 AAGGUGCUC AAGGUGUC 1588

1216 UGGGCUGG CUGAUGAG X CGAA ACACCUUG 1281 CAAGGUGUC CCAGCCCA 1589

1245 AUUCUUGA CUGAUGAG X CGAA AGCCUGGG 1282 CCCAGGCUU UCAAGAAU 1590

1246 CAUUCUUG CUGAUGAG X CGAA AAGCCUGG 1283 CCAGGCUUU CAAGAAUG 1591

1247 UCAUUCUU CUGAUGAG X CGAA AAAGCCUG 1284 CAGGCUUUC AAGAAUGA 1592

1268 GUCUUCCU CUGAUGAG X CGAA AGCACCUG 1285 CAGGUGCUC AGGAAGAC 1593

1286 AAGAUGUU CUGAUGAG X CGAA ACAUGUCG 1286 CGACAUGUC AACAUCUU 1594

1292 AACAGCAA CUGAUGAG X CGAA AUGUUGAC 1287 GUCAACAUC UUGCUGUU 1595

1294 UAAACAGC CUGAUGAG X CGAA AGAUGUUG 1288 CAACAUCUU GCUGUUUA 1596

1300 AGCCCAUA CUGAUGAG X CGAA ACAGCAAG 1289 CUUGCUGUU UAUGGGCU 1597

1301 AAGCCCAU CUGAUGAG X CGAA AACAGCAA 1290 UUGCUGUUU AUGGGCUU 1598

1302 GAAGCCCA CUGAUGAG X CGAA AAACAGCA 1291 UGCUGUUUA UGGGCUUC 1599

1309 GGGUCAUG CUGAUGAG X CGAA AGCCCAUA 1292 UAUGGGCUU CAUGACCC 1600

1310 CGGGUCAU CUGAUGAG X CGAA AAGCCCAU 1293 AUGGGCUUC AUGACCCG 1601

1327 UGAUGGCA CUGAUGAG X CGAA AUCCCGGC 1294 GCCGGGAUU UGCCAUCA 1602

1328 AUGAUGGC CUGAUGAG X CGAA AAUCCCGG 1295 CCGGGAUUU GCCAUCAU 1603

1334 UGUGUGAU CUGAUGAG X CGAA AUGGCAAA 1296 UUUGCCAUC AUCACACA 1604

1337 CACUGUGU CUGAUGAG X CGAA AUGAUGGC 1297 GCCAUCAUC ACACAGUG 1605

1357 AGAGGCUG CUGAUGAG X CGAA AGCCCUCA 1298 UGAGGGCUC CAGCCUCU 1606

1364 UGAUGGUA CUGAUGAG X CGAA AGGCUGGA 1299 UCCAGCCUC UACCAUCA 1607

1366 GGUGAUGG CUGAUGAG X CGAA AGAGGCUG 1300 CAGCCUCUA CCAUCACC 1608

1371 AUGCAGGU CUGAUGAG X CGAA AUGGUAGA 1301 UCUACCAUC ACCUGCAU 1609

1396 CCAUGUCG CUGAUGAG X CGAA AGCGUGUG 1302 CACACGCUU CGACAUGG 1610

1397 ACCAUGUC CUGAUGAG X CGAA AAGCGUGU 1303 ACACGCUUC GACAUGGU 1611

1406 AUGAGCUG CUGAUGAG X CGAA ACCAUGUC 1304 GACAUGGUC CAGCUCAU 1612

1412 ACGUCGAU CUGAUGAG X CGAA AGCUGGAC 1305 GUCCAGCUC AUCGACGU 1613

1415 GCCACGUC CUGAUGAG X CGAA AUGAGCUG 1306 CAGCUCAUC GACGUGGC 1614

1450 CAUGGAGG CUGAUGAG X CGAA AGUCCAUG 1307 CAUGGACUA CCUCCAUG 1615

1454 UUGGCAUG CUGAUGAG X CGAA AGGUAGUC 1308 GACUACCUC CAUGCCAA 1616

1469 CGGUGGAU CUGAUGAG X CGAA AUGUUCUU 1309 AAGAACAUC AUCCACCG 1617

1472 UCUCGGUG CUGAUGAG X CGAA AUGAUGUU 1310 AACAUCAUC CACCGAGA 1618

1482 AGACUUGA CUGAUGAG X CGAA AUCUCGGU 1311 ACCGAGAUC UCAAGUCU 1619

1484 UUAGACUU CUGAUGAG X CGAA AGAUCUCG 1312 CGAGAUCUC AAGUCUAA 1620 SEQ ID. SEQ

Pos RZ No. Substrate ID. No.

1489 UGUUGUUA CUGAUGAG X CGAA ACUUGAGA 1313 UCUCAAGUC UAACAACA 1621

1491 GAUGUUGU CUGAUGAG X CGAA AGACUUGA 1314 UCAAGUCUA ACAACAUC 1622

1499 UGUAGGAA CUGAUGAG X CGAA AUGUUGUU 1315 AACAACAUC UUCCUACA 1623

1501 CAUGUAGG CUGAUGAG X CGAA AGAUGUUG 1316 CAACAUCUU CCUACAUG 1624

1502 UCAUGUAG CUGAUGAG X CGAA AAGAUGUU 1317 AACAUCUUC CUACAUGA 1625

1505 CCCUCAUG CUGAUGAG X CGAA AGGAAGAU 1318 AUCUUCCUA CAUGAGGG 1626

1517 UUCACCGU CUGAUGAG X CGAA AGCCCCUC 1319 GAGGGGCUC ACGGUGAA 1627

1529 AAGUCACC CUGAUGAG X CGAA AUCUUCAC 1320 GUGAAGAUC GGUGACUU 1628

1537 CCAAGCCA CUGAUGAG X CGAA AGUCACCG 1321 CGGUGACUU UGGCUUGG 1629

1538 GCCAAGCC CUGAUGAG X CGAA AAGUCACC 1322 GGUGACUUU GGCUUGGC 1630

1543 CUGUGGCC CUGAUGAG X CGAA AGCCAAAG 1323 CUUUGGCUU GGCCACAG 1631

1560 GCUCCAUC CUGAUGAG X CGAA AGUCUUCA 1324 UGAAGACUC GAUGGAGC 1632

1582 GCUGCUCC CUGAUGAG X CGAA AGGGCUGG 1325 CCAGCCCUU GGAGCAGC 1633

1594 CAGAUCCU CUGAUGAG X CGAA AGGGCUGC 1326 GCAGCCCUC AGGAUCUG 1634

1600 ACAGCACA CUGAUGAG X CGAA AUCCUGAG 1327 CUCAGGAUC UGUGCUGU 1635

1628 UGCAUACG CUGAUGAG X CGAA AUCACCUC 1328 GAGGUGAUC CGUAUGCA 1636

1632 GUCCUGCA CUGAUGAG X CGAA ACGGAUCA 1329 UGAUCCGUA UGCAGGAC 1637

1651 GGAAGCUG CUGAUGAG X CGAA AGGGGUUC 1330 GAACCCCUA CAGCUUCC 1638

1657 CUGACUGG CUGAUGAG X CGAA AGCUGUAG 1331 CUACAGCUU CCAGUCAG 1639

1658 UCUGACUG CUGAUGAG X CGAA AAGCUGUA 1332 -_ UACAGCUUC CAGUCAGA 1640

1663 AGACGUCU CUGAUGAG X CGAA ACUGGAAG 1333 CUUCCAGUC AGACGUCU 1641

1670 UAGGCAUA CUGAUGAG X CGAA ACGUCUGA 1334 UCAGACGUC UAUGCCUA 1642

1672 CGUAGGCA CUGAUGAG X CGAA AGACGUCU 1335 AGACGUCUA UGCCUACG 1643

1678 CAACCCCG CUGAUGAG X CGAA AGGCAUAG 1336 CUAUGCCUA CGGGGUUG 1644

1685 UAGAGCAC CUGAUGAG X CGAA ACCCCGUA 1337 UACGGGGUU GUGCUCUA 1645

1691 AGCUCGUA CUGAUGAG X CGAA AGCACAAC 1338 GUUGUGCUC UACGAGCU 1646

1693 UAAGCUCG CUGAUGAG X CGAA AGAGC CA 1339 UGUGCUCUA CGAGCUUA 1647

1700 CCAGUCAU CUGAUGAG X CGAA AGCUCGUA 1340 UACGAGCUU AUGACUGG 1648

1701 GCCAGUCA CUGAUGAG X CGAA AAGCUCGU 1341 ACGAGCUUA UGACUGGC 1649

1711 AAGGCAGU CUGAUGAG X CGAA AGCCAGUC 1342 GACUGGCUC ACUGCCUU 1650

1719 GUGGCUGU CUGAUGAG X CGAA AGGCAGUG 1343 CACUGCCUU ACAGCCAC 1651

1720 UGUGGCUG CUGAUGAG X CGAA AAGGCAGU 1344 ACUGCCUUA CAGCCACA 1652

1730 CGGCAGCC CUGAUGAG X CGAA AUGUGGCU 1345 AGCCACAUU GGCUGCCG 1653

1748 AUAAAGAU CUGAUGAG X CGAA AUCUGGUC 1346 GACCAGAUU AUCUUUAU 1654

1749 CAUAAAGA CUGAUGAG X CGAA AAUCUGGU 1347 ACCAGAUUA UCUUUAUG 1655

1751 ACCAUAAA CUGAUGAG X CGAA AUAAUCUG 1348 CAGAUUAUC UUUAUGGU 1656

1753 CCACCAUA CUGAUGAG X CGAA AGAUAAUC 1349 GAUUAUCUU UAUGGUGG 1657

1754 CCCACCAU CUGAUGAG X CGAA AAGAUAAU 1350 AUUAUCUUU AUGGUGGG 1658

1755 GCCCACCA CUGAUGAG X CGAA AAAGAUAA 1351 UUAUCUUUA UGGUGGGC 1659

1771 GGGACAGA CUGAUGAG X CGAA AGCCACGG 1352 CCGUGGCUA UCUGUCCC 1660

1773 CGGGGACA CUGAUGAG X CGAA AUAGCCAC 1353 GUGGCUAUC UGUCCCCG 1661

1777 GGUCCGGG CUGAUGAG X CGAA ACAGAUAG 1354 CUAUCUGUC CCCGGACC 1662

1787 AUUUUGCU CUGAUGAG X CGAA AGGUCCGG 1355 CCGGACCUC AGCAAAAU 1663

1796 UUGCUGGA CUGAUGAG X CGAA AUUUUGCU 1356 AGCAAAAUC UCCAGCAA 1664

1798 AGUUGCUG CUGAUGAG X CGAA AGAUUUUG 1357 CAAAAUCUC CAGCAACU 1665

1834 GGCAGUCA CUGAUGAG X CGAA ACAGCAGG 1358 CCUGCUGUC UGACUGCC 1666

1844 UGGAACUU CUGAUGAG X CGAA AGGCAGUC 1359 GACUGCCUC AAGUUCCA 1667

1849 CCCGCUGG CUGAUGAG X CGAA ACUUGAGG 1360 CCUCAAGUU CCAGCGGG 1668

1850 UCCCGCUG CUGAUGAG X CGAA AACUUGAG 1361 CUCAAGUUC CAGCGGGA 1669

1871 UGGGGGAA CUGAUGAG X CGAA AGGGGCCG 1362 CGGCCCCUC UUCCCCCA 1670

1873 UCUGGGGG CUGAUGAG X CGAA AGAGGGGC 1363 GCCCCUCUU CCCCCAGA 1671

1874 AUCUGGGG CUGAUGAG X CGAA AAGAGGGG 1364 CCCCUCUUC CCCCAGAU 1672

1883 GUGGCCAG CUGAUGAG X CGAA AUCUGGGG 1365 CCCCAGAUC CUGGCCAC 1673

1895 AGCAGCUC CUGAUGAG X CGAA AUUGUGGC 1366 GCCACAAUU GAGCUGCU 1674 SEQ ID. SEQ

Pos RZ No. Substrate ID. No.

