CA2324421A1 - Method and reagents for the treatment of diseases or conditions related to molecules involved in angiogenic responses - Google Patents

Abstract

Nucleic acid molecule which modulates the synthesis, expression and/or stability of an mRNA encoding for angiogenic factors selected from aryl hydrocarbon nuclear transport (ARNT), intergrin subunit beta 3 (.beta.3), integrin subunit alpha 6 (.alpha.6) and tie - 2RNA. This invention further provides a treatment for indications related to angiogenesis using the nucleic acid molecules.

Description

. CA 02324421 2000-09-26. ' DEMANDES OU BREVETS VOLUMtNEUX
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PlOTE:.For additional voiumes~piease contact~the Canadian Patent Office DESCRIPTION
Method And Rea ents For The Treatment Of Diseases Or Conditions Related To Molecules Involved In An io enic Res onses 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 angiogenic factors and receptors involved in the regulation of angiogenesis.
The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
The formation of blood vessels in vertebrates can be described in two embyronic stages. During the first stage, known as vasculogenesis, yolk sac splanchnopleuric mesenchyme differentiates into vascular progenitor cells and then to blood island aggregates which are primitive blood cells surrounded by fused endothelial progenitors (angioblasts). These blood islands then fuse and go on to form a vascular plexus which supplies nutrients to the embryo (Merenmies et al., 1997, Gel1 Growth & Development 8, 3-10). The next vascular developmental step is known as angiogenesis. From the vessels formed during vasculogenesis, new blood vessels sprout, elongate and develop into capillary loop formations of endothelial cells. It is a highly complex event involving local basement membrane disruption, endothelial cell proliferation, migration and microvessel morphogenesis (Rak et al., 1995, Anti-Cancer Drugs 6, 3-18). Organs such as the brain and kidney are vascularized through the angiogenic process (Dumont et al., 1995, Developmental Dynamics 203, 80-92).

2 Angiogenesis has been described to occur through two mechanisms, vascular sprouting and intussusception.
Intussusception of pre-existing vessels occur after proliferation of endothelial cells producing a wide lumen.
Through the utilization of transcapillary pillars or posts of extracellular matrix, the lumen is split to form two vessels (Risau, 1997, Nature 386, 671-674). Sprouting angiogenesis also originates from pre-existing blood vessels and consists of new blood vessels sprouting, elongating and developing into capillary loop formations of endothelial cells. It is a highly complex event involving disruption of extracellular matrix, endothelial cell proliferation, chemotaxic migration and microvessel morphogenesis (Rak, supra). Many factors regulating positive and negative control of angiogenesis have been reported demonstrating the sophistication of this process.
An example of an angiogenic factor is Vascular Endothelial Growth Factor receptor (VEGFr) which has been shown to be specific to endothelial cells and is discussed in Pavco et al., Int. PCT Pub. No. WO 97/15662.
Unlike vasculogenesis, angiogenesis not only occurs in embyronic development, but can also occur throughout the lifespan of the organism during such events as wound healing, bone repair, inflammation, and female menstral cycles. Local delivery of oxygen and nutrients and the removal of waste requires a complex system of blood vessels which has the ability to adapt as the tissue requirements changes. Involvement of a large number of positive and negative factors in angiogenic regulation demonstrates the complexity of this process. When the balance between upregulating factors and downregulating factors is disrupted in favor of increased angiogenesis, disease states have been known to occur.
Many factors have been identified which contribute to increased angiogenesis including:

3 PCT/US99/06507 1) ryl Hydrocarbon Nuclear Transporter (ARNT): ARNT
(also known as HIF-1(3) forms heterodimers with several factors including HIF-a(Maxwell et al., 1997, Proc. Natl.
Acad. Sci. USA 94, 8109-8209). When HIF-a and ARNT complex together, they form a complex called HIF-1. HIF -1 is believed to be regulate genes involved in the response to oxygen deprivation. ARNT -/- embryonic stem cells fail to induce VEGF expression in response to hypoxia. ARNT -/-mice are not viable beyond embryonic day 10.5. Like VEGF
knockout mice, these embryos show defective angiogenesis of the yolk sac (Maltepe et al., 1997, Nature 386, 403-907) .
Hepatoma cells containing an ARNT mutation that is functionally deficient in dimerizing with HIF-lashows greatly reduced VEGF expression in response to hypoxia compared to normal cells (Wood et al., 1996, J. Biol.
Chem. 271, 15117-15123). Tumor xenografts derived from these cells show reduced vascularity and approximately 2=
fold reduced tumor growth rates (Maxwell et al., 1997, supra) .
2) Tie-2: Tie-2 (also known as Tek), is a tyrosine kinase protein receptor which consists of 1122 amino acids and is produced in endothelial (Merenmies et al., 1997, Cell Growth & Differentiation 8, 3-10) as well as early hematopoeitic cells (Maisonpierree et al., 1993, Oncogene 8, 1631-1637). Tie-2 expression has been demonstrated in mice, rats and humans. The human gene is thought to be located on chromosome 9p21 (Dumont et al., 1994, Genes &
Development 8, 1897-1909). Tie-2 homozygous mutant endothelial cells were examined using anti-PECAM
monoclonal antibody (Sato et al., 1997, Nature 376, 70-74). All of the homozygous mutants were dead within 10.5 days with obvious deformities in the head and heart present by day 9.5. In addition, large vessels were indistinguishable from small vessels and no capillary sprouts were seen in the brain. These observations suggested that Tie-2 plays an important role in angiogenesis rather than vasculogenesis. The earlier effects of Tie-2 mutant compared to the Tie-1 mutant indicates separate roles for the two RTK's in angiogenesis.
Ligands to Tie-2 have been discovered and named' angiopoietin 1 and 2 (angl and 2) (Davis, S. et al., 1993, Cell 87, 1161; Maisonpierre, P.C. et a1.,1997, Science, 277, 55-60). Both factors consist of an NH2-terminal coiled-coil domain as well as a COOH-terminal fibrinogen-like domain. Angl binds to Tie-2/Tek but not Tie-1 and stimulates angiogenesis through autophosphorylation. Ang2 is a 496 amino acid polypeptide whose human and mouse homologs are 85$ identical. Autophosphorylation caused by Angl binding to the Tie-2 receptor can be blocked with the addition of Ang2. The Tie-2 receptor is unusual in that it utilizes both positive and negative control mechanisms.
3) Integrins: Integrins are a family of cell adhesion and migration mediating proteins that are comprised of at least 15 alpha and 8 beta subunits that are expressed as a number of different a(3 non-covalently bound heterodimers on cell surfaces (Varner, 1997, Regulation of Angiogenesis, ed I.D Goldberg & E.M. Rosen, 361-390; Brooks, 1996, Eur J Cancer 14, 2423-2429). Each combination of integrin subunits is thought to have angiogenic capabilities, for example a6(31 has been implicated in capillary tube formation Additionally, distinct integrins allow for the attachment to many different extracellular matrix (ECM) components including fibronectin, vitronectin, laminin and collagen (Stromblad & Cheresh, 1996, Chemistry & Biochemistry 3, 881-885).
Integrin production has been shown to be induced by a number a stimuli including intracellular pH increases, calcium concentration, inositol lipid synthesis, tyrosine phosphorylation of a focal contact associated tyrosine kinase, and activation of p34/cdc2 and cyclin A (Varner &
Cheresh, 1996, Curr Op in Cell Bio1 8,724-730).
a"[33 a 160kDa protein is the most well characterized 5 molecule of the integrin family and is believed to play a large role in angiogenesis (Varner, 1997, supra). a~(33 binds the largest number of ECM components of all known heterodimers indicating any cell with these molecules on the cell surface could adhere to or migrate on almost any of the ECM components (Varner, 1997, supra). When vascular endothelial cells are in their quiescent state very little a~(33 is expressed, but is highly upregulated in several pathological conditions including neoplasms.
Antagonists to a"(33 can inhibit angiogenesis in the chick chorioallentoic membrane (CAM) model and in SCID mice and even reduce the tumor volume. When antibodies are administered for A"(33, apoptosis is observed in the proliferating vascular vessels. This has led to suggestions that a"~33 provides a survival signal for vascular cells allowing for continued proliferation (Stromblad & Cheresh, 1996, supra; Varner, 1997 supra).
Other angiogenic targets are included and their characteristics are defined in the following references, all of which are incorporated herein by reference in their entirety: Methionine Aminopeptidase: (Arfin et al., 1995, PNAS 92, 7714-7718 (Genbank Accession No. U29607) ; Sin, N. et.al., 1997, PNAS 94, 6099-6103; Griffith et al., 1997, Chem Biol. 4(6), 461-471); Transcription factor Ets-1: (Iwasaka, C. et a1. 1996. J. Cell Phys.iol. 169, 522-531:
Chen, Z. et al. ,1997, Cancer Res. 57, 2013-2019;
Hultgardh-Nilsson A, et al., 1996, Circ Res. 78(4), 589-595; Reddy et al., 1988, Oncogene Res. 3 (3), 239-246 (Genbank accession No. X14798)); Platelet-derived endothelial cell growth factor and its receptor (PD-ECGF &
PD-ECGFr): (Furukawa, T. et al., 1992, Nature 356, 668;

Moghaddam, A. et al., 1995, Proc. Natl. Acad. Sci.; Clark, R.A.F. et a1. ,1996, Am J. Pathol. 148, 1407; Hoshina, T.M., et al., 1995, Int. J. Cancer 64, 79-82; Nakanishi, A.K., et al., 1992, J. Biol. Chem 267, 20311-20316;
Finnis et al., unpublished (Genbank accession No. M63193);
Transforming Growth factors (TGFs): (Schreiber et al., 1986, Science 232, 1250; Maione, T.E. and Sharpe, R.J.,1990, Trends Pharm. Sci., 11, 457-461; Noma et al., 1991, Growth Factors 9 (4), 247-255; Sukurai (unpublished) (Genbank accession No. AB009356);Transformin growth factor receptor: (Miyazono, K.,1996, Nippon Yakurigaku Zasshu 107, 133-140; Mahooti-Brooks. et a1.,1996, J. Clin.
Invest. 97, 1436-1446; Lopez-Casillas et al., 1991, Cell 67 (4 ) , 797-805; Lopez-Casillas et a1. , 1991, Cell 67 (4 ) , 785-795 (Genbank Accession No. L07594); Angiogenin: (Fett et al., 1985, Biochemistry 24, 5480-5486; Bicknell &
Vallee, 1988, PNAS 85, 5961-5965; Vallee & Riordan,1988, Adv. Exp. Med. Bio.I 234, 41-53; Shapiro & Vallee,1987, PNAS 84, 2238-2241; Shapiro et a.I.,1986, Biochemistry 25, 3527-3532; Olson et al., 1994, Cancer Res. 54, 4576-4579;
Kurachi et al., 1985, Biochemistry 24, 5494-5499; Kurachi et al., 1985, Biochemistry 24 (20), 5494-5499(Genbank Accession No. M11567)); Tumor necrosis factor receptor:
(Naismith et a1.,1995,. J. Inflamm 47, 1-7; Loetscher et al., 1990, Cell 61, 351-359; Himmler et al., 1990, DNA
Cell Biol. 9, 705-715 (Genbank Accession No. M63121 M75861); Endothelial cell stimulating an io enesis factor (ESAF): (Brown & Weiss,1988,. Ann. Rheum. Dis., 47, 881-885); Interleukin-8 (IL-8): (Elner et al., 1991, , Am J. Pathol. 139, 977-988; Strieter et a1.,1992, Am. J.
Pathol. 191, 1279-1284; Mukaida et al., 1989, J. Immunol.
143 (4), 1366-1371(Genbank Accession No. M28130));
Angiopoietin 1: (Davis, S. et a1.,1996, Cel~ 87, 1161;
Iwama, A. et a1.,1993, Biochem Biophys. Res. Commun. 195, 301; Dumont, D.J. et al.,1995, Genes Dev 8, 1897; Sato, T.N. et a1.,1995, Nature 376, 70; Suri, C. et al., 1996) Cell 87, 1171(Genbank Accession No. U83508)); Angiopoietin 2: (Maisonpierre, et a1.,1997, Science, 277, 55-60;
Hanahan, 1997, Science 277, 48-50; Genbank Accession No.
AF004327 (unpublished));Insulin-like growth factor (IGF-1): (Warren, R.S. et a1.,1996, J. Biol. Chem. 271, 29483-29488; Grant et.al., 1993,Diabetologia 36, 282-291;
Nicosia et al., 1994, Am. J. Pathol. 145, 1023-1029;
Steenbergh et al., Biochem. Biophys. Res. Commun. 175, 507-514 (Genbank Accession: X57025); Insulin-like growth factor receptor (IGF-lr): (Ullrich et al., 1986, EMBO J.
5, 2503-2512 (Genbank Accession No. X04439 M24599); B61:
(Pandey, A. et al., 1995, Science 268, 567-569; Holzman et al., 1990, Mol. Cell. Biol. 10, 5830-5838 (Genbank Accession No. M57730 M37476); B61 receptor (Eck): (Pandey, A. et al., 1995, Science 268, 567-569; Lindberg & Hunter, 1990, Mol. Cell. Biol. 10 (12), 6316-6324 (Genbank Accession No. M59371 M36395); Protein kinase C: (Morris et al., 1988, Cell Physiol. 23, C318-C322; Oikawa, T. et al., 20. 1992, J. Antibiot. 45, 1155-1160; Finkenzeller. et al., 1992, Cancer Res. 52, 4821-4823; Kubo et al., 1987, FEBS
Lett. 223 (1), 138-142 (Genbank Accession No. X06318 M27545): ); SH2 domain (Guo, D. et al., 1995, J. Biol.
Chem 270, 6729-6733) a. Phospholipase c-g:(Guo, D. at al., 1995, J. Biol.
Chem 270, 6729-6733; Rhee, S.G. et al. (1992) J. Biol.
Chem 267, 12393-12396; Burgess et al., 1990, Mol. Cell.
Biol. 10, 4770-4777 (Genbank Accession No. M34667)) b. Phosphatidylinositol 3 kinase (PI-3): (Downs, C.P.
et a1.,1991, Cell Signalling 3, 501-513; Genbank accession No. 229090; Genbank accession No. 296973) c. Ras GTPase activating protein (GAP): (Trahey, M.
et a1.,1987, Science 238, 542-545; Guo, D. et al., 1995, J. Biol. Chem 270, 6729-6733; Trahey et al., 1988, Science 242, 1697-1700 (Genbank accession No. M23612)) d. Oncogene adaptor protein Nck:(Park & Rhee, 1992, Mol. Cell. Biol. 12, 5816-5823; Johnson, 1990, Nucleic Acids Res. 18 (4), 1048 (Genbank accession No. X17576));
Granulocyte Colony-Stimulating Factor: (Devlin et al., 1987, J. Leukoc. Biol. 41, 302-306 (Genbank accession No.
M17706)); Hepatocyte growth factor: (Miyazawa et al., 1991, Eur. J. Biochem. 197 (1), 15-22 (Genbank accession No. X57574); Proliferin: (Groskopf et al., 1997, Endocrinology 138(7), 2835-2840; Jackson D, et al., 1994, Science. 266(5190), 1581-1584; Volpert et al., 1996 , Endocrinology 137(9): 3871-3876); Placental growth factor:
(Kodama et al., 1997, Eur J Gynaecol Oncol.; 18(6), 508 510; Ziche et al., 1997, Lab Invest. 76(4), 517-531; Relf et al., 1997, Cancer Res. 57(5), 963-969; Genbank accession No. Y09268) Summary Of The Invention The invention features the use of enzymatic nucleic acid molecules and methods for their use to down regulate or inhibit the expression of angiogenic factors.
Specifically, the enzymatic nucleic acids of the present invention are used as a treatment for indications relating to angiogenesis including but not limited to cancer, age related macular degeneration (ARMD), diabetic retinopathy, inflammation, arthritis, psoriasis and the like.
In a preferred embodiment, the invention features enzymatic nucleic acid molecules that cleave RNAs encoding angiogenic selected from a group comprising: Tie-2, integrin subunit X33, integrin subunit a6, and aryl hydrocarbon nuclear transporter (ARNT).
By "inhibit" it is meant that the activity of the cleaved RNA is reduced below that observed in the absence of the nucleic acid. In one embodiment, inhibition with ribozymes preferably is below that level observed in the presence of an enzymatically inactive. RNA molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA.
By "angiogenic factors" is meant a peptide molecule which is involved in a process or pathway necessary for the formation of novel blood vessels.
In another preferred embodiment, the invention features the use of enzymatic nucleic acids that cleave the RNAs encoded by angiogenic factors selected from a group comprising: Methionine Aminopeptidase; Ets-1 Transcription factor; integrins; platelet derived endothelial cell growth factor (PD-ECGF); PD-ECGF
receptor; Transforming Growth factors (TGFs); Transforming growth factor receptor; Angiogenin; Endothelial cell stimulating angiogenesis factor (ESAF); Interleukin-8 (IL-8); Angiopoietin 1 and 2; TIE-1; insulin-like growth factor (IGF-1); insulin-like growth factor receptor (IGF-lr); B61; B61 receptor (Eck); Protein kinase C; an SH2 domain. (e.g. Phospholipase c-g, Phosphatidylinositol 3 kinase (PI-3), Ras GTPase activating protein (GAP);
Oncogene adaptor protein Nck; Granulocyte Colony-Stimulating Factor; Hepatocyte growth factor; Proliferin;
and Placental growth factor.
By "enzymatic nucleic acid" it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing. 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 5 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, endo-10 ribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not meant to be limiting and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it have 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 (Cech et al., U.S. Patent No. 4,987,071 Cech et al., 1988, JAMA).
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 1).
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 IO bases out of 14 may be base-paired. Such arms are shown generally in Figure 1. 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 arms) 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).
By DNAzyme is meant, an enzymatic nucleic acid molecule lacking a 2'-OH group.
In one of the preferred embodiments, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis 8 virus, group I intron, group II intron or RNase P RNA
(in association with an RNA guide sequence), Neurospora VS
RNA or DNAzymes. 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 Cel.I 35, 849; Forster and Altman, 1990, Science 299, 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: of the Group I intron by Cech et al:, U.S.
Patent 4,987,071 and of DNAzymes by Usman et al., International PCT Publication No. WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.
Bio. 2, 655: Santoro et al., 1997, PNAS 94, 4262. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S.
Patent No. 4,987,071).
By "equivalent" RNA to Tie-2, integrin subunit (33, integrin subunit a6, or ARNT is meant to include those naturally occurring RNA molecules having homology (partial or complete) to Tie-2, integrin subunit (33, integrin subunit a6, or ARNT or encoding for proteins with similar function as Tie-2, integrin subunit (33, integrin subunit a6, or ARNT 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 "homology" is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.

By "complementarity" is meant a nucleic acid molecules that can form hydrogen bonds) with another nucleic acid 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 enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target RNAs encoding Tie-2, integrin subunit (33, integrin subunit a6, or ARNT proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
By "highly conserved sequence region" is meant a nucleotide sequence of one or more regions in a nucleic acid molecule does not vary significantly from one generation to the other or from one biological system to the other.
Such ribozymes are useful for the prevention of the diseases and conditions including cancer, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome and any other diseases or conditions that are related to the levels of Tie-2, integrin subunit ~i3, integrin subunit a6, or ARNT
activity in a cell or tissue.
By "related" is meant that the inhibition of Tie-2, integrin subunit X33, integrin subunit a6, and/or ARNT RNAs and thus reduction in the level respective protein activity will relieve to some extent the symptoms of the disease or condition.
In preferred embodiments, the ribozyrnes have binding arms which are complementary to the target sequences in Tables III-X. Examples of such ribozymes are also shown in Tables III-X. Tables III and IV display target sequences and ribozymes for ARNT, Tables V and VI display target sequences and ribozymes for Tie-2, tables VII and VIII display target sequences and ribozymes for integrin subunit alpha 6, and tables IX and X display target sequences and ribozymes for integrin subunit beta 3.
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.
Alternatively, the ribozymes are DNAzymes. 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. Chemically synthesized RNA molecules also include RNA molecules assembled together from various fragments of RNA using a chemical or an enzymatic ligation method.
In a preferred embodiment, ribozymes are added directly, or can be complexed with cationic lipids, 5 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 10 preferred embodiment, the ribozyme is administered to the site of Tie-2, integrin subunit (33, integrin subunit a6, or ARNT expression (e. g. tumor cells, endothelial cells) in an appropriate liposomal vehicle.
In another aspect of the invention, ribozymes that 15 cleave target molecules and inhibit Tie-2, integrin subunit (33, integrin subunit a6, or ARNT 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 ribazymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target RNA. 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 WO 99/50403 PC'T/US99/06507 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 enzymatic nucleic acid molecules 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 Tie-2, integrin subunit X33, integrin subunit a6, or ARNT, 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. 394-786, 849-910, 1612 2312, 2381-2448, 3588-4726, 4821-4914, 5702-6488, and 6569-6648. Examples of such ribozymes are shown as Seq.
I.D. Nos.l-393, 787-848, 911-1611, 2313-2380, 2449-3587, 4727 -4820. 4915-5701, and 6489-6568. Other sequences may be present which do not interfere with such cleavage.
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.
Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. 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., I, 273). RNas~ 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. W0 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 z 1 base). Helix 1, 4 or may also be extended by 2 or more base pairs (e.g., 4 -

5 20 base pairs) to stabilize the ribozyme structure,' and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is Z
2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et al., 1996, Nucleic Acids & Mol.
eiol., 10, 129; Chowrira et al., US Patent No. 5,631,359).
Figure 2 is a diagrammatic representation of a hammerhead ribozyme targeted against Tie-2 at position 1037.
Enzymatic Nucleic Acid Molecules Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. 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 cleavage and ligation of phosphodiester 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 a1.,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; Santoro et al., 1997, Proc. Natl. Acad.
Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye &
Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra;
Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in traps (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of some of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA
target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the WO 99/50403 PCT/US99/0650'1 ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.
In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on 5 the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme.
10 Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and 15 efficient cleavage achieved in vitro (Zaug et al., 324, Nature 429 1986 ; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92;_ Haseloff and Gerlach, 339 Nature 585, 1988; Cech, 260 JAMA 3030, 1988;
20 and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).
Because of their sequence-specificity, trans-cleaving ribozymes 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). Ribozymes 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.
Ribozymes that cleave the specified sites in Tie-2, integrin subunit (33, integrin subunit a6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs represent a novel therapeutic approach to treat cancer, macular degeneration, diabetic retinopathy, inflammation, psoriasis and other diseases. Applicant indicates that ribozymes are able to inhibit the activity of Tie-2;
integrin subunit (33; integrin subunit a6; and aryl hydrocarbon nuclear transporter (ARNT) 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 Tie-2, integrin subunit (33, integrin subunit a6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs may be readily designed and are within the scope of the invention.
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. 5,525,968 and hereby incorporated by reference herein in totality.
Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.
The sequence of human Tie-2, integrin subunit (33, integrin subunit a6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm.
Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables III-X (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.
Hammerhead or hairpin 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 structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA
message. The binding arms are complementary to the target site sequences described above.
Ribozyme Synthesis 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;
Sullenger 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). 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 W093/23569, and Sullivan et al., PCT
W094/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids Symp.
Ser., 27, 15-6; Taira 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. In a non-limiting example, small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 umol 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 uL of 0.1 M = 16.3 pmol) of phosphoramidite and a 29-fold excess of S-ethyl tetrazole (238 uL of 0.25 M = 59.5 umol) 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 RNA 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:MeCN:H20/3:1:1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder.
The base-deprotected oligoribonucleotide was resuspended in anhydrous TEA~HF/NMP solution (250 uL of a solution of l.5mL N-methylpyrrolidinone, 750 uL 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 TEAR 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 RNA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.

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).
5 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 10 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 15 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).
20 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 25 resuspended in water.
The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Tables III-X. 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

WO 99/50403 PCTlUS99/06507 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 III-X 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.
Optimizing Ribozyme Activity Catalytic activity of the ribozymes 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.