1912 UGGGGAGU CUGAUGAG X CGAA ACCGUUGC 1367 GCAACGGUC ACUCCCCA 1675

1916 AUCUUGGG CUGAUGAG X CGAA AGUGACCG 1368 CGGUCACUC CCCAAGAU 1676

1925 CUCCGCUC CUGAUGAG X CGAA AUCUUGGG 1369 CCCAAGAUU GAGCGGAG 1677

1939 AGGGUUCC CUGAUGAG X CGAA AGGCACUC 1370 GAGUGCCUC GGAACCCU 1678

1948 GGUGCAAG CUGAUGAG X CGAA AGGGUUCC 1371 GGAACCCUC CUUGCACC 1679

1951 UGCGGUGC CUGAUGAG X CGAA AGGAGGGU 1372 ACCCUCCUU GCACCGCA 1680

1975 AGGCAGGC CUGAUGAG X CGAA ACUCAUCG 1373 CGAUGAGUU GCCUGCCU 1681

1988 GCGCUGAG CUGAUGAG X CGAA AGGCAGGC 1374 GCCUGCCUA CUCAGCGC ^•1682

1991 GCUGCGCU CUGAUGAG X CGAA AGUAGGCA 1375 UGCCUACUC AGCGCAGC 1683

2006 UAAGGCAC CUGAUGAG X CGAA AGGCGGGC 1376 GCCCGCCUU GUGCCUUA 1684

2013 CGGGGCCU CUGAUGAG X CGAA AGGCACAA 1377 UUGUGCCUU AGGCCCCG 1685

2014 GCGGGGCC CUGAUGAG X CGAA AAGGCACA 1378 UGUGCCUUA GGCCCCGC 1686

2044 AGGGCUGA CUGAUGAG X CGAA AUUGGCUC 1379 GAGCCAAUC UCAGCCCU 1687

2046 GGAGGGCU CUGAUGAG X CGAA AGAUUGGC 1380 GCCAAUCUC AGCCCUCC 1688

2053 UUGGCGUG CUGAUGAG X CGAA AGGGCUGA 1381 UCAGCCCUC CACGCCAA 1689

2069 UGGUGGGC CUGAUGAG X CGAA AGGCUCCU 1382 AGGAGCCUU GCCCACCA 1690

2084 CGAACAUU CUGAUGAG X CGAA AUUGGCUG 1383 CAGCCAAUC AAUGUUCG 1691

2090 CAGAGACG CUGAUGAG X CGAA ACAUUGAU 1384 AUCAAUGUU CGUCUCUG 1692

2091 GCAGAGAC CUGAUGAG X CGAA AACAUUGA 1385 UCAAUGUUC GUCUCUGC 1693

2094 AGGGCAGA CUGAUGAG X CGAA ACGAACAU 1386 AUGUUCGUC UCUGCCCU 1694

2096 UCAGGGCA CUGAUGAG X CGAA AGACGAAC 1387 GUUCGUCUC UGCCCUGA 1695

2113 GGGAUCCU CUGAUGAG X CGAA AGGCAGCA 1388 UGCUGCCUC AGGAUCCC 1696

2119 GAAUGGGG CUGAUGAG X CGAA AUCCUGAG 1389 CUCAGGAUC CCCCAUUC 1697

2126 GGGUGGGG CUGAUGAG X CGAA AUGGGGGA 1390 UCCCCCAUU CCCCACCC 1698

2127 AGGGUGGG CUGAUGAG X CGAA AAUGGGGG 1391 CCCCCAUUC CCCACCCU 1699

2151 CACAUGGG CUGAUGAG X CGAA ACCCCCUC 1392 GAGGGGGUC CCCAUGUG 1700

2162 AACUGGAA CUGAUGAG X CGAA AGCACAUG 1393 CAUGUGCUU UUCCAGUU 1701

2163 GAACUGGA CUGAUGAG X CGAA AAGCACAU 1394 AUGUGCUUU UCCAGUUC 1702

2164 AGAACUGG CUGAUGAG X CGAA AAAGCACA 1395 UGUGCUUUU CCAGUUCU 1703

2165 AAGAACUG CUGAUGAG X CGAA AAAAGCAC 1396 GUGCUUUUC CAGUUCUU 1704

2170 UCCAGAAG CUGAUGAG X CGAA ACUGGAAA 1397 UUUCCAGUU CUUCUGGA 1705

2171 UUCCAGAA CUGAUGAG X CGAA AACUGGAA 1398 UUCCAGUUC UUCUGGAA 1706

2173 AAUUCCAG CUGAUGAG X CGAA AGAACUGG 1399 CCAGUUCUU CUGGAAUU 1707

2174 CAAUUCCA CUGAUGAG X CGAA AAGAACUG 1400 CAGUUCUUC UGGAAUUG 1708

2181 GGUCCCCC CUGAUGAG X CGAA AUUCCAGA 1401 UCUGGAAUU GGGGGACC 1709

2214 AUGGAGGA CUGAUGAG X CGAA ACAGGGGG 1402 CCCCCUGUC UCCUCCAU 1710

2216 UGAUGGAG CUGAUGAG X CGAA AGACAGGG 1403 CCCUGUCUC CUCCAUCA 1711

2219 AAAUGAUG CUGAUGAG X CGAA AGGAGACA 1404 UGUCUCCUC CAUCAUUU 1712

2223 AACCAAAU CUGAUGAG X CGAA AUGGAGGA 1405 UCCUCCAUC AUUUGGUU 1713

2226 GGAAACCA CUGAUGAG X CGAA AUGAUGGA 1406 UCCAUCAUU UGGUUUCC 1714

2227 AGGAAACC CUGAUGAG X CGAA AAUGAUGG 1407 CCAUCAUUU GGUUUCCU 1715

2231 CAAGAGGA CUGAUGAG X CGAA ACCAAAUG 1408 CAUUUGGUU UCCUCUUG 1716

2232 CCAAGAGG CUGAUGAG X CGAA AACCAAAU 1409 AUUUGGUUU CCUCUUGG 1717

2233 GCCAAGAG CUGAUGAG X CGAA AAACCAAA 1410 UUUGGUUUC CUCUUGGC 1718

2236 AAAGCCAA CUGAUGAG X CGAA AGGAAACC 1411 GGUUUCCUC UUGGCUUU 1719

2238 CCAAAGCC CUGAUGAG X CGAA AGAGGAAA 1412 UUUCCUCUU GGCUUUGG 1720

2243 UAUCCCCA CUGAUGAG X CGAA AGCCAAGA 1413 UCUUGGCUU UGGGGAUA 1721

2244 GUAUCCCC CUGAUGAG X CGAA AAGCCAAG 1414 CUUGGCUUU GGGGAUAC 1722

2251 UUUAGAAG CUGAUGAG X CGAA AUCCCCAA 1415 UUGGGGAUA CUUCUAAA 1723

2254 AAAUUUAG CUGAUGAG X CGAA AGUAUCCC 1416 GGGAUACUU CUAAAUUU 1724

2255 AAAAUUUA CUGAUGAG X CGAA AAGUAUCC 1417 GGAUACUUC UAAAUUUU 1725

2257 CCAAAAUU CUGAUGAG X CGAA AGAAGUAU 1418 AUACUUCUA AAUUUUGG 1726

2261 GCUCCCAA CUGAUGAG X CGAA AUUUAGAA 1419 UUCUAAAUU UUGGGAGC 1727

2262 AGCUCCCA CUGAUGAG X CGAA AAUUUAGA 1420 UCUAAAUUU UGGGAGCU 1728 SEQ ID. SEQ

Pos RZ No. Substrate ID. No.

2263 GAGCUCCC CUGAUGAG X CGAA AAAUUUAG 1421 CUAAAUUUU GGGAGCUC 1729

2271 AGAUGGAG CUGAUGAG X CGAA AGCUCCCA 1422 UGGGAGCUC CUCCAUCU 1730

2274 UGGAGAUG CUGAUGAG X CGAA AGGAGCUC 1423 GAGCUCCUC CAUCUCCA 1731

2278 CCAUUGGA CUGAUGAG X CGAA AUGGAGGA 1424 UCCUCCAUC UCCAAUGG 1732

2280 AGCCAUUG CUGAUGAG X CGAA AGAUGGAG 1425 CUCCAUCUC CAAUGGCU 1733

2294 CUGCCACA CUGAUGAG X CGAA AUCCCAGC 1426 GCUGGGAUU UGUGGCAG 1734

2295 CCUGCCAC CUGAUGAG X CGAA AAUCCCAG 1427 CUGGGAUUU GUGGCAGG 1735

2307 CUGAGUGG CUGAUGAG X CGAA AUCCCUGC 1428 GCAGGGAUU CCACUCAG 1736

2308 UCUGAGUG CUGAUGAG X CGAA AAUCCCUG 1429 CAGGGAUUC CACUCAGA 1737

2313 GAGGUUCU CUGAUGAG X CGAA AGUGGAAU 1430 AUUCCACUC AGAACCUC 1738

2321 AUUCCAGA CUGAUGAG X CGAA AGGUUCUG 1431 CAGAACCUC UCUGGAAU 1739

2323 AAAUUCCA CUGAUGAG X CGAA AGAGGUUC 1432 GAACCUCUC UGGAAUUU 1740

2330 CAGGCACA CUGAUGAG X CGAA AUUCCAGA 1433 UCUGGAAUU UGUGCCUG 1741

2331 UCAGGCAC CUGAUGAG X CGAA AAUUCCAG 1434 CUGGAAUUU GUGCCUGA 1742

2347 UCCAGUGG CUGAUGAG X CGAA AGGCACAU 1435 AUGUGCCUU CCACUGGA 1743

2348 AUCCAGUG CUGAUGAG X CGAA AAGGCACA 1436 UGUGCCUUC CACUGGAU 1744

2357 AACCCCAA CUGAUGAG X CGAA AUCCAGUG 1437 CACUGGAUU UUGGGGUU 1745

2358 GAACCCCA CUGAUGAG X CGAA AAUCCAGU 1438 ACUGGAUUU UGGGGUUC 1746

2359 GGAACCCC CUGAUGAG X CGAA AAAUCCAG 1439 CUGGAUUUU GGGGUUCC 1747

2365 GUGCUGGG CUGAUGAG X CGAA ACCCCAAA 1440 UUUGGGGUU CCCAGCAC 1748

2366 GGUGCUGG CUGAUGAG X CGAA AACCCCAA 1441 UUGGGGUUC CCAGCACC 1749

2385 CCCCCCAA CUGAUGAG X CGAA AUCCACAU 1442 AUGUGGAUU UUGGGGGG 1750

2386 ACCCCCCA CUGAUGAG X CGAA AAUCCACA 1443 UGUGGAUUU UGGGGGGU 1751

2387 GACCCCCC CUGAUGAG X CGAA AAAUCCAC 1444 GUGGAUUUU GGGGGGUC 1752

2395 ACAAAAGG CUGAUGAG X CGAA ACCCCCCA 1445 UGGGGGGUC CCUUUUGU 1753

2399 AGACACAA CUGAUGAG X CGAA AGGGACCC 1446 GGGUCCCUU UUGUGUCU 1754

2400 GAGACACA CUGAUGAG X CGAA AAGGGACC 1447 GGUCCCUUU UGUGUCUC 1755

2401 GGAGACAC CUGAUGAG X CGAA AAAGGGAC 1448 GUCCCUUUU GUGUCUCC 1756

2406 GCGGGGGA CUGAUGAG X CGAA ACACAAAA 1449 UUUUGUGUC UCCCCCGC 1757

2408 UGGCGGGG CUGAUGAG X CGAA AGACACAA 1450 UUGUGUCUC CCCCGCCA 1758

2418 AGUCCUUG CUGAUGAG X CGAA AUGGCGGG 1451 CCCGCCAUU CAAGGACU 1759

2419 GAGUCCUU CUGAUGAG X CGAA AAUGGCGG 1452 CCGCCAUUC AAGGACUC 1760

2427 AAAGAGAG CUGAUGAG X CGAA AGUCCUUG 1453 CAAGGACUC CUCUCUUU 1761

2430 AAGAAAGA CUGAUGAG X CGAA AGGAGUCC 1454 GGACUCCUC UCUUUCUU 1762

2432 UGAAGAAA CUGAUGAG X CGAA AGAGGAGU 1455 ACUCCUCUC UUUCUUCA 1763

2434 GGUGAAGA CUGAUGAG X CGAA AGAGAGGA 1456 UCCUCUCUU UCUUCACC 1764

2435 UGGUGAAG CUGAUGAG X CGAA AAGAGAGG 1457 CCUCUCUUU CUUCACCA 1765

2436 UUGGUGAA CUGAUGAG X CGAA AAAGAGAG 1458 CUCUCUUUC UUCACCAA 1766

2438 UCUUGGUG CUGAUGAG X CGAA AGAAAGAG 1459 cucuuucuu CACCAAGA 1767

2439 UUCUUGGU CUGAUGAG X CGAA AAGAAAGA 1460 ucuuucuuc ACCAAGAA 1768

Where "X" represents stem II region of a HH ribozyme (Hertel et al. 1992 Nucleic Acids Res. 20: 3252). The length of stem II may be > 2 base-pairs. Table XV: Human A-rα/Hairpin Ribozyme and Target Sequence nt. Position Ribozyme Sequen :e SEQ ID. No. Target Sequence SEQ ID. No.

69 CACAGA AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1769 AGGCG GAC UCUGUG 1841

117 CGCUAC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1770 CGGCG GCU GUAGCG 1842

120 CGCCGC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1771 CGGCU GUA GCGGCG 1843

151 GGGCUG AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1772 CACCU GCC CAGCCC 1844

156 AGGUGG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1773 GCCCA GCC CCACCU 1845

167 AAGAUG AGAA GAGG ACCAGAGAAACA X GUACAUUACCUGGUA 1774 CCUCA GCC CAUCUU 1846

268 UUGUUG AGAA GGUA ACCAGAGAAACA X GUACAUUACCUGGUA 1775 UACCU GCC CAACAA 1847

296 AUCCCG AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1776 UGACU GUC CGGGAU 1848

366 CCACAC AGAA GUCC ACCAGAGAAACA X GUACAUUACCUGGUA 1777 GGACU GCU GUGUGG 1849

381 UGAUGA AGAA GUAG ACCAGAGAAACA X GUACAUUACCUGGUA 1778 CUACC GAC UCAUCA 1850

410 GUCCCA AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1779 UCACU GCC UGGGAC 1851

478 AUGGUC AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1780 GUCCC GCU GACCAU 1852

481 UGCAUG AGAA GCGG ACCAGAGAAACA X GUACAUUACCUGGUA 1781 CCGCU GAC CAUGCA 1853

516 ACGCCA AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1782 CUUCA GCC UGGCGU 1854

537 ACUUAA AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1783 CUUCU GCC UUAAGU 1855

550 CCAUGG AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1784 UUUCU GUU CCAUGG 1856

564 UUUGGC AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1785 CUUCC GUU GCCAAA 1857

620 AACACA AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 1786 CCACA GUC UGUGUU 1858

652 UGGUAG AGAA GUUG ACCAGAGAAACA X GUACAUUACCUGGUA 1787 CAACA GUU CUACCA 1859

714 UCAGGG AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1788 GAACC GCC CCCUGA 1860

750 UGCGGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1789 UCCCA GCC CCCGCA 1861

794 GGCUGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1790 UCCCU GCC CCAGCC 1862

825 ACGUGG AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 1791 CAUCC GCU CCACGU 1863

866 CAUGGG AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 1792 CCACG GCC CCCAUG 1864

892 CCAGUG AGAA GGAU ACCAGAGAAACA X GUACAUUACCUGGUA 1793 AUCCA GCU CACUGG 1865

917 GGCAGC AGAA GUGC ACCAGAGAAACA X GUACAUUACCUGGUA 1794 GCACU GAU GCUGCC 1866

923 ACUACC AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1795 AUGCU GCC GGUAGU 1867

927 CUCUAC AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1796 UGCCG GUA GUAGAG 1868

969 UGGCUG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1797 CCCCA GCC CAGCCA 1869

1049 CUUGUC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1798 UGGCC GAU GACAAG 1870

1126 UUCAGC AGAA GCAC ACCAGAGAAACA X GUACAUUACCUGGUA 1799 GUGCA GCU GCUGAA 1871

1129 CUCUUC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1800 CAGCU GCU GAAGAG 1872

1219 GCUGUG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1801 UCCCA GCC CACAGC 1873

1226 CUGCUC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 1802 CCACA GCU GAGCAG 1874

1297 CCCAUA AGAA GCAA ACCAGAGAAACA X GUACAUUACCUGGUA 1803 UUGCU GUU UAUGGG 1875

1318 AAUCCC AGAA GGGU ACCAGAGAAACA X GUACAUUACCUGGUA 1804 ACCCG GCC GGGAUU 1876

1359 GGUAGA AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1805 CUCCA GCC UCUACC 1877

1408 UCGAUG AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1806 GUCCA GCU CAUCGA 1878

nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

1429 UGGGCA AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1807 CGGCA GAC UGCCCA 1879

1433 GCCCUG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 1808 AGACU GCC CAGGGC 1880

1576 UCCAAG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1809 GCCCA GCC CUUGGA 1881

1588 CCUGAG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1810 GAGCA GCC CUCAGG 1882

1616 CACCUC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1811 UGGCA GCU GAGGUG 1883

1629 CCUGCA AGAA GAUC ACCAGAGAAACA X GUACAUUACCUGGUA 1812 GAUCC GUA UGCAGG 1884

1653 ACUGGA AGAA GUAG ACCAGAGAAACA X GUACAUUACCUGGUA 1813 CUACA GCU UCCAGU 1885

1664 AUAGAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1814 AGUCA GAC GUCUAU 1886

1714 CUGUAA AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1815 UCACU GCC UUACAG 1887

1734 GGUCAC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1816 UGGCU GCC GUGACC 1888

1744 AAGAUA AGAA GGUC ACCAGAGAAACA X GUACAUUACCUGGUA 1817 GACCA GAU UAUCUU 1889

1774 UCCGGG AGAA GAUA ACCAGAGAAACA X GUACAUUACCUGGUA 1818 UAUCU GUC CCCGGA 1890

1781 GCUGAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1819 CCCCG GAC CUCAGC 1891

1806 CCUUGG AGAA GUUG ACCAGAGAAACA X GUACAUUACCUGGUA 1820 CAACU GCC CCAAGG 1892

1828 UCAGAC AGAA GGCG ACCAGAGAAACA X GUACAUUACCUGGUA 1821 CGCCU GCU GUCUGA 1893

1831 CAGUCA AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 1822 CUGCU GUC UGACUG 1894

1835 GAGGCA AGAA GACA ACCAGAGAAACA X GUACAUUACCUGGUA 1823 UGUCU GAC UGCCUC 1895

1839 ACUUGA AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1824 UGACU GCC UCAAGU 1896

1864 AAGAGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1825 GAGCG GCC CCUCUU 1897

1879 GCCAGG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1826 CCCCA GAU CCUGGC 1898

1900 CGUUGC AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1827 GAGCU GCU GCAACG 1899

1967 CAACUC AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1828 AGGCC GAU GAGUUG 1900

1979 UAGGCA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1829 UGCCU GCC UGCCUA 1901

1983 UGAGUA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1830 UGCCU GCC UACUCA 1902

1997 AAGGCG AGAA GCGC ACCAGAGAAACA X GUACAUUACCUGGUA 1831 GCGCA GCC CGCCUU 1903

2001 GCACAA AGAA GGCU ACCAGAGAAACA X GUACAUUACCUGGUA 1832 AGCCC GCC UUGUGC 1904

2020 GGCUUG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1833 GCCCC GCC CAAGCC 1905

2047 GUGGAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1834 UCUCA GCC CUCCAC 1906

2097 CAUCAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1835 UCUCU GCC CUGAUG 1907

2102 GGCAGC AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1836 GCCCU GAU GCUGCC 1908

2108 UCCUGA AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1837 AUGCU GCC UCAGGA 1909

2167 CAGAAG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1838 UUCCA GUU CUUCUG 1910

2211 GGAGGA AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1839 CCCCU GUC UCCUCC 1911

2337 AGGCAC AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1840 UGCCU GAU GUGCCU 1912

Where "X" represents stem IV region of a Haiφin ribozyme. The length of stem IV may be 2 base-pairs.