7; 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).
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into enzymatic nucleic acid molecules 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 enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et a1. Nature 1990, 394, 565-568; Pieken et a1. Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et a1. 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.
Nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are 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, WO 99/50403 PC'T/US99/06507 14090) . Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme.
Therapeutic ribozymes delivered exogenously must optimally be 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.
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.
By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1' carbon of b-D-ribo-furanose.
By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
Various modifications to ribozyme structure can be made to enhance the utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
Administration of Ribozymes Sullivan et al., PCT WO 94/02595, describes the general methods for delivery of enzymatic RNA molecules .
Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by 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 5 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 10 detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al., PCT W093/23569 which have been incorporated by reference herein.
The molecules of the instant invention can be used as 15 pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the 20 invention can be administered (e. g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for 25 formation of liposomes can be followed. The compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and 30 the like.
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

WO 99/50403 PC'T/US99/06507 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 absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, 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 drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug 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 (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer an method for increasing the accumulation of drugs in target tissues. This class of drug 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 drug (Lasic et a1.
Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem.
Pharm. Bull. 2995, 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 a1.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, 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 incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs 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 incorporated 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 incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1949. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.
Id.
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 0.1 mg/kg . and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
Alternatively, the enzymatic nucleic acid molecules 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, 9, 45; all of the references are hereby incorporated 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 incorporated in their totality by reference herein).
In another aspect of the invention, enzymatic nucleic acid molecules that cleave target molecules are expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) 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 RNA. 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 5 desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect the invention features, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention 10 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 another aspect the invention features, the 15 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 20 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 25 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), 30 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, 35 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. U S A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U. S. 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 adenovirus 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 incorporated by reference herein.
The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA
vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, I996, supra).

WO 99/50403 PCT/US99l06507 In yet another aspect the invention features 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 molecules 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 regions 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 framed 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.
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 Tie-2, integrin subunit b3, integrin subunit a6, and aryl hydrocarbon nuclear transporter (ARNT). The methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA
targets required for angiogenesis. 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 Ribozvme Cleavaae Sites in TIE-2 The sequence of human Tie-2 was screened for accessible sites using a computer folding algorithm.
Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead and/or hairpin ribozyme cleavage sites were identified. The sequences of these cleavage sites are shown in tables V
VI.

Example 2: Selection of Ribozyme Cleavage Sites in Human_ mr~_~ Drtn To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in Tie-2 RNA, 20 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of Tie-2 (Ziegler et al., 1993, Oncogene

8 (3), 663-670 (Genbank sequence HUMTEKRPTK accession number: M69238) 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 structure. 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. An example of a ribozyme targeted to Tie-2 is shown in figure 2.
Example 3: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of TIE-2 RNA
Ribozymes of the hammerhead or hairpin 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 WO 99/50403 PC'T/US99/06507 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 5 U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also 10 synthesized 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 15 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 incorporated herein by 20 reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table V-VI.
Example 4: Ribozyme Cleavage of TIE-2 RNA Target in vitro Ribozymes targeted to the human Tie-2 RNA are 25 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 Tie-2 mRNA are given in Table V.
30 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-HC1, pH 7.5 at 37°C, 10 mM MgCl2) 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 37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme 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 °
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.
Use of Ribozymes Targeting TIE-2 The rate of tumor growth is believed to be a function of blood supplied and therefore a function of angiogenesis (Rak, Supra; Blood & Zetter, 1990, Biochimica et Biophysica Acta 1032, 89-118). Elevated levels of a number of these angiogenic factors including Tie-2;
integrin subunit (i3; integrin subunit a6; and aryl hydrocarbon nuclear transporter have been reported in a number of cancers. Thus, inhibition of expression of these angiogenic factors (for example using ribozymes) would potentially reduce that rate of growth of these tumors. The use of ribozymes would be desirable over such therapies as chemotherapeutics since, chemotherapeutic compounds such as doxorubicin because of its highly specific inhibition and reduction of the likelihood for side effects. 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. Tumor angiogenesis and other indications are discussed below.
Indications 1) Tumor angiogenesis: Angiogenesis has been shown to be necessary for tumors to grow into pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Treatment 38, 109-119). In addition, it allows tumor cells to travel through the circulatory system during metastasis. Increased levels of gene expression of a number of angiogenic factors such as vascular endothelial growth factor (VEGF) have been reported in vascularized and edema-associated brain tumors (Beckman et al., 1993 J. Clini. Invest. 91, 153). A more direct demostration of the role of VEGF in tumor angiogenesis was demonstrated by Jim Kim et al., 1993 Nature 362,841 wherein, monoclonal antibodies against VEGF
were successfully used to inhibit the growth of rhabdomyosarcoma, glioblastoma multiforme cells in nude mice. Similarly, expression of a dominant negative mutated form of the flt-1 VEGF receptor inhibits vascularization induced by human glioblastoma cells in nude mice (Millauer et al., 1999, Nature 367, 576).
2) Ocular diseases: Neovascularization has been shown to cause or exacerbate ocular diseases including but not limited to, macular degeneration, neovascular glaucoma, diabetic retinopathy, myopic degeneration, and trachoma (Norrby, 1997, APMIS 105, 417-437). Aiello et al., 1994 New Engl. J. Med. 331, 1480, showed that the ocular fluid, of a majority of patients suffering from diabetic retinopathy and other retinal disorders, contains a high concentration of VEGF. Miller et al., 1994 Am. J.
Pathol. 145, 574, reported elevated levels of VEGF mRNA in patients suffering from retinal ischemia. These observations support a direct role for VEGF in ocular diseases. Other factors including those that stimulate VEGF synthesis may also contribute to these indications.
3) Dermatological Disorders: Many indications have been identified which may by angiogenesis dependent including but not limited to psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, and Osler-Weber-Rendu syndrome (Norrby, supra).
Intradermal injection of the angiogenic factor b-FGF
demonstrated angiogenesis in nude mice (Weckbecker et al., 1992, Angiogenesis: Key principles-Science-Technology Medicine, ed R. Steiner) Detmar et al., 1999 J. Exp.
Med. 180, 1191 reported that VEGF and its receptors were over-expressed in psoriatic skin and psoriatic dermal microvessels, suggesting that VEGF plays a significant role in psoriasis.
4) Rheumatoid arthritis: Immunohistochemistry and in situ hybridization studies on tissues from the joints of patients suffering from rheumatoid arthritis show an increased level of VEGF and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341). Additionally, Koch et al., 1994 J. Immunol. 152, 4149, found that VEGF-specific antibodies were able to significantly reduce the mitogenic activity of synovial tissues from patients suffering from rheumatoid arthritis. These observations support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors including those of the present invention may also be involved in arthritis.

Animal Models There are several animal models in which the anti-angiogenesis effect of nucleic acids of the present invention, such as ribozymes, directed against ARNT RNAs can be tested. Typically a corneal model has been used to study angiogenesis in rat and rabbit since recruitment of vessels can easily be followed in this normally avascular tissue (Pandey et al., 1995 Science 268: 567-569). In these models, a small Teflon or Hydron disk pretreated with an angiogenic compound is inserted into a pocket surgically created in the cornea. Angiogenesis is monitored 3 to 5 days later. Ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the disk as well, or dropwise to the eye over the time course of the experiment. In another eye model, hypoxia has been shown to cause both increased expression of VEGF
and neovascularization in the retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909; Shweiki et al., 1992 J. Clin. Invest. 91: 2235-2243).
Another animal model that addresses neovascularization involves Matrigel, an extract of basement membrane that becomes a solid gel when injected subcutaneously (Passaniti et al., 1992 Lab. Invest. 67:
519-528). When the Matrigel is supplemented with angiogenesis factors, vessels grow into the Matrigel over a period of 3 to 5 days and angiogenesis can be assessed.
Again, ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the Matrigel.
Several animal models exist for screening of anti angiogenic agents. These include corneal vessel formation following corneal injury (Burger et al., 1985 Cornea 4:
35-41; Lepri, et al., 1994 J. Ocular Pharmacol. 10: 273 280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243-1252) or intracorneal growth factor implant (Grant et al., 1993 Diabetologia 36: 282-291; Pandey et a1. 1995 supra;

Zieche et al., 1992 Lab. Invest. 67: 711-715), vessel growth into Matrigel matrix containing growth factors (Passaniti et al., 1992 supra), female reproductive organ neovascularization following hormonal manipulation 5 (Shweiki et al., 1993 Clin. Invest. 91: 2235-2243), several models involving inhibition of tumor growth in highly vascularized solid tumors (0'Reilly et al., 1994 Cell 79: 315-328; Senger et al., 1993 Cancer and Metas.
Rev. 12: 303-329; Takahasi et al., 1994 Cancer Res. 54:
10 4233-4237; Kim et al., 1993 supra), and transient hypoxia-induced neovascularization in the mouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909).
The cornea model, described in Pandey et al. supra, is the most common and well characterized anti-angiogenic 15 agent efficacy screening model. This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin). The corneal model would utilize the intrastromal corneal implantation of a Teflon pellet 20 soaked in a angiogenic compound-Hydron solution to recruit blood vessels toward the pellet which can be quantitated using standard microscopic and image analysis techniques.
To evaluate their anti-angiogenic efficacy, ribozymes are applied topically to the eye or bound within Hydron on the 25 Teflon pellet itself. This avascular cornea as well as the Matrigel (see below) provide for low background assays. While the corneal model has been performed extensively in the rabbit, studies in the rat have also been conducted.
30 The mouse model (Passaniti et al., supra) is a non-tissue model which utilizes Matrigel, an extract of basement membrane (Kleinman et al., 1986) or Millipore~
filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to 35 injection. Upon subcutaneous administration at body WO 99/50403 PCT/US99/0650~

temperature, the Matrigel or Millipore~ filter disk forms a solid implant. An angiogenic compound would be embedded in the Matrigel or Millipore~ filter disk which would be used to recruit vessels within the matrix of the Matrigel or Millipore~ filter disk that can be processed histologically for endothelial cell specific vWF (factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or hemoglobin content. Like the cornea, the Matrigel or Millipore~ filter disk are avascular; however, it is not tissue. In the Matrigel or Millipore~ filter disk model, ribozymes are administered within the matrix of the Matrigel or Millipore~ filter disk to test their anti-angiogenic efficacy. Thus, delivery issues in this model, as with delivery of ribozymes by Hydron- coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the ribozyme within the respective matrix.
These models offer a distinct advantage over several other angiogenic models listed previously. The ability to use VEGF as a pro-angiogenic stimulus in both models is highly desirable since ribozymes will target only VEGFr RNA. In other words, the involvement of other non-specific types of stimuli in the cornea and Matrigel models is not advantageous from the standpoint of understanding the pharmacologic mechanism by which the anti-VEGFr RNA ribozymes produce their effects. In addition, the models will allow for testing the specificity of the anti-VEGFr RNA ribozymes by using either a- or bFGF as a pro-angiogenic factor. Vessel recruitment using FGF should not be affected in either model by anti-VEGFr RNA ribozymes. Other models of angiogenesis including vessel formation in the female reproductive system using hormonal manipulation (Shweiki et al., 1993 supra); a variety of vascular solid tumor models which involve indirect correltations with angiogenesis (O'Reilly et al., 1994 supra; Senger et al., 1993 supra; Takahasi et al., 1994 supra; Kim et al., 1993 supra); and retinal neovascularization following transient hypoxia (Pierce et al., 1995 supra) were not selected for efficacy screening due to their non-specific nature, although there is a correlation between VEGF and angiogenesis in these models.
Other model systems to study tumor angiogenesis is reviewed by Folkman, 1985 Adv. Cancer. Res.. 43, 175.
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 10' 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 RNA reduction).
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.
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 Tie-2;
integrin subunit ~i3; integrin subunit a6; and/or aryl hydrocarbon nuclear transporter 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 structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA
structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (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 RNAs associated with Tie-2;
integrin subunit (33; integrin subunit a6; and/or aryl hydrocarbon nuclear transporter 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 5 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 10 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 15 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 20 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 25 changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., Tie-2; integrin subunit (33; integrin subunit a6; ARNT) is adequate to establish risk. If probes of comparable specific activity are used for both 30 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.

WO 99/50403 PCT/US99l06507 Additional Uses Potential usefulness of sequence-specific enzymatic 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.
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 [1,2] .
~ Complete kinetic framework established for one ribozyme [3, 9, 5~ s] .
Michel, Francois; Westhof, Eric. Slippery substrates. Nat.
Struct. Biol. (1994), 1(1), 5-7.
Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J.
Mol. Biol. (1994), 235(9), 1206-17.
Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the WO 99/50403 PCT/US99/0650?

~ Studies of ribozyme folding and substrate docking underway ['-, 8, 9] .
reaction of an RNA substrate complementary to the active site.
Biochemistry (1990), 29(44), 10159-71.
°- 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.
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.
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.
'- 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.
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.
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.

~ Chemical modification investigation of important residues well established [lo, ii] .
~ 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" ~i-galactosidase message by the ligation of new (3-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 [
~ Reaction mechanism: possible attack by M2+-OH to generate cleavage products with 3'-OH and 5'-phosphate.
to 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), 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.
i2 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).

~ 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 5 therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA
~19 15~
i ~ Important phosphate and 2' OH contacts recently identified [ls,1']
10 Group II Introns Size: >1000 nucleotides.
~ Trans cleavage of target RNAs recently demonstrated [18 ls] .
1' Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C., 1883-) (1990), 249(4970), 783-6. -is yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.
is Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA
(1995), 1(2), 210-18.
1' 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), 12510-19.
ie 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.

~ 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.
~ Only natural ribozyme with demonstrated participation in DNA cleavage [io~21] in addition to RNA
cleavage and ligation.
~ Major structural features largely established through phylogenetic comparisons [2z].
~ Important 2' OH contacts beginning to be identified [?3]
'-s 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), 39(9), 2965-77.
Zimmerly, Steven; Guo, Huatao; Eskes, Robert: Yang, Jian Perlman, Philip S.~ Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility.
Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
Zi Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.
Zz Michel, Francois~ Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 935-61.
z3 Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie.
Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science (Washington, D. C.) (1996), 271(5254), 1410-13.

WO 99/50403 PC'T/US99/06507 ~ Kinetic framework under development 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 VS RNA.
Hammerhead Ribozyme (see text for references) ~ Size: ~13 to 90 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.
24 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), 31-99.
zs Guo, Hans C. T.: Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS
RNA. EMBO J. (1995), 14(2), 368-76.

~ 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA 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) [2g]
~ Complete kinetic framework established for two or more ribozymes [29] .
~ Chemical modification investigation of important residues well established [30].
Hairpin Ribozyme ~ Size: ~50 nucleotides.
zs 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.
2' McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403.
?8 Long, D., Uhlenbeck, 0., Hertel, K. Ligation with hammerhead ribozymes. US Patent No. 5,633,133.
?9 Hertel, K.J., Herschlag, D., Uhlenbeck, 0. 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.
3° Beigelman, L:, et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

~ 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 f-: ; ,-J
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.
3z 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 gerzal-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.
39 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.

~ Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [3s]
~ Complete kinetic framework established for one 5 ribozyme [36] .
~ Chemical modification investigation of important residues begun [3'~ 3e~ .
Hepatitis Delta Virus (HDV) Ribozyme ~ Size: ~60 nucleotides.
10 ~ Trans cleavage of target RNAs demonstrated [39].
as 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.
3s Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.
3' 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), 39(12), 4068-76.
3a 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(9), 573-B1.
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.

~ 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 [9i]
'-° 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), 934-6.
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 umol RNA Synthesis Cycle Reagent Equivalents Amount Time*

Phosphoramidites 6.5 163 uL 2.5 S-Ethyl Tetrazole 23.8 238 uL 2.5 Acetic Anhydride 100 233 uL 5 sec N-Methyl Imidazole 186 233 uL 5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec Acetonitrile NA 6.67 mL NA

* Wait time does not include contact time during delivery.

TABLE III: HAMMERHEAD RIBOZYME AND SITE SEQUENCES FOR ARNT
Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGCCGCCA CUCCCACU

CGAA

AGGAGCCG CCACUGGG

CGAA

AUGCCACC UGCGGCCA

CGAA

CGAA

AUGUCAUU AGAUGUAC

CGAA

ACAUCUGA CCAUCACU

CGAA

AUGGUACA ACUGGGUC

CGAA

ACCCAGUG CAGCCAUU

CGAA

AUGGCUGG GCCUCUGG

CGAA

AGGCAAUG UGGAAACU

CGAA

AGUUUCCA UGGACCUG

CGAA

AUUCCAGG CAAGGUGG

CGAA

AAUUCCAG AAGGUGGA

CGAA

AUGGCUCC GUCCAGAG

CGAA

ACAAUGGC CAGAGGGC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGCCCUCU UUAAGCGG

CGAA

AUAGCCCU AAGCGGCG

CGAA

AAUAGCCC AGCGGCGA

CGAA

AUCCAGCC UUGAUGAU

CGAA

AAUCCAGC UGAUGAUG

CGAA

AAAUCCAG GAUGAUGA

CGAA

ACUGUUCC AAUUUUUG

CGAA

AUUUACUG UUUGAGGU

CGAA

O

CGAA

AAAUUUAC UGAGGUGU

CGAA

AAAAUUUA GAGGUGUG

CGAA

AUCAUCAU AGAUGUCU

CGAA

ACAUCUGA UAACGAUA

CGAA

AGACAUCU ACGAUAAG

CGAA

AUCGUUAG AGGAGCGG

CGAA

ACCGCUCC UGCCAGGU

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AACCGCUC GCCAGGUC

CGAA

ACCUGGCA GGAUGAUG

CGAA

AGCUCUGC UGCGGAUA

CGAA

CGAA

AGUCUCUC GCCAGGGA

CGAA

AUUUUCCC ACAGUGAA

CGAA

AUUUCACU GAACGGCG

CGAA

AGGCUGUC CAUCACAG

CGAA

O

CGAA

ACAGUUCU AGAUAUGG

CGAA

AUCUGACA UGGUACCC

ACCAUAUC CCCACCUG

CGAA

ACAGGUGG GUGCCCUG

CGAA

O

AGCCAGGG GAAAACCA

CGAA

AGCUUGUC ACCAUCUU

CGAA

AUGGUUAG UUACGCAU

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGAUGGUU ACGCAUGG

CGAA

AAGAUGGU CGCAUGGC

CGAA

ACUGCCAU UCUCACAU

CGAA

AACUGCCA CUCACAUG

CGAA

AAACUGCC UCACAUGA

CGAA

AGAAACUG ACAUGAAG

CGAA

ACUUCAUG CUUGCGGG

CGAA

AGGACUUC GCGGGGAA

CGAA

O

CGAA

AGCCAUCA CUAUAAGC

CGAA

AGGAGCCA UAAGCCGU

AUAGGAGC AGCCGUCU

CGAA

ACGGCUUA UUUCCUCA

CGAA

AGACGGCU UCCUCACU

CGAA

AAGACGGC CCUCACUG

CGAA

AAAGACGG CUCACUGA

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGGAAAGA ACUGAUCA

CGAA

AUCAGUGA AGGAACUG

CGAA

AUGUUUCA UGAUCUUG

CGAA

O

CGAA

AUCAAAUG UUGGAGGC

CGAA

AGAUCAAA GGAGGCAG

CGAA

AGCCAUCU UCUGUUUA

CGAA

AAGCCAUC CUGUUUAU

CGAA

O

CGAA

ACAGAAAG UAUUGUCU

CGAA

AACAGAAA AUUGUCUC

CGAA

AAACAGAA UUGUCUCA

CGAA

AUAAACAG GUCUCAUG

CGAA

ACAAUAAA UCAUGUGA

CGAA

AGACAAUA AUGUGAGA

CGAA

ACACCACC UGUGUCUG

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

ACACAUAC UGACUCCG

CGAA

AGUCAGAC CGUGACUC

CGAA

AGUCACGG CUGUUUUG

CGAA

ACAGGAGU UUGAACCA

CGAA

AACAGGAG UGAACCAG

CGAA

AAACAGGA GAACCAGC

CGAA

ACUGUGGC UGAAUGGU

CGAA

ACCAUUCA UGGCAGCA

CGAA

O

CGAA

AGUGUGCU UAUGAUCA

CGAA

AGAGUGUG UGAUCAGG

CGAA

AUCAUAGA AGGUGCAC

CGAA

AUCCACAU AACUUCGU

CGAA

AGUUUAUC CGUGAGCA

CGAA

AAGUUUAU GUGAGCAG

CGAA

AGCUGCUC UCCACUUC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AAGCUGCU CCACUUCA

CGAA

AAAGCUGC CACUUCAG

CGAA

AGUGGAAA CAGAAAAU

CGAA

AAGUGGAA AGAAAAUG

CGAA

ACGCCCUG UCCUGGAU

CGAA

AUACGCCC CUGGAUCU

CGAA

AUCCAGGA UAAAGACU

CGAA

AGAUCCAG AAGACUGG

CGAA

CGAA

ACUGCUGA UUCCAUGA

CGAA

AGACUGCU CCAUGAGA

AAGACUGC CAUGAGAA

CGAA

ACACAUUC UGGGCUCA

CGAA

AGCCCAUA AAGGAGAU

CGAA

AUCUCCUU GUUUAUUU

CGAA

ACGAUCUC UAUUUGCC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AACGAUCU AUUUGCCG

CGAA

AAACGAUC UUUGCCGA

CGAA

AUAAACGA UGCCGAAU

CGAA

CGAA

ACUGCCAC GCUCUGUG

CGAA

AGCUACUG UGUGGACC

CGAA

ACUGGGUC UCUGUGAA

CGAA

AACUGGGU CUGUGAAU

CGAA

CGAA

AUUCACAG GGCUGAGC

CGAA

AGCUCAGC UGUGAGGA

CGAA

AAGCUCAG GUGAGGAA

CGAA

AGUCCAUU GGCUCUGU

CGAA

AGCCAAGU UGUAAAGG

CGAA

ACAGAGCC AAGGAUGG

CGAA

AGGUUCCC ACUUCGUG

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGUGAGGU CGUGGUGG

CGAA

AAGUGAGG GUGGUGGU

CGAA

ACCACCAC CACUGCAC

CGAA

ld AGCCUGUG CAUCAAGG

CGAA

AUGUAGCC AAGGCCUG

CGAA

ACACCUGC UCCCUCCC

CGAA

AACACCUG CCCUCCCA

CGAA

AAACACCU CCUCCCAG

CGAA

CGAA

ACUUGCUU UUGCCUAG

CGAA

AACUUGCU UGCCUAGU

CGAA

AAACUUGC GCCUAGUG

CGAA

AGGCAAAA GUGGCCAU

CGAA

AUGGCCAC GGCAGAUU

CGAA

AUCUGCCA GCAGGUAA

CGAA

ACCUGCAA ACUAGUUC

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGUUACCU GUUCUCCC

CGAA

ACUAGUUA CUCCCAAC

CGAA

AACUAGUU UCCCAACU

CGAA

AGAACUAG CCAACUGU

CGAA

ACAGUUGG CAGACAUG

CGAA

ACUCAUGU AUGUUUGU

CGAA

ACAUUAGU UGUCAACC

CGAA

AACAUUAC GUCAACCA

CGAA

ACAAACAU AACCAACA

CGAA

ACUCUGUU CAUCUCCC

CGAA

AACUCUGU AUCUCCCG

CGAA

AUGAACUC UCCCGACA

CGAA

AGAUGAAC CCGACACA

CGAA

AUGUUGUG GAGGGUAU

CGAA

ACCCUCAA UCUUCACU

CGAA

AUACCCUC UUCACUUU

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AGAUACCC CACUUUUG

CGAA

AAGAUACC ACUUUUGU

CGAA

AGUGAAGA UUGUGGAU

CGAA

O

CGAA

AAAGUGAA GUGGAUCA

CGAA

AUGCACAA ACCGCUGU

CGAA

AGCCACAC CUGUUGGC

CGAA

ACAGUAGC GGCUACCA

CGAA

CGAA

AGUUCCUG UUAGGAAA

CGAA

AGAGUUCC AGGAAAGA

CGAA

AAGAGUUC GGAAAGAA

CGAA

AUUCUUUC UUGUAGAA

CGAA

AUAUUCUU GUAGAAUU

CGAA

ACAAUAUU GAAUUCUG

CGAA

AUUCUACA CUGUCAUC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AAUUCUAC UGUCAUCC

CGAA

ACAGAAUU AUCCUGAA

CGAA

AUGACAGA CUGAAGAC

CGAA

AGCUGCUG CUAAGAGA

CGAA

AAGCUGCU UAAGAGAC

CGAA

AGAAGCUG AGAGACAG

CGAA

AGCUGUCU CCAACAGG

CGAA

AAGCUGUC CAACAGGU

CGAA

O

CGAA

AUUUCACU AAAAGGCC

CGAA

AAUUUCAC AAAGGCCA

CGAA

ACAGCACU UGUCAUGU

GGAA

ACAGACAG AUGUUCCG

CGAA

ACAUGACA CCGGUUCC

CGAA

AACAUGAC CGGUUCCG

CGAA

ACCGGAAC CCGGUCUA

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AACCGGAA CGGUCUAA

CGAA

ACCGGAAC UAAGAACC

CGAA

AGACCGGA AGAACCAA

CGAA

CGAA

AGCUGGUU CUUUACUU

CGAA

AGGAGCUG UACUUUCC

CGAA

AAGGAGCU ACUUUCCA

CGAA

AAAGGAGC CUUUCCAG

CGAA

CGAA

AAGUAAAG CCAGAACC

CGAA

AAAGUAAA CAGAACCC

CGAA

AGGGUUCU ACUCAGAU

CGAA

AAGGGUUC CUCAGAUG

CGAA

AGUAAGGG AGAUGAAA

CGAA

AUUUCAUC GAGUACAU

CGAA

ACUCAAUU CAUCAUCU

WO 99/50403 PC'T/US99/06507 Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No:

CGAA

AUGUACUC AUCUGUAC

CGAA

AUGAUGUA UGUACCAA

CGAA

ACAGAUGA CCAACACC

CGAA

AGUUCUUC UAGCCAAG

CGAA

AGAGUUCU GCCAAGAA

CGAA

AGGCCGUG CACUCUCC

CGAA

AGUGUAGG UCCAACAC

CGAA

AGAGUGUA CAACACAA

CGAA

O

CGAA

AGUUGUGG GGUCCCAC

CGAA

ACCUAGUU CCACAGCU

CGAA

AGCUGUGG AUUUACCC

CGAA

AUUAGCUG UACCCCUG

CGAA

AAUUAGCU ACCCCUGG

CGAA

AAAUUAGC CCCCUGGA

CGAA

AGCCCAUC AGGACAGC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AUUCUGUU GGACAUGG

CGAA

ACCAUGUC CCAGGAAG

CGAA

AGCUGGCC CAAUCAUU

CGAA

IO AUUGUAGC AUUCCCAG

CGAA

AUGAUUGU CCCAGGUG

CGAA

AAUGAUUG CCAGGUGG

CGAA

ACCACCUG CAGCCUGU

CGAA

AACCACCU AGCCUGUG

CGAA

O

CGAA

ACUUCUCA AGAUGGUU

CGAA

ACCAUCUG UAUUUGCC

S CGAA

AACCAUCU AUUUGCCC

CGAA

AAACCAUC UUUGCCCA

CGAA

AUAAACCA UGCCCAGG

CGAA

AAUAAACC GCCCAGGA

CGAA

AUCCUGGG GAGAUCCA

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AUCUCUAU CAAGAUUU

CGAA

AUCUUGGA UUCAGAAA

CGAA

AAUCUUGG UCAGAAAU

CGAA

AAAUCUUG CAGAAAUC

CGAA

AAAAUCUU AGAAAUCU

CGAA

AUUUCUGA UAUCACAA

CGAA

AGAUUUCU UCACAACA

CGAA

AUAGAUUU ' ACAACAUC

CGAA

O

CGAA

AUCCGCAU AGAGUAAA

CGAA

ACUCUGAU AAGGCAUC

CGAA

AUGCCUUU UCCUCCAG

CGAA

AGAUGCCU CUCCAGCA

CGAA

AGGAGAUG CAGCACUG

CGAA

ACAGUGCU CCUGCCAC

CGAA

AGCUGUUG UUCUCCCA

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

AUAGCUGU CUCCCAGG

AAUAGCUG UCCCAGGG

AGAAUAGC CCAGGGCA

AUGUGUUG CCCUCCUA

AAUGUGUU CCUCCUAC

AGGGAAUG CUACCCCC

AGGAGGGA CCCCCCGG

AUUCUCUG UCAGGAAU

a AAAUUCUC AGGAAUAG

AUUCCUGA GUGGCCUA

AGGCCACU GCCCCUCC

AGGGGCUA CUGUAACC

ACAGGAGG ACCAUUGU

AUGGUUAC GUCCAGCC

ACAAUGGU CAGCCAUC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AUGGCUGG AGCUUCUG

CGAA

AGCUGAUG CUGCAGGA

CGAA

AAGCUGAU UGCAGGAC

CGAA

CGAA

AUCUGGGC UCCCGCCA

CGAA

AAUCUGGG CCCGCCAC

CGAA

AAAUCUGG CCGCCACU

CGAA

AGUGGCGG CAACCCCA

CGAA

CGAA

AGGGGUCC CUACCCGC

CGAA

AGUAGGGG CCCGCUCA

CGAA

AGCGGGUA AGGCUUUU

CGAA

AGCCUGAG UUCUGCCC

CGAA

AAGCCUGA UCUGCCCA

CGAA

AAAGCCUG CUGCCCAG

CGAA

AAAAGCCU UGCCCAGC

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

AGCCACCU CCCAGGCU

AGCCUGGG CUGCUAAG

AGCAGUAG AGACUCGU

AGUCUUAG GUACUUCC

ACGAGUCU CUUCCCAG

AGUACGAG CGCAGUUU

AAGUACGA CCAGUUUG

ACUGGGAA UGGUGUGG

O

AGCUGCCC UCAGACUC

AAGCUGCC CAGACUCC

AAAGCUGC AGACUCCA

AGUCUGAA CAUCCUCC

AUGGAGUC CUCCUUCA

AGGAUGGA CUUCAGCU

AGGAGGAU CAGCUCCA

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

AAGGAGGA AGCUCCAU

AGCUGAAG CAUGUCCC

ACAUGGAG CCUCCCUG

AGGGACAU CCUGGUGC

AUGCAGUU GCCUGGUG

AGGCAGCA CCCUAGUC

AGGGUAGG GUCUCACC

ACUAGGGU UCACCAAU

O

AUUGGUGA GUGGAUCU

AUCCACGA UAACUUUG

AGAUCCAC ACUUUGCU

AGUUAGAU UGCUCCUG

AAGUUAGA GCUCCUGA

AGCAAAGU CUGAGACU

AUUGUCCU CCAGACAC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AAUUGUCC CAGACACG

CGAA

ACACCCAC UGGCCACA

CGAA

AGGCUGCU AUCAUCGU

CGAA

AUGAGGCU AUCGUUCA

CGAA

AUGAUGAG GUUCAAGU

CGAA

ACGAUGAU CAAGUUCU

GGAA

AACGAUGA AAGUUCUA

CGAA

ACUUGAAC CUAGUGAG

CGAA

AACUUGAA UAGUGAGC

CGAA

AGAACUUG GUGAGCAA

CGAA

ACAUGUUG CAACAACC

CGAA

AACAUGUU AACAACCG

CGAA

ACCUCAGG UUCCAGGA

CGAA

AGACCUCA CCAGGAGA

CGAA

AAGACCUC CAGGAGAU

CGAA

ACAGCAUC CAUGCUGG

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

AUCUCCCA AGAGCAAC

AGCUGUUG CAACAAUG

AUUCUUCA CCCUGAUC

AAUUCUUC CCUGAUCU

AUCAGGGA UAACUAUG

AGAUCAGG ACUAUGUU

AGUUAGAU UGUUUCCC

ACAUAGUU UCCCCCCU

AACAUAGU CCCCCCUU

AAACAUAG CCCCCUUU

AGGGGGGA UUCAGAAU

AAGGGGGG UCAGAAUA

AAAGGGGG CAGAAUAG

~pGGGG AGAAUAGA

AUUCUGAA GAACUAUU

AGUUCUAU UUGGGGUG

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AUAGUUCU GGGGUGAG

CGAA

AUCCUCAC AGGGGUGG

CGAA

AUUUUUUC ACUGUUUG

CGAA

CGAA

AACAGUGA GUUUUUAA

CGAA

ACAAACAG UUUAAAAA

CGAA

AACAAACA UUAAAAAG

CGAA

AAACAAAC UAAAAAGC

CGAA

CGAA

AAAAACAA AAAAGCAA

CGAA

AUUUGCUU UUUCUGUA

AGAUUUGC UCUGUAAA

CGAA

AAGAUUUG CUGUAAAC

CGAA

O

Ap,AGAUUU UGUAAACA

CGAA

ACAGAAAG AACAGAAU

CGAA

AUUCUGUU AAAGUUCC

Posi- Seq. Seq. I.D.

tion RZ I.D. Substrate No.
No.

CGAA

ACUUUUAU CCUCUCCC

CGAA

AACUUUUA CUCUCCCU

CGAA

AGGAACUU UCCCUUCC

CGAA

AGAGGAAC CCUUCCCU

CGAA

AGGGAGAG CCCUUCCC

CGAA

AAGGGAGA CCUUCCCU

CGAA

AGGGAAGG CCCUCACC

CGAA

AAGGGAAG CCUCACCC

CGAA

O

CGAA

ACAUGUCA CCCCCUUU

CGAA

AGGGGGUA UCCCUUCU

CGAA

AAGGGGGU CCCUUCUG

CGAA

AAAGGGGG CCUUCUGG

CGAA

AGGGAAAG CUGGCUGU

CGAA

AAGGGAAA UGGCUGUU

CGAA

ACAGCCAG CCCCUGCU

Posi- Seq. Seq.
I.D.

tion RZ I.D. Substrate No.
No.

CGAA

AACAGCCA CCCUGCUC

CGAA

AGCAGGGG UGUUGCCU

CGAA

ACAGAGCA GCCUCCUA

CGAA

AGGCAACA CUAAGGUA

CGAA

AGGAGGCA AGGUAACA

CGAA

ACCUUAGG ACAUUUAU

CGAA

AUGUUACC UAUAAAAA

CGAA

AAUGUUAC AUAAAAAA

CGAA

AAAUGUUA UAAAAAAA

CGAA

AUAAAUGU AAAAAAAA

TABLE I V: HAIRPIN RIBOZYME SEøUENCES AND TARGET SITES FOR ARNT
Posi- Seq. Seq.
I.D.

tion RZ No. Substrate I.D.
No.

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

X GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

Posi- Seq. Seq.
I.D.

tionRZ No. Substrate I.D.
No.

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

Posi- Seq. Seq.
I.D.

tionRZ No. Substrate I.D.
No.

X GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

r 1?54CUCUG AGAA GCAU ACCAGAGAAACA826 UGCG GAU CAGAGU 888 GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

X GUACAUUACCUGGUA

Posi- Seq. Seq.
I.D.

tionRZ No. Substrate I.D.
No.

X GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

X GUACAUUACCUGGUA

GUACAUUACCUGGUA

TABLE V: HAMMERHEAD RIBOZYMES AND TARGET SITES FOR TIE
Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA ACAGCACA CCUUCUUG

X CGAA AACAGCAC CUUCUUGC

X CGAA AGGAACAG CUUGCCUC

X CGAA AAGGAACA UUGCCUCU

X CGAA AGAAGGAA GCCUCUAA

X CGAA AGAGGCAA ACUUGUAA

X CGAA AGUUAGAG GUAAACAA

X CGAA ACAAGUUA AACAAGAC

X CGAA ACGUCUUG CUAGGACG

X CGAA AGCAUCGU AUGGAAAG

X CGAA ACUUUCCA ACAAACCG

X CGAA ACCCAGCG UUUGAAAG

X CGAA AACCCAGC UUGAAAGG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAACCCAG UGAAAGGA

X CGAA AAAACCCA GAAAGGAU

X CGAA AUCCUUUC CUUGGGAC

X CGAA AGGUCCCA AUGCACAU

X CGAA AUGUGCAU UGUGGAAA

X CGAA AAUGUGCA GUGGAAAC

X CGAA AUCUCUCC UGGGGAAG

X CGAA AGUCCAUG UUUAGCCA

X CGAA AGAGUCCA UAGCCAGC

X CGAA AAGAGUCC AGCCAGCU

X CGAA AAAGAGUC GCCAGCUU

168 AGAGAACU CUGAUGAG939 ~ AGCCAGCUU 1640 X CGAA AGCUGGCU AGUUCUCU

X CGAA AAGCUGGC GUUCUCUG

X CGAA ACUAAGCU CUCUGUGG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AACUAAGC UCUGUGGA

X CGAA AGAACUAA UGUGGAGU

X CGAA ACUCCACA AGCUUGCU

X CGAA AGCUGACU GCUCCUUU

X CGAA AGCAAGCU CUUUCUGG

X CGAA AGGAGCAA UCUGGAAC

X CGAA AAGGAGCA CUGGAACU

X CGAA AAAGGAGC UGGAACUG

X CGAA AUCAAGUC UUGAUCAA

X CGAA AGAUCAAG GAUCAAUU

X CGAA AUCAAGAU AAUUCCCU

X CGAA AUUGAUCA CCCUACCU

X CGAA AAUUGAUC CCUACCUC

X CGAA AGGGAAUU CCUCUUGU

X CGAA AGGUAGGG UUGUAUCU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGAGGUAG GUAUCUGA

X CGAA ACAAGAGG UCUGAUGC

X CGAA AUACAAGA UGAUGCUG

X CGAA AGAUGUUU UCACCUGC

X CGAA AGAGAUGU ACCUGCAU

X CGAA AUGCAGGU GCCUCUGG

X CGAA AGGCAAUG UGGGUGGC

2~ X CGAA RUGGGCUC ACCAUAGG

X CGAA AUGGUGAU GGAAGGGA

X CGAA AGUCCCUU UGAAGCCU

X CGAA AAGUCCCU GAAGCCUU

X CGAA AGGCUUCA AAUGAACC

X CGAA AAGGCUUC AUGAACCA

X CGAA AUCCUGGU CGCUGGAA

X CGAA ACUUCCAG ACUCAAGA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AACUUCCA CUCAAGAU

X CGAA AGUAACUU AAGAUGUG

X CGAA AGCCCAUU AAAAAGUU

X CGAA ACUUUUUU GUUUGGAA

X CGAA ACAACUUU UGGAAGAG

X CGAA AACAACUU GGAAGAGA

X CGAA AGCCUUUU GUAAGAUC

X CGAA ACUAGCCU AGAUCAAU

X CGAA AGCACCAU AUUUCUGU

X CGAA AAGCACCA UUUCUGUG

44g UUCACAGA CUGAUGAG985 GUGCUUAUU 1686 X CGAA AUAAGCAC UCUGUGAA

X CGAA AAUAAGCA CUGUGAAG

X CGAA AAAUAAGC UGUGAAGG

X CGAA ACUCGCCC CGAGGAGA

X CGAA AACUCGCC GAGGAGAG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUGCCUC AGGAUACG

X CGAA AUCCUGAU CGAACCAU

X CGAA ACGCAUCU AACAAGCU

X CGAA AGCUUGUU CCUUCCUA

X CGAA AAGCUUGU CUUCCUAC

X CGAA AGGAAGCU CCUACCAG

X CGAA AAGGAAGC CUACCAGC

X CGAA AGGAAGGA CCAGCUAC

X CGAA AGUAGCUG UAACUAUG

X CGAA AAGUAGCU AACUAUGA

X CGAA AAAGUAGC ACUAUGAC

X CGAA AGUUAAAG UGACUGUG

X CGAA AUCUCCCU ACGUGAAC

X CGAA AUGUUCAC UCUUUCAA

X CGAA AUAUGUUC UUUCAAAA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGAUAUGU UCAAAAAG

X CGAA AAGAUAUG CAAAAAGG

X CGAA AAAGAUAU AAAAAGGU

X CGAA ACCUUUUU UUGAUUAA

X CGAA AUACCUUU GAUUAAAG

X CGAA AUCAAUAC AAAGAAGA

X CGAA AAUCAAUA AAGAAGAA

613 UUUUUGUA CUGAUGAG1013 GCAGUGAUU 1?14 X CGAA AUCACUGC UACAAAAA

X CGAA AAAUCACU CAAAAAUG

X CGAA ACCAUUUU CCUUCAUC

X CGAA AACCAUUU CUUCAUCC

X CGAA AGGAACCA CAUCCAUU

X CGAA AAGGAACC AUCCAUUC

X CGAA AUGAAGGA CAUUCAGU

X CGAA AUGGAUGA CAGUGCGC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAUGGAUG AGUGCCCC

X CGAA ACUUCAUG CCUGAUAU

X CGAA AUCAGGUA UUCUAGAA

X CGAA AUAUCAGG CUAGAAGU

X CGAA AAUAUCAG UAGAAGUA

X CGAA AGAAUAUC GAAGUACA

X CGAA ACUUCUAG CACCUGCC

X CGAA AGGCAGGU AUGCUCAG

X CGAA AGCAUGAG AGCCCCAG

X CGAA ACACUCCA CUCGGCCA

X CGAA AGUACACU GGCCAGGU

X CGAA ACCUGGCC UAUAGGAG

X CGAA AUACCUGG UAGGAGGA

X CGAA AUAUACCU GGAGGAAA

X CGAA AGGUUUCC UUCACCUC

X CGAA AGAGGUUU CACCUCGG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAGAGGUU ACCUCGGC

X CGAA AGGUGAAG GGCCUUCA

X CGAA AGGCCGAG CACCAGGC

X CGAA AAGGCCGA ACCAGGCU

X CGAA AUCAGCCU GUCCGGAG

X CGAA ACUAUCAG CGGAGAUG

X CGAA AUGGUUGC UCUGUACU

X CGAA AGAUGGUU UGUACUGC

X CGAA AGCAGUAC GUAUGAAC

X CGAA ACAAGCAG UGAACAAU

g4q UCAUGGCA CUGAUGAG1099 AAUGGUGUC 1750 X CGAA ACACCAUU UGCCAUGA

X CGAA AUCUUCAU CUGGAGAA

X CGAA AUGCAUUC UGCCCUCC

X CGAA AAUGCAUU GCCCUCCU

X CGAA AGGGCAAA CUGGGUUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA ACCCAGGA UAUGGGAA

X CGAA AACCCAGG AUGGGAAG

X CGAA AAACCCAG UGGGAAGG

X CGAA AGCCUUCU GUGAACUG

X CGAA ACGUGUGC UGGCAGAA

X CGAA AACGUGUG GGCAGAAC

X CGAA AGUUCUGC GUAAAGAA

X CGAA ACAAGUUC AAGAAAGG

X CGAA AGACUUGC AUGUGUUC

X CGAA AAGACUUG UGUGUUCU

ggq GGAGACAG CUGAUGAG1065 UUAUGUGUU 1766 X CGAA ACACAUAA CUGUCUCC

X CGAA AACACAUA UGUCUCCC

X CGAA ACAGAACA UCCCUGAC

X CGAA AGACAGAA CCUGACCC

X CGAA AGGGGUCA UGGGUGUU

WO 99/50403 PCT/US99/0650~

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA ACACCCAU CCUGUGCC

X CGAA AACACCCA CUGUGCCA

X CGAA ACCCUUCC UGCAGUGC

X CGAA ACCAGGGU UUUACGGG

X CGAA AACCAGGG UUACGGGC

X CGAA AAACCAGG UACGGGCC

X CGAA AAAACCAG ACGGGCCA

X CGAA AAAAACCA CGGGCCAG

X CGAA AUCUGGCC GUAAGCUU

X CGAA ACAAUCUG AGCUUAGG

X CGAA AGCUUACA AGGUGCAG

X CGAA AAGCUUAC GGUGCAGC

X CGAA AUCACACA GCUUCCAA

X CGAA AGCGAUCA CCAAGGAU

X CGAA AAGCGAUC CAAGGAUG

X CGAA ACAUCCUU UCUGCUCU

WO 99/50403 PC'f/US99/06507 Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGACAUCC UGCUCUCC

X CGAA AGCAGAGA UCCAGGAU

X CGAA AGAGCAGA CAGGAUGG

X CGAA AGCCCCUG CAGUGUGA

X CGAA AUGCCUUC CCGAGGAU

X CGAA AUCUUUGG GUGGAUUU

X CGAA AUCCACUA UGCCAGAU

X CGAA AAUCCACU GCCAGAUC

X CGAA AUCUGGCA AUAUAGAA

X CGAA AUGAUCUG UAGAAGUA

X CGAA AUAUGAUC GAAGUAAA

X CGAA ACUUCUAU AACAGUGG

X CGAA ACCACUGU AAUUUAAU

X CGAA AUUUACCA UAAUCCCA

X CGAA AAUUUACC AAUCCCAU

X CGAA AAAUUUAC AUCCCAUU

WO 99/50403 PC'T/US99/06507 Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUAAAUU CCAUUUGC

X CGAA AUGGGAUU UGCAAAGC

X CGAA AAUGGGAU GCAAAGCU

X CGAA AGCUUUGC CUGGCUGG

X CGAA AAGCUUUG UGGCUGGC

X CGAA AGCGGCCA CCUACUAA

X CGAA AGGUAGCG CUAAUGAA

X CGAA AGUAGGUA AUGAAGAA

X CGAA AGCACUGU CAUCCAAA

X CGAA AUGGAGCA CAAAAGAC

X CGAA AGUCUUUU UAACCAUA

X CGAA AAGUCUUU AACCAUAC

X CGAA AAAGUCUU ACCAUACG

X CGAA AUGGUUAA CGGAUCAU

X CGAA AUCCGUAU AUUUCUCA

X CGAA AUGAUCCG UCUCAGUA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAUGAUCC CUCAGUAG

X CGAA AAAUGAUC UCAGUAGC

X CGAA AGAAAUGA AGUAGCCA

X CGAA ACUGAGAA GCCAUAUU

X CGAA AUGGCUAC UUCACCAU

X CGAA AUAUGGCU CACCAUCC

X GGAA AAUAUGGC ACCAUCCA

X CGAA AUGGUGAA CACCGGAU

X CGAA AUCCGGUG CUCCCCCC

X CGAA AGGAUCCG CCCCCUGA

X CGAA AGUCAGGG AGGAGUUU

X CGAA ACUCCUGA UGGGUCUG

X CGAA AACUCCUG GGGUCUGC

X CGAA ACCCAAAC UGCAGUGU

X CGAA AGGGCUUU CAACAUUU

X CGAA AAGGGCUU AACAUUUC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUGUUGAA UCUGUUAA

X CGAA AAUGUUGA CUGUUAAA

X CGAA AAAUGUUG UGUUAAAG

X CGAA ACAGAAAU AAAGUUCU

X CGAA AACAGAAA AAGUUCUU

X CGAA ACUUUAAC CUUCCAAA

X CGAA AACUUUAA UUCCAAAG

X CGAA AGAACUUU CCAAAGCC

X CGAA AUCACGUU GACACUGG

X CGAA AUGUCCAG ACUUUGCU

X CGAA AGUUAUGU UGCUGUCA

X CGAA AAGUUAUG GCUGUCAU

X CGAA ACAGCAAA AUCAACAU

X CGAA AUGACAGC AACAUCAG

X CGAA AUGUUGAU AGCUCUGA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGCUGAUG UGAGCCUU

X CGAA AGGCUCAG ACUUUGGG

X CGAA AAGGCUCA CUUUGGGG

X CGAA AGUAAGGC UGGGGAUG

X CGAA AAGUAAGG GGGGAUGG

X CGAA AUUGGUCC AAAUCCAA

X CGAA AUUUGAUU CAAGAAGC

X CGAA AGCUUCUU CUAUACAA

X CGAA AAGCUUCU UAUACAAA

X CGAA AGAAGCUU UACAAACC

X CGAA AUAGAAGC CAAACCCG

X CGAA ACGGGUUU AAUCACUA

X CGAA AACGGGUU AUCACUAU

X CGAA AUUAACGG ACUAUGAG

X CGAA AGUGAUUA UGAGGCUU

X CGAA AGCCUCAU GGCAACAU

Seq. I.p. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUGUUGCC UUCAAGUG

X CGAA AUAUGUUG CAAGUGAC

X CGAA AAUAUGUU AAGUGACA

X CGAA AUCUCAUU GUUACACU

X CGAA ACAAUCUC ACACUCAA

X CGAA AACAAUCU CACUCAAC

X CGAA AGUGUAAC AACUAUUU

X CGAA AGUUGAGU UUUGGAAC

X CGAA AUAGUUGA UGGAACCU

X CGAA AAUAGUUG GGAACCUC

X CGAA AGGUUCCA GGACAGAA

X CGAA AUUCUGUC UGAACUCU

X CGAA AGUUCAUA UGUGUGCA

X CGAA ACCAGUUG CGUCGUGG

X CGAA ACGGACCA GUGGAGAG

X CGAA AUGCCCUU CUGGACCU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGCGUCUC CACAACAG

X CGAA AAGCGUCU ACAACAGC

X CGAA AGCUGUUG CUAUCGGA

X CGAA AAGCUGUU UAUCGGAC

X CGAA AGAAGCUG UCGGACUC

X CGAA AUAGAAGC GGACUCCC

X CGAA AGUCCGAU, CCUCCUCC

X CGAA AGGGAGUC CUCCAAGA

X CGAA ACCUCUUG UAAAUCUC

X CGAA AGACCUCU AAUCUCCU

X CGAA AUUUAGAC UCCUGCCU

X CGAA AGAUUUAG CUGCCUAA

X CGAA AGGCAGGA AAAGUCAG

X CGAA ACUUUUAG AGACCACU

X CGAA AGUGGUCU UAAAUUUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGAGUGGU AAUUUGAC