Table XVI: Hammerhead Ribozyme Sites for B-raf nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

17 CGGGCGGG CUGAUGAG X CGAA AGGGGGCC 1913 GGCCCCCUC CCCGCCCG 2354

38 CGGGGCCC CUGAUGAG X CGAA AGCGGCCG 1914 CGGCCGCUC GGGCCCCG 2355

50 AUAACCGA CUGAUGAG X CGAA AGCCGGGG 1915 CCCCGGCUC UCGGUUAU 2356

52 UUAUAACC CUGAUGAG X CGAA AGAGCCGG 1916 CCGGCUCUC GGUUAUAA 2357

56 CAUCUUAU CUGAUGAG X CGAA ACCGAGAG 1917 CUCUCGGUU AUAAGAUG 2358

57 CCAUCUUA CUGAUGAG X CGAA AACCGAGA 1918 UCUCGGUUA UAAGAUGG 2359

59 CGCCAUCU CUGAUGAG X CGAA AUAACCGA 1919 UCGGUUAUA AGAUGGCG 2360

113 GUUGAACA CUGAUGAG X CGAA AGCCUGGC 1920 GCCAGGCUC UGUUCAAC 2361

117 CCCCGUUG CUGAUGAG X CGAA ACAGAGCC 1921 GGCUCUGUU CAACGGGG 2362

118 UCCCCGUU CUGAUGAG X CGAA AACAGAGC 1922 GCUCUGUUC AACGGGGA 2363

165 CAGCCGAA CUGAUGAG X CGAA AGGCCGCG 1923 CGCGGCCUC UUCGGCUG 2364

167 CGCAGCCG CUGAUGAG X CGAA AGAGGCCG 1924 CGGCCUCUU CGGCUGCG 2365

168 CCGCAGCC CUGAUGAG X CGAA AAGAGGCC 1925 GGCCUCUUC GGCUGCGG 2366

187 UCCUCCGG CUGAUGAG X CGAA AUGGCAGG 1926 CCUGCCAUU CCGGAGGA 2367

188 CUCCUCCG CUGAUGAG X CGAA AAUGGCAG 1927 CUGCCAUUC CGGAGGAG 2368

206 UUGUUUGA CUGAUGAG X CGAA AUUCCACA 1928 UGUGGAAUA UCAAACAA 2369

208 AUUUGUUU CUGAUGAG X CGAA AUAUUCCA 1929 UGGAAUAUC AAACAAAU 2370

220 GUCAACUU CUGAUGAG X CGAA AUCAUUUG 1930 CAAAUGAUU AAGUUGAC 2371

221 UGUCAACU CUGAUGAG X CGAA AAUCAUUU 1931 AAAUGAUUA AGUUGACA 2372

225 CCUGUGUC CUGAUGAG X CGAA ACUUAAUC 1932 GAUUAAGUU GACACAGG 2373

239 GGCCUCUA CUGAUGAG X CGAA AUGUUCCU 1933 AGGAACAUA UAGAGGCC 2374

241 AGGGCCUC CUGAUGAG X CGAA AUAUGUUC 1934 GAACAUAUA GAGGCCCU 2375

250 UUGUCCAA CUGAUGAG X CGAA AGGGCCUC 1935 GAGGCCCUA UUGGACAA 2376

252 AUUUGUCC CUGAUGAG X CGAA AUAGGGCC 1936 GGCCCUAUU GGACAAAU 2377

261 CCCCACCA CUGAUGAG X CGAA AUUUGUCC 1937 GGACAAAUU UGGUGGGG 2378

262 UCCCCACC CUGAUGAG X CGAA AAUUUGUC 1938 GACAAAUUU GGUGGGGA 2379

275 UGGUGGAU CUGAUGAG X CGAA AUGCUCCC 1939 GGGAGCAUA AUCCACCA 2380

278 UGAUGGUG CUGAUGAG X CGAA AUUAUGCU 1940 AGCAUAAUC CACCAUCA 2381

285 GAUAUAUU CUGAUGAG X CGAA AUGGUGGA 1941 UCCACCAUC AAUAUAUC 2382

289 UCCAGAUA CUGAUGAG X CGAA AUUGAUGG 1942 CCAUCAAUA UAUCUGGA 2383

291 CCUCCAGA CUGAUGAG X CGAA AUAUUGAU 1943 AUCAAUAUA UCUGGAGG 2384

293 GGCCUCCA CUGAUGAG X CGAA AUAUAUUG 1944 CAAUAUAUC UGGAGGCC 2385

303 AUUCUUCA CUGAUGAG X CGAA AGGCCUCC 1945 GGAGGCCUA UGAAGAAU 2386

312 UGCUGGUG CUGAUGAG X CGAA AUUCUUCA 1946 UGAAGAAUA CACCAGCA 2387

325 AGUGCAUC CUGAUGAG X CGAA AGCUUGCU 1947 AGCAAGCUA GAUGCACU 2388

334 CUUUGUUG CUGAUGAG X CGAA AGUGCAUC 1948 GAUGCACUC CAACAAAG 2389

354 AUUCCAAU CUGAUGAG X CGAA ACUGUUGU 1949 ACAACAGUU AUUGGAAU 2390

355 GAUUCCAA CUGAUGAG X CGAA AACUGUUG 1950 CAACAGUUA UUGGAAUC 2391

357 GAGAUUCC CUGAUGAG X CGAA AUAACUGU 1951 ACAGUUAUU GGAAUCUC 2392

363 UCCCCAGA CUGAUGAG X CGAA AUUCCAAU 1952 AUUGGAAUC UCUGGGGA 2393

365 GUUCCCCA CUGAUGAG X CGAA AGAUUCCA 1953 UGGAAUCUC UGGGGAAC 2394

383 AACAGAAA CUGAUGAG X CGAA AUCAGUUC 1954 GAACUGAUU UUUCUGUU 2395

384 AAACAGAA CUGAUGAG X CGAA AAUCAGUU 1955 AACUGAUUU UUCUGUUU 2396

385 GAAACAGA CUGAUGAG X CGAA AAAUCAGU 1956 ACUGAUUUU UCUGUUUC 2397

386 AGAAACAG CUGAUGAG X CGAA AAAAUCAG 1957 CUGAUUUUU CUGUUUCU 2398

387 UAGAAACA CUGAUGAG X CGAA AAAAAUCA 1958 UGAUUUUUC UGUUUCUA 2399

391 GAGCUAGA CUGAUGAG X CGAA ACAGAAAA 1959 UUUUCUGUU UCUAGCUC 2400

392 AGAGCUAG CUGAUGAG X CGAA AACAGAAA 1960 UUUCUGUUU CUAGCUCU 2401

393 CAGAGCUA CUGAUGAG X CGAA AAACAGAA 1961 UUCUGUUUC UAGCUCUG 2402

395 UGCAGAGC CUGAUGAG X CGAA AGAAACAG 1962 CUGUUUCUA GCUCUGCA 2403

399 UUGAUGCA CUGAUGAG X CGAA AGCUAGAA 1963 UUCUAGCUC UGCAUCAA 2404

405 UAUCCAUU CUGAUGAG X CGAA AUGCAGAG 1964 CUCUGCAUC AAUGGAUA 2405

413 UGUAACGG CUGAUGAG X CGAA AUCCAUUG 1965 CAAUGGAUA CCGUUACA 2406 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

418 GAAGAUGU CUGAUGAG X CGAA ACGGUAUC 1966 GAUACCGUU ACAUCUUC 2407

419 AGAAGAUG CUGAUGAG X CGAA AACGGUAU 1967 AUACCGUUA CAUCUUCU 2408

423 AGGAAGAA CUGAUGAG X CGAA AUGUAACG 1968 CGUUACAUC UUCUUCCU 2409

425 AGAGGAAG CUGAUGAG X CGAA AGAUGUAA 1969 UUACAUCUU CUUCCUCU 2410

426 AAGAGGAA CUGAUGAG X CGAA AAGAUGUA 1970 UACAUCUUC uuccucuu 2411

428 AGAAGAGG CUGAUGAG X CGAA AGAAGAUG 1971 CAUCUUCUU ccucuucu 2412

429 UAGAAGAG CUGAUGAG X CGAA AAGAAGAU 1972 AUCUUCUUC CUCUUCUA 2413

432 GGCUAGAA CUGAUGAG X CGAA AGGAAGAA 1973 UUCUUCCUC UUCUAGCC 2414

434 AAGGCUAG CUGAUGAG X CGAA AGAGGAAG 1974 CUUCCUCUU CUAGCCUU 2415

435 AAAGGCUA CUGAUGAG X CGAA AAGAGGAA 1975 uuccucuuc UAGCCUUU 2416

437 UGAAAGGC CUGAUGAG X CGAA AGAAGAGG 1976 CCUCUUCUA GCCUUUCA 2417

442 AGCACUGA CUGAUGAG X CGAA AGGCUAGA 1977 UCUAGCCUU UCAGUGCU 2418

443 UAGCACUG CUGAUGAG X CGAA AAGGCUAG 1978 CUAGCCUUU CAGUGCUA 2419

444 GUAGCACU CUGAUGAG X CGAA AAAGGCUA 1979 UAGCCUUUC AGUGCUAC 2420

451 GAUGAAGG CUGAUGAG X CGAA AGCACUGA 1980 UCAGUGCUA CCUUCAUC 2421

455 AAGAGAUG CUGAUGAG X CGAA AGGUAGCA 1981 UGCUACCUU CAUCUCUU 2422

456 AAAGAGAU CUGAUGAG X CGAA AAGGUAGC 1982 GCUACCUUC AUCUCUUU 2423

459 CUGAAAGA CUGAUGAG X CGAA AUGAAGGU 1983 ACCUUCAUC UCUUUCAG 2424

461 AACUGAAA CUGAUGAG X CGAA AGAUGAAG 1984 CUUCAUCUC UUUCAGUU 2425

463 AAAACUGA CUGAUGAG X CGAA AGAGAUGA 1985 UCAUCUCUU UCAGUUUU 2426

464 AAAAACUG CUGAUGAG X CGAA AAGAGAUG 1986 CAUCUCUUU CAGUUUUU 2427

465 GAAAAACU CUGAUGAG X CGAA AAAGAGAU 1987 AUCUCUUUC AGUUUUUC 2428

469 UUUUGAAA CUGAUGAG X CGAA ACUGAAAG 1988 CUUUCAGUU UUUCAAAA 2429

470 AUUUUGAA CUGAUGAG X CGAA AACUGAAA 1989 UUUCAGUUU UUCAAAAU 2430

471 GAUUUUGA CUGAUGAG X CGAA AAACUGAA 1990 UUCAGUUUU UCAAAAUC 2431

472 GGAUUUUG CUGAUGAG X CGAA AAAACUGA 1991 UCAGUUUUU CAAAAUCC 2432

473 GGGAUUUU CUGAUGAG X CGAA AAAAACUG 1992 CAGUUUUUC AAAAUCCC 2433

479 AUCUGUGG CUGAUGAG X CGAA AUUUUGAA 1993 UUCAAAAUC CCACAGAU 2434

510 UUUGUGGU CUGAUGAG X CGAA ACUUGGGG 1994 CCCCAAGUC ACCACAAA 2435

524 UCUAACGA CUGAUGAG X CGAA AGGUUUUU 1995 AAAAACCUA UCGUUAGA 2436

526 ACUCUAAC CUGAUGAG X CGAA AUAGGUUU 1996 AAACCUAUC GUUAGAGU 2437

529 AAGACUCU CUGAUGAG X CGAA ACGAUAGG 1997 CCUAUCGUU AGAGUCUU 2438

530 GAAGACUC CUGAUGAG X CGAA AACGAUAG 1998 CUAUCGUUA GAGUCUUC 2439

535 GGCAGGAA CUGAUGAG X CGAA ACUCUAAC 1999 GUUAGAGUC UUCCUGCC 2440

537 UGGGCAGG CUGAUGAG X CGAA AGACUCUA 2000 UAGAGUCUU CCUGCCCA 2441

538 UUGGGCAG CUGAUGAG X CGAA AAGACUCU 2001 AGAGUCUUC CUGCCCAA 2442

565 CUUGCAGG CUGAUGAG X CGAA ACCACUGU 2002 ACAGUGGUA CCUGCAAG 2443

583 CGGACUGU CUGAUGAG X CGAA ACUCCACA 2003 UGUGGAGUU ACAGUCCG 2444

584 UCGGACUG CUGAUGAG X CGAA AACUCCAC 2004 GUGGAGUUA CAGUCCGA 2445

589 CUGUCUCG CUGAUGAG X CGAA ACUGUAAC 2005 GUUACAGUC CGAGACAG 2446

599 UUUCUUUA CUGAUGAG X CGAA ACUGUCUC 2006 GAGACAGUC UAAAGAAA 2447

601 GCUUUCUU CUGAUGAG X CGAA AGACUGUC 2007 GACAGUCUA AAGAAAGC 2448

626 UGGGAUUA CUGAUGAG X CGAA ACCUCUCA 2008 UGAGAGGUC UAAUCCCA 2449

628 UCUGGGAU CUGAUGAG X CGAA AGACCUCU 2009 AGAGGUCUA AUCCCAGA 2450

631 CACUCUGG CUGAUGAG X CGAA AUUAGACC 2010 GGUCUAAUC CCAGAGUG 2451

649 AUUCUGUA CUGAUGAG X CGAA ACAGCACA 2011 UGUGCUGUU UACAGAAU 2452

650 AAUUCUGU CUGAUGAG X CGAA AACAGCAC 2012 GUGCUGUUU ACAGAAUU 2453

651 GAAUUCUG CUGAUGAG X CGAA AAACAGCA 2013 UGCUGUUUA CAGAAUUC 2454

658 CCAUCCUG CUGAUGAG X CGAA AUUCUGUA 2014 UACAGAAUU CAGGAUGG 2455

659 UCCAUCCU CUGAUGAG X CGAA AAUUCUGU 2015 ACAGAAUUC AGGAUGGA 2456

682 UCCCAACC CUGAUGAG X CGAA AUUGGUUU 2016 AAACCAAUU GGUUGGGA 2457

686 AGUGUCCC CUGAUGAG X CGAA ACCAAUUG 2017 CAAUUGGUU GGGACACU 2458

698 CCAGGAAA CUGAUGAG X CGAA AUCAGUGU 2018 ACACUGAUA UUUCCUGG 2459

700 AGCCAGGA CUGAUGAG X CGAA AUAUCAGU 2019 ACUGAUAUU UCCUGGCU 2460

701 AAGCCAGG CUGAUGAG X CGAA AAUAUCAG 2020 CUGAUAUUU CCUGGCUU 2461 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

702 UAAGCCAG CUGAUGAG X CGAA AAAUAUCA 2021 UGAUAUUUC CUGGCUUA 2462

709 UCUCCAGU CUGAUGAG X CGAA AGCCAGGA 2022 UCCUGGCUU ACUGGAGA 2463

710 UUCUCCAG CUGAUGAG X CGAA AAGCCAGG 2023 CCUGGCUUA CUGGAGAA 2464

723 CCACAUGC CUGAUGAG X CGAA AUUCUUCU 2024 AGAAGAAUU GCAUGUGG 2465

738 CAUUCUCC CUGAUGAG X CGAA ACACUUCC 2025 GGAAGUGUU GGAGAAUG 2466

748 GUAAGUGG CUGAUGAG X CGAA ACAUUCUC 2026 GAGAAUGUU CCACUUAC 2467

749 UGUAAGUG CUGAUGAG X CGAA AACAUUCU 2027 AGAAUGUUC CACUUACA 2468

754 UGUGUUGU CUGAUGAG X CGAA AGUGGAAC 2028 GUUCCACUU ACAACACA 2469

755 GUGUGUUG CUGAUGAG X CGAA AAGUGGAA 2029 UUCCACUUA CAACACAC ^■2470

768 UUCGUACA CUGAUGAG X CGAA AGUUGUGU 2030 ACACAACUU UGUACGAA 2471

769 UUUCGUAC CUGAUGAG X CGAA AAGUUGUG 2031 CACAACUUU GUACGAAA 2472

772 GUUUUUCG CUGAUGAG X CGAA ACAAAGUU 2032 AACUUUGUA CGAAAAAC 2473

783 AGGUGAAA CUGAUGAG X CGAA ACGUUUUU 2033 AAAAACGUU UUUCACCU 2474

784 AAGGUGAA CUGAUGAG X CGAA AACGUUUU 2034 AAAACGUUU UUCACCUU 2475

785 UAAGGUGA CUGAUGAG X CGAA AAACGUUU 2035 AAACGUUUU UCACCUUA 2476

786 CUAAGGUG CUGAUGAG X CGAA AAAACGUU 2036 AACGUUUUU CACCUUAG 2477

787 GCUAAGGU CUGAUGAG X CGAA AAAAACGU 2037 ACGUUUUUC ACCUUAGC 2478

792 AAAAUGCU CUGAUGAG X CGAA AGGUGAAA 2038 UUUCACCUU AGCAUUUU 2479

793 CAAAAUGC CUGAUGAG X CGAA AAGGUGAA 2039 UUCACCUUA GCAUUUUG 2480

798 AGUCACAA CUGAUGAG X CGAA AUGCUAAG 2040 CUUAGCAUU UUGUGACU 2481

799 AAGUCACA CUGAUGAG X CGAA AAUGCUAA 2041 UUAGCAUUU UGUGACUU 2482

800 AAAGUCAC CUGAUGAG X CGAA AAAUGCUA 2042 UAGCAUUUU GUGACUUU 2483

807 UUCGACAA CUGAUGAG X CGAA AGUCACAA 2043 UUGUGACUU UUGUCGAA 2484

808 UUUCGACA CUGAUGAG X CGAA AAGUCACA 2044 UGUGACUUU UGUCGAAA 2485

809 CUUUCGAC CUGAUGAG X CGAA AAAGUCAC 2045 GUGACUUUU GUCGAAAG 2486

812 CAGCUUUC CUGAUGAG X CGAA ACAAAAGU 2046 ACUUUUGUC GAAAGCUG 2487

823 CCCUGGAA CUGAUGAG X CGAA AGCAGCUU 2047 AAGCUGCUU UUCCAGGG 2488

824 ACCCUGGA CUGAUGAG X CGAA AAGCAGCU 2048 AGCUGCUUU UCCAGGGU 2489

825 AACCCUGG CUGAUGAG X CGAA AAAGCAGC 2049 GCUGCUUUU CCAGGGUU 2490

826 AAACCCUG CUGAUGAG X CGAA AAAAGCAG 2050 CUGCUUUUC CAGGGUUU 2491

833 ACAGCGGA CUGAUGAG X CGAA ACCCUGGA 2051 UCCAGGGUU UCCGCUGU 2492

834 GACAGCGG CUGAUGAG X CGAA AACCCUGG 2052 CCAGGGUUU CCGCUGUC 2493

835 UGACAGCG CUGAUGAG X CGAA AAACCCUG 2053 CAGGGUUUC CGCUGUCA 2494

842 ACAUGUUU CUGAUGAG X CGAA ACAGCGGA 2054 UCCGCUGUC AAACAUGU 2495

854 AAAUUUAU CUGAUGAG X CGAA ACCACAUG 2055 CAUGUGGUU AUAAAUUU 2496

855 GAAAUUUA CUGAUGAG X CGAA AACCACAU 2056 AUGUGGUUA UAAAUUUC 2497

857 GUGAAAUU CUGAUGAG X CGAA AUAACCAC 2057 GUGGUUAUA AAUUUCAC 2498

861 GCUGGUGA CUGAUGAG X CGAA AUUUAUAA 2058 UUAUAAAUU UCACCAGC 2499

862 CGCUGGUG CUGAUGAG X CGAA AAUUUAUA 2059 UAUAAAUUU CACCAGCG 2500

863 ACGCUGGU CUGAUGAG X CGAA AAAUUUAU 2060 AUAAAUUUC ACCAGCGU 2501

872 UGUACUAC CUGAUGAG X CGAA ACGCUGGU 2061 ACCAGCGUU GUAGUACA 2502

875 UUCUGUAC CUGAUGAG X CGAA ACAACGCU 2062 AGCGUUGUA GUACAGAA 2503

878 AACUUCUG CUGAUGAG X CGAA ACUACAAC 2063 GUUGUAGUA CAGAAGUU 2504

886 AUCAGUGG CUGAUGAG X CGAA ACUUCUGU 2064 ACAGAAGUU CCACUGAU 2505

887 CAUCAGUG CUGAUGAG X CGAA AACUUCUG 2065 CAGAAGUUC CACUGAUG 2506

901 UCAUAAUU CUGAUGAG X CGAA ACACACAU 2066 AUGUGUGUU AAUUAUGA 2507

902 GUCAUAAU CUGAUGAG X CGAA AACACACA 2067 UGUGUGUUA AUUAUGAC 2508

905 UUGGUCAU CUGAUGAG X CGAA AUUAACAC 2068 GUGUUAAUU AUGACCAA 2509

906 GUUGGUCA CUGAUGAG X CGAA AAUUAACA 2069 UGUUAAUUA UGACCAAC 2510

916 AGCAAAUC CUGAUGAG X CGAA AGUUGGUC 2070 GACCAACUU GAUUUGCU 2511

920 AAACAGCA CUGAUGAG X CGAA AUCAAGUU 2071 AACUUGAUU UGCUGUUU 2512

921 CAAACAGC CUGAUGAG X CGAA AAUCAAGU 2072 ACUUGAUUU GCUGUUUG 2513

927 UGGAGACA CUGAUGAG X CGAA ACAGCAAA 2073 UUUGCUGUU UGUCUCCA 2514

928 UUGGAGAC CUGAUGAG X CGAA AACAGCAA 2074 UUGCUGUUU GUCUCCAA 2515

931 AACUUGGA CUGAUGAG X CGAA ACAAACAG 2075 CUGUUUGUC UCCAAGUU 2516 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

933 AGAACUUG CUGAUGAG X CGAA AGACAAAC 2076 GUUUGUCUC CAAGUUCU 2517

939 GUUCAAAG CUGAUGAG X CGAA ACUUGGAG 2077 CUCCAAGUU CUUUGAAC 2518

940 UGUUCAAA CUGAUGAG X CGAA AACUUGGA 2078 UCCAAGUUC UUUGAACA 2519

942 GGUGUUCA CUGAUGAG X CGAA AGAACUUG 2079 CAAGUUCUU UGAACACC 2520

943 UGGUGUUC CUGAUGAG X CGAA AAGAACUU 2080 AAGUUCUUU GAACACCA 2521

958 UCCUGUGG CUGAUGAG X CGAA AUUGGGUG 2081 CACCCAAUA CCACAGGA 2522

975 CUGCUAAG CUGAUGAG X CGAA ACGCCUCU 2082 AGAGGCGUC CUUAGCAG 2523

978 UCUCUGCU CUGAUGAG X CGAA AGGACGCC 2083 GGCGUCCUU AGCAGAGA 2524

979 GUCUCUGC CUGAUGAG X CGAA AAGGACGC 2084 GCGUCCUUA GCAGAGAC '2525

994 CCAGAUGU CUGAUGAG X CGAA AGGGCAGU 2085 ACUGCCCUA ACAUCUGG 2526

999 AUGAUCCA CUGAUGAG X CGAA AUGUUAGG 2086 CCUAACAUC UGGAUCAU 2527

1005 AAGGGGAU CUGAUGAG X CGAA AUCCAGAU 2087 AUCUGGAUC AUCCCCUU 2528

1008 CGGAAGGG CUGAUGAG X CGAA AUGAUCCA 2088 UGGAUCAUC CCCUUCCG 2529

1013 GGGUGCGG CUGAUGAG X CGAA AGGGGAUG 2089 CAUCCCCUU CCGCACCC 2530

1014 CGGGUGCG CUGAUGAG X CGAA AAGGGGAU 2090 AUCCCCUUC CGCACCCG 2531

1026 UAGAGUCC CUGAUGAG X CGAA AGGCGGGU 2091 ACCCGCCUC GGACUCUA 2532

1032 GCCCAAUA CUGAUGAG X CGAA AGUCCGAG 2092 CUCGGACUC UAUUGGGC 2533

1034 GGGCCCAA CUGAUGAG X CGAA AGAGUCCG 2093 CGGACUCUA UUGGGCCC 2534

1036 UGGGGCCC CUGAUGAG X CGAA AUAGAGUC 2094 GACUCUAUU GGGCCCCA 2535

1048 CUGGUGAG CUGAUGAG X CGAA AUUUGGGG 2095 CCCCAAAUU CUCACCAG 2536

1049 ACUGGUGA CUGAUGAG X CGAA AAUUUGGG 2096 CCCAAAUUC UCACCAGU 2537

1051 GGACUGGU CUGAUGAG X CGAA AGAAUUUG 2097 CAAAUUCUC ACCAGUCC 2538

1058 AGGAGACG CUGAUGAG X CGAA ACUGGUGA 2098 UCACCAGUC CGUCUCCU 2539

1062 UUGAAGGA CUGAUGAG X CGAA ACGGACUG 2099 CAGUCCGUC UCCUUCAA 2540

1064 UUUUGAAG CUGAUGAG X CGAA AGACGGAC 2100 GUCCGUCUC CUUCAAAA 2541

1067 GGAUUUUG CUGAUGAG X CGAA AGGAGACG 2101 CGUCUCCUU CAAAAUCC 2542

1068 UGGAUUUU CUGAUGAG X CGAA AAGGAGAC 2102 GUCUCCUUC AAAAUCCA 2543

1074 UUGGAAUG CUGAUGAG X CGAA AUUUUGAA 2103 UUCAAAAUC CAUUCCAA 2544

1078 GGAAUUGG CUGAUGAG X CGAA AUGGAUUU 2104 AAAUCCAUU CCAAUUCC 2545

1079 UGGAAUUG CUGAUGAG X CGAA AAUGGAUU 2105 AAUCCAUUC CAAUUCCA 2546

1084 GGCUGUGG CUGAUGAG X CGAA AUUGGAAU 2106 AUUCCAAUU CCACAGCC 2547

1085 GGGCUGUG CUGAUGAG X CGAA AAUUGGAA 2107 UUCCAAUUC CACAGCCC 2548

1095 CUGGUCGG CUGAUGAG X CGAA AGGGCUGU 2108 ACAGCCCUU CCGACCAG 2549

1096 GCUGGUCG CUGAUGAG X CGAA AAGGGCUG 2109 CAGCCCUUC CGACCAGC 2550

1115 AUUUCGAU CUGAUGAG X CGAA AUCUUCAU 2110 AUGAAGAUC AUCGAAAU 2551

1118 UUGAUUUC CUGAUGAG X CGAA AUGAUCUU 2111 AAGAUCAUC GAAAUCAA 2552

1124 CCCAAAUU CUGAUGAG X CGAA AUUUCGAU 2112 AUCGAAAUC AAUUUGGG 2553

1128 GUUGCCCA CUGAUGAG X CGAA AUUGAUUU 2113 AAAUCAAUU UGGGCAAC 2554

1129 CGUUGCCC CUGAUGAG X CGAA AAUUGAUU 2114 AAUCAAUUU GGGCAACG 2555

1146 CUGAUGAG CUGAUGAG X CGAA AUCGGUCU 2115 AGACCGAUC CUCAUCAG 2556

1149 GAGCUGAU CUGAUGAG X CGAA AGGAUCGG 2116 CCGAUCCUC AUCAGCUC 2557

1152 UGGGAGCU CUGAUGAG X CGAA AUGAGGAU 2117 AUCCUCAUC AGCUCCCA 2558

1157 CACAUUGG CUGAUGAG X CGAA AGCUGAUG 2118 CAUCAGCUC CCAAUGUG 2559

1169 UGUGUUUA CUGAUGAG X CGAA AUGCACAU 2119 AUGUGCAUA UAAACACA 2560

1171 AUUGUGUU CUGAUGAG X CGAA AUAUGCAC 2120 GUGCAUAUA AACACAAU 2561

1180 ACAGGUUC CUGAUGAG X CGAA AUUGUGUU 2121 AACACAAUA GAACCUGU 2562

1189 UCAAUAUU CUGAUGAG X CGAA ACAGGUUC 2122 GAACCUGUC AAUAUUGA 2563

1193 GUCAUCAA CUGAUGAG X CGAA AUUGACAG 2123 CUGUCAAUA UUGAUGAC 2564

1195 AAGUCAUC CUGAUGAG X CGAA AUAUUGAC 2124 GUCAAUAUU GAUGACUU 2565

1203 CUCUAAUC CUGAUGAG X CGAA AGUCAUCA 2125 UGAUGACUU GAUUAGAG 2566

1207 UGGUCUCU CUGAUGAG X CGAA AUCAAGUC 2126 GACUUGAUU AGAGACCA 2567

1208 UUGGUCUC CUGAUGAG X CGAA AAUCAAGU 2127 ACUUGAUUA GAGACCAA 2568

1221 CACCACGA CUGAUGAG X CGAA AUCCUUGG 2128 CCAAGGAUU UCGUGGUG 2569

1222 UCACCACG CUGAUGAG X CGAA AAUCCUUG 2129 CAAGGAUUU CGUGGUGA 2570

1223 AUCACCAC CUGAUGAG X CGAA AAAUCCUU 2130 AAGGAUUUC GUGGUGAU 2571 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