X CGAA AUUUAGAG UGACCUGG

X CGAA AAUUUAGA GACCUGGC

X CGAA AUUGGUUG UUUCCAAG

X CGAA AUAUUGGU UCCAAGCU

X CGAA AAUAUUGG CCAAGCUC

X CGAA AAAUAUUG CAAGCUCG

X CGAA AGCUUGGA GGAAGAUG

X CGAA AGUCAUCU UUAUGUUG

X CGAA AAGUCAUC UAUGUUGA

X CGAA AAAGUCAU AUGUUGAA

18?2 CUUCAACA CUGAUGAG1209 UGACUUUUA 1910 X CGAA AAAAGUCA UGUUGAAG

X CGAA ACAUAAAA GAAGUGGA

X CGAA ACCUUCUC UGUGCAAA

X CGAA AUCACUUU AGCAGAAU

X CGAA AUUCUGCU UUAAAGUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUAUUCUG AAAGUUCC

X CGAA AAUAUUCU AAGUUCCA

X CGAA ACUUUAAU CCAGGCAA

X CGAA AACUUUAA CAGGCAAC

1938 CCGAAGUC CUGAUGAG1218 ' AGGCAACUU 1919 X CGAA AGUUGCCU GACUUCGG

X CGAA AGUCAAGU CGGUGCUA

X CGAA AAGUCAAG GGUGCUAC

X CGAA AGCACCGA CUUAACAA

X CGAA AAGUAGCA ACAACUUA

X CGAA AGUUGUUA ACAUCCCA

X CGAA AAGUUGUU CAUCCCAG

X CGAA AUGUAAGU CCAGGGAG

X CGAA ACUGCUCC CGUGGUCC

X CGAA ACCACGUA CGAGCUAG

X CGAA AGCUCGGA GAGUCAAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA ACUCUAGC AACACCAA

X CGAA AUCUUCAC UCACUGCU

X CGAA AGAUCUUC ACUGCUUG

X CGAA AGGGUCCA AGUGACAU

X CGAA AAGGGUCC GUGACAUU

X CGAA AUGUCACU CUUCCUCC

X CGAA AAUGUCAC UUCCUCCU

X CGAA AGAAUGUC CCUCCUCA

X CGAA AAGAAUGU CUCCUCAA

X CGAA AGGAAGAA CUCAACCA

X CGAA AGGAGGAA AACCAGAA

X CGAA AUGUUUUC AAGAUUUC

X CGAA AUCUUGAU UCCAACAU

X CGAA AAUCUUGA CCAACAUU

X CGAA AAAUCUUG CAACAUUA

WO 99/50403 PC'TJU599/06507 Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUGUUGGA ACACACUC

X CGAA AAUGUUGG CACACUCC

X CGAA AGUGUGUA CUCGGCUG

X CGAA AGGAGUGU GGCUGUGA

X CGAA AUCACAGC UCUUGGAC

X CGAA AAUCACAG CUUGGACA

X CGAA AAAUCACA UUGGACAA

X CGAA AGAAAUCA GGACAAUA

X CGAA AUUGUCCA UUGGAUGG

X CGAA AUAUUGUC GGAUGGCU

X CGAA AGCCAUCC UUCUAUUU

X CGAA AUAGCCAU CUAUUUCU

X CGAA AAUAGCCA UAUUUCUU

X CGAA AGAAUAGC UUUCUUCU

X CGAA AUAGAAUA UCUUCUAU

X CGAA AAUAGAAU CUUCUAUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAAUAGAA UUCUAUUA

X CGAA AGAAAUAG CUAUUACU

X CGAA AAGAAAUA UAUUACUA

X CGAA AGAAGAAA UUACUAUC

X CGAA AUAGAAGA ACUAUCCG

X CGAA AAUAGAAG CUAUCCGU

X CGAA AGUAAUAG UCCGUUAC

X CGAA AUAGUAAU CGUUACAA

X CGAA AACGGAUA CAAGGUUC

X CGAA ACCUUGUA CAAGGCAA

X CGAA AACCUUGU AAGGCAAG

X CGAA ACGUGCUG GAUGUGAA

X CGAA AUCUUCAC AAGAAUGC

X CGAA AUGGUGGC AUUCAGUA

X CGAA AUGAUGGU CAGUAUCA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAUGAUGG AGUAUCAG

X CGAA ACUGAAUG UCAGCUCA

X CGAA AUACUGAA AGCUCAAG

X CGAA AGCUGAUA AAGGGCCU

X CGAA AGGCCCUU GAGCCUGA

X CGAA AUGCUGUU CCAGGUGG

X CGAA AUGUCCAC UUUGCAGA

X CGAA AAUGUCCA UUGCAGAG

X CGAA AAAAUGUC GCAGAGAA

X CGAA AUGUUGUU GGGUCAAG

X CGAA ACCCUAUG AAGCAACC

X CGAA AGGCUGGG UUCUCAUG

X CGAA AAGGCUGG UCUCAUGA

X CGAA AAAGGCUG CUCAUGAA

X CGAA AAAAGGCU UCAUGAAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGAAAAGG AUGAACUG

X CGAA AGGGUCAC CCAGAAUC

X CGAA AUUCUGGG UCAAGCAC

X CGAA AGAUUCUG AAGCACCA

X CGAA AGGUCCGC GGAGGGGG

X CGAA AGCAGCAU AUAGCCAU

X CGAA AAGCAGCA UAGCCAUC

X CGAA AUAAGCAG GCCAUCCU

X CGAA AUGGCUAU CUUGGCUC

X CGAA AGGAUGGC GGCUCUGC

X CGAA AGCCAAGG UGCUGGAA

X CGAA ACAGCACA GGCCUUUC

X CGAA AGGCCAAC UCUGAUCA

X CGAA AAGGCCAA CUGAUCAU

X CGAA AAAGGCCA UGAUCAUA

X CGAA AUCAGAAA AUAUUGCA

Seq. I.D. Seq. I.D.

Position R2 No. Substrate No.

X CGAA AUGAUCAG UUGCAAUU

X CGAA AUAUGAUC GCAAUUGA

X CGAA AUUGCAAU GAAGAGGG

X CGAA AGGCUUGG CCAAAACG

X CGAA AAGGCUUG CAAAACGU

X CGAA ACUGCACA CAACUCAG

X CGAA AACUGCAC AACUCAGG

X CGAA AGUUGAAC AGGGACUC

X CGAA AGUCCCUG UGGCCCUA

X CGAA AGGGCCAG AACAGGAA

X CGAA ACCUUCCU AAAAACAA

X CGAA AUCUGGGU CUACAAUU

X CGAA AGGAUCUG CAAUUUAU

X CGAA AUUGUAGG UAUCCAGU

X CGAA AAUUGUAG AUCCAGUG

X CGAA AAAUUGUA UCCAGUGC

Seq. I.D. Seq. I.D.

Position R2 No. Substrate No.

X CGAA AUAAAUUG CAGUGCUU

X CGAA AGCACUGG GACUGGAA

X CGAA AUGUCAUU AAAUUUCA

lO X CGAA AUUUGAUG UCAAGAUG

X CGAA AAUUUGAU CAAGAUGU

X CGAA AAAUUUGA AAGAUGUG

X CGAA AUCACAUC GGGGAGGG

X CGAA AUUGCCCU UUGGCCAA

X CGAA AAAUUGCC GGCCAAGU

X CGAA ACUUGGCC CUUAAGGC

X CGAA AACUUGGC UUAAGGCG

X CGAA AGAACUUG AAGGCGCG

X CGAA AAGAACUU AGGCGCGC

X CGAA AUGCGCGC AAGAAGGA

X CGAA ACCCAUCC ACGGAUGG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AACCCAUC CGGAUGGA

X CGAA AUGGCAGC AAAAGAAU

X CGAA AUUCUUUC UGCCUCCA

X CGAA AGGCAUAU CAAAGAUG

X CGAA AUCAUCUU ACAGGGAC

X CGAA AGUCCCUG UGCAGGAG

X CGAA AAGUCCCU GCAGGAGA

X CGAA ACUUCCAG CUUUGUAA

X CGAA AGAACUUC UGUAAACU

X CGAA AAGAACUU GUAAACUU

X CGAA ACAAAGAA AACUUGGA

X CGAA AGUUUACA GGACACCA

X CGAA AUGGUGUC CAAACAUC

2803 AGAUUGAU CUGAUGAG13,56 CCAAACAUC 2057 X CGAA AUGUUUGG AUCAAUCU

X CGAA AUGAUGUU AAUCUCUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUGAUGA UCUUAGGA

X CGAA AGAUUGAU UUAGGAGC

X CGAA AGAGAUUG AGGAGCAU

X CGAA AAGAGAUU GGAGCAUG

X CGAA AUGUUCAC GAGGCUAC

X CGAA AGCCUCGA CUUGUACC

X CGAA AGUAGCCU GUACCUGG

X CGAA ACAAGUAG CCUGGCCA

X CGAA ACUCAAUG CGCGCCCC

X CGAA AGGUUUCC CUGGACUU

X CGAA AAGGUUUC UGGACUUC

X CGAA AGUCCAGA CCUUCGCA

X CGAA AAGUCCAG CUUCGCAA

X CGAA AGGAAGUC CGCAAGAG

X CGAA AAGGAAGU GCAAGAGC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUGCUGGG UGCCAUUG

X CGAA AAUGCUGG GCCAUUGC

X CGAA AUGGCAAA GCCAAUAG

X CGAA AUUGGCAA GCACCGCG

X CGAA ACGCGGUG CACACUGU

X CGAA ACAGUGUG CUCCCAGC

2961 GCUGCUGG CUGAUGAG1380 ACUGUCCUC 2087.

X CGAA AGGACAGU CCAGCAGC

X CGAA AGCUGCUG CUUCACUU

X CGAA AAGGAGCU ACUUCGCU

X CGAA AGUGAAGG CGCUGCCG

X CGAA AAGUGAAG GCUGCCGA

X CGAA AGUCCAUG CUUGAGCC

~

X CGAA AGUAGUCC GAGCCAAA

X CGAA ACUGUUUU UAUCCACA

X CGAA AACUGUUU AUCCACAG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAACUGUU UCCACAGG

X CGAA AUAAACUG CACAGGGA

X CGAA AUCCCUGU UGGCUGCC

X CGAA AUGUUUCU UUAGUUGG

X CGAA AAUGUUUC UAGUUGGU

X CGAA AAAUGUUU AGUUGGUG

X CGAA AAAAUGUU GUUGGUGA

X CGAA ACUAAAAU GGUGAAAA

X CGAA AUUUUUGC GCAGAUUU

X CGAA AUCUGCUA UUGGAUUG

X CGAA AAUCUGCU UGGAUUGU

309? GACAAUCC CUGAUGAG1902 GCAGAUUUU 2103 X CGAA AAAUCUGC GGAUUGUC

X CGAA AUCCAAAA GUCCCGAG

X CGAA ACAAUCCA CCGAGGUC

X CGAA ACCUCGGG AAGAGGUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA ACACCUCU CGUGAAAA

X CGAA AGCCUUCC CCAGUGCG

X CGAA AUGGCCAU GAGUCACU

X CGAA ACUCGAUG ACUGAAUU

X CGAA AUUCAGUG ACAGUGUG

X CGAA AAUUCAGU CAGUGUGU

X CGAA ACACACUG CACAACCA

X CGAA ACAUCACU UGGUCCUA

X CGAA ACCAUACA CUAUGGUG

X CGAA AGGACCAU UGGUGUGU

X CGAA ACACACCA ACUAUGGG

X CGAA AACACACC CUAUGGGA

X CGAA AGUAACAC UGGGAGAU

X CGAA AUCUCCCA GUUAGCUU

X CGAA ACAAUCUC AGCUUAGG

X CGAA AACAAUCU GCUUAGGA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGCUAACA AGGAGGCA

X CGAA AAGCUAAC GGAGGCAC

X CGAA AGGGUGUG CUGCGGGA

X CGAA AGUCAUCC GUGCAGAA

X CGAA AGUUCUGC UACGAGAA

X CGAA AGAGUUCU CGAGAAGC

X CGAA AGCCCUGG CAGACUGG

X CGAA ACACCUCA UGAUCUAA

X CGAA AUCAUACA UAAUGAGA

X CGAA AGAUCAUA AUGAGACA

X CGAA AGGCUUCU AUGAGAGG

X CGAA AAGGCUUC UGAGAGGC

X CGAA AUGGCCUC AUUUGCCC

X CGAA AUGAUGGC UGCCCAGA

X CGAA AAUGAUGG GCCCAGAU

X CGAA AUCUGGGC UUGGUGUC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUAUCUGG GGUGUCCU

X CGAA ACACCAAU CUUAAACA

X CGAA AGGACACC AAACAGAA

X CGAA AAGGACAC AACAGAAU

X CGAA ACAUUCUG AGAGGAGC

X CGAA AACAUUCU GAGGAGCG

X CGAA AGGUCUUU CGUGAAUA

X CGAA AUUCACGU CCACGCUU

X CGAA AGCGUGGU UAUGAGAA

X CGAA AAGCGUGG AUGAGAAG

X CGAA AAAGCGUG UGAGAAGU

X CGAA ACUUCUCA UACUUAUG

X CGAA AACUUCUC ACUUAUGC

X CGAA AAACUUCU CUUAUGCA

X CGAA AGUAAACU AUGCAGGA

X CGAA AAGUAAAC UGCAGGAA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUCCUGC GACUGUUC

X CGAA ACAGUCAA CUGCUGAA

X CGAA AACAGUCA UGCUGAAG

X CGAA AGGCCGCU GGACAGAA

X CGAA AUGUUCUG UGUAUACC

X CGAA ACAGAUGU UACCCUCU

X CGAA AUACAGAU CCCUCUGU

X CGAA AGGGUAUA UGUUUCCC

X CGAA ACAGAGGG UCCCUUUC

X CGAA AACAGAGG CCCUUUCA

X CGAA AAACAGAG CCUUUCAC

X CGAA AGGGAAAC UCACUGGC

X CGAA AAGGGAAA CACUGGCA

X CGAA AAAGGGAA ACUGGCAU

3576 CAGUUGUC CUGAUGAG146$ GAGACCCUU 2169 X CGAA AGGGUCUC GACAACUG

X CGAA AGGCAUGU UGCCAAAG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUCACAUC UAUAAGUG

X CGAA AUAUCACA UAAGUGUA

X CGAA AUAUAUCA AGUGUACA

X CGAA ACACUUAU CAUAUGUG

X CGAA AUGUACAC UGUGCUGG

X CGAA AUUCCAGC CUAACAAG

X CGAA AAUUCCAG UAACAAGU

X CGAA AGAAUUCC ACAAGUCA

X CGAA AUGACUUG GGUUAAUA

X CGAA ACCUAUGA AAUAUUUA

X CGAA AACCUAUG AUAUUUAA

X CGAA AUUAACCU UUUAAGAC

X CGAA AUAUUAAC UAAGACAC

X CGAA AAUAUUAA AAGACACU

X CGAA AAAUAUUA AGACACUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUUUUCA UAAGUGAU

X CGAA AGAUUUUU AGUGAUAU

X CGAA AUCACUUA UAAAUCAG

X CGAA AUAUCACU AAUCAGAU

X CGAA AUUUAUAU AGAUUCUU

X CGAA AUCUGAUU CUUCUCUC

X CGAA AAUCUGAU UUCUCUCU

X CGAA AGAAUCUG CUCUCUCA

3?08 AUGAGAGA CUGAUGAG1499 AGAUUCUUC 2195 X CGAA AAGAAUCU UCUCUCAU

X CGAA AGAAGAAU UCUCAUUU

X CGAA AGAGAAGA UCAUUUUA

X CGAA AGAGAGAA AUUUUAUC

X CGAA AUGAGAGA UUAUCCCU

X CGAA AAUGAGAG UAUCCCUC

X CGAA AAAUGAGA AUCCCUCA

X CGAA AAAAUGAG UCCCUCAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUAAAAUG CCUCACCU

X CGAA AGGGAUAA ACCUGUAG

X CGAA ACAGGUGA GCAUGCCA

X CGAA ACUGGCAU CCGUUUCA

X CGAA ACGGGACU UCAUUUAG

X CGAA AACGGGAC CAUUUAGU

X CGAA AAACGGGA AUUUAGUC

X CGAA AUGAAACG UAGUCAUG

X CGAA AAAUGAAA GUCAUGUG

X CGAA ACUAAAUG AUGUGACC

X CGAA AGUGGUCA UGUCUUGU

X CGAA ACAGAGUG UUGUGUUU

X CGAA AGACAGAG GUGUUUCC

X CGAA ACACAAGA UCCACAGC

3783 GGCUGUGG CUGAUGAG151? CUUGUGUUU 2218 X CGAA AACACAAG CCACAGCC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAACACAA CACAGCCU

X CGAA ACUUGCAG CAGUCCAG

X CGAA AACUUGCA AGUCCAGG

X CGAA ACUGAACU CAGGAUGC

X CGAA AGCAUCCU ACAUCUAA

X CGAA AUGUUAGC UAAAAAUA

X CGAA AGAUGUUA AAAAUAGA

X CGAA AUUUUUAG GACUUAAA

X CGAA AGUCUAUU AAAUCUCA

X CGAA AAGUCUAU AAUCUCAU

X CGAA AUUUAAGU UCAUUGCU

X CGAA AGAUUUAA AUUGCUUA

X CGAA AUGAGAUU GCUUACAA

X CGAA AGCAAUGA ACAAGCCU

X CGAA AAGCAAUG CAAGCCUA

X CGAA AGGCUUGU AGAAUCUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AUUCUUAG UUUAGAGA

X CGAA AGAUUCUU UAGAGAAG

X CGAA AAGAUUCU AGAGAAGU

X CGAA AAAGAUUC GAGAAGUA

X CGAA ACUUCUCU UACAUAAG

X CGAA AUACUUCU CAUAAGUU

X CGAA AUGUAUAC AGUUUAGG

X CGAA ACUUAUGU UAGGAUAA

X CGAA AAACUUAU GGAUAAAA

X CGAA AUCCUAAA AAAUAAUG

X CGAA AUUUUAUC AUGGGAUU

X CGAA AUCCCAUU UUCUUUUC

X CGAA AAUCCCAU UCUUUUCU

X CGAA AAAUCCCA CUUUUCUU

X CGAA AAAAUCCC UUUUCUUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGAAAAUC UUCUUUUC

X CGAA AAGAAAAU UCUUUUCU

X CGAA AAAGAAAA CUUUUCUC

X CGAA AAAAGAAA UUUUCUCU

X CGAA AGAAAAGA UUCUCUGG

X CGAA AAGAAAAG UCUCUGGU

X CGAA AAAGAAAA CUCUGGUA

X CGAA AAAAGAAA UCUGGUAA

X CGAA ACCAGAGA AUAUUGAC

X CGAA AUUACCAG UUGACUUG

X CGAA AUAUUACC GACUUGUA

X CGAA AGUCAAUA GUAUAUUU

X CGAA ACAAGUCA UAUUUUAA

X CGAA AUACAAGU UUUUAAGA

X CGAA AUAUACAA UUAAGAAA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AAUAUACA UAAGAAAU

X CGAA AAAUAUAC AAGAAAUA

X CGAA AAAAUAUA AGAAAUAA

X CGAA AUUUCUUA ACAGAAAG

X CGAA AUGUCACC UGGGAGAC

X CGAA AAUGUCAC GGGAGACA

X CGAA AUGUCACA UAUAUAUU

X CGAA AAUGUCAC AUAUAUUG

X CGAA AAAUGUCA UAUAUUGA

X CGAA AUAAAUGU UAUUGAAU

X CGAA AUAUAAAU UUGAAUUA

X CGAA AUAUAUAA GAAUUAAU

X CGAA AUUCAAUA AAUAUCCC

X CGAA AAUUCAAU AUAUCCCU

X CGAA AUUAAUUC UCCCUACA

X CGAA AUAUUAAU CCUACAUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGGGAUAU CAUGUAUU

X CGAA ACAUGUAG UUGCACAU

X CGAA AUACAUGU GCACAUUG

X CGAA AUGUGCAA GUAAAAAG

X CGAA ACAAUGUG AAAAGUUU

X CGAA ACUUUUUA UUAGUUUp X CGAA AACUUUUU UAGUUUUG

X CGAA AAACUUUU AGUUUUGA

X CGAA AAAACUUU GUUUUGAU

X CGAA ACUAAAAC UUGAUGAG

X CGAA AACUAAAA UGAUGAGU

X CGAA AAACUAAA GAUGAGUU

X CGAA ACUCAUCA GUGAGUUU

X CGAA ACUCACAA UACCUUGU

X CGAA AACUCACA ACCUUGUA

X CGAA AAACUCAC CCUUGUAU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X CGAA AGGUAAAC GUAUACUG

X CGAA ACAAGGUA UACUGUAG

X CGAA AUACAAGG CUGUAGGC

X CGAA ACAGUAUA GGCACACU

X CGAA AGUGUGCC UGCACUGA

X CGAA AAGUGUGC GCACUGAU

X CGAA AUCAGUGC UAUCAUGA

X CGAA AUAUCAGU UCAUGAGU

X CGAA AUUCACUC AAUGUCUU

X CGAA ACAUUUAU UUGCCUAC

X CGAA AGACAUUU GCCUACUC

X CGAA AGGCAAGA CUCAAAAA

X CGAA AGUAGGCA AAAAAAAA

TABLE VI: HAIRPIN RIZOZYMES AND TRARGET SITES FOR TIE-2 Seq. Seq. I.D.

PositionRZ I.D. SubstrateNo.
No.

GUU

X GUACAUUACCUGGUA CCUUCU

GCU

X GUACAUUACCUGGUA GGGUUU

GCU

X GUACAUUACCUGGUA UAGUUC

GCU

GAU

X GUACAUUACCUGGUA GCUGAA

GCU

X GUACAUUACCUGGUA GGAAGU

GCC

X GUACAUUACCUGGUA UCAUGC

GCC

X GUACAUUACCUGGUA CCAGGA

GCC

X GUACAUUACCUGGUA UUCACC

GUA

X GUACAUUACCUGGUA CUGCUU

GCU

X GUACAUUACCUGGUA UGUAUG

GUC

X GUACAUUACCUGGUA UCCCUG

GAC

X GUACAUUACCUGGUA CCCUAU

GAU

X GUACAUUACCUGGUA UGUAAG

1094 UGUUGC AGAA GCAC ACCAGAGAAACA232? GUGCA 2395 GCU

X GUACAUUACCUGGUA GCAACA

Seq. Seq. I.D.

PositionRZ I.D. SubstrateNo.
No.