1239 CUGUGGUU CUGAUGAG X CGAA AUCCUCCA 2131 UGGAGGAUC AACCACAG 2572

1250 AGCAGACA CUGAUGAG X CGAA ACCUGUGG 2132 CCACAGGUU UGUCUGCU 2573

1251 UAGCAGAC CUGAUGAG X CGAA AACCUGUG 2133 CACAGGUUU GUCUGCUA 2574

1254 GGGUAGCA CUGAUGAG X CGAA ACAAACCU 2134 AGGUUUGUC UGCUACCC 2575

1259 AGGGGGGG CUGAUGAG X CGAA AGCAGACA 2135 UGUCUGCUA CCCCCCCU 2576

1272 CAGGUAAU CUGAUGAG X CGAA AGGCAGGG 2136 CCCUGCCUC AUUACCUG 2577

1275 AGCCAGGU CUGAUGAG X CGAA AUGAGGCA 2137 UGCCUCAUU ACCUGGCU 2578

1276 GAGCCAGG CUGAUGAG X CGAA AAUGAGGC 2138 GCCUCAUUA CCUGGCUC 2579

1284 UAGUUAGU CUGAUGAG X CGAA AGCCAGGU 2139 ACCUGGCUC ACUAACUA ^■ 2580

1288 ACGUUAGU CUGAUGAG X CGAA AGUGAGCC 2140 GGCUCACUA ACUAACGU 2581

1292 UUUCACGU CUGAUGAG X CGAA AGUUAGUG 2141 CACUAACUA ACGUGAAA 2582

1305 AUUUCUGU CUGAUGAG X CGAA AGGCUUUC 2142 GAAAGCCUU ACAGAAAU 2583

1306 GAUUUCUG CUGAUGAG X CGAA AAGGCUUU 2143 AAAGCCUUA CAGAAAUC 2584

1314 GUCCUGGA CUGAUGAG X CGAA AUUUCUGU 2144 ACAGAAAUC UCCAGGAC 2585

1316 AGGUCCUG CUGAUGAG X CGAA AGAUUUCU 2145 AGAAAUCUC CAGGACCU 2586

1325 UUCUCGCU CUGAUGAG X CGAA AGGUCCUG 2146 CAGGACCUC AGCGAGAA 2587

1341 AUGAAGAU CUGAUGAG X CGAA ACUUCCUU 2147 AAGGAAGUC AUCUUCAU 2588

1344 AGGAUGAA CUGAUGAG X CGAA AUGACUUC 2148 GAAGUCAUC UUCAUCCU 2589

1346 UGAGGAUG CUGAUGAG X CGAA AGAUGACU 2149 AGUCAUCUU CAUCCUCA 2590

1347 CUGAGGAU CUGAUGAG X CGAA AAGAUGAC 2150 GUCAUCUUC AUCCUCAG 2591

1350 CUUCUGAG CUGAUGAG X CGAA AUGAAGAU 2151 AUCUUCAUC CUCAGAAG 2592

1353 UGUCUUCU CUGAUGAG X CGAA AGGAUGAA 2152 UUCAUCCUC AGAAGACA 2593

1367 UUUCAUUC CUGAUGAG X CGAA AUUCCUGU 2153 ACAGGAAUC GAAUGAAA 2594

1381 CGUCUACC CUGAUGAG X CGAA AGUGUUUU 2154 AAAACACUU GGUAGACG 2595

1385 GUCCCGUC CUGAUGAG X CGAA ACCAAGUG 2155 CACUUGGUA GACGGGAC 2596

1395 CAUCACUC CUGAUGAG X CGAA AGUCCCGU 2156 ACGGGACUC GAGUGAUG 2597

1406 AAUCUCCC CUGAUGAG X CGAA AUCAUCAC 2157 GUGAUGAUU GGGAGAUU 2598

1414 CCAUCAGG CUGAUGAG X CGAA AUCUCCCA 2158 UGGGAGAUU CCUGAUGG 2599

1415 CCCAUCAG CUGAUGAG X CGAA AAUCUCCC 2159 GGGAGAUUC CUGAUGGG 2600

1429 CCCACUGU CUGAUGAG X CGAA AUCUGCCC 2160 GGGCAGAUU ACAGUGGG 2601

1430 UCCCACUG CUGAUGAG X CGAA AAUCUGCC 2161 GGCAGAUUA CAGUGGGA 2602

1447 CCAGAUCC CUGAUGAG X CGAA AUUCUUUG 2162 CAAAGAAUU GGAUCUGG 2603

1452 AUGAUCCA CUGAUGAG X CGAA AUCCAAUU 2163 AAUUGGAUC UGGAUCAU 2604

1458 UUCCAAAU CUGAUGAG X CGAA AUCCAGAU 2164 AUCUGGAUC AUUUGGAA 2605

1461 CUGUUCCA CUGAUGAG X CGAA AUGAUCCA 2165 UGGAUCAUU UGGAACAG 2606

1462 ACUGUUCC CUGAUGAG X CGAA AAUGAUCC 2166 GGAUCAUUU GGAACAGU 2607

1471 CCCUUGUA CUGAUGAG X CGAA ACUGUUCC 2167 GGAACAGUC UACAAGGG 2608

1473 UUCCCUUG CUGAUGAG X CGAA AGACUGUU 2168 AACAGUCUA CAAGGGAA 2609

1512 UCACAUUC CUGAUGAG X CGAA ACAUUUUC 2169 GAAAAUGUU GAAUGUGA 2610

1529 CUGAGGUG CUGAUGAG X CGAA AGGUGCUG 2170 CAGCACCUA CACCUCAG 2611

1535 UAACUGCU CUGAUGAG X CGAA AGGUGUAG 2171 CUACACCUC AGCAGUUA 2612

1542 AGGCUUGU CUGAUGAG X CGAA ACUGCUGA 2172 UCAGCAGUU ACAAGCCU 2613

1543 AAGGCUUG CUGAUGAG X CGAA AACUGCUG 2173 CAGCAGUUA CAAGCCUU 2614

1551 CAUUUUUG CUGAUGAG X CGAA AGGCUUGU 2174 ACAAGCCUU CAAAAAUG 2615

1552 UCAUUUUU CUGAUGAG X CGAA AAGGCUUG 2175 CAAGCCUUC AAAAAUGA 2616

1564 AGUACUCC CUGAUGAG X CGAA ACUUCAUU 2176 AAUGAAGUA GGAGUACU 2617

1570 UUCCUGAG CUGAUGAG X CGAA ACUCCUAC 2177 GUAGGAGUA CUCAGGAA 2618

1573 GUUUUCCU CUGAUGAG X CGAA AGUACUCC 2178 GGAGUACUC AGGAAAAC 2619

1595 GAGUAGGA CUGAUGAG X CGAA AUUCACAU 2179 AUGUGAAUA UCCUACUC 2620

1597 AAGAGUAG CUGAUGAG X CGAA AUAUUCAC 2180 GUGAAUAUC CUACUCUU 2621

1600 AUGAAGAG CUGAUGAG X CGAA AGGAUAUU 2181 AAUAUCCUA CUCUUCAU 2622

1603 CCCAUGAA CUGAUGAG X CGAA AGUAGGAU 2182 AUCCUACUC UUCAUGGG 2623

1605 AGCCCAUG CUGAUGAG X CGAA AGAGUAGG 2183 CCUACUCUU CAUGGGCU 2624

1606 UAGCCCAU CUGAUGAG X CGAA AAGAGUAG 2184 CUACUCUUC AUGGGCUA 2625

1614 UUGUGGAA CUGAUGAG X CGAA AGCCCAUG 2185 CAUGGGCUA UUCCACAA 2626 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

1616 CUUUGUGG CUGAUGAG X CGAA AUAGCCCA 2186 UGGGCUAUU CCACAAAG 2627

1617 GCUUUGUG CUGAUGAG X CGAA AAUAGCCC 2187 GGGCUAUUC CAC AAGC 2628

1637 GGUAACAA CUGAUGAG X CGAA AGCCAGUU 2188 AACUGGCUA UUGUUACC 2629

1639 UGGGUAAC CUGAUGAG X CGAA AUAGCCAG 2189 CUGGCUAUU GUUACCCA 2630

1642 CACUGGGU CUGAUGAG X CGAA ACAAUAGC 2190 GCUAUUGUU ACCCAGUG 2631

1643 CCACUGGG CUGAUGAG X CGAA AACAAUAG 2191 CUAUUGUUA CCCAGUGG 2632

1662 ACAAGCUG CUGAUGAG X CGAA AGCCCUCA 2192 UGAGGGCUC CAGCUUGU 2633

1668 GGUGAUAC CUGAUGAG X CGAA AGCUGGAG 2193 CUCCAGCUU GUAUCACC 2634

1671 GAUGGUGA CUGAUGAG X CGAA ACAAGCUG 2194 CAGCUUGUA UCACCAUC 2635

1673 GAGAUGGU CUGAUGAG X CGAA AUACAAGC 2195 GCUUGUAUC ACCAUCUC 2636

1679 GAUAUGGA CUGAUGAG X CGAA AUGGUGAU 2196 AUCACCAUC UCCAUAUC 2637

1681 AUGAUAUG CUGAUGAG X CGAA AGAUGGUG 2197 CACCAUCUC CAUAUCAU 2638

1685 CUCAAUGA CUGAUGAG X CGAA AUGGAGAU 2198 AUCUCCAUA UCAUUGAG 2639

1687 GUCUCAAU CUGAUGAG X CGAA AUAUGGAG 2199 CUCCAUAUC AUUGAGAC 2640

1690 UUGGUCUC CUGAUGAG X CGAA AUGAUAUG 2200 CAUAUCAUU GAGACCAA 2641

1701 UCAUCUCA CUGAUGAG X CGAA AUUUGGUC 2201 GACCAAAUU UGAGAUGA 2642

1702 AUCAUCUC CUGAUGAG X CGAA AAUUUGGU 2202 ACCAAAUUU GAGAUGAU 2643

1711 AUAAGUUU CUGAUGAG X CGAA AUCAUCUC 2203 GAGAUGAUC AAACUUAU 2644

1717 AUAUCUAU CUGAUGAG X CGAA AGUUUGAU 2204 AUCAAACUU AUAGAUAU 2645

1718 AAUAUCUA CUGAUGAG X CGAA AAGUUUGA 2205 UCAAACUUA UAGAUAUU 2646

1720 GCAAUAUC CUGAUGAG X CGAA AUAAGUUU 2206 AAACUUAUA GAUAUUGC 2647

1724 UCGUGCAA CUGAUGAG X CGAA AUCUAUAA 2207 UUAUAGAUA UUGCACGA 2648

1726 UGUCGUGC CUGAUGAG X CGAA AUAUCUAU 2208 AUAGAUAUU GCACGACA 2649

1754 GUGUAAGU CUGAUGAG X CGAA AUCCAUGC 2209 GCAUGGAUU ACUUACAC 2650

1755 CGUGUAAG CUGAUGAG X CGAA AAUCCAUG 2210 CAUGGAUUA CUUACACG 2651

1758 UGGCGUGU CUGAUGAG X CGAA AGUAAUCC 2211 GGAUUACUU ACACGCCA 2652

1759 UUGGCGUG CUGAUGAG X CGAA AAGUAAUC 2212 GAUUACUUA CACGCCAA 2653

1770 GGAUGAUU CUGAUGAG X CGAA ACUUGGCG 2213 CGCCAAGUC AAUCAUCC 2654

1774 CUGUGGAU CUGAUGAG X CGAA AUUGACUU 2214 AAGUCAAUC AUCCACAG 2655

1777 UCUCUGUG CUGAUGAG X CGAA AUGAUUGA 2215 UCAAUCAUC CACAGAGA 2656

1789 UUACUCUU CUGAUGAG X CGAA AGGUCUCU 2216 AGAGACCUC AAGAGUAA 2657

1796 UAUAUUAU CUGAUGAG X CGAA ACUCUUGA 2217 UCAAGAGUA AUAAUAUA 2658

1799 AAAUAUAU CUGAUGAG X CGAA AUUACUCU 2218 AGAGUAAUA AUAUAUUU 2659

1802 AAGAAAUA CUGAUGAG X CGAA AUUAUUAC 2219 GUAAUAAUA UAUUUCUU 2660

1804 UGAAGAAA CUGAUGAG X CGAA AUAUUAUU 2220 AAUAAUAUA UUUCUUCA 2661

1806 CAUGAAGA CUGAUGAG X CGAA AUAUAUUA 2221 UAAUAUAUU UCUUCAUG 2662

1807 UCAUGAAG CUGAUGAG X CGAA AAUAUAUU 2222 AAUAUAUUU CUUCAUGA 2663

1808 UUCAUGAA CUGAUGAG X CGAA AAAUAUAU 2223 AUAUAUUUC UUCAUGAA 2664

1810 UCUUCAUG CUGAUGAG X CGAA AGAAAUAU 2224 AUAUUUCUU CAUGAAGA 2665

1811 GUCUUCAU CUGAUGAG X CGAA AAGAAAUA 2225 UAUUUCUUC AUGAAGAC 2666

1822 UUUACUGU CUGAUGAG X CGAA AGGUCUUC 2226 GAAGACCUC ACAGUAAA 2667

1828 CCUAUUUU CUGAUGAG X CGAA ACUGUGAG 2227 CUCACAGUA AAAAUAGG 2668

1834 AAAUCACC CUGAUGAG X CGAA AUUUUUAC 2228 GUAAAAAUA GGUGAUUU 2669

1841 UAGACCAA CUGAUGAG X CGAA AUCACCUA 2229 UAGGUGAUU UUGGUCUA 2670

1842 CUAGACCA CUGAUGAG X CGAA AAUCACCU 2230 AGGUGAUUU UGGUCUAG 2671

1843 GCUAGACC CUGAUGAG X CGAA AAAUCACC 2231 GGUGAUUUU GGUCUAGC 2672

1847 UGUAGCUA CUGAUGAG X CGAA ACCAAAAU 2232 AUUUUGGUC UAGCUACA 2673

1849 ACUGUAGC CUGAUGAG X CGAA AGACCAAA 2233 UUUGGUCUA GCUACAGU 2674

1853 UUUCACUG CUGAUGAG X CGAA AGCUAGAC 2234 GUCUAGCUA CAGUGAAA 2675

1863 UCCAUCGA CUGAUGAG X CGAA AUUUCACU 2235 AGUGAAAUC UCGAUGGA 2676

1865 ACUCCAUC CUGAUGAG X CGAA AGAUUUCA 2236 UGAAAUCUC GAUGGAGU 2677

1878 ACUGAUGG CUGAUGAG X CGAA ACCCACUC 2237 GAGUGGGUC CCAUCAGU 2678

1883 UUCAAACU CUGAUGAG X CGAA AUGGGACC 2238 GGUCCCAUC AGUUUGAA 2679

1887 ACUGUUCA CUGAUGAG X CGAA ACUGAUGG 2239 CCAUCAGUU UGAACAGU 2680

1888 AACUGUUC CUGAUGAG X CGAA AACUGAUG 2240 CAUCAGUUU GAACAGUU 2681 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