GCU

X GUACAUUACCUGGUA CUCCAG

GAU

X GUACAUUACCUGGUA CAUAUA

GAU

X GUACAUUACCUGGUA GGGACA

GAU

X GUACAUUACCUGGUA CAUUUC

GUA

X GUACAUUACCUGGUA GCCAUA

GAU

X GUACAUUACCUGGUA CCUCCC

GAC

X GUACAUUACCUGGUA UCAGGA

GCU

X GUACAUUACCUGGUA CUGAGC

GUC

X GUACAUUACCUGGUA ' GUGGAG

GCU

X GUACAUUACCUGGUA UCUAUC

GAC

X GUACAUUACCUGGUA UCCCUC

GCC

X GUACAUUACCUGGUA UAAAAG

GAC

X GUACAUUACCUGGUA CACUCU

GUA

X GUACAUUACCUGGUA CGUGGU

GCU

X GUACAUUACCUGGUA UGGACC

GCU

X GUACAUUACCUGGUA GUGAUU

Seq. Seq. I.D.

PositionRZ I.D. SubstrateNo.
No.

GUA

X GUACAUUACCUGGUA UCAGCU

GCU

X GUACAUUACCUGGUA CAAGGG

GCC

X GUACAUUACCUGGUA UUUUCU

GAC

X GUACAUUACCUGGUA CUCGGA

GCU

X GUACAUUACCUGGUA UAUAGC

GCU

X GUACAUUACCUGGUA GGAAUG

GCC

X GUACAUUACCUGGUA UGACUG

GAC

X GUACAUUACCUGGUA UGUGCU

GUU

X GUACAUUACCUGGUA GGCCUU

GAU

X GUACAUUACCUGGUA CAUAUU

GCU

X GUACAUUACCUGGUA GUGCAG

GUU

X GUACAUUACCUGGUA CAACUC

GAU

X GUACAUUACCUGGUA CCUACA

GAU

3a X GUACAUUACCUGGUA GGAUGC

GAC

X GUACAUUACCUGGUA CCAGCA

GUC

X GUACAUUACCUGGUA CUCCCA

Seq. Seq. I.D.

PositionRZ I.D. SubstrateNo.
No.

GCU

X GUACAUUACCUGGUA CCUUCA

GCC

X GUACAUUACCUGGUA GACGUG

GAC

X GUACAUUACCUGGUA GUGGCC

GUU

X GUACAUUACCUGGUA UAUCCA

GAU

X GUACAUUACCUGGUA UUUGGA

GCC

X GUACAUUACCUGGUA CCAGGG

GAC

X GUACAUUACCUGGUA UGGAGA

GUU

X GUACAUUACCUGGUA CUGCUG

GCU

X GUACAUUACCUGGUA GAAGAA

GCC

X GUACAUUACCUGGUA UAGGAC

GUA

X GUACAUUACCUGGUA UACCCU

GUU

X GUACAUUACCUGGUA UCCCUU

GCU

X GUACAUUACCUGGUA GAGAAA

GAU

X GUACAUUACCUGGUA UCUUCU

GUA

X GUACAUUACCUGGUA GCAUGC

GUC

X GUACAUUACCUGGUA CCGUUU

Seq. Seq. I.D.

PositionRZ I.D. SubstrateNo.
No.

GUU

X GUACAUUACCUGGUA UCAUUU

GUC

X GUACAUUACCUGGUA UUGUGU

GCC

X GUACAUUACCUGGUA UGCAAG

GUC

X GUACAUUACCUGGUA CAGGAU

GUA

X GUACAUUACCUGGUA GGCACA

TABLE VI I: HAMMERHEAD RIBOZYME AND TARGET SITE SEQUENCES FOR INTEGRIN

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACGGUCGC CCGGGGGU

X

CGAA ACCGCAGC GCAGCAGC

X

CGAA AGGCUGCC GGACCCAG

X

CGAA ACCUGCAG CCCGCUCC

X

CGAA AGCGGGGA CCCUCCCC

X

CGAA AGGGGAGC CCCGUGCG

X

CGAA ACGCACGG CGCCCAUG

X

CGAA AGCACAGC GCUCUACC

X

CGAA AGCAAGCA UACCUGUC

178. CCGACAGG CUGAUGAG 2458 CUUGCUCUA 3597 X

CGAA AGAGCAAG CCUGUCGG

X

CGAA ACAGGUAG GGCGGGGC

X

CGAA AGCCCCGC CUGUCCCG

X

CGAA ACAGGAGC CCGGCUCG

X

CGAA AGCCGGGA GGCGCAGC

X

CGAA AGGCUGCG CAACUUGG

Seq. Seq. I.D.
T.D.

Position RZ No. Substrate No.

X

CGAA AAGGCUGC AACUUGGA

X

CGAA AGUUGAAG GGACACUC

X

CGAA AGUGUCCA GGGAGGAC

X

CGAA AUCACGUU CGGAAAUA

X

CGAA AUUUCCGG UGGAGACC

X

CGAA AGGCUCCC UUCGGCUU

X

CGAA AGAGGCUC CGGCUUCU

X

CGAA AAGAGGCU GGCUUCUC

X

CGAA AGCCGAAG CUCGCUGG

X

CGAA AAGCCGAA UCGCUGGC

X

CGAA AGAAGCCG GCUGGCCA

X

CGAA ACAGCCGC GCUCGUGG

X

CGAA AGCAACAG GUGGGGGC

X

CGAA AGCGCUUC CCACUGCA

X

CGAA AAGCGCUU CACUGCAG

X

CGAA ACAGCCCU CAGCUGCG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUGUCGCA ACCGCCCG

X

CGAA AUCCGCGU GAGUUUGA

X

CGAA ACUCGAUC UGAUAACG

X

CGAA AACUCGAU GAUAACGA

X

CGAA AUCAAACU ACGAUGCU

X

CGAA ACGUGGGG AGAAAGCA

X

CGAA AUCUUCCU AGUGGAUG

X

CGAA ACCCCCAU ACCGUCCA

X

X

CGAA ACCUUGGC CAGGGGGC

X

CGAA ACCUUGCC GUGACAUG

X

CGAA AGCACAUG ACCGAUAU

X

CGAA AUCGGUGA UGAAAAAA

X

CGAA ACAUGCUG AAUACGAA

X

CGAA AACAUGCU AUACGAAG

X

CGAA AUUAACAU CGAAGCAG

WO 99/50403 PC'T/US99/06507 Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUUCCUGC CCGAGACA

X

CGAA AUGUCUCG UUUGGGCG

X

CGAA AGAUGUCU UGGGCGGU

X

CGAA AAGAUGUC GGGCGGUG

X

CGAA ACACCGCG AUGUCCUG

X

CGAA AACACCGC UGUCCUGA

X

CGAA ACAUAACA CUGAGUCA

X

CGAA ACUCAGGA AGAAUCUC

X

CGAA AUUCUGAC UCAGGAUU

X

CGAA AGAUUCUG AGGAUUGA

X

CGAA AUCCUGAG GAAGACGA

X

CGAA AUCGUCUU UGGAUGGG

X

CGAA AUCUCCCC GGAGCUUU

X

CGAA AGCUCCAA UUGUGAUG

X

CGAA AAGCUCCA UGUGAUGG

X

CGAA AAAGCUCC GUGAUGGG

Seq. Seq. I.D.
I.D.

Position R2 No. Substrate No.

X

CGAA AUCGCCCA GAGAGGCC

X

CGAA AUUUCUCA UGGCUCUU

X

CGAA AAUUUCUC GGCUCUUG

X

CGAA AGCCAAAU UUGCCAGC

X

CGAA AGAGCCAA GCCAGCAA

X

CGAA ACACCUUG GCAGCUAC

X

CGAA AGCUGCUA CUUUUACU

X

CGAA AGUAGCUG UUACUAAA

X

X

CGAA AAAGUAGC ACUAAAGA

X

CGAA AAAAGUAG CUAAAGAC

X

CGAA AGUAAAAG AAGACUUU

X

CGAA AGUCUUUA UCAUUACA

X

CGAA AAGUCUUU CAUUACAU

X

CGAA AAAGUCUU AUUACAUU

X

CGAA AUGAAAGU ACAUUGUA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAUGAAAG CAUUGUAU

X

CGAA AUGUAAUG GUAUUUGG

X

CGAA ACAAUGUA UUUGGAGC

X

CGAA AUACAAUG UGGAGCCC

X

CGAA AAUACAAU GGAGCCCC

X

CGAA ACCCGGGG CUUAUAAC

X

CGAA AGUACCCG AUAACUGG

778 UCCAGUUA CUGAUGAG 2,535 GGGUACUUA 3674 X

CGAA AAGUACCC UAACUGGA

X

X

CGAA AUCCCUUU GUUCGUGU

X

CGAA ACAAUCCC CGUGUAGA

79g CUCUACAC CUGAUGAG 2539 GGAUUGUUC 3678 X

CGAA AACAAUCC GUGUAGAG

X

CGAA ACACGAAC GAGCAAAA

X

CGAA AUUCUUUU ACACUUUU

X

CGAA AGUGUUAU UUUUUGAC

X

CGAA AAGUGUUA UUUUGACA ~
I

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AAAGUGUU UUUGACAU

X

CGAA AAAAGUGU UUGACAUG

X

CGAA AAAAAGUG UGACAUGA

X

CGAA AAAAAAGU GACAUGAA

X

CGAA AUGUUCAU UUUGAAGA

X

CGAA AGAUGUUC UGAAGAUG

X

CGAA AAGAUGUU GAAGAUGG

X

CGAA AGGCCCAU AUGAAGUU

X

CGAA AAGGCCCA UGAAGUUG

X

CGAA ACUUCAUA GGUGGAGA

X

CGAA ACUUUCAU UCGUUCCU

gg3 ACAGGAAC CUGAUGAG 2555 GAAAGUCUC 3694 X

CGAA AGACUUUC GUUCCUGU

X

CGAA ACGAGACU CCUGUUCC

X

CGAA AACGAGAC CUGUUCCU

X

CGAA ACAGGAAC CCUGCUAA

X

CGAA AACAGGAA CUGCUAAC

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGCAGGAA ACAGUUAC

X

CGAA ACUGUUAG ACUUAGGU

X

CGAA AACUGUUA CUUAGGUU

X

CGAA AGUAACUG AGGUUUUU

X

CGAA AAGUAACU GGUUUUUC

X

CGAA ACCUAAGU UUUCUUUG

X

CGAA AACCUAAG UUCUUUGG

X

CGAA AAACCUAA UCUUUGGA

X

CGAA AAAACCUA CUUUGGAC

X

CGAA AAAAACCU UUUGGACU

930 UGAG(JCCA CUGAUGAG2570 GUUUUUCUU 3709 X

CGAA AGAAAAAC UGGACUCA

X

CGAA AAGAAAAA GGACUCAG

X

CGAA AGUCCAAA AGGGAAAG

X

CGAA ACCUUUCC UUGUUUCU

X

CGAA AUACCUUU GUUUCUAA

X

CGAA ACAAUACC UCUAAAGA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AACAAUAC CUAAAGAU

X

CGAA AAACAAUA UAAAGAUG

X

CGAA AGAAACAA AAGAUGAG

X

CGAA AUCUCAUC ACUUUUGU

X

CGAA AGUGAUCU UUGUAUCU

X

CGAA AAGUGAUC UGUAUCUG

X

CGAA AAAGUGAU GUAUCUGG

X

CGAA ACAAAAGU UCUGGUGC

X

X

CGAA AGCACCAG CCAGAGCC

X

CGAA AUUGGCUC ACAGUGGA

X

CGAA ACCACGGC UUGCUGAA

X

CGAA AACCACGG UGCUGAAG

X

CGAA AAACCACG GCUGAAGA

X

CGAA ACUUCAUG UGCACAUC

X

CGAA AUGUGCAG UCCUCCCU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGAUGUGC CUCCCUGA

X

CGAA AGGAGAUG CCUGAGCA

X

CGAA AUGUGCUC UUCGAUGG

X

CGAA AUAUGUGC CGAUGGAG

X

CGAA AAUAUGUG GAUGGAGA

X

CGAA ACCUUCUC UGGCCUCU

X

CGAA AGGCCAGA UUCAUUUG

X

CGAA AGAGGCCA CAUUUGGC

X

CGAA AAGAGGCC AUUUGGCU

X

CGAA AUGAAGAG UGGCUAUG

X

CGAA AAUGAAGA GGCUAUGA

X

CGAA AGCCAAAU UGAUGUGG

X

CGAA AGGUCCAC AACAAGGA

X

CGAA AUCUUGCC UAGUUAUU

X

CGAA AUAUCUUG GUUAUUGG

X

CGAA ACUAUAUC AUUGGAGC

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AACUAUAU UUGGAGCC

X

CGAA AUAACUAU GGAGCCCC

X

CGAA ACUGUGGG UUUUGAUA

X

CGAA AUACUGUG UUGAUAGA

X

CGAA AAUACUGU UGAUAGAG

X

CGAA AAAUACUG GAUAGAGA

X

CGAA AUCAAAAU GAGAUGGA

X

CGAA ACUUCUCC GGAGGUGC

X

X

CGAA ACAUACAC UACAUGAA

X

CGAA AGACAUAC CAUGAACC

X

CGAA AUUCCAUC AUGUGAAG

X

CGAA AUUGGCUU CGUCUUAA

X

CGAA AAUUGGCU GUCUUAAU

X

CGAA ACGAAUUG UUAAUGGA

X

CGAA AGACGAAU AAUGGAAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AAGACGAA AUGGAACC

X

CGAA AUCUUUGG CUAUGUUU

X

CGAA AAUCUUUG UAUGUUUG

X

CGAA AGAAUCUU UGUUUGGC

X

CGAA ACAUAGAA UGGCAUUG

X

CGAA AACAUAGA GGCAUUGC

X

CGAA AUGCCAAA GCAGUAAA

X

CGAA ACUGCAAU AAAAAUAU

1296 AUCUCCAA CUGAUGAG 2632 UAAAAAi~IUA3771 X

X

CGAA AUAUUUUU GGAGAUAU

X

CGAA AUCUCCAA UUAAUCAA

X

CGAA AUAUCUCC AAUCAAGA

X

CGAA AAUAUCUC AUCAAGAU

X

CGAA AUUAAUAU AAGAUGGC

X

CGAA AGCCAUCU CCCAGAUA

X

CGAA AUCUGGGU UUGCAGUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUAUCUGG GCAGUUGG

X

CGAA ACUGCAAU GGAGCUCC

X

CGAA AGCUCCAA CGUAUGAU

X

CGAA ACGGAGCU UGAUGACU

X

CGAA AGUCAUCA GGGAAAGG

X

CGAA ACCUUUCC UUUAUCUA

X

CGAA AACCUUUC UUAUCUAU

X

CGAA AAACCUUU UAUCUAUC

X

X

CGAA AAAAACCU UCUAUCAU

X

CGAA AUAAAAAC UAUCAUGG

X

CGAA AGAUAAAA UCAUGGAU

1377 AGAUCCAU CUGAUGAG 2652 UUAUCUAUC 3?91 X

CGAA AUAGAUAA AUGGAUCU

X

CGAA AUGCAUGA UGCAAAUG

X

CGAA AUUCCAUU AAUACCAA

X

CGAA AUUUAUUC CCAAACCA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACCUGUGU CUCAAGGG

X

CGAA AACCUGUG UCAAGGGU

1421 . AUACCCUU CUGAUGAG 2658 CAGGUUCUC 3797 X

CGAA AGAACCUG AAGGGUAU

X

CGAA ACCCUUGA UAUCACCU

X

CGAA AUACCCUU UCACCUUA

X

CGAA AUAUACCC ACCUUAUU

X

CGAA AGGUGAUA AUUUUGGA

X

CGAA AAGGUGAU UUUUGGAU

X

CGAA AUAAGGUG UUGGAUAU

X

CGAA AAUAAGGU UGGAUAUU

X

CGAA AAAUAAGG GGAUAUUC

1947 CAAUUGAA CUGAUGAG 266'7 UUUUGGAUA 3806 X

CGAA AUCCAAAA UUCAAUUG

X

CGAA AUAUCCAA CAAUUGCU

X

CGAA AAUAUCCA AAUUGCUG

X

CGAA AUUGAAUA GCUGGAAA

X

CGAA AGGUCCAU GAUCGAAA

WO 99/50403 PCT/US99/0650?

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUCAAGGU GAAAUUCC

X

CGAA AUUUCGAU CCUACCCU

X

CGAA AAUUUCGA CUACCCUG

X

CGAA AGGAAUUU CCCUGAUG

X

CGAA ACAUCAGG GCUGUUGG

X

CGAA ACAGCAAC GGUUCCCU

X

CGAA ACCAACAG CCCUCUCA

X

CGAA AACCAACA CCUCUCAG

X

2~ CGAA AGGGAACC UCAGAUUC

X

CGAA AGAGGGAA AGAUUCAG

X

CGAA AUCUGAGA CAGUAAGU

X

CGAA AAUCUGAG AGUAACUA

X

CGAA ACUGAAUC ACUAUUUU

X

CGAA AGUUACUG UUUUCAGA

X

CGAA AUAGUUAC UUCAGAUC

X

CGAA AAUAGUUA UCAGAUCC

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAAUAGUU CAGAUCCC

X

CGAA AAAAUAGU AGAUCCCG

X

CGAA AUCUGAAA CCGGCCUG

X

CGAA AUCACAGG AAUAUUCA

X

CGAA AAUCACAG AUAUUCAG

X

CGAA AUUAAUCA UUCAGAAA

X

CGAA AUAUUAAU CAGAAAAC

X

CGAA AAUAUUAA AGAAAACC

X

CGAA AUGGUUUU ACAGUAAC

X

CGAA ACUGUGAU ACUCCUAA

X

CGAA AGUUACUG CUAACAGA

X

CGAA AGGAGUUA ACAGAAUU

X

CGAA AUUCUGUU GACCUCCG

X

CGAA AGGUCAAU CGCCAGAA

X

CGAA AGGCGCCC GUGGGAUA

X

CGAA AUCCCACU UGCCUCCA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGGCAUAU CAGGUUAA

X

CGAA ACCUGGAG AAAUCCUG

X

CGAA AACCUGGA AAUCCUGU

X

CGAA AUUUAACC CUGUUUUG

X

CGAA ACAGGAUU UUGAAUAU

X

CGAA AACAGGAU UGAAUAUA

X

CGAA AAACAGGA GAAUAUAC

X

CGAA AUUCAAAA UACUGCUA

X

CGAA AUAUUCAA CUGCUAAC

X

CGAA AGCAGUAU ACCCCGCU

X

CGAA ACCAGCGG AUAAUCCU

X

CGAA AACCAGCG UAAUCCUU

X

CGAA AUAACCAG AUCCUUCA

X

CGAA AUUAUAAC CUUCAAUA

X

CGAA AGGAUUAU CAAUAUCA

X

CGAA AAGGAUUA AAUAUCAA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUUGAAGG UCAAUUGU

X

CGAA AUAUUGAA AAUUGUGG

X

CGAA AUUGAUAU GUGGGCAC

X

CGAA AGUGUGCC GAAGCUGA

X

CGAA AUUUUCUU UGGGCUAU

X

CGAA AGCCCAGA UCCUCAAG

X

CGAA AUAGCCCA CUCAAGAG

X

CGAA AGGAUAGC AAGAGUUC

X

X

CGAA AACUCUUG AGUUUCGA

X

CGAA ACUGAACU UCGAAACC

X

CGAA AACUGAAC CGAAACCA

1769 UUGGUUUC CUGAUGAG,X2732 UUCAGUUUC 3871 CGAA AAACUGAA GAAACCAA

X

CGAA ACCUUGGU CUGAGCCC

X

CGAA AACCUUGG UGAGCCCA

X

CGAA AUUUGGGC UACUCAAG

WO 99/50403 PC'T/US99/06507 Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUAUUUGG CUCAAGAA

X

CGAA AGUAUAUU AAGAACUA

1802 UUCAGAGU CUGAUGAG 2738 CAAGAACUA 387?
X

CGAA AGUUCUUG ACUCUGAA

X

CGAA AGUUAGUU UGAAGAGG

X

CGAA AGCCACAG CAGGAUAA

X

CGAA AUCCUGUA AUAUCAGA

X

CGAA AUUAUCCU UCAGAGAU

X

CGAA AUAUUAUC AGAGAUAA

X

X

CGAA ACGCAGUU CCAUUCCC

X

CGAA AUGGGACG CCCAUAAC

X

CGAA AAUGGGAC CCAUAACU

X

CGAA AUGGGAAU ACUGCCUC

X

CGAA AGGCAGUU AGUGGAGA

X

CGAA AUCUCCAC CAAGAGCC

X

CGAA AGCUUGGC UCGUAGGC

WO 99/50403 ' PCT/US99/06507 Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AGAGCUUG GUAGGCGA

X

CGAA ACGAGAGC GGCGAGUG

X

CGAA AUUCACUC CACUUCCA

X

CGAA AAUUCACU ACUUCCAG

X

CGAA AGUGAAUU CCAGAAGU

X

CGAA AAGUGAAU CAGAAGUU

X

CGAA ACUUCUGG CUUCCAAU

X

CGAA AACUUCUG UUCCAAUU

X

X

CGAA AAGAACUU CAAUUCUG

X

CGAA AUUGGAAG ~ CUGAAUUC

X

CGAA AAUUGGAA UGAAUUCA

X

CGAA AUUCAGAA CAGAUGAA

X

CGAA AAUUCAGA AGAUGAAC

X

CGAA AGCUGUCU AUAUUGAU

X

CGAA AUGAGCUG UUGAUGUU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUAUGAGC GAUGUUCA

X

CGAA ACAUCAAU CACUUCUU

X

CGAA AACAUCAA ACUUCUUA

X

CGAA AGUGAACA CUUAAAAG

X

CGAA AAGUGAAC UUAAAAGA

X

CGAA AGAAGUGA AAAAGAGG

X

CGAA AAGAAGUG AAAGAGGG

X

CGAA ACAUUGUC UGUAACAG

X

CGAA ACAUACAU ACAGCAAC

X

CGAA AGGUUGCU AAACUAGA

2055 UUCUAGUU CUGAUGAG 2?78 GCAACCUUA 3917 X

CGAA AAGGUUGC AACUAGAA

X

CGAA AGUUUAAG GAAUAUAA

X

CGAA AUUCUAGU UAAAUUUU

X

CGAA AUAUUCUA AAUUUUGC

X

CGAA AUUUAUAU UUGCACCC

X

CGAA AAUUUAUA UGCACCCG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAAUUUAU GCACCCGA

X

CGAA AUUUCCUU ~ AAGACAAA

X

CGAA AUUUGUCU UUCUUAUU

X

CGAA AAUUUGUC UCUUAUUU

X

CGAA AAAUUUGU CUUAUUUA

X

CGAA AAAAUUUG UUAUUUAC

X

CGAA AGAAAAUU AUUUACCA

X

CGAA AAGAAAAU UUUACCAA

X

X

CGAA AAUAAGAA ACCAAUUC

X

CGAA AAAUAAGA CCAAUUCA

CGAA AUUGGUAA CAAAAAGG

X

CGAA AAUUGGUA AAAAAGGU

X

CGAA ACACCUUU CCAGAACU

X

CGAA AGUUCUGG GUUCUAAA

X

CGAA ACUAGUUC CUAAAAGA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AACUAGUU UAAAAGAU

X

CGAA AGAACUAG AAAGAUCA

X

CGAA AUCUUUUA AGAAGGAU

X

CGAA AUCCUUCU UUGCUUUA

X

CGAA AUAUCCUU GCUUUAGA

X

CGAA AGCAAUAU UAGAAAUA

X

CGAA AAGCAAUA AGAAAUAA

X

CGAA AAAGCAAU GAAAUAAC

X

X

CGAA AGGGCUGU CCAACCCA

X

CGAA AAGGGCUG CAACCCAA

X

CGAA AUUCCUUG CCACAAAA

X

CGAA AGCCUCAU AACUGAUU

X

CGAA AUCAGUUU GCAACGUU

X

CGAA ACGUUGCA UCCAGACA

X

CGAA AACGUUGC CCAGACAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AAACGUUG CAGACACU

X

CGAA AGUGUCUG UAACCUAU

X

CGAA AAGUGUCU AACCUAUU

X

CGAA AAAGUGUC ACCUAUUC

X

CGAA AGGUUAAA UUCUGCAU

X

CGAA AUAGGUUA CUGCAUAU

X

CGAA AAUAGGUU UGCAUAUA

X

CGAA AUGCAGAA UAGAGAAC

X

CGAA AUAUGCAG GAGAACUG

X

CGAA AGCCCUCA UCCCUGAG

X

CGAA AAGCCCUC CCCUGAGA

X

CGAA AAAGCCCU CCUGAGAA

X

CGAA ACUGUUUC GAGUUGUG

X

CGAA ACUCAACU GUGUUGCC

X

CGAA ACACAACU GCCAACCA

X

CGAA AGCCAUUC GCAAGCUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AGCUCACA GGAAAUCC

X

CGAA AUUUCCGA CUUUUAAA

X

CGAA AGGAUUUC UUAAAAGA

2374 UUCUUUUA CUGAUGAG 2835 AAAUCCUUU 39?4 X

CGAA AAGGAUUU UAAAAGAA

X

CGAA AAAGGAUU AApAG~

X

CGAA AAAAGGAU AAAGAAAU

X

CGAA AUUUCUUU CAAAUGUC

X

CGAA AAUUUCUU AAAUGUCA

X

CGAA ACAUUUGA ACUUUUUA

X

CGAA AGUGACAU UUUAUUUG

X

CGAA AAGUGACA UUAUUUGG

X

CGAA AAAGUGAC UAUUUGGU

X

CGAA AAAAGUGA AUUUGGUU

X

CGAA AAAAAGUG UUUGGUUU

X

CGAA AUAAAAAG UGGUUUUA

X

CGAA AAUAAAAA GGUUUUAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACCAAAUA UUAAGUAC

X

CGAA AACCAAAU UAAGUACA

X

CGAA AAACCAAA AAGUACAA

X

CGAA AAAACCAA AGUACAAC

X

CGAA ACUUAAAA CAACUGAA

X

CGAA ACUUCAGU ACCUUUGA

X

CGAA AGGUGACU UGACACCC

X

CGAA AAGGUGAC GACACCCC

X

X

CGAA AUAUGGGG UGGAUAUU

X

, CGAA AUCCAGAU UUAAUCUG

X

CGAA AUAUCCAG AAUCUGAA

X

CGAA AAUAUCCA AUCUGAAG

X

CGAA AUUAAUAU UGAAGUUA

X

CGAA ACUUCAGA P .GAAACAA

X

CGAA AACUUCAG GAAACAAC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUUGCUUG AAGAUAAU

X

CGAA AUCUUGAU AUUUGGCU

X

CGAA AUUAUCUU UGGCUCCA

X

CGAA AAUUAUCU GGCUCCAA

X

CGAA AGCCAAAU CAAUUACA

X

CGAA AUUGGAGC ACAGCUAA

X

CGAA AAUUGGAG CAGCUAAA

X

CGAA AGCUGUAA AAGCAAAA

X

CGAA ACCACUUU AUUGAACU

X

CGAA AACCACUU UUGAACUG

X

CGAA AUAACCAC GAACUGCU

X

CGAA AGCAGUUC UUAUCGGU

X

CGAA AAGCAGUU UAUCGGUC

X

CGAA AAAGCAGU AUCGGUCU

X

CGAA AAAAGCAG UCGGUCUC

X

CGAA AUAAAAGC GGUCUCGG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACCGAUAA UCGGGAGU

X

CGAA AGACCGAU GGGAGUUG

X

CGAA ACUCCCGA GCUAAACC

X

CGAA AGCAACUC AACCUUCC

X

CGAA AGGUUUAG CCCAGGUG

X

CGAA AAGGUUUA CCAGGUGU

X

CGAA ACACCUGG UUUUGGAG

X

CGAA AUACACCU UUGGAGGU

X

CGAA AAUACACC UGGAGGUA

X

CGAA AAAUACAC GGAGGUAC

X

CGAA ACCUCCAA CAGUUGUU

X

CGAA ACUGUACC GUUGGCGA

X

CGAA ACAACUGU GGCGAGCA

X

CGAA AGCUUGCU UGAAAUCU

X

CGAA AUUUCAUA UGAAGAUG

X

CGAA ACUUCCCA UAAUAGAG

WO 99/50403 PCTNS99/0650'I

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AACUUCCC AAUAGAGU

X

CGAA AAACUUCC AUAGAGUA

X

CGAA AUUAAACU GAGUAUGA

X

CGAA ACUCUAUU UGAAUUCA

X

CGAA AUUCAUAC CAGGGUAA

X

CGAA AAUUCAUA AGGGUAAU

X

CGAA ACCCUGAA AUAAACUU

X

CGAA AUUACCCU AACUUAGG

X

CGAA AGUUUAUU AGGUAAAC

X

CGAA AAGUUUAU GGUAAACC

X

CGAA ACCUAAGU AACCUCUU

X

CGAA AGGUUUAC UUACAAAC

X

CGAA AGAGGUUU ACAAACCU

X

CGAA AAGAGGUU CAAACCUC

X

CGAA AGGUUUGU GGCACAGC

X

CGAA AGGUUGCU GAACAUUC

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUGUUCAA CAGUGGCC

X

CGAA AAUGUUCA AGUGGCCA

X

CGAA AUUUCUUU AGCAAUGG

X

CGAA AAUUUCUU GCAAUGGG

X

CGAA ACCAUUUC GCUUUAUU

X

CGAA AGCAACCA UAUUUGGU

X

CGAA AAGCAACC AUUUGGUG

X

CGAA AAAGCAAC UUUGGUGA

X

CGAA AUAAAGCA UGGUGAAA

X

CGAA AAUAAAGC GGUGAAAG

X

CGAA ACUUUCAC GAAUCCAA

X

CGAA AUUCUACU CAAAGGAU

X

CGAA AUCCUUUG GGAAAAGG

X

CGAA ACCUUUUC ACUUGUGA

X

CGAA AGUUACCU GUGAGCCA

X

CGAA AUCUCCUU AACUCCCU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGUUUAUC CCUGAACC

X

CGAA AGGUUCAG ACGGAGUC

X

CGAA ACUCCGUU UCACAACU

X

CGAA AGACUCCG ACAACUCA

X

CGAA AGUUGUGA AAGAAAGA

X

CGAA AUUUCCCG ACUGAAAA

X

CGAA AAUUUCCC CUGAAAAA

X

CGAA AUCUGUUU GAUGAUAA

X

X

CGAA AUUUUCUG UUCUUUAU

X

CGAA AAUUUUCU UCUUUAUU

X

CGAA AAAUUUUC CUUUAUUU

X

CGAA AAAAUUUU UUUAUUUG

X

CGAA AGAAAAUU UAUUUGCU

X

CGAA AAGAAAAU AUUUGCUG

X

GGAA AAAGAAAA UUUGCUGA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUAAAGAA UGCUGAAA

X

CGAA AAUAAAGA GCUGAAAG

X

CGAA AUUUUCUU CCAGACUC

X

CGAA AGUCUGGU UUAACUGU

X

CGAA AGAGUCUG AACUGUAG

X

CGAA AAGAGUCU ACUGUAGC

X

CGAA ACAGUUAA GCGUGAAC

X

CGAA AUGUUCAC AGAUGCCC

X

CGAA ACGCCUUG UCUUAUUU

X

CGAA AGACGCCU UUAUUUUG

X

CGAA AGAGACGC AUUUUGCG

X

CGAA AAGAGACG UUUUGCGC

X

CGAA AUAAGAGA UUGCGCUC

X

CGAA AAUAAGAG UGCGCUCG

X

CGAA AAAUAAGA GCGCUCGA

X

CGAA AGCGCAAA GAGGUUAU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACCUCGAG AUGGAACA

X

CGAA AACCUCGA UGGAACAG

X

CGAA AUGUGCUG UCUAGAGG

X

CGAA AAUGUGCU CUAGAGGA

X

CGAA AAAUGUGC UAGAGGAA

X

CGAA AGAAAUGU GAGGAAUA

X

CGAA AUUCCUCU UUCCAAAC

X

CGAA AUAUUCCU CCAAACUG

X

X

CGAA AGUUCAGU CUUGGACA

X

CGAA AGUAGUUC GGACAUUC

X

CGAA AUGUCCAA CUCAUGCG

X

CGAA AAUGUCCA UCAUGCGA

X

CGAA AGAAUGUC AUGCGAGC

X

CGAA AGGCUCGC CAUUGAUG

X

CGAA AAGGCUCG AUUGAUGU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUGAAGGC GAUGUGAC

X

CGAA AUUUUCGG UCAGGCUG

X

CGAA AUAUUUUC AGGCUGCC

X

CGAA AGUGCCUG AGGUUCGA

X

CGAA ACCUGAGU CGAGUGAC

X

CGAA AACCUGAG GAGUGACU

X

CGAA ACACAGUC UCCCUCAA

X

CGAA AACACAGU CCCUCAAA

X

X

CGAA AGGGAAAC AAAGACUG

X

CGAA ACAGUCUU GCUCAGUA

X

CGAA AGCUACAG AGUAUUCG

X

CGAA ACUGAGCU UUCGGGAG

X

CGAA AUACUGAG CGGGAGUA

X

CGAA AAUACUGA GGGAGUAC

X

CGAA ACUCCCGA CCUUGGUG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGGUACUC GGUGGAUC

X

CGAA AUCCACCA AUCCUAGU

X

CGAA AUGAUCCA CUAGUGGC

X

CGAA AGGAUGAU GUGGCUAU

X

CGAA AGCCACUA UUCUCGCU

X

CGAA AUAGCCAC CUCGCUGG

X

CGAA AAUAGGCA UCGCUGGG

X

CGAA AGAAUAGC GCUGGGAU

X

CGAA AUCCCAGC UUGAUGCU

X

CGAA AGAUCCCA GAUGCUUG

X

CGAA AGCAUCAA GCUUUAUU

X

CGAA AGCAAGCA UAUUAGUG

X

CGAA AAGCAAGC AUUAGUGU

X

CGAA AAAGCAAG UUAGUGUU

X

CGAA AUAAAGCA AGUGUUUA

X

CGAA AAUAAAGC GUGUUUAU

Seq. I.D. Seq. I.D.

Position RZ . No. Substrate No.

X

CGAA ACACUAAU UAUACUAU

X

CGAA AACACUAA AUACUAUG

X

CGAA AAACACUA UACUAUGG

X

CGAA AUAAACAC CUAUGGAA

X

CGAA AGUAUAAA UGGAAGUG

X

CGAA ACCACACU UCUUCAAG

X

CGAA AACCACAC CUUCAAGA

X

CGAA AAACCACA UUCAAGAG

X

CGAA AGAAACCA CAAGAGAA

X

CGAA AAGAAACC AAGAGAAA

X

CGAA AUUUCUCU AGAAAGAU

X

CGAA AUCUUUCU AUUAUGAU

X

CGAA AUGAUCUU AUGAUGCC

X

CGAA AAUGAUCU UGAUGCCA

X

CGAA AUGUGGCA UCACAAGG

X

CGAA AUAUGUGG ACAAGGCU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUCUCAGC CAUGCUCA

X

CGAA AGCAUGGA AGCCAUCU

X

CGAA AUGGCUGA UGAUAAAG

X

CGAA AUCAGAUG AAGAGAGG

X

CGAA AGCCUCUC ACUUCUGA

X

CGAA AAGCCUCU CUUCUGAU

X

CGAA AGUAAGCC CUGAUGCA

X

CGAA AAGUAAGC UGAUGCAU

X

CGAA AUGCAUCA GUAUUGAU

X

CGAA ACUAUGCA UUGAUCUA

X

CGAA AUACUAUG GAUCUACU

X

CGAA AUCAAUAC UACUUCUG

X

CGAA AGAUCAAU CUUCUGUA

X

CGAA AGUAGAUC CUGUAAUU

3382 CAAUUACA CUGAUGAG 3038 AUCUACUUC 4I7?
X

CGAA AAGUAGAU UGUAAUUG

X

CGAA ACAGAAGU AUUGUGUG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUUACAGA GUGUGGAU

X

CGAA AUCCACAC CUUUAAAC

X

CGAA AAUCCACA UUUAAACG

X

CGAA AGAAUCCA UAAACGCU

X

CGAA AAGAAUCC AAACGCUC

X

CGAA AAAGAAUC AACGCUCU

X

CGAA AGCGUUUA UAGGUACG

X

CGAA AGAGCGUU GGUACGAU

X

CGAA ACCUAGAG CGAUGACA

X

CGAA ACACUGUC CCCCGAUA

X

CGAA AACACUGU CCCGAUAC

X

CGAA AUCGGGGA CCAUGCUG

X

CGAA ACAGCAUG AGGAUCCG

X

CGAA AUCCUUAC CGGAAAGA

X

CGAA AUCUCUCG AAAGAUGA

X

CGAA ACUUUUCA UAUUGAUA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUACUUUU UUGAUAAC

X

CGAA AUAUACUU GAUAACCU

X

CGAA AUCAAUAU ACCUUGAA

X

CGAA AGGUUAUC GAAAAAAA

X

CGAA AUCCACUG ACAAAGUG

X

CGAA AGCUUUCA CUCAUAGC

X

CGAA AGUAGCUU AUAGCGGG

X

CGAA AUGAGUAG GCGGGGGC

X

2 0 CGAA AGGCCCCC F~AAAAAHA

X

CGAA AGCUUUUU CACAGUAC

X

CGAA AAGCUUUU ACAGUACC

X

CGAA ACUGUGAA CCCAAACU

X

CGAA AGCAGUUU UUUCCAAC

X

CGAA AAGCAGUU UUCCAACU

X

CGAA AAAGCAGU UCCAACUC

X

CGAA AAAAGCAG CCAACUCA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAAAAGCA CAACUCAG

X

CGAA AGUUGGAA AGAAAUUC

X

CGAA AUUUCUGA CAAUUUGG

X

CGAA AAUUUCUG AAUUUGGA

X

CGAA AUUGAAUU UGGAUUUA

X

CGAA AAUUGAAU GGAUUUAA

X

CGAA AUCCAAAU UAAAAGCC

X

CGAA AAUCCAAA AAAAGCCU

X

X

CGAA AGCAGGCU AAUCCCUG

X

CGAA AUUGAGCA CCUGAGGA

X

CGAA AUCAGUCC UCAGAGUG

X

CGAA AAUCAGUC CAGAGUGA

X

CGAA AAAUCAGU AGAGUGAC

X

CGAA AGUCACUC CACACAGU

X

CGAA ACUGUGUG CGAACCUA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGGUUCGU CAGUUUUA

X

CGAA ACUGUAGG UUAACUGU

X

CGAA AACUGUAG UAACUGUG

X

CGAA AAACUGUA AACUGUGG

X

CGAA AAAACUGU ACUGUGGA

X

CGAA AUCCACAG UUGUUACG

X

CGAA AUAUCCAC GUUACGUA

370? GGCUACGU CUGAUGAG 3095 GAUAUUGUU 4234 X

CGAA ACAAUAUC ACGUAGCC

X

CGAA AACAAUAU CGUAGCCU

X

CGAA ACGUAACA GCCUAAGG

X

CGAA AGGCUACG AGGCUCCU

X

CGAA AGCCUUAG CUGUUUUG

X

CGAA ACAGGAGC UUGCACAG

X

CGAA AACAGGAG UGCACAGC

X

CGAA AAACAGGA GCACAGCC

X

CGAA AUUUGGCU UAAAACUG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAUUUGGC AAAACUGU

X

CGAA AAAUUUGG AAACUGUU

X

CGAA ACAGUUUU GGAAUGGA

X

CGAA AUCCAUUC UUUCUUUA

X

CGAA AAUCCAUU UUCUUUAA

X

CGAA AAAUCCAU UCUUUAAC

X

CGAA AAAAUCCA CUUUAACU

X

CGAA AAAAAUCC UUUAACUG

X

2 p CGAA AGAAAAAU UAACUGCC

X

CGAA AAGAAAAA AACUGCCG

X

CGAA AAAGAAAA ACUGCCGU

X

CGAA ACGGCAGU AUUUAACU

X

CGAA AUUACGGC UAACUUUC

X

CGAA AAUUACGG AACUUUCU

X

CGAA AAAUUACG ACUUUCUG

X

CGAA AGUUAAAU UCUGGGUU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAGUUAAA CUGGGUUG

X

CGAA AAAGUUAA UGGGUUGC

X

CGAA ACCCAGAA GCCUUUGU

X

CGAA AGGCAACC UGUUUUUG

X

CGAA AAGGCAAC GUUUUUGG

X

CGAA ACAAAGGC UUUGGCGU

X

CGAA AACAAAGG UUGGCGUG

3808 CCACGCCA CUGAUGAG 312? CUUUGUUUU 4266 X

CGAA AAACAAAG UGGCGUGG

X

CGAA AAAACAAA GGCGUGGC

X

CGAA AGUCAGCC ACAUCAUG

X

CGAA AAGUCAGC CAUCAUGU

X

CGAA AUGUAAGU AUGUGUUG

X

CGAA ACACAUGA GGGGAAGG

X

CGAA ACUGGGCA GCACUCAG

X

CGAA AGUGCAAC AGGUGACA

X

CGAA AUGUCACC CUCCAGAU

WO 99/50403 PCT/US99/0650?

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGGAUGUC CAGAUAGU

X

CGAA AUCUGGAG GUGUAGCU

X

CGAA ACACUAUC GCUGAGGA

X

CGAA AGGUGCCU CACUCACC

X

CGAA AGUGUAGG ACCUGCAC

X

CGAA AGUGCAGG ACAGAGUG

X

CGAA ACGGCCAC CUAACCUC

X

CGAA AGGACGGC ACCUCGGG

X

CGAA AGGUUAGG GGGCCUGC

X

CGAA ACGUCUGC CAUCACGU

X

CGAA AUGGACGU ACGUUAGC

X

CGAA ACGUGAUG AGCUGUCC

X

CGAA AACGUGAU GCUGUCCC

X

CGAA ACAGCUAA CCACAUCA

X

CGAA AUGUGGGA ACAAGACU

X

CGAA AGUCUUGU UGCCAUUG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUGGCAUA GGGGUAGU

X

CGAA ACCCCAAU GUUGUGUU

X

CGAA ACUACCCC GUGUUUCA

X

CGAA ACACAACU UCAACGGA

X

CGAA AACACAAC CAACGGAA

X

CGAA AAACACAA AACGGAAA

X

CGAA ACAGCACU UUAAACUA

X

CGAA AGACAGCA AAACUAAA

X

CGAA AAGACAGC AACUAAAU

X

CGAA AGUUUAAG AAUGUGCA

X

CGAA AUUGCACA GAAGGUGA

X

CGAA ACAUCACC GCCAUCCU

X

CGAA AUGGCAAC CUACCGUC

X

CGAA AGGAUGGC CCGUCUUU

X

CGAA ACGGUAGG UUUUCCUG

X

CGAA AGACGGUA UUCCUGUU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAGACGGU UCCUGUUU

X

CGAA AAAGACGG CCUGUUUC

X

CGAA AAAAGACG CUGUUUCC

X

CGAA ACAGGAAA UCCUAGCU

X

CGAA AACAGGAA CCUAGCUG

X

CGAA AAACAGGA CUAGCUGU

X

CGAA AGGAAACA GCUGUGUG

X

CGAA AUUCACAC CCUGCUCA

X

X

CGAA ACGUGAGC AAAUGCAU

X

CGAA AUGCAUUU CAAGUUUC

q128 GAGAAUGA CUGAUGAG 3179 AUACAAGUU 4318 X

CGAA ACUUGUAU UCAUUCUC

X

CGAA AACUUGUA CAUUCUCC

X

CGAA AAACUUGU AUUCUCCC

X

CGAA AUGAAACU CUCCCUUU

X

CGAA AAUGAAAC UCCCUUUC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AGAAUGAA CCUUUCAC

X

CGAA AGGGAGAA UCACUAAA

X

CGAA AAGGGAGA CACUAAAA

X

CGAA AAAGGGAG ACUAAAAA

X

CGAA AGUGAAAG AAAACACA

X

CGAA AGUCUGUU GAAUGCUA

X

CGAA AGCAUUCA GUUAUACU

X

CGAA ACUAGCAU AUACUUAU

X

CGAA AACUAGCA UACUUAUU

X

CGAA AUAACUAG CUUAUUUG

X

CGAA AGUAUAAC AUUUGUAU

X

CGAA AAGUAUAA UUUGUAUA

X

CGAA AUAAGUAU UGUAUAUG

X

CGAA AAUAAGUA GUAUAUGG

X

CGAA ACAAAUAA UAUGGUAU

X

CGAA AUACAAAU UGGUAUUU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACCAUAUA UUUAUUUU

X

CGAA AUACCAUA UAUUUUUU

X

CGAA AAUACCAU AUUUUUUC

X

CGAA AAAUACCA UUUUUUCU

X

CGAA AUAAAUAC UUUUCUUU

X

CGAA AAUAAAUA UUUCUUUU

X

CGAA AAAUAAAU UUCUUUUC

X

CGAA AAAAUAAA UCUUUUCU

X

X

CGAA AAAAAAUA UUUUCUUU

X

CGAA AGAAAAAA UUCUUUAC

X

CGAA AAGAAAAA UCUUUACA

X

CGAA AAAGAAAA CUUUACAA

X

CGAA AAAAGAAA UUUACAAA

X

CGAA AGAAAAGA UACAAACC

X

CGAA AAGAAAAG ACAAACCA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAAGAAAA CAAACCAU

X

CGAA AUGGUUUG UUGUUAUU

X

CGAA AAUGGUUU UGUUAUUG

X

CGAA AAAUGGUU GUUAUUGA

X

CGAA ACAAAAUG AUUGACUA

X

CGAA AACAAAAU UUGACUAA

X

CGAA AUAACAAA GACUAACA

X

CGAA AGUCAAUA ACAGGCCA

X

CGAA ACUCUUUG UCCAGUUU

X

CGAA AGACUCUU CAGUUUAC

X

CGAA ACUGGAGA UACCCUUC

X

CGAA AACUGGAG ACCCUUCA

X

CGAA AAACUGGA CCCUUCAG

X

CGAA AGGGUAAA CAGGUUGG

X

CGAA AAGGGUAA AGGUUGGU

X

CGAA ACCUGAAG GGUUUAAU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACCAACCU UAAUCAAU

X

CGAA AACCAACC AAUCAAUC

X

CGAA AAACCAAC AUCAAUCA

X

CGAA AUUAAACC AAUCAGAA

X

CGAA AUUGAUUA AGAAUUAG

X

CGAA AUUCUGAU AGAAUUAG

X

CGAA AAUUCUGA GAAUUAGA

X

CGAA AUUCUAAU AGAGCAUG

X

X

CGAA ACCCUCCC AUCACUAU

X

CGAA AUGACCCU ACUAUGAC

X

CGAA AGUGAUGA UGACCUAA

X

CGAA AGGUCAUA AAUUAUUU

X

CGAA AUUUAGGU AUUUACUG

X

CGAA AAUUUAGG UUUACUGC

X

CGAA AUAAUUUA UACUGCAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAUAAUUU ACUGCAAA

X

CGAA AAAUAAUU CUGCAAAA

X

CGAA AUUUUCUU UUUAUAAA

X

CGAA AGAUUUUC UAUAAAUG

X

CGAA AAGAUUUU AUAAAUGU

X

CGAA AAAGAUUU UAAAUGUA

X

CGAA AUAAAGAU AAUGUACC

X

CGAA ACAUUUAU CCAGAGAG

X

X

CGAA ACAACUCU UUAAUAAC

X

CGAA AACAACUC UAAUAACU

X

CGAA AAACAACU AAUAACUU

X

CGAA AAAACAAC AUAACUUA

X

CGAA AUUAAAAC ACUUAUCU

X

CGAA AGUUAUUA AUCUAUAA

X

CGAA AAGUUAUU UCUAUAAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUAAGUUA UAUAAACU