1896 AUCCAGAC CUGAUGAG X CGAA ACUGUUCA 2241 UGAACAGUU GUCUGGAU 2682

1899 UGGAUCCA CUGAUGAG X CGAA ACAACUGU 2242 ACAGUUGUC UGGAUCCA 2683

1905 ACAAAAUG CUGAUGAG X CGAA AUCCAGAC 2243 GUCUGGAUC CAUUUUGU 2684

1909 AUCCACAA CUGAUGAG X CGAA AUGGAUCC 2244 GGAUCCAUU UUGUGGAU 2685

1910 CAUCCACA CUGAUGAG X CGAA AAUGGAUC 2245 GAUCCAUUU UGUGGAUG 2686

1911 CCAUCCAC CUGAUGAG X CGAA AAAUGGAU 2246 AUCCAUUUU GUGGAUGG 2687

1930 AUUCUGAU CUGAUGAG X CGAA ACUUCUGG 2247 CCAGAAGUC AUCAGAAU 2688

1933 UGCAUUCU CUGAUGAG X CGAA AUGACUUC 2248 GAAGUCAUC AGAAUGCA 2689

1946 UGGAUUUU CUGAUGAG X CGAA AUCUUGCA 2249 UGCAAGAUA AAAAUCCA 2690

1952 GCUGUAUG CUGAUGAG X CGAA AUUUUUAU 2250 AUAAAAAUC CAUACAGC 2691

1956 GAAAGCUG CUGAUGAG X CGAA AUGGAUUU 2251 AAAUCCAUA CAGCUUUC 2692

1962 CUGACUGA CUGAUGAG X CGAA AGCUGUAU 2252 AUACAGCUU UCAGUCAG 2693

1963 UCUGACUG CUGAUGAG X CGAA AAGCUGUA 2253 UACAGCUUU CAGUCAGA 2694

1964 AUCUGACU CUGAUGAG X CGAA AAAGCUGU 2254 ACAGCUUUC AGUCAGAU 2695

1968 AUACAUCU CUGAUGAG X CGAA ACUGAAAG 2255 CUUUCAGUC AGAUGUAU 2696

1975 AAUGCAUA CUGAUGAG X CGAA ACAUCUGA 2256 UCAGAUGUA UAUGCAUU 2697

1977 CAAAUGCA CUGAUGAG X CGAA AUACAUCU 2257 AGAUGUAUA UGCAUUUG 2698

1983 CAAUCCCA CUGAUGAG X CGAA AUGCAUAU 2258 AUAUGCAUU UGGGAUUG 2699

1984 ACAAUCCC CUGAUGAG X CGAA AAUGCAUA 2259 UAUGCAUUU GGGAUUGU 2700

1990 UACAGAAC CUGAUGAG X CGAA AUCCCAAA 2260 UUUGGGAUU GUUCUGUA 2701

1993 UCAUACAG CUGAUGAG X CGAA ACAAUCCC 2261 GGGAUUGUU CUGUAUGA 2702

1994 UUCAUACA CUGAUGAG X CGAA AACAAUCC 2262 GGAUUGUUC UGUAUGAA 2703

1998 UCAAUUCA CUGAUGAG X CGAA ACAGAACA 2263 UGUUCUGUA UGAAUUGA 2704

2004 CAGUCAUC CUGAUGAG X CGAA AUUCAUAC 2264 GUAUGAAUU GAUGACUG 2705

2019 AAUAAGGU CUGAUGAG X CGAA ACUGUCCA 2265 UGGACAGUU ACCUUAUU 2706

2020 GAAUAAGG CUGAUGAG X CGAA AACUGUCC 2266 GGACAGUUA CCUUAUUC 2707

2024 GUUUGAAU CUGAUGAG X CGAA AGGUAACU 2267 AGUUACCUU AUUCAAAC 2708

2025 UGUUUGAA CUGAUGAG X CGAA AAGGUAAC 2268 GUUACCUUA UUCAAACA 2709

2027 GAUGUUUG CUGAUGAG X CGAA AUAAGGUA 2269 UACCUUAUU CAAACAUC 2710

2028 UGAUGUUU CUGAUGAG X CGAA AAUAAGGU 2270 ACCUUAUUC AAACAUCA 2711

2035 CUGUUGUU CUGAUGAG X CGAA AUGUUUGA 2271 UCAAACAUC AACAACAG 2712

2053 AUAAAAAU CUGAUGAG X CGAA AUCUGGUC 2272 GACCAGAUA AUUUUUAU 2713

2056 ACCAUAAA CUGAUGAG X CGAA AUUAUCUG 2273 CAGAUAAUU UUUAUGGU 2714

2057 CACCAUAA CUGAUGAG X CGAA AAUUAUCU 2274 AGAUAAUUU UUAUGGUG 2715

2058 CCACCAUA CUGAUGAG X CGAA AAAUUAUC 2275 GAUAAUUUU UAUGGUGG 2716

2059 CCCACCAU CUGAUGAG X CGAA AAAAUUAU 2276 AUAAUUUUU AUGGUGGG 2717

2060 UCCCACCA CUGAUGAG X CGAA AAAAAUUA 2277 UAAUUUUUA UGGUGGGA 2718

2076 GAGACAGG CUGAUGAG X CGAA AUCCUCGU 2278 ACGAGGAUA CCUGUCUC 2719

2082 GAUCUGGA CUGAUGAG X CGAA ACAGGUAU 2279 AUACCUGUC UCCAGAUC 2720

2084 GAGAUCUG CUGAUGAG X CGAA AGACAGGU 2280 ACCUGUCUC CAGAUCUC 2721

2090 CUUACUGA CUGAUGAG X CGAA AUCUGGAG 2281 CUCCAGAUC UCAGUAAG 2722

2092 ACCUUACU CUGAUGAG X CGAA AGAUCUGG 2282 CCAGAUCUC AGUAAGGU 2723

2096 CCGUACCU CUGAUGAG X CGAA ACUGAGAU 2283 AUCUCAGUA AGGUACGG 2724

2101 UUACUCCG CUGAUGAG X CGAA ACCUUACU 2284 AGUAAGGUA CGGAGUAA 2725

2108 UGGACAGU CUGAUGAG X CGAA ACUCCGUA 2285 UACGGAGUA ACUGUCCA 2726

2114 GGCUUUUG CUGAUGAG X CGAA ACAGUUAC 2286 GUAACUGUC CAAAAGCC 2727

2133 CUGCCAUU CUGAUGAG X CGAA AUCUCUUC 2287 GAAGAGAUU AAUGGCAG 2728

2134 UCUGCCAU CUGAUGAG X CGAA AAUCUCUU 2288 AAGAGAUUA AUGGCAGA 2729

2149 UUCUUUUU CUGAUGAG X CGAA AGGCACUC 2289 GAGUGCCUC AAAAAGAA 2730

2176 UGGGGAAA CUGAUGAG X CGAA AGUGGUCU 2290 AGACCACUC UUUCCCCA 2731

2178 UUUGGGGA CUGAUGAG X CGAA AGAGUGGU 2291 ACCACUCUU UCCCCAAA 2732

2179 AUUUGGGG CUGAUGAG X CGAA AAGAGUGG 2292 CCACUCUUU CCCCAAAU 2733

2180 AAUUUGGG CUGAUGAG X CGAA AAAGAGUG 2293 CACUCUUUC CCCAAAUU 2734

2188 GAGGCGAG CUGAUGAG X CGAA AUUUGGGG 2294 CCCCAAAUU CUCGCCUC 2735

2189 AGAGGCGA CUGAUGAG X CGAA AAUUUGGG 2295 CCCAAAUUC UCGCCUCU 2736 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

2191 AUAGAGGC CUGAUGAG X CGAA AGAAUUUG 2296 CAAAUUCUC GCCUCUAU 2737

2196 GCUCAAUA CUGAUGAG X CGAA AGGCGAGA 2297 UCUCGCCUC UAUUGAGC 2738

2198 CAGCUCAA CUGAUGAG X CGAA AGAGGCGA 2298 UCGCCUCUA UUGAGCUG 2739

2200 AGCAGCUC CUGAUGAG X CGAA AUAGAGGC 2299 GCCUCUAUU GAGCUGCU 2740

2217 UUGGCAAU CUGAUGAG X CGAA AGCGGGCC 2300 GGCCCGCUC AUUGCCAA 2741

2220 UUUUUGGC CUGAUGAG X CGAA AUGAGCGG 2301 CCGCUCAUU GCCAAAAA 2742

2230 CUGCGGUG CUGAUGAG X CGAA AUUUUUGG 2302 CCAAAAAUU CACCGCAG 2743

2231 ACUGCGGU CUGAUGAG X CGAA AAUUUUUG 2303 CAAAAAUUC ACCGCAGU 2744

2244 AGGGUUCU CUGAUGAG X CGAA AUGCACUG 2304 CAGUGCAUC AGAACCCU ^■2745

2253 GAUUCAAG CUGAUGAG X CGAA AGGGUUCU 2305 AGAACCCUC CUUGAAUC 2746

2256 CCCGAUUC CUGAUGAG X CGAA AGGAGGGU 2306 ACCCUCCUU GAAUCGGG 2747

2261 ACCAGCCC CUGAUGAG X CGAA AUUCAAGG 2307 CCUUGAAUC GGGCUGGU 2748

2270 UGUUUGGA CUGAUGAG X CGAA ACCAGCCC 2308 GGGCUGGUU UCCAAACA 2749

2271 CUGUUUGG CUGAUGAG X CGAA AACCAGCC 2309 GGCUGGUUU CCAAACAG 2750

2272 UCUGUUUG CUGAUGAG X CGAA AAACCAGC 2310 GCUGGUUUC CAAACAGA 2751

2285 UAGACUAA CUGAUGAG X CGAA AUCCUCUG 2311 CAGAGGAUU UUAGUCUA 2752

2286 AUAGACUA CUGAUGAG X CGAA AAUCCUCU 2312 AGAGGAUUU UAGUCUAU 2753

2287 UAUAGACU CUGAUGAG X CGAA AAAUCCUC 2313 GAGGAUUUU AGUCUAUA 2754

2288 AUAUAGAC CUGAUGAG X CGAA AAAAUCCU 2314 AGGAUUUUA GUCUAUAU 2755

2291 AGCAUAUA CUGAUGAG X CGAA ACUAAAAU 2315 AUUUUAGUC UAUAUGCU 2756

2293 CAAGCAUA CUGAUGAG X CGAA AGACUAAA 2316 UUUAGUCUA UAUGCUUG 2757

2295 CACAAGCA CUGAUGAG X CGAA AUAGACUA 2317 UAGUCUAUA UGCUUGUG 2758

2300 AGAAGCAC CUGAUGAG X CGAA AGCAUAUA 2318 UAUAUGCUU GUGCUUCU 2759

2306 UUUUGGAG CUGAUGAG X CGAA AGCACAAG 2319 CUUGUGCUU CUCCAAAA 2760

2307 UUUUUGGA CUGAUGAG X CGAA AAGCACAA 2320 UUGUGCUUC UCCAAAAA 2761

2309 UGUUUUUG CUGAUGAG X CGAA AGAAGCAC 2321 GUGCUUCUC CAAAAACA 2762

2323 CCUGCCUG CUGAUGAG X CGAA AUGGGUGU 2322 ACACCCAUC CAGGCAGG 2763

2337 ACGCACCA CUGAUGAG X CGAA AUCCCCCU 2323 AGGGGGAUA UGGUGCGU 2764

2346 GGACAGGA CUGAUGAG X CGAA ACGCACCA 2324 UGGUGCGUU UCCUGUCC 2765

2347 UGGACAGG CUGAUGAG X CGAA AACGCACC 2325 GGUGCGUUU CCUGUCCA 2766

2348 GUGGACAG CUGAUGAG X CGAA AAACGCAC 2326 GUGCGUUUC CUGUCCAC 2767

2353 UUUCAGUG CUGAUGAG X CGAA ACAGGAAA 2327 UUUCCUGUC CACUGAAA 2768

2379 CUCUCCUG CUGAUGAG X CGAA ACUCUCUC 2328 GAGAGAGUU CAGGAGAG 2769

2380 ACUCUCCU CUGAUGAG X CGAA AACUCUCU 2329 AGAGAGUUC AGGAGAGU 2770

2389 UUUGUUGC CUGAUGAG X CGAA ACUCUCCU 2330 AGGAGAGUA GCAACAAA 2771

2406 UGUUCAUU CUGAUGAG X CGAA AUUUUCCU 2331 AGGAAAAUA AAUGAACA 2772

2416 AGCAAACA CUGAUGAG X CGAA AUGUUCAU 2332 AUGAACAUA UGUUUGCU 2773

2420 UAUAAGCA CUGAUGAG X CGAA ACAUAUGU 2333 ACAUAUGUU UGCUUAUA 2774

2421 AUAUAAGC CUGAUGAG X CGAA AACAUAUG 2334 CAUAUGUUU GCUUAUAU 2775

2425 UAACAUAU CUGAUGAG X CGAA AGCAAACA 2335 UGUUUGCUU AUAUGUUA 2776

2426 UUAACAUA CUGAUGAG X CGAA AAGCAAAC 2336 GUUUGCUUA UAUGUUAA 2777

2428 AUUUAACA CUGAUGAG X CGAA AUAAGCAA 2337 UUGCUUAUA UGUUAAAU 2778

2432 UUCAAUUU CUGAUGAG X CGAA ACAUAUAA 2338 UUAUAUGUU AAAUUGAA 2779

2433 AUUCAAUU CUGAUGAG X CGAA AACAUAUA 2339 UAUAUGUUA AAUUGAAU 2780

2437 UUUUAUUC CUGAUGAG X CGAA AUUUAACA 2340 UGUUAAAUU GAAUAAAA 2781

2442 GAGUAUUU CUGAUGAG X CGAA AUUCAAUU 2341 AAUUGAAUA AAAUACUC 2782

2447 AAAGAGAG CUGAUGAG X CGAA AUUUUAUU 2342 AAUAAAAUA CUCUCUUU 2783

2450 AAAAAAGA CUGAUGAG X CGAA AGUAUUUU 2343 AAAAUACUC UCUUUUUU 2784

2452 AAAAAAAA CUGAUGAG X CGAA AGAGUAUU 2344 AAUACUCUC UUUUUUUU 2785

2454 UAAAAAAA CUGAUGAG X CGAA AGAGAGUA 2345 UACUCUCUU UUUUUUUA 2786

2455 UUAAAAAA CUGAUGAG X CGAA AAGAGAGU 2346 ACUCUCUUU UUUUUUAA 2787

2456 CUUAAAAA CUGAUGAG X CGAA AAAGAGAG 2347 CUCUCUUUU UUUUUAAG 2788

2457 CCUUAAAA CUGAUGAG X CGAA AAAAGAGA 2348 ucucuuuuu UUUUAAGG 2789

2458 ACCUUAAA CUGAUGAG X CGAA AAAAAGAG 2349 cucuuuuuu UUUAAGGU 2790

2459 CACCUUAA CUGAUGAG X CGAA AAAAAAGA 2350 ucuuuuuuu UUAAGGUG 2791

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

Table XVII: Human B-rα/Hairpin Ribozyme and Target Sequence nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