X

CGAA AGAUAAGU UAAACUAU

X

CGAA AUAGAUAA AACUAUAA

X

CGAA AGUUUAUA UAACCUCU

X

CGAA AUAGUUUA ACCUCUCC

X

CGAA AGGUUAUA UCCUUCAU

X

CGAA AGAGGUUA CUUCAUGA

X

CGAA AGGAGAGG CAUGACAG

X

CGAA AAGGAGAG AUGACAGC

X

CGAA AGGCUGUC CACCCCAC

X

CGAA ACCUUUUG UAAGAAAU

X

CGAA AACCUUUU AAGAAAUA

X

CGAA AAACCUUU AGAAAUAG

X

CGAA AUUUCUUA GAAUUAUA

X

CGAA AUUCUAUU AUAACUGU

X

CGAA AAUUCUAU UAACUGUA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUAAUUCU ACUGUAAA

X

CGAA ACAGUUAU AAGAUGUU

X

CGAA ACAUCUUU UAUUUCAG

X

CGAA AACAUCUU AUUUCAGG

X

CGAA AAACAUCU UUUCAGGC

X

CGAA AUAAACAU UCAGGCAU

X

CGAA AAUAAACA CAGGCAUU

X

CGAA AAAUAAAC AGGCAUUG

X

CGAA AUGCCUGA GGAUAUUU

X

CGAA AUCCAAUG UUUUUUAC

X

CGAA AUAUCCAA UUUUACUU

X

CGAA AAUAUCCA UUUACUUU

X

CGAA AAAUAUCC UUACUUUA

X

CGAA AAAAUAUC UACUUUAG

X

' CGAA AAAAAUAU ACUUUAGA

X

CGAA AAAAAAUA CUUUAGAA

WO 99!50403 PCT/US99/06507 Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGUAAAAA UAGAAGCC

X

CGAA AAGUAAAA AGAAGCCU

X

CGAA AAAGUAAA GAAGCCUG

X

CGAA AUGCAGGC AUGUUUCU

X

CGAA ACAUUAUG UCUGGAUU

X

CGAA AACAUUAU CUGGAUUU

X

CGAA AAACAUUA UGGAUUUA

X

CGAA AUCCAGAA UACAUACU

X

CGAA AAUCCAGA ACAUACUG

X

CGAA AAAUCCAG CAUACUGU

X

CGAA AUGUAAAU CUGUAACA

45qg CUGAAUGU CUGAUGAG 3307 CAUACUGUA 9446 X

CGAA ACAGUAUG ACAUUCAG

X

CGAA AUGUUACA CAGGAAUU

X

CGAA AAUGUUAC AGGAAUUC

X

CGAA AUUCCUGA CUUGGAGA

X

CGAA AAUUCCUG UUGGAGAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGAAUUCC GGAGAAGA

X

CGAA ACCCAUCU UAUUCACU

X

CGAA AACCCAUC AUUCACUG

X

CGAA AAACCCAU UUCACUGA

X

CGAA AUAAACCC CACUGAAG

X

CGAA AAUAAACC ACUGAACU

X

CGAA AGUUCAGU UAGUGCGG

X

CGAA AGAGUUCA GUGCGGUU

X

2 a CGAA ACCGCACU UACUCACU

X

CGAA AACCGCAC ACUCACUG

X

CGAA AAACCGCA GUCACUGC

X

CGAA AGUAAACC ACUGCUGC

X

CGAA AUUUGCAG CUGUAUAU

X

CGAA ACAGUAUU UAUUCAGG

X

CGAA AUACAGUA UUCAGGAC

X

CGAA AUAUACAG CAGGACUU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAUAUACA AGGACUUG

X

CGAA AGUCCUGA GAAAGAAA

X

CGAA AGGCAUUC UGGAACUA

X

CGAA AGUUCCAU GUGGAUCC

X

CGAA AUCCACUA CAAACUGA

X

CGAA AUCAGUUU CAGUAUAA

X

CGAA ACUGGAUC UAAGACUA

X

CGAA AUACUGGA AGACUACU

X

CGAA AGUCUUAU CUGAAUCU

X

CGAA AUUCAGUA UGCUACCA

X

CGAA AGCAGAUU CCAAAACA

X

CGAA ACUGUUUU AAUCAGUG

X

CGAA AACUGUUU AUCAGUGA

X

CGAA AUUAACUG AGUGAGUC

X

CGAA ACUCACUG GAGUGUUC

X

CGAA ACACUCGA CUAUUUUU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AACACUCG UAUUUUUU

X

CGAA AGAACACU UUUUUUGU

4799 AAACAAAA CUGAUGAG 3346 UGUUCUAUU 49$5 X

CGAA AUAGAACA UUUUGUUU

X

CGAA AAUAGAAC UUUGUUUU

X

CGAA AAAUAGAA UUGUUUUG

4797 ACAAAACA CUGAUGAG 3399 UCUAUUUUU 498$
X

CGAA AAAAUAGA UGUUUUGU

X

CGAA AAAAAUAG GUUUUGUU

X

CGAA ACAAAAAA UUGUUUCC

X

X

CGAA AAACAAAA GUUUCCUC

X

CGAA ACAAAACA UCCUCCCC

X

CGAA AACAAAAC CCUCCCCU

X

CGAA AAACAAAA CUCCCCUA

X

CGAA AGGAAACA CCCUAUCU

X

CGAA AGGGGAGG UCUGUAUU

X

CGAA AUAGGGGA UGUAUUCC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACAGAUAG UUCCCAAA

X

CGAA AUACAGAU CCCAAAAA

X

CGAA AAUACAGA CCAAAAAU

X

CGAA AUUUUUGG ACUUUGGG

X

CGAA AAUUUUUG CUUUGGGG

X

CGAA AGUAAUUU UGGGGCUA

X

CGAA AAGUAAUU GGGGCUAA

X

CGAA AGCCCCAA AUUUAACA

X

2~ CGAA AUUAGCCC UAACAAGA

X

CGAA AAUUAGCC AACAAGAA

X

CGAA AAAUUAGC ACAAGAAC

X

CGAA AGUUCUUG UAAAUUGU

X

CGAA AAGUUCUU AAAUUGUG

X

CGAA AAAGUUCU AAUUGUGU

X

CGAA AUUUAAAG GUGUUUUA

X

CGAA ACACAAUU UUAAUUGU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AACACAAU UAAUUGUA

X

CGAA AAACACAA AAUUGUAA

4825 UUUACAAU CUGAUGAG 3378 UGUGUUUUA 451?
X

CGAA AAAACACA AUUGUAAA

X

CGAA AUUAAAAC GUAAAAAU

X

CGAA ACAAUUAA AAAAUGGC

X

CGAA AUUCCACC AUUACUCU

X

CGAA AAUUCCAC UUACUCUA

X

CGAA AUAAUUCC ACUCUAUA

X

CGAA AAUAAUUC CUCUAUAC

X

CGAA AGUAAUAA UAUACAUU

X

CGAA AGAGUAAU UACAUUCA

X

CGAA AUAGAGUA CAUUCAAC

X

CGAA AUGUAUAG CAACAGAG

X

CGAA AAUGUAUA AACAGAGA

X

CGAA AUUCAGUC GAUAUGAA

X

CGAA AUCUAUUC UGAAAGCU

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUCAGCUU UUUUUUAA

X

CGAA AAUCAGCU UUUUUAAU

X

CGAA AAAUCAGC UUUUAAUU

X

CGAA AAAAUCAG UUUAAUUA

X

CGAA AAAAAUCA UUAAUUAC

X

CGAA AAAAAAUC UAAUUACC

X

CGAA AAAAAAAU AAUUACCA

X

CGAA APIA AUUACCAU

X

X

CGAA AAUUAAAA CCAUGCUU

X

CGAA AGCAUGGU CACAAUGU

X

CGAA AAGCAUGG ACAAUGUU

X

CGAA ACAUUGUG AAGUUAUA

X

CGAA AACAUUGU AGUUAUAU

X

CGAA ACUUAACA AUAUGGGG

X

CGAA AACUUAAC UAUGGGGA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AUAACUUA UGGGGAGC

X

CGAA AGCACCUG AUUUGUUU

X

CGAA AUUAGCAC UGUUUUGG

X

CGAA AAUUAGCA GUUUUGGA

X

CGAA ACAAAUUA UUGGAUAU

X

CGAA AACAAAUU UGGAUAUA

X

CGAA AAACAAAU GGAUAUAG

X

CGAA AUCCAAAA UAGUAUAA

X

CGAA AUAUCCAA GUAUAAGC

X

CGAA ACUAUAUC UAAGCAGU

X

CGAA AUACUAUA AGCAGUGU

q.ggl AAAACACA CUGAUGAG 3419 AGCAGUGUC 4558 X

CGAA ACACUGCU UGUGUUUU

X

CGAA ACACAGAC UUGAAAGA

X

CGAA AACACAGA UGAAAGAA

X

CGAA AAACACAG GAAAGAAU

X

CGAA AUUCUUUC GAACACAG

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACUGUGUU UGUAGUGC

X

CGAA AACUGUGU GUAGUGCC

X

CGAA ACAAACUG GUGCCACU

X

CGAA ACAGUGGC GUUUUGGG

X

CGAA ACAACAGU UUGGGGGG

X

CGAA AACAACAG UGGGGGGG

X

CGAA AAACAACA GGGGGGGG

X

CGAA AGCCCCCC UUUUUCUU

X

CGAA AAGCCCCC UUUUCUUU

X

CGAA AAAGCCCC UUUCUUUU

X

CGAA AAAAGCCC UUCUUUUU

X

CGAA AAAAAGCC UCUUUUUC

X

CGAA AAAAAAGC CUUUUUGC

X

CGAA AAAAAAAG UUUUUCCG

505? UCCGGAAA CUGAUGAG 3438 UUUUUUCUU 4577 X

CGAA AGAAAAAA UUUCCGGA

X

CGAA AAGAAAAA UUCCGGAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AAAGAAAA UCCGGAAA

X

CGAA AAAAGAAA CCGGAAAA

X

GGAA AAAAAGAA CGGAAAAU

X

CGAA AUUUUCCG CUUAAACC

X

CGAA AGGAUUUU AAACCUUA

X

CGAA AAGGAUUU AACCUUAA

X

CGAA AGGUUUAA AAGAUACU

X

CGAA AAGGUUUA AGAUACUA

X

X

CGAA AGUAUCUU AGGACGUU

X

CGAA ACGUCCUU GUUUUGGU

X

CGAA ACAACGUC UUGGUUGU

X

CGAA AACAACGU UGGUUGUA

X

CGAA AAACAACG GGUUGUAC

X

CGAA ACCAAAAC GUACUUGG

X

CGAA ACAACCAA CUUGGAAU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AGUACAAC GGAAUUCU

X

CGAA AUUCCAAG CUUAGUCA

X

CGAA AAUUCCAA UUAGUCAC

X

CGAA AGAAUUCC AGUCACAA

X

CGAA AAGAAUUC GUCACAAA

X

CGAA ACUAAGAA ACAAAAUA

X

CGAA AUUUUGUG UAUUUUGU

X

CGAA AUAUUUUG UUUUGUUU

X

2 d CGAA AUAUAUUU UUGUUUAC

X

CGAA AAUAUAUU UGUUUACA

X

CGAA AAAUAUAU GUUUACAA

X

CGAA ACAAAAUA UACAAAAA

X

CGAA AACAAAAU ACAAAAAU

X

CGAA AAACAAAA CAAAAAUU

X

CGAA AUUUUUGU UCUGUAAA

X

CGAA AAUUUUUG CUGUAAAA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA AAAUUUUU UGUAAAAC

X

CGAA ACAGAAAU AAACAGGU

X

CGAA ACCUGUUU AUAACAGU

X

CGAA AACCUGUU UAACAGUG

X

CGAA AUAACCUG ACAGUGUU

5178 AGACUUUA CUGAUGAG 347? AACAGUGUU 4616 X

CGAA ACACUGUU UAAAGUCU

X

CGAA AACACUGU AAAGUCUC

X

CGAA AAACACUG AAGUCUCA

X

CGAA ACUUUAAA UCAGUUUC

X

CGAA AGACUUUA AGUUUCUU

X

CGAA ACUGAGAC UCUUGCUU

X

CGAA AACUGAGA CUUGCUUG

X

CGAA AAACUGAG UUGCUUGG

X

CGAA AGAAACUG GCUUGGGG

X

CGAA AGCAAGAA GGGGAACU

X

CGAA AGUUCCCC GUGUCCCU

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACACAAGU CCUAAUGU

X

CGAA AGGGACAC AUGUGUUA

X

CGAA ACACAUUA AGAUUGCU

X

CGAA AACACAUU GAUUGCUA

X

CGAA AUCUAACA GCUAGAUU

X

CGAA AGCAAUCU GAUUGCUA

X

CGAA AUCUAGCA GCUAAGGA

X

CGAA AGCAAUCU AGGAGGUG

X

X

CGAA AGUAUCAG GACAGUUU

X

CGAA ACUGUCAA UUUUAGAC

X

CGAA AACUGUCA UUUAGACC

X

CGAA AAACUGUC UUAGACCU

X

CGAA AAAACUGU UAGACCUG

X

CGAA AAAAACUG AGACCUGU

X

CGAA AAAAAACU GACCUGUG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACACAGGU ACUAAAAA

X

CGAA AACACAGG CUAAAAAA

X

CGAA AGUAACAC AAAAAAAG

X

CGAA ACAUUCAU GGAAAAGG

X

CGAA ACACCCUU GGGAGGGU

X

CGAA ACCACCCU AACAAAGA

X

CGAA ACAUCUUU AUGGUGUU

X

CGAA AACAUCUU UGGUGUUU

X

CGAA ACACCAUA UAGACUUA

X

CGAA AACACCAU AGACUUAU

X

CGAA AAACACCA GACUUAUG

X

CGAA AGUCUAAA AUGGUUGU

X

CGAA AAGUCUAA UGGUUGUU

X

CGAA ACCAUAAG GUUAAAAA

536? ACAUUUUU CUGAUGAG 3518 AUGGUUGUU 4657 X

CGAA ACAACCAU AAAAAUGU

X

CGAA AACAACCA AAAAUGUC

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACAUUUUU AUCUCAAG

X

CGAA AUGACAUU UCAAGUCA

X

CGAA AGAUGACA AAGUCAAG

X

CGAA ACUUGAGA AAGUCACU

X

CGAA ACUUGACU ACUGGUCU

X

CGAA ACCAGUGA UGUUUGCA

X

CGAA ACAGACCA UGCAUUUG

X

CGAA AACAGACC GCAUUUGA

X

CGAA AUGCAAAC UGAUACAU

X

CGAA AAUGCAAA GAUACAUU

X

CGAA AUCAAAUG CAUUUUUG

X

CGAA AUGUAUCA UUUGUACU

X

CGAA AAUGUAUC UUGUACUA

X

CGAA AAAUGUAU UGUACUAA

X

CGAA AAAAUGUA GUACUAAC

X

CGAA ACAAAAAU CUAACUAG

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AGUACAAA ACUAGCAU

X

CGAA AGUUAGUA GCAUUGUA

X

CGAA AUGCUAGU GUAAAAUU

X

CGAA ACAAUGCU AAAUUAUU

X

CGAA AUUUUACA AUUUCAUG

X

CGAA AAUUUUAC UUUCAUGA

X

CGAA AUAAUUUU UCAUGAUU

X

CGAA AAUAAUUU CAUGAUUA

X

X

CGAA AUCAUGAA AGAAAUUA

X

CGAA AAUCAUGA GAAAUUAC

X

CGAA AUUUCUAA ACCUGUGG

X

CGAA AAUUUCUA CCUGUGGA

X

CGAA AUCCACAG UUUGUAUA

X

CGAA AUAUCCAC UGUAUAAA

X

CGAA AAUAUCCA GUAUAAAA

WO 99/50403 PC'f/US99/0650'I

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA ACAAAUAU UAAAAGUG

X

CGAA AUACAAAU AAAGUGUG

5993 AAAP~AAUU CUGAUGAG3554 UGUGAAAUA 4693 X

CGAA AUUUCACA AAUUUUUU

X

CGAA AUUUAUUU UUUUAUAA

X

CGAA AAUUUAUU UUUAUAAA

X

CGAA AAAUUUAU UUAUAAAA

X

CGAA AAAAUUUA UAUAAAAG

X

CGAA AAAAAUUU AUAAAAGU

X

CGAA AAAAAAUU UAAAAGUG

X

CGAA AUAAAAAA AAAGUGUU

5512 AAACAAUG CUGAUGAG 3562 AAAAGUGUU 9?O1 X

CGAA ACACUUUU CAUUGUUU

X

CGAA AACACUUU AUUGUUUC

X

CGAA AUGAACAC GUUUCGUA

X

CGAA ACAAUGAA UCGUAACA

X

CGAA AACAAUGA CGUAACAC

X

CGAA AAACAAUG GUAACACA

Seq. I.D. Seq. I.D.

Position RZ No. Substrate No.

X

CGAA ACGAAACA ACACAGCA

X

CGAA AUGCUGUG GUAUAUGU

X

CGAA ACAAUGCU UAUGUGAA

X

CGAA AUACAAUG UGUGAAGC

X

CGAA AGUUUGGU UAAAAUUA

X

CGAA AGAGUUUG AAAUUAUA

X

CGAA AUUUUAGA AUAAAUGA

X

CGAA AAUUUUAG UAAAUGAC

X

CGAA AUAAUUUU AAUGACAA

X

CGAA AUUCAGGU AUCUAUUU

X

CGAA AAUUCAGG UCUAUUUC

X

CGAA AUAAUUCA UAUUUCAU

X

CGAA AGAUAAUU UUUCAUCA

X

CGAA AUAGAUAA UCAUCAAA

X

CGAA AAUAGAUA CAUCAAAA

X

CGAA AAAUAGAU AUCAAAAA

Seq. Seq. I.D.
I.D.

Position RZ No. Substrate No.

X

CGAA AUGAAAUA AAAAAAAA

X

CGAA AGUUUUUU UAUGGGCA

X

CGAA AAGUUUUU AUGGGCAC

X

CGAA AAAGUUUU UGGGCACA

WO 99/50403 PCT/US99/Ob507 TABLE VI I I: HAIRPIN RIBOZYME AND TARGET SEQUENCES FOR

Seq. Seq. I.D.
I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X CCGGGG

GUACAUUACCUGGUA

ACCAGAGAAACA X GCAGCA

GUACAUUACCUGGUA

ACCAGAGAAACA X UCGGAC

GUACAUUACCUGGUA

ACCAGAGAAACA X CCAGCC

GUACAUUACCUGGUA

gg GCUCCG AGAA GGGU 4731 ACCCA GCC 4825 ACCAGAGAAACA X CGGAGC

GUACAUUACCUGGUA

ACCAGAGAAACA X GCUGCA

GUACAUUACCUGGUA

ACCAGAGAAACA X GCAGGU

GUACAUUACCUGGUA

ACCAGAGAAACA X CCCCUC

GUACAUUACCUGGUA

ACCAGAGAAACA X CAUGGC

GUACAUUACCUGGUA

ACCAGAGAAACA X GCCGGG

GUACAUUACCUGGUA

Seq. Seq.
I.D. I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X GGGCAG

GUACAUUACCUGGUA

ACCAGAGAAACA X GUGCUU

GUACAUUACCUGGUA

ACCAGAGAAACA X GGCGGG

GUACAUUACCUGGUA

ACCAGAGAAACA X CCGGCU

GUACAUUACCUGGUA

ACCAGAGAAACA X CGGCGC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUCAAC

GUACAUUACCUGGUA

ACCAGAGAAACA X UCUCGC

GUACAUUACCUGGUA

ACCAGAGAAACA X CGAGGA

GUACAUUACCUGGUA

ACCAGAGAAACA X GUUGCU

GUACAUUACCUGGUA

3a 328 ACGAGC AGAA GCCG 4796 CGGCU GUU 4840 ACCAGAGAAACA X GCUCGU

GUACAUUACCUGGUA

Seq. I.D. Seq.
I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X CAGCUG

GUACAUUACCUGGUA

ACCAGAGAAACA X GCGACA

GUACAUUACCUGGUA

GUACAUUACCUGGUA

ACCAGAGAAACA X CGAGUU

GUACAUUACCUGGUA

ACCAGAGAAACA X CCCACG

GUACAUUACCUGGUA

ACCAGAGAAACA X CAGAGC

ACCAGAGAAACA X CCUGCU

GUACAUUACCUGGUA

ACCAGAGAAACA X UUUUGA

GUACAUUACCUGGUA

ACCAGAGAAACA X GGAAUA

GUACAUUACCUGGUA

ACCAGAGAAACA X GGAGCU

GUACAUUACCUGGUA

Seq. I.D. Seq.
I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X UGAUGA

GUACAUUACCUGGUA

ACCAGAGAAACA X GUUGCU

GUACAUUACCUGGUA

ACCAGAGAAACA X GGUUCC

GUACAUUACCUGGUA

ACCAGAGAAACA X UCAGUA

GUACAUUACCUGGUA

ACCAGAGAAACA X CCCGGC

GUACAUUACCUGGUA

ACCAGAGAAACA X UGUGAU

GUACAUUACCUGGUA

ACCAGAGAAACA X UUGAAU

GUACAUUACCUGGUA

ACCAGAGAAACA X GGUUAU

GUACAUUACCUGGUA

ACCAGAGAAACA X UCGAAA

GUACAUUACCUGGUA

ACCAGAGAAACA X UCAGUG

GUACAUUACCUGGUA

Seq. Seq. I.D.
I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X GAACCC

GUACAUUACCUGGUA

ACCAGAGAAACA X CAUAUU

GUACAUUACCUGGUA

ACCAGAGAAACA X CUUCCA

GUACAUUACCUGGUA

ACCAGAGAAACA X UGCAAC

GUACAUUACCUGGUA

ACCAGAGAAACA X GAGUUG

GUACAUUACCUGGUA

ACCAGAGAAACA X UGUGAG

ACCAGAGAAACA X UUUAUC

GUACAUUACCUGGUA

ACCAGAGAAACA X UCGGGA

GUACAUUACCUGGUA

ACCAGAGAAACA X GUUGGC

GUACAUUACCUGGUA

ACCAGAGAAACA X UCUUAA

GUACAUUACCUGGUA

Seq. Seq.
I.D. I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X GCGUGA

GUACAUUACCUGGUA

ACCAGAGAAACA X GCCCGC

GUACAUUACCUGGUA

ACCAGAGAAACA X GCGGGG

GUACAUUACCUGGUA

ACCAGAGAAACA X GCUGCC

GUACAUUACCUGGUA

ACCAGAGAAACA X GCCGAA

GUACAUUACCUGGUA

ACCAGAGAAACA X GAAAAU

ACCAGAGAAACA X GCUCAG

GUACAUUACCUGGUA

ACCAGAGAAACA X UUCGGG

GUACAUUACCUGGUA

ACCAGAGAAACA X GCAUAG

GUACAUUACCUGGUA

ACCAGAGAAACA X CCCAAA

GUACAUUACCUGGUA

Seq. I.D. Seq.
I.D.

Position RZ No. Substrate No ACCAGAGAAACA X UUUUCC

GUACAUUACCUGGUA

ACCAGAGAAACA X CAAUCC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUCAGA

GUACAUUACCUGGUA

ACCAGAGAAACA X CGAACC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUAACU

GUACAUUACCUGGUA

ACCAGAGAAACA X UUGCAC

GUACAUUACCUGGUA

ACCAGAGAAACA X GGAAUG

GUACAUUACCUGGUA

GUACAUUACCUGGUA

ACCAGAGAAACA X UUACAU

GUACAUUACCUGGUA

3gg7 CAACUG AGAA GGCC 9796 GGCCU GCC 4890 ACCAGAGAAACA X CAGUUG

GUACAUUACCUGGUA

Seq. Seq. I.D.
I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X GCACUC

GUACAUUACCUGGUA

ACCAGAGAAACA X CUAACC

GUACAUUACCUGGUA

ACCAGAGAAACA X GCGCAG

GUACAUUACCUGGUA

ACCAGAGAAACA X GUCCAU

GUACAUUACCUGGUA

ACCAGAGAAACA X CCACAU

GUACAUUACCUGGUA

ACCAGAGAAACA X UUAAAC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUUUCC

GUACAUUACCUGGUA

ACCAGAGAAACA X UCCUAG

GUACAUUACCUGGUA

ACCAGAGAAACA X CACGUC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUGAAU

GUACAUUACCUGGUA

Seq. I.D. Seq. I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X UACCCU

GUACAUUACCUGGUA

ACCAGAGAAACA X UCCACC

GUACAUUACCUGGUA

ACCAGAGAAACA X UACUCA

GUACAUUACCUGGUA

ACCAGAGAAACA X GCAAAU

GUACAUUACCUGGUA

ACCAGAGAAACA X UAUUCA

GUACAUUACCUGGUA

ACCAGAGAAACA X CCAGUA

ACCAGAGAAACA X UAAGAC

GUACAUUACCUGGUA

GUACAUUACCUGGUA

ACCAGAGAAACA X UUUUUU

GUACAUUACCUGGUA

ACCAGAGAAACA X UGUAGU

GUACAUUACCUGGUA

Seq. Seq.
I.D. I.D.