9 GGAGGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 2795 UCCCG GCC CCCUCC 2846

20 CUGUCG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 2796 UCCCC GCC CGACAG 2847

31 CCGAGC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 2797 CAGCG GCC GCUCGG 2848

34 GGCCCG AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 2798 CGGCC GCU CGGGCC 2849

46 ACCGAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 2799 CCCCG GCU CUCGGU 2850

114 CCGUUG AGAA GAGC ACCAGAGAAACA X GUACAUUACCUGGUA 2800 GCUCU GUU CAACGG 2851

149 CGGGCC AGAA GGCG ACCAGAGAAACA X GUACAUUACCUGGUA 2801 CGCCG GCC GGCCCG 2852

153 GCCGCG AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 2802 GGCCG GCC CGCGGC 2853

160 CGAAGA AGAA GCGG ACCAGAGAAACA X GUACAUUACCUGGUA 2803 CCGCG GCC UCUUCG 2854

169 GUCCGC AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 2804 CUUCG GCU GCGGAC 2855

175 GGCAGG AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 2805 CUGCG GAC CCUGCC 2856

379 AGAAAA AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 2806 GAACU GAU UUUUCU 2857

388 GCUAGA AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 2807 UUUCU GUU UCUAGC 2858

466 UUGAAA AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 2808 UUUCA GUU UUUCAA 2859

484 UGCCAC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 2809 CCACA GAU GUGGCA 2860

540 UUGUUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2810 UUCCU GCC CAACAA 2861

586 GUCUCG AGAA GUAA ACCAGAGAAACA X GUACAUUACCUGGUA 2811 UUACA GUC CGAGAC 2862

596 UCUUUA AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2812 AGACA GUC UAAAGA 2863

612 CUCAUC AGAA GUGC ACCAGAGAAACA X GUACAUUACCUGGUA 2813 GCACU GAU GAUGAG 2864

646 UCUGUA AGAA GCAC ACCAGAGAAACA X GUACAUUACCUGGUA 2814 GUGCU GUU UACAGA 2865

819 UGGAAA AGAA GCUU ACCAGAGAAACA X GUACAUUACCUGGUA 2815 AAGCU GCU UUUCCA 2866

836 UUUGAC AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 2816 UUUCC GCU GUCAAA 2867

891 ACACAC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 2817 CCACU GAU GUGUGU 2868

924 GAGACA AGAA GCAA ACCAGAGAAACA X GUACAUUACCUGGUA 2818 UUGCU GUU UGUCUC 2869

988 UGUUAG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2819 AGACU GCC CUAACA 2870

1021 GUCCGA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 2820 CACCC GCC UCGGAC 2871

1027 AAUAGA AGAA GAGG ACCAGAGAAACA X GUACAUUACCUGGUA 2821 CCUCG GAC UCUAUU 2872

1055 GAGACG AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 2822 CACCA GUC CGUCUC 2873

1059 GAAGGA AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 2823 AGUCC GUC UCCUUC 2874

1089 CGGAAG AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 2824 CCACA GCC CUUCCG 2875

1097 CUGCUG AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 2825 CUUCC GAC CAGCAG 2876

1105 AUCUUC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 2826 CAGCA GAU GAAGAU 2877

1142 AUGAGG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2827 AGACC GAU CCUCAU 2878

1153 AUUGGG AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 2828 CAUCA GCU CCCAAU 2879

1267 UAAUGA AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 2829 CCCCU GCC UCAUUA 2880

1417 CUGCCC AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2830 UUCCU GAU GGGCAG 2881

1425 ACUGUA AGAA GCCC ACCAGAGAAACA X GUACAUUACCUGGUA 2831 GGGCA GAU UACAGU 2882

1468 CUUGUA AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 2832 GAACA GUC UACAAG 2883

nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

1664 GAUACA AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 2833 CUCCA GCU UGUAUC 2884

1734 UGUGCA AGAA GUCG ACCAGAGAAACA X GUACAUUACCUGGUA 2834 CGACA GAC UGCACA 2885

1884 UGUUCA AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 2835 CAUCA GUU UGAACA 2886

1893 CCAGAC AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 2836 GAACA GUU GUCUGG 2887

1958 ACUGAA AGAA GUAU ACCAGAGAAACA X GUACAUUACCUGGUA 2837 AUACA GCU UUCAGU 2888

1969 AUAUAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 2838 AGUCA GAU GUAUAU 2889

1995 AAUUCA AGAA GAAC ACCAGAGAAACA X GUACAUUACCUGGUA 2839 GUUCU GUA UGAAUU 2890

2079 UCUGGA AGAA GGUA ACCAGAGAAACA X GUACAUUACCUGGUA 2840 UACCU GUC UCCAGA 2891

2086 ACUGAG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 2841 CUCCA GAU CUCAGU 2892

2111 CUUUUG AGAA GUUA ACCAGAGAAACA X GUACAUUACCUGGUA 2842 UAACU GUC CAAAAG 2893

2205 CGGGCC AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 2843 GAGCU GCU GGCCCG 2894

2213 GCAAUG AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 2844 GGCCC GCU CAUUGC 2895

2350 UCAGUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2845 UUCCU GUC CACUGA 2896

Where "X" represents stem IV region of a Haiφin ribozyme. The length of stem IV may be > 2 base-pairs.

Table XVIII. Hammerhead (HH) Ribozyme target with sequence homology between c-raf and A-raf

Table XIX. Hammerhead Ribozyme Target with sequence homology between c-raf and B-raf

Table XX.

Experimental Ribozyme Dose Sample Size per

Group Activity/Target (mg/kg/day) dose

RPI.4610 Ac velflt-1 1,3, 10,30,100 10

RPI.4611 lna.ctive/flt-1 1,3,10,30,100 10

RPI.4733 Acύve/flk-1 1,3,10,30,100 10

RPI.4734 lnactive/flk-1 1,3,10,30,100 10

Saline NA 12 μl/day 10

Claims

Claims

1. A method for identification of a nucleic acid molecule capable of modulating a process in a biological system comprising the steps of:

a) introducing a random library of a nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence, into said biological system under conditions suitable for modulating said process; and

b) determining the nucleotide sequence of at least a portion of the substrate binding domain of said nucleic acid catalyst from a said biological system in which the process has been modulated.

2. A method for identifying one or more nucleic acid molecules involved in a process in a biological system comprising the steps of:

a) providing a library of a nucleic acid catalyst, with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence, to said biological system under conditions suitable for said process to be altered;

b) identifying any said nucleic acid catalyst present in said biological system where said process has been altered by said any said nucleic acid catalyst; and

c) determining the nucleotide sequence of at least a portion of the binding arm of said any said nucleic acid catalyst to allow said identification of said nucleic acid molecule involved in said process in said biological system.

3. A method for identification of a nucleic acid catalyst capable of modulating a process in a biological system comprising the steps of:

a) introducing a random library of a nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence, into said biological system under conditions suitable for modulating said process; and

b) identifying said nucleic acid catalyst from said biological system in which the process has been modulated.

4. The method of any of claims 1-3, wherein said biological system is a bacterial cell.

5. The method of any of claims 1-3, wherein said biological system is of plant origin.

6. The method of any of claims 1-3, wherein said biological system is of mammalian origin.

7. The method of any of claims 1-3, wherein said biological system is of yeast origin.

8. The method of any of claims 1-3, wherein said biological system is Drosophila.

9. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a hammerhead motif.

10. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a haiφin motif.

11. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a hepatitis delta virus ribozyme motif.

12. The method of any of claims 1-3, wherein said nucleic acid catalyst is in group I intron, group II intron, VS ribozyme or RNase P ribozyme motif.

13. The method of any of claims 1-3, wherein said process is selected from the group consisting of growth, proliferation, apoptosis, moφhology, angiogenesis, differentiation, migration, viral multiplication, drug resistance, signal transduction, cell cycle regulation, temperature sensitivity and chemical sensitivity.

14. The method of any of claims 1-3, wherein said random library of nucleic acid catalysts is encoded by an expression vector in a manner which allows expression of said nucleic acid catalysts.

15. The method of claim 14, wherein said expression vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) a gene encoding at least one said nucleic acid catalyst; and

wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst.

16. The method of claim 14, wherein said expression vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an open reading frame;

d) a gene encoding at least one said nucleic acid catalyst, wherein said gene is operably linked to the 3'-end of said open reading frame; and

wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst.

17. The method of claim 14, wherein said expression vector comprises: a) a transcription initiation region;

b) a transcription termination region;

c) an intron;

d) a gene encoding at least one said nucleic acid catalyst; and

wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst.

18. The method of claim 14, wherein said expression vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an intron;

d) an open reading frame;

e) a gene encoding at least one said nucleic acid catalyst, wherein said gene is operably linked to the 3'-end of said open reading frame; and

wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst.

19. The method of claim 14, wherein said expression vector is derived from a retrovirus.

20. The method of claim 14, wherein said expression vector is derived from an adenovirus. -. „-, l o3

21. The method of claim 14, wherein said expression vector is derived from an adeno-associated virus.

22. The method of claim 14, wherein said expression vector is derived from an alphavirus.

23. The method of claim 14, wherein said expression vector is derived from a bacterial plasmid.

24. The method of claim 14, wherein said expression vector is operable linked to a RNA polymerase II promoter element.

25. The method of claim 14, wherein said expression vector is operable linked to a RNA polymerase III promoter element.

26. The method of claim 25, wherein said RNA polymerase III promoter is derived from a transfer RNA gene.

27. The method of claim 25, wherein said RNA polymerase III promoter is derived from a U6 small nuclear RNA gene.

28. The method of claim 25, wherein the nucleic acid catalyst comprises a sequence at its 5 '-end homologous to the terminal 27 nucleotides of encoded by said U6 small nuclear RNA gene.

29. The method of claim 28, wherein said RNA polymerase III promoter is derived from a TRZ RNA gene.

30. The method of any of claims 1-3, wherein said biological system is of an eukaryotic origin.

31. The method of any of claims 1-3, wherein said biological system is of an prokaryotic origin. -. _R .

32. The method of any of claims 1-3, wherein said biological system is of an archaebacterial origin.

33. The method of any of claims 1-3, wherein said substrate binding domain of the nucleic acid catalyst is of length sufficient to form a stable interaction with a target sequence.

34. The method of claim 33, wherein said substrate binding domain is of length between 12 and 100 nucleotides.

35. The method of claim 33, wherein said substrate binding domain is of length between 14 and 24 nucleotides.

36. The method of any of claims 1-3, wherein said nucleic acid catalyst comprises one substrate binding arm.

37. The method of any of claims 1-3, wherein said nucleic acid catalyst comprises two substrate binding arms.

38. The method of claim 37, wherein said substrate binding arms are of similar length.

39. The method of claim 37, wherein said substrate binding arms are of different length.

40. A nucleic acid molecule with an endonuclease activity having the formula III:

^ (N) o— C— G — A — A — A M— 3'

L

^ (N) n— G — A — G — Nγ— A — G — C4— U3— Q^~ 5'

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are l o5

integers greater than or equal to 1, wherein if (N)₀ and (N)_n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; represents a chemical linkage; and A, C, U and G represent adenosine, cytidine, uridine and guanosine nucleotides, respectively.

41. A nucleic acid molecule with catalytic activity having the formula IV:

^M\ / Q ⁵

A Z3

Z4

A G

A A

G Z7

A^G c • G

(N)o (N)n

\ /

L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)₀ and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2 '-methylthiomethyl uridine; Z4 is 2'-C-allyl uridine; Z7 is 6- methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively. l o6

42. A nucleic acid molecule with catalytic activity having the formula V:

3' M_χ / Q ⁵

A Z3

Z4

A G

A A

G C^ Z7

A^G c • G

(N)o (N)n

\ / L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)₀ and (N)_n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2 '-methylthiomethyl uridine; Z4 is 2 '-methylthiomethyl cytidine;

Z7 is 6-methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

3. A nucleic acid molecule with catalytic activity having the formula VI:

3'

^M\ / Q

A Z3

Z4

A

G

A A

G _ΛG ^Z7

A c • G

(N)o (N)n

\ /

L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)₀ and (N)_n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2 '-methylthiomethyl uridine; Z4 is 2 '-methylthiomethyl cytidine;

Z7 is 2'-C- allyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

l oo

44. A nucleic acid molecule with catalytic activity having the formula VII:

^M\ / Q ⁵

A Z3

Z4

A G

A A

G Z7

A^G c • G

(N)o (N)n

\ / L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)₀ and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2 '-methylthiomethyl uridine; Z4 is 2 '-methylthiomethyl cytidine;

Z7 is pyridine-4-one; and represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

45. The nucleic acid molecules of any of claims 40-44, wherein said (N)₀ and (N)_n are nucleotides and said o and n are integers greater than or equal to 3.

46. The nucleic acid molecules of any of claims 40-44, wherein said L is nucleotide linker.

47. The nucleic acid molecule of any of claims 40-44, wherein said nucleic acid cleaves a separate nucleic acid molecule. -. _RQ

48. The nucleic acid molecule of claim 47, wherein said separate nucleic acid molecule is RNA.

49. The nucleic acid molecule of claim 47, wherein said nucleic acid comprises between 12 and 100 bases complementary to said separate nucleic acid molecule.

50. The nucleic acid molecule of claim 47, wherein said nucleic acid comprises between 14 and 24 bases complementary to said separate nucleic acid molecule.

51. A cell including the nucleic acid molecule of any of claims 40-44.

52. The cell of claim 17, wherein said cell is a mammalian cell.

53. The cell of claim 18, wherein said cell is a human cell.

54. An expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecule of any of claims 40-44, in a manner which allows expression of that nucleic acid molecule.

55. A cell including the expression vector of claim 54.

56. The cell of claim 55, wherein said cell is a mammalian cell.

57. The cell of claim 55, wherein said cell is a human cell.

58. A pharmaceutical composition comprising the nucleic acid molecule of any of claims 40-44.

59. A method for modulating expression of a gene in a plant cell by administering to said cell the nucleic acid molecule of any of claims 40-44.

60. A method for modulating expression of gene in a mammalian cell by administering to said cell the nucleic acid molecule of any of claims 40-44. , _QQ

61. A method of cleaving a separate nucleic acid comprising, contacting the nucleic acid molecule of any of claims 40-44with said separate nucleic acid molecule under conditions suitable for the cleavage of said separate nucleic acid molecule.

62. The method of claim 61, wherein said cleavage is carried out in the presence of a divalent cation.

63. The method of claim 62, wherein said divalent cation is Mg2⁺.

64. The nucleic acid molecule of claims 40-44, wherein said nucleic acid is chemically synthesized.

65. The expression vector of claim 54, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) a gene encoding at least one said nucleic acid molecule; and

wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

66. The expression vector of claim 54, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an open reading frame;

d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 '-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

67. The expression vector of claim 59, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an intron;

d) a gene encoding at least one said nucleic acid molecule; and

wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

68. The expression vector of claim 59, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an intron;

d) an open reading frame;

e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and

wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

9. A method for identifying variants of a nucleic acid catalyst comprising the steps of:

a) selecting at least three positions within said nucleic acid catalyst to be varied with a predetermined group of different nucleotides;

b) synthesizing a first class of different pools of said nucleic acid catalyst, wherein the number of pools synthesized is equal to the number of nucleotides in the predetermined group of different nucleotides, wherein at least one of the positions to be varied in each pool comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides;

c) testing the different pools of said nucleic acid catalyst under conditions suitable for said pools to show a desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps;

d) synthesizing a second class of different pools of nucleic acid catalyst, wherein at least one of the positions to be varied in each of the second class of different pools comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides;

e) testing the second class of different pools of said nucleic acid catalyst under conditions suitable for showing desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps; and

f) repeating the process similar to steps d and e until every position selected in said nucleic acid catalyst to be varied is made constant.

70. A method for identifying novel nucleic acid molecules in a biological system, comprising the steps of:

a) synthesizing a pool of nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence;

b) testing the pools of nucleic acid catalyst under conditions suitable for showing a desired effect in said biological system and identifying the catalyst showing said desired effect;

c) using an oligonucleotide, comprising the sequence of the substrate binding domain of the nucleic acid catalyst showing said desired activity, as a probe, screening said biological system for nucleic acid molecules complementary to said probe; and

d) isolating and sequencing said complementary nucleic acid molecules.

71. A compound having the formula I:

O O O

-O- -O - -O - ^■OR

O- O- O-

wherein R is independently any nucleoside selected from the group consisting of 2'-O-methyl-2,6-diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine riboside; 2'-(N-alanyl) amino-2'-deoxy-uridine; 2'-(N-phenylalanyl)amino-2'-deoxy-uridine; 2'- deoxy -2'-(N-β-alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'- O-amino-uridine; 2 '-O-methylthiomethyl adenosine; 2 '-O-methylthiomethyl cytidine ; 2'- O-methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-Deoxy-2'-(N-histidyl) amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N-β-carboxamidine-β- alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(Ν-β-alanyl)-guanosine; and 2'-O-amino- adenosine.

72. A process for incoφoration of the compounds of claim 71 into an oligonucleotide comprising the step of contacting said compound with a mixture comprising a nucleic acid template, an RNA polymerase enzyme, and an enhancer of modified nucleotide triphosphate incoφoration, under conditions suitable for the incoφoration of said compound into said oligonucleotide.

73. The process of claim 72, wherein said RNA polymerase is a T7 RNA polymerase.

74. The process of claim 72, wherein said RNA polymerase is a mutant T7 RNA polymerase.

75. The process of claim 72, wherein said RNA polymerase is a SP6 RNA polymerase.

76. The process of claim 72, wherein said RNA polymerase is a mutant SP6 RNA polymerase.

77. The process of claim 72, wherein said RNA polymerase is a T3 RNA polymerase.

78. The process of claim 72, wherein said RNA polymerase is a mutant T3 RNA polymerase.

79. The process of claim 72, wherein said enhancer of modified nucleotide triphosphate incoφoration is selected from the group consisiting of LiCl, methanol, polyethylene glycol, diethyl ether, propanol, methylamine, and ethanol.

80. A process for the synthesis of a pyrimidine nucleotide triphosphate comprising the steps of:

(a) monophosphorylation, wherein a pyrimidine nucleoside is contacted with a mixture comprising a phosphorylating reagent, a trialkyi phosphate and -. _gt.

dimethylaminopyridine, under conditions suitable for the formation of a pyrimidine nucleotide monophosphate; and

(b) pyrophosphorylation, wherein said pyrimidine monophosphate from step (a) is contacted with a pyrophosphorylating reagent under conditions suitable for the formation of said pyrimidine nucleoside triphosphate.

81. The process of claim 80, wherein said pyrimidine nucleoside triphosphate is uridine triphosphate.

82. The process of claim 80, wherein said uridine triphosphate has a 2'-sugar modification.

83. The process of claim 82, wherein said uridine triphosphate is 2'-O- methylthiomethyl uridine triphosphate.

84. The process of claim 80, wherein said phosphorylating agent is selected from the group consisting of phosphorus oxychloride, phospho-tris-triazolides and phospho-tris-triimidazolides.

85. A process of claim 80, wherein said trialkylphosphate is triethyl phosphate.

86. The process of claim 80, wherein said pyrophosphorylating reagent is tributyl ammonium pyrophosphate.

87. The process of claim 72, wherein said oligonucleotide is RNA.

88. The process of claim 72, wherein said oligonucleotide is an nucleic acid catalyst.

89. The process of claim 72, wherein said oligonucleotide is an aptamer.

90. A kit for synthesis of an oligonucleotide comprising an RNA polymerase, an enhancer of modified nucleotide triphosphate incoφoration and at least one compound of claim 71. -.

19o

91. A kit for synthesis of an oligonucleotide comprising a DNA polymerase, an enhancer of modified nucleotide triphosphate incoφoration and at least one compound of claim 71.

92. The kit of claim 90, wherein said RNA polymerase is a bacteriophage T7 RNA polymerase.

93. The kit of claim 90, wherein said RNA polymerase is a bacteriophage SP6 RNA polymerase.

94. The kit of claim 90, wherein said RNA polymerase is a bacteriophage T3 RNA polymerase.

95. The kit of claim 90, wherein said RNA polymerase is a mutant T7 RNA polymerase.

96. The kit of claim 90 or 91, wherein said kit comprises at least two compounds of claim 71.

97. A compound having the formula II:

wherein, R, is OH, O-R3, wherein R₃ is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester, C-R , wherein R is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester, halo, NHR₄ wherein R₄ is independently a moiety selected from a group consisting of alkyl (Cl-22), acyl

(Cl-22), substituted or unsubstituted aryl), or ΟCH₂SCH₃ (methylthiomethyl),

ΟNHR₅ where R₅ is independently a moiety selected from a group consisting of

H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester, ON=R₆, where R₆ is independently pyridoxal residue, pyridoxal-5 -phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl , alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl;

B is independently a nucleotide base or its analog or hydrogen;

X is independently a phosphorus-containing group; and

R₂ is independently blocking group or a phosphorus-containing group.

98. The compound of claim 97, wherein said compound is a nucleotide.

99. The compound of claim 97, wherein said compound is a nucleotide-tri- phosphate.

100. A polynucleotide comprising the compound of claim 97 at one or more positions.

101. The polynucleotide of claim 100, wherein said polynucleotide is an enzymatic nucleic acid.

102. The enzymatic nucleic acid of claim 101, wherein said nucleic acid is in a hammerhead configuration.

103. The enzymatic nucleic acid of claim 102, wherein said nucleic acid is in a haiφin configuration.

104. The enzymatic nucleic acid of claim 102, wherein said nucleic acid is in a hepatitis delta virus, group I intron, VS RNA, group II intron or RNase P RNA configuration.

105. The compound of claim 97, wherein said compound is xylo riboadenosine. -. _g„

106. The compound of claim 97, wherein said compound is xylo riboguanosine.

107. The compound of claim 97, wherein said compound is xylo ribonucleoside phosphoramidite.

108. The compound of claim 107, wherein said compound is xylo riboguanosine phosphoramidite.

109. The compound of claim 107, wherein said compound is xylo riboadenosine phosphoramidite.

110. A mammalian cell comprising the compound of claim 97.

111. The mammalian cell of claim 14, wherein said cell is a human cell.

112. A mammalian cell comprising the compound of claim 101.

113. The mammalian cell of claim 112, wherein said cell is a human cell.

114. A method of making a polynucleotide of claim 100.

115. A method of modulating gene expression using a polynucleotide of claim 100.

116. A pharmaceutical composition comprising a compound of claim 97.

117. A pharmaceutical composition comprising a polynucleotide of claim 101.

118. The compound of claim 97, wherein said compound is used as an antiviral agent.

119. A process for the synthesis of a xylo ribonucleoside phosphoramidite comprising the steps of:

a) oxidation of a 2' and 5'-protected ribonucleoside using an oxidant followed by reduction using a reducing agent under conditions suitable for the formation of 2' and 5'-protected xylofuranosyl nucleoside; and b) phosphitylation under conditions suitable for the formation of xylofuranosyl nucleoside phosphoramidite.;

120. The process of claim 119, wherein said oxidantion is carried out in the presence of chromium oxide, pyridine, and aceticanhydride.

121. The process of claim 119, wherein said oxidantion is carried out in the presence of dimethylsulfoxide and aceticanhydride.

122. The process of claim 119, wherein said oxidantion is carried out in the presence of Dess-Martin reagent (periodinane).

123. The process of claim 119, wherein said reduction is carried out in the presence of triacetoxy sodium borohydride.

124. The process of claim 119, wherein said reduction is carried out in the presence of sodium borohydride

125. The process of claim 119, wherein said reduction is carried out in the presence of lithium borohydride,

126. A process for one pot deprotection of RNA comprising protecting groups, comprising the steps of:

a) contacting said RNA with a mixture of anhydrous alkylamine, trialkylamine and a polar organic reagent in a predetermined proportions, at room temperature for about between 30 and 100 min under conditions suitable for the removal of nucleic acid base and phosphate protecting groups from said RNA; and

b) contacting the resulting RNA from step a with an anhydrous triethylamine^»hydrogen fluoride at about between 50 °C-70 °C under conditions suitable for the removal of a 2'-OH protecting group.

127. The process of claim 126 wherein said RNA is an enzymatic RNA molecule.

128. The process of claim 128, wherein said enzymatic RNA molecule is in a hammerhead motif.

129 The process of claim 126, wherein said polar organic reagent is dimethylsulfoxide

130. The process of claim 126, wherein said anhydrous alkylamine is anhydrous methyl amine.

131. The process of claim 126, wherein said anhydrous alkylamine is anhydrous ethylamine.

132. The process of claim 126, wherein said trialkylamine is triethylamine.

133. The process of claim 129, wherein the predetermined proportion of anhydrous alkylamine, trialkylamine and dimethylsulfoxide in said mixture is 10, 3 and 13, respectively.

134. A process for one pot deprotection of RNA comprising protecting groups, comprising the steps of:

a) contacting said RNA with a mixture of anhydrous methylamine, triethylamine and dimethylsulfoxide in proportions of 10, 3 and 13, respectively, at room temperature for about 90 min under conditions suitable for the removal of nucleic acid base and phosphate protecting groups from said RNA; and

b) contacting the resulting RNA from step a with an anhydrous triethylamine'hydrogen fluoride at about between 65 °C under conditions suitable for the removal of a 2'-OH protecting group.

135 The process of claim 134 wherein said RNA is an enzymatic RNA molecule.

136. The process of claim 135, wherein said enzymatic RNA molecule is in a hammerhead motif. 137 A process for one pot deprotection of RNA comprising protecting groups, comprising the steps of:

a) contacting said RNA with a mixture of anhydrous alkylamine and a polar organic reagent in a predetermined proportions, at room temperature for about between 30 and 100 min under conditions suitable for the removal of nucleic acid base and phosphate protecting groups from said RNA; and

b) contacting the resulting RNA with a anhydrous triethylamine'hydrogen fluoride at about between 50 °C-70 °C under conditions suitable for the removal of 2'-OH protecting group.

138. The process of claim 137 wherein said RNA is an enzymatic RNA molecule.

139. The process of claim 138, wherein said enzymatic RNA molecule is in a hammerhead motif.

140. The process of claim 137, wherein said polar organic reagent is dimethylsulfoxide

141. The process of claim 137, wherein said anhydrous alkylamine is anhydrous methyl amine.

142. The process of claim 137, wherein said anhydrous alkylamine is anhydrous ethylamine.

143. An nucleic acid catalyst with RNA cleaving activity, wherein said nucleic acid catalyst modulates the expression of a Raf gene.

144. The nucleic acid catalyst of claim 143, wherein said nucleic acid catalyst is in a hammerhead configuration.

145. The nucleic acid catalyst of claim 144, wherein said nucleic acid catalyst comprises a stem II region of length greater than or equal to 2 base pairs.

146. The nucleic acid catalyst of claim 143, wherein said nucleic acid catalyst is in a haiφin configuration.

147. The nucleic acid catalyst of claim 143, wherein said enzymatic nucleic acid is in a hepatitis δ virus, group I intron, group II intron, VS nucleic acid or RNase P nucleic acid configuration.

148. The enzymatic nucleic acid of claim 146, wherein said nucleic acid catalyst comprises a stem II region of length between three and seven base-pairs.

149. The nucleic acid catalyst of claim 143, wherein said nucleic acid comprises between 12 and 100 bases complementary to said RNA.

150. The nucleic acid catalyst of claim 143, wherein said nucleic acid comprises between 14 and 24 bases complementary to said mRNA.

151. The nucleic acid catalyst of claim 144, wherein said nucleic acid catalyst consists essentially of any sequence defined as Seq ID Nos 502-1102, 1153-1460 and 1913-2353.

152. A mammalian cell including an nucleic acid catalyst of any of claim 143.

153. The mammalian cell of claim 152, wherein said mammalian cell is a human cell.

154. An expression vector comprising nucleic acid sequence encoding at least one nucleic acid catalyst of claim 143, in a manner which allows expression of that nucleic acid catalyst.

155. A mammalian cell including an expression vector of claim 154.

156. The mammalian cell of claim 155, wherein said mammalian cell is a human cell.

157. A method for treatment of cancer, restenosis, psoriasis and rheumatoid arthritis comprising the step of administering to a patient the nucleic acid catalyst of claim 143.

158. A method for treatment of cancer, restenosis, psoriasis and rheumatoid arthritis comprising the step of administering to a patient the expression vector of claim 154.

159. A method for treatment of cancer comprising the steps of: a) isolating cells from a patient; b) administering to said cells the nucleic acid catalyst of claim 143; and c) introducing said cells back into said patient.

160. A pharmaceutical composition comprising the nucleic acid catalyst of claim 143.

161. A method of treatment of a patient having a condition associated with the level of c-raf, wherein said patient is administered the nucleic acid catalyst of claim 143.

162. A method of treatment of a patient having a condition associated with the level of c-raf, comprising contacting cells of said patient with the nucleic acid molecule of claim 143, and further comprising the use of one or more drug therapies.

163. The nucleic acid catalyst of claim 144, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid comprises a 2'-C-allyl modification at position No. 4 of said nucleic acid, and wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a 3'- end modification.

164. The enzymatic nucleic acid of claim 163, wherein said nucleic acid comprises a 3'-3' linked inverted ribose moiety at said 3' end.

165. The nucleic acid catalyst of claim 144, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid molecule comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid comprises a 2'-amino modification at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid molecule comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid comprises a 3'- end modification.

166. The nucleic acid catalyst of claim 144, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid molecule comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid molecule comprises an abasic substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid molecule comprises a 3 '-end modification.

167. The nucleic acid catalyst of claim 144, wherein said nucleic acid molecule comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid molecule comprises a 6-methyl uridine substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid molecule comprises at least ten 2'-O-methyl modifications, and wherein said nucleic acid molecule comprises a 3' end modification.

168. A method for modulating expression of c-raf gene in a mammalian cell by administering to said cell the nucleic acid catalyst of claim 143.

169. A method of cleaving a separate RNA molecule comprising, contacting the nucleic acid catalyst of claim 143 with said separate RNA molecule under conditions suitable for the cleavage of said separate RNA molecule.

170. The method of claim 169, wherein said cleavage is carried out in the presence of a divalent cation.

171. The method of claim 170, wherein said divalent cation is Mg2⁺.

172. The nucleic acid molecule of claim 143, wherein said nucleic acid is chemically synthesized.

173. The expression vector of claim 154, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) a gene encoding at least one said nucleic acid molecule; and

wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

174. The expression vector of claim 154, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an open reading frame;

d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and

wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

175. The expression vector of claim 154, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an intron; 2Uo

d) a gene encoding at least one said nucleic acid molecule; and

wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

176. The expression vector of claim 154, wherein said vector comprises:

a) a transcription initiation region;

b) a transcription termination region;

c) an intron;

d) an open reading frame;

e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and

wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

177. The nucleic acid catalyst of claim 144, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 1-501, 1461-1768 and 2354-2794.

178. The nucleic acid catalyst of claim 146, wherein said nucleic acid catalyst consists essentially of any sequence defined as Seq ID Nos 1003-1077, 1769-1840 and 2795-2845.

179. The nucleic acid catalyst of claim 146, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 1078-1152, 1841-1912 and 2846-2896.

180. The nucleic acid catalyst of claim 144, wherein said enzymatic nucleic acid comprises sequences complementary to any of sequences defined as Seq ID Nos 2897-2956.

181. The nucleic acid catalyst of claim 143, wherein said Raf gene is c-Raf-1 gene.

182. The nucleic acid catalyst of claim 143, wherein said Raf gene is A-Raf gene.

183. The nucleic acid catalyst of claim 143, wherein said Raf gene is B-Raf gene.

184. The nucleic acid catalyst of claim 143, wherein said enzymatic nucleic acid is a DNA enzyme.

185. The nucleic acid catalyst of claim 143, wherein said enzymatic nucleic acid comprises at least one 2'-sugar modification.

186. The nucleic acid catalyst of claim 143, wherein said enzymatic nucleic acid comprises at least one nucleic acid base modification.

187. The nucleic acid catalyst of claim 143, wherein said enzymatic nucleic acid comprises at least one phosphorothioate modification.

188. A method of treatment of a systemic disease in a patient comprising the step of systemically administering to said patient a nucleic acid catalyst which specifically cleaves RNA associated with said disease, under conditions in which said RNA in said patient is cleaved and a therapeutic result is attained.

189. The method of claim 188, wherein said disease is selected from the group consisiting of cancer, inflammation, psoriasis, non-hepatic ascites and infectious disease.

190. The method of claim 189, wherein said treatment of said cancer is characterised by a decrease in tumor metastasis. 20o

191. The method of claim 189, wherein said treatment of said cancer is characterised by a decrease in tumor volume.

192. The method of claim 189, wherein said treatment of said cancer is characterised by a decrease in the progression of primary tumor.

193. The method of claim 188, wherein said nucleic acid catalyst is chemically modified.

194. The method of claim 188, wherein said nucleic acid catalyst is in a hammerhead motif.

195. The method of claim 194, wherein said hammerhead nucleic acid catalyst comprises 2'-C-allyl modification at position 4, phosphorothioate linkages at four

5 '-terminal positions and inverted abasic nucleotide at the 3 '-end of said nucleic acid molecule.

196. The method of claim 188, wherein said systemic administration is by intravenous administration of said nucleic acid catalyst into said patient.

197. The method of claim 188, wherein said systemic administration is by a bolus administration of said nucleic acid catalyst into said patient.

198. The method of claim 188, wherein said systemic administration is by continuous infusion of said nucleic acid catalyst into said patient.