PositionRZ No. Substrate No ACCAGAGAAACA X GUUUUG

GUACAUUACCUGGUA

ACCAGAGAAACA X UCUUGC

GUACAUUACCUGGUA

ACCAGAGAAACA X UUUUAG

GUACAUUACCUGGUA

ACCAGAGAAACA X UGCAUU

GUACAUUACCUGGUA

TABLE IX: HAMMERHEAD RIBOZYME AND TARGET SEQUENCES FOR

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

ACCACAUC UUGCCCUC

CGAA

AGACCACA GCCCUCAA

CGAA

AGGGCAAG AACAGGUA

CGAA

ACCUGUUG GGUAGUCU

CGAA

ACCUACCU GUCUACCG

CGAA

ACUACCUA UACCGGAA

q2 UUUUCCGG CUGAUGAG X 9921 GGUAGUCUA 5708 CGAA

AGACUACC CCGGAAAA

CGAA

AGUUUGGU AGGCAAGA

CGAA

AUUUUUUU AGUGAAUA

CGAA

AAUUUUUU GUGAAUAA

CGAA

AUUCACUA AUAAAGGA

CGAA

AUUAUUCA AAGGACUG

CGAA

ACCGGUUC CAGAGAAG

CGAA

AACCGGUU AGAGAAGG

CGAA

AUGCCUUC CAGCAGAU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAUGCCUU AGCAGAUG

CGAA

ACAUCUGC UGCCAGUC

CGAA

AACAUCUG GCCAGUCA

CGAA

ACUGGCAA AAAUGAAU

CGAA

AUUCAUUU AAAGUGUG

CGAA

AAUUCAUU AAGUGUGA

CGAA

AGUUUCAU GAGGUAGU

CGAA

ACCUCGAG GUGGGUGA

CGAA

CGAA

AUUCUUGG CAGCGAAA

CGAA

ACCCUGUU UCCCAGGA

CGAA

AGACCCUG CCAGGAGG

CGAA

ACCCUUCC CGGAGAGG

CGAA

AGCCUGUG CUGGCCUU

GGAA

AGGCCAGG UCUAAGCA

CGAA

AAGGCCAG CUAAGCAC

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAAGGCCA UAAGCACA

CGAA

AGAAAGGC AGCACACC

CGAA

ACUGGGCA GCGGACCC

CGAA

ACCCGCCU CUCGUGGG

CGAA

AGGACCCG GUGGGCGA

CGAA

AUUGCUCC GUUUCCCA

CGAA

ACUAUUGC UCCCACCG

CGAA

AACUAUUG CCCACCGC

CGAA

CGAA

AGCGGUGG CCUCUCAG

CGAA

AGGGAGCG UCAGGCGC

2 5 3gg CUGCGCCU CUGAUGAG X 4957 CUCCCUCUC 5744 CGAA

AGAGGGAG AGGCGCAG

CGAA

ACCCUGCG UAGAGAAG

CGAA

AGACCCUG GAGAAGCG

CGAA

AUCCCCUC UAGAGAAG

CGAA

AGAUCCCC GAGAAGCC

WO 99/50403 PC'f/US99/06507 Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

ACUCGCGC CGCGGCCC

CGAA

ACGGGGCG GCGUCCCA

CGAA

ACGCAACG CCACCCAC

496 GGGGAGGG CUGAUGAG X 9965 CACCG.CGUC 5752 CGAA

ACGCGGUG CCCUCCCC

CGAA

AGGGGACG CCCUCCCC

CGAA

AGGGGAGG CCCUCCCG

CGAA

AGGGGAGG CCGCUGCG

CGAA

AGCGGCCG UGGGCGAC

CGAA

2~ ACGCCCGC GGCGUAGG

CGAA

ACGCCAAC GGAGGUGA

CGAA

AGCCUCAC CGGCUCGG

CGAA

AGCCGGAG GGCAGCGU

CGAA

ACGCUGCC GCAGCUGC

CGAA

AUCCUGGG UGCGCCCC

CGAA

ACCGGGGC AAGUUGCG

CGAA

ACUUGACC GCGGACUU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AGUCCGCA GGAGCCGG

CGAA

ACCAGUCC CGCNCGUC

CGAA

ACGNGCGG UGCGUGGG

CGAA

AUUCCCAC CNCGUGUC

CGAA

ACACGNGN CUGGCUGG

CGAA

ACCGNGCC GGANCCGG

CGAA

AGGUNCCC CCUGGCCC

CGAA

AAGGUNCC CUGGCCCG

CGAA

CGAA

AGGACCCG CGAGACGC

CGAA

AUGGCUUC AGCCAGGC

2 5 g16 CGGCCGGG CUGAUGAG X 4989 GANNNCCUU 5776 CGAA

AGGNNNUC CCCGGCCG

CGAA

AAGGNNNU CCGGCCGC

CGAA

AUGCGCCC UCUGAGCC

CGAA

AGAUGCGC UGAGCCCC

CGAA

AGCGCGGG ACCCGGGG

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

ACGCGCGC GCGGGUGN

CGAA

ANCACCCG CUGGUCGG

CGAA

ACCAGGAN GGNCCAAG

CGAA

AGCCCCAC CCGGGGGU

CGAA

AAGCCCCA CGGGGGUU

CGAA

ACCCCCGG GUUCCCGC

CGAA

ACAACCCC CCCGCCCC

CGAA

AACAACCC CCGCCCCU

CGAA

CGAA

ACAGGGCA ACUUCCUG

CGAA

AGUUACAG CCUGGGUG

CGAA

AAGUUACA CUGGGUGA

CGAA

ACCCGCGC UACAUUUC

CGAA

AACCCGCG ACAUUUCC

CGAA

AAACCCGC CAUUUCCC

CGAA

AUGUAAAC UCCCCACA

WO 99/50403 PC'T/US99/06507 Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAUGUAAA CCCCACAU

CGAA

AAAUGUAA CCCACAUU

CGAA

AUGUGGGG UCCAAUUU

CGAA

AAUGUGGG CCAAUUUC

CGAA

AAAUGUGG CAAUUUCU

CGAA

AUUGGAAA UCUCCUGU

CGAA

AAUUGGAA CUCCUGUU

CGAA

AAAUUGGA UCCUGUUA

1150 CGUAACAG CUGAUGAG X 5018 CAAUUUCUC 580.5 CGAA

AGAAAUUG CUGUUACG

CGAA

ACAGGAGA ACGCUUUC

CGAA

AACAGGAG CGCUUUCU

CGAA

AGCGUAAC UCUCCAGA

CGAA

AAGCGUAA CUCCAGAA

CGAA

~AGCGUA UCCAGAAG

CGAA

AGAAAGCG CAGAAGGU

CGAA

ACCUUCUG UUUUCUUU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AACCUUCU UUUCUUUC

CGAA

AAACCUUC UUCUUUCC

CGAA

AAAACCUU UCUUUCCU

CGAA

AAAAACCU CUUUCCUU

CGAA

AAAAAACC UUUCCUUU

CGAA

AGAAAAAA UCCUUUUU

CGAA

AAGAAAAA CCUUUUUU

CGAA

AAAGAAAA CUUUUUUC

CGAA

2 d AGGAAAGA UUUUCUUU

CGAA

AAGGAAAG UUUCUUUC

CGAA

AAAGGAAA UUCUUUCU

CGAA

AAAAGGAA UCUUUCUU

CGAA

AAAAAGGA CUUUCUUU

CGAA

~ppp~GG UUUCUUUC

CGAA

AGAAAAAA UCUUUCUU

CGAA

AAGAAAAA CUUUCUUU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAAGAAAA UUUCUUUC

CGAA

AGAAAGAA UCUUUCUU

CGAA

AAGAAAGA CUUUCUUU

CGAA

AAAGAAAG UUUCUUUU

CGAA

AGAAAGAA UCUUUUUU

CGAA

AAGAAAGA CUUUUUUU

CGAA

AAAGAAAG UUUUUUUA

CGAA

AGAAAGAA UUUUUACC

CGAA

CGAA

AAAGAAAG UUUACCUU

CGAA

AAAAGAAA UUACCUUC

CGAA

AAAAAGAA UACCUUCA

CGAA

AAAAAAGA ACCUUCAA

CGAA

SAG CCUUCAAC

CGAA

AGGUAAP.A CAACAUAC

CGAA

AAGGUAAA AACAUACU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AUGUUGAA CUCCUGCG

CGAA

AGUAUGUU CUGCGGGG

CGAA

ACCCCGCA GUUUUGGA

CGAA

ACAACCCC UUGGAGCA

CGAA

AACAACCC UGGAGCAG

CGAA

AAACAACC GGAGCAGG

CGAA

AGCCUCAU UGCCUCCU

CGAA

AAGCCUCA GCCUCCUC

CGAA

CGAA

AGGAGGCA CAGUGUCC

CGAA

ACACUGGA CCCAGGUG

CGAA

AGGCACCG UGCUCCCA

CGAA

AGCAGAGG CCAGGGCA

CGAA

AUUUUUCG UCUAGUGU

CGAA

AGAUUUUU UAGUGUAU

CGAA

AGAGAUUU GUGUAUUC

Posi- Seq. I.D. Seq. I.D.
' tion RZ No Substrate No.
.

CGAA

ACACUAGA UUCGGGGA

CGAA

AUACACUA CGGGGAAC

CGAA

AAUACACU GGGGAACC

CGAA

AGCCUUUU CCUUGGGC

CGAA

AGGGAGCC GGGCCGGU

CGAA

AUCCCACC CUUGGCUU

CGAA

AGGAUCCC GGCUUUGU.

CGAA

AGCCAAGG UGUCUCUG

CGAA

2~ AAGCCAAG GUCUCUGG

CGAA

ACAAAGCC UCUGGCUG

CGAA

AGACAAAG UGGCUGCU

CGAA

ACGGUGUG AGCCGUCA

CGAA

ACGGCUGA AGGGCAAU

CGAA

CGAA

AUGCCAAU CGGCCUCU

CGAA

AAUGCCAA GGCCUCUU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AGGCCGAA UUUGGUAC

CGAA

AGAGGCCG UGGUACUG

CGAA

AAGAGGCC GGUACUGG

CGAA

ACCAAAGA CUGGGGAC

CGAA

ACCCCGGG GCUGCCCG

CGAA

ACCACGGG CUCUCUGA

CGAA

AGGACCAC UCUGAGUC

CGAA

AGAGGACC UGAGUCCU

CGAA

ACUCAGAG CUUGGUGA

CGAA

AGGACUCA GGUGAUUU

CGAA

AUCACCAA UUGCCUGG

CGAA

AAUCACCA UGCCUGGG

CGAA

AAAUCACC GCCUGGGC

CGAA

AGCCAGGG CUGGUCUG

CGAA

ACCAGGAG UGCUGGGG

CGAA

AGGCGGCC UGCCUCAG

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

15$1 CAUCCUCU CUGAUGAG X 5106 CUCUGCCUC 5893 CGAA

AGGCAGAG AGAGGAUG

CGAA

ACAUGCAC AGUAUUUU

CGAA

ACUUACAU UUUUUAAU

CGAA

AUACUUAC UUUAAUAA

CGAA

AAUACUUA UUAAUAAA

CGAA

AAAUACUU UAAUAAAA

CGAA

AP.AAUACU AAUAAAAA

CGAA

AAAAAUAC AUAAAAAC

CGAA

AUUAAAAA AAAACUGU

CGAA

ACAGUUUU GUACUCGU

CGAA

ACUACAGU CUCGUAAA

CGAA

AGUACUAC GUAAAACA

CGAA

ACGAGUAC AAACAAUC

CGAA

AUUGUUUU UACACCCU

CGAA

AGAUUGUU CACCCUGC

CGAA

AUCCCUUC UGUUAUUU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAUCCCUU GUUAUUUU

CGAA

ACAAAUCC AUUUUAUU

CGAA

AACAAAUC UUUUAUUU

CGAA

AUAACAAA UUAUUUUA

CGAA

AAUAACAA UAUUUUAU

CGAA

AAAUAACA AUUUUAUU

CGAA

AAAAUAAC UUUUAUUA

CGAA

AUAAAAUA UUAUUAUU

CGAA

2 O p,AU~U UAUUAUUU

CGAA

AAAUAAAA AUUAUUUA

CGAA

AAAAUAAA UUAUUUAU

CGAA

AUAAAAUA AUUUAUUU

CGAA

AAUAAAAU UUUAUUUA

CGAA

AUAAUAAA UAUUUAUU

CGAA

AAUAAUAA AUUUAUUU

CGAA

AAAUAAUA UUUAUUUA

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AUAAAUAA UAUUUAUU

CGAA

AAUAAAUA AUUUAUUU

CGAA

AAAUAAAU UUUAUUUA

CGAA

1 O AUAAAU~ UAUUUAUU

CGAA

AAUAAAUA AUUUAUUU

CGAA

AAAUAAAU UUUAUUUA

CGAA

AUAAAUAA UAUUUAUU

CGAA

AAUAAAUA AUUUAUUU

CGAA

2 O ~Up~,pU UUUAUUUU

CGAA

AUAAAUAA UAUUUUUG

CGAA

AAUAAAUA AUUUUUGA

CGAA

AAAUAAAU UUUUUGAG

CGAA

AUAAAUAA UUUGAGAC

CGAA

~Up,~.~UA UUGAGACG

CGAA

AAAUAAAU UGAGACGG

CGAA

~U~A GAGACGGA

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

ACUCCGUC UUGCUCUG

CGAA

AGACUCCG GCUCUGUC

CGAA

AGCAAGAC UGUCGCCC

CGAA

ACAGAGCA GCCCAGGC

CGAA

ACCCACCA CUCGGCUC

CGAA

AACCCACC UCGGCUCA

CGAA

AGAACCCA GGCUCACU

CGAA

AGCCGAGA ACUGCAAC

CGAA

CGAA

AAGUUGCA UGCCUCCU

CGAA

AGGCAGAA CUGGGUUU

CGAA

ACCCAGGA UAAGCGAU

CGAA

AACCCAGG AAGCGAUU

CGAA

AAACCCAG AGCGAUUC

CGAA

AUCGCUUA CUUCUGGC

CGAA

AAUCGCUU UUCUGGCU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AGAAUCGC CUGGCUCA

CGAA

AAGAAUCG UGGCUCAG

CGAA

AGCCAGAA AGCCUCCC

CGAA

AGGCUGAG CCGAGUAG

CGAA

ACUCGGGA GCUGGGAU

CGAA

AUCCCAGC ACAGGCGC

CGAA

AAUCCCAG CAGGCGCC

CGAA

AGCCGGCC AUUUUUGU

CGAA

AUUAGCCG UUUGUAUU

CGAA

AAUUAGCC UUGUAUUU

CGAA

AAAUUAGC UGUAUUUU

CGAA

AAAAUUAG GUAUUUUU

CGAA

ACAAAAAU UUUUUAGU

CGAA

AUACAAAA UUUAGUAG

CGAA

AAUACAAA UUAGUAGA

CGAA

AAAUACAA UAGUAGAG

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate ~ No.

CGAA

AAAAUACA AGUAGAGA

CGAA

AAAAAUAC GUAGAGAC

CGAA

ACUAAAAA GAGACGCG

CGAA

ACCGCGUC UCACCAUG

CGAA

AACCGCGU CACCAUGU

CGAA

AAACCGCG ACCAUGUU

CGAA

ACAUGGUG GGCCAGGC

CGAA

ACCAGCCU UGGAGCUC

CGAA

AGCUCCAG CUGGCCUC

CGAA

AGGCCAGG AAGUGAUC

CGAA

AUCACUUG CGCCCACC

CGAA

AGGUGGGC AGCCUCCC

CGAA

AGGCUGAG CCAAAGUG

CGAA

AUUCCCAG CAGGCGUG

CGAA

AUCCUGGC UAUUUUAA

CGAA

AAUCCUGG AUUUUAAA

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAAUCCUG UUUUAAAA

CGAA

AUAAAUCC UUAAAAAG

CGAA

AAUAAAUC UAAAAAGG

CGAA

AAAUAAAU AAAAAGGG

CGAA

AAAAGGGA

CGAA

AUCUUCCC UGUUGAUA

CGAA

AAUCUUCC GUUGAUAA

CGAA

ACAAAUCU GAUAAAUU

CGAA

AUCAACAA AAUUCACU

CGAA

AUUUAUCA CACUUCAA

CGAA

AAUUUAUC ACUUCAAA

CGAA

AGUGAAUU CAAAGAUA

CGAA

AAGUGAAU AAAGAUAA

CGAA

A UCUUUGA A ACUAUUC

U

A GUUUAUC U UCGAAAA

U

A UAGUUUA C GAAAAUA

WO 99/50403 r PCT/US99/06507 Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

2049 GUAUUUUC CUGAUGAG X , 5218 AAACUAUUC 6005 CGAA

AAUAGUUU GAAAAUAC

CGAA

AUUUUCGA CUUUAGUG

CGAA

AGUAUUUU UAGUGAUU

CGAA

AAGUAUUU AGUGAUUC

CGAA

AAAGUAUU GUGAUUCC

CGAA

AUCACUAA CCCGUCAA

CGAA

AAUCACUA CCGUCAAG

CGAA

ACGGGAAU AAGACUCU

CGAA

CGAA

AGAGUCUU CUGUGUAU

CGAA

AAGAGUCU UGUGUAUG

2pgg UCUAUACA CUGAUGAG X 5229 UUCUGUGUA 6016 CGAA

ACACAGAA UGUAUAGA

CGAA

ACAUACAC UAGACGUA

CGAA

AUACAUAC GACGUAUA

CGAA

ACGUCUAU UAACUCAU

CGAA

AUACGUCU ACUCAUUC

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AGUUAUAC AUUCUGGA

CGAA

AUGAGUUA CUGGACAG

CGAA

AAUGAGUU UGGACAGG

CGAA

AUCCUUGC UCUUUUUU

CGAA

AUAUCCUU UUUUUUUG

CGAA

AGAUAUCC UUUUUGUU

CGAA

AAGAUAUC UUUUGUUU

CGAA

AAAGAUAU UUUGUUUG

CGAA

AAAAGAUA UUGUUUGU

CGAA

AAAAAGAU UGUUUGUU

CGAA

AAAP~APrGA GUUUGUUU

CGAA

ACAAAAAA UGUUUGUU

CGAA

AACAAAAA GUUUGUUU

CGAA

ACAAACAA UGUUUGUU

CGAA

AACAAACA GUUUGUUU

CGAA

ACAAACAA UGUUUUGA

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AACAAACA GUUUUGAG

CGAA

ACAAACAA UUGAGAUG

CGAA

AACAAACA UGAGAUGG

CGAA

AAACAAAC GAGAUGGA

CGAA

AGUCCAUC UCGCUGUC

CGAA

AGAGUCCA GCUGUCGC

CGAA

ACAGCGAG GCCAGGCU

CGAA

AGCCUGGC GAGUGCAG

CGAA

AUCGCGCC UCAGCUCA

CGAA

AAUCGCGC CAGCUCAC

CGAA

AAAUCGCG. AGCUCACU

CGAA

AGCUGAAA ACUGCAAC

CGAA

AGGUUGCA CGCUUCCC

CGAA

AGCGGAGG CCCGGGUU

CGAA

AAGCGGAG CCGGGUUC

CGAA

ACCCGGGA CAAGCGAU

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AACCCGGG AAGCGAUU

CGAA

AUCGCUUG CUCCUGCC

CGAA

AAUCGCUU UCCUGCCU

CGAA

AGAAUCGC CUGCCUCA

CGAA

AGGCAGGA AGCCUCCC

CGAA

AGGCUGAG CCGAGUAG

CGAA

ACUCGGGA GCUGGGAU

22?2 GUGCCUGU CUGAUGAG X 5273 GCUGGGAUU 6060 CGAA

AUCCCAGC ACAGGCAC

CGAA

CGAA

AGGGCGUG CUAAUUUU

CGAA

AGUAGGGC AUUUUUGA

CGAA

AUUAGUAG UUUGAUUU

CGAA

AAUUAGUA UUGAUUUU

CGAA

~UUAGU UGAUUUUU

2305 UAAAAAUC CUGAUGAG X 5280 CUAAUUUUU 606?
CGAA

AAAAUUAG GAUUUUUA

CGAA

AUCAAAAA G ~
UUUAGUA

Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AAUCAAAA UUAGUAGA

CGAA

AAAUCAAA UAGUAGAG

CGAA

AAAAUCAA AGUAGAGA

CGAA

AAAAAUCA GUAGAGAC

CGAA

ACUAAAAA GAGACGGG

CGAA

AUCCCGUC UCCCCAUG

CGAA

AAUCCCGU CCCCAUGU

CGAA

AAAUCCCG CCCAUGUU

CGAA

ACAUGGGG GGCCAGGA

CGAA

AUCAUCCU UCGAUCUC

CGAA

AGAUCAUC GAUCUCUU

CGAA

AUCGAGAU UCUUGACC

CGAA

AGAUCGAG UUGACCCC

CGAA

AGAGAUCG GACCCCGU

CGAA

AUCACGGG AGCCUGCC

CGAA

AGGCAGGC GGCCUCCC

WO 99/50403 PCTlUS99/06507 Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AGGCCAAG CCAAAGUG

CGAA

AUCCCAGC ACAGGCGU

CGAA

AAUCCCAG CAGGCGUG

CGAA

ACCCUUGG UCUUGAAG

CGAA

AUACCCUU UUGAAGGA

CGAA

AGAUACCC GAAGGAGG

CGAA

AUCCCUCC ACAGUUGA

CGAA

AAUCCCUC CAGUUGAU

CGAA

CGAA

AUCAACUG UGUAGAGG

CGAA

ACAUAUCA GAGGAAUA

CGAA

AUUCCUCU UUGCAGUG

CGAA

AUAUUCCU GCAGUGGU

CGAA

ACCACUGC AUUGCUGC

CGAA

AACCACUG UUGCUGCA

CGAA

AUAACCAC GCUGCAUU

WO 99/50403 PCT/US99/0650'1 Posi- Seq. I.D. Seq. I.D.

tion RZ No. Substrate No.

CGAA

AUGCAGCA UCCUAUGU

CGAA

AAUGCAGC CCUAUGUG

CGAA

AAAUGCAG CUAUGUGA

CGAA

AGGAAAUG UGUGACUG

CGAA

AGUCCCAG AAACAGAU

CGAA

AUCUGUUU AGCUGAUA

CGAA

AUCAGCUG GUGUUAGC

CGAA

ACACUAUC AGCGUGCA

CGAA

CGAA

ACUGCUCA UGAUGACU

CGAA

AGUCAUCA UGACACAG

CGAA

AUUUCUGU AGAAUCUC

CGAA

AUUCUUAU UCCAGCAU

CGAA

AGAUUCUU CAGCAUUC

CGAA

AUGCUGGA CUGCCCUG

CGAA

AAUGCUGG UGCCCUGG

. CA 02324421 2000-09-26 DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDS OU CE BREVET
COMPREND PLUS D'UN TOME.
CEC! EST LE TOME ~ DE -NOTE: Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets -THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ~ OF tT
NOTE:.For additional voiumes~please contact'the Canadian Patent Ofif~ce

Claims (59)

Claims

1. An enzymatic nucleic acid molecule with RNA
cleaving activity, wherein said enzymatic nucleic acid molecule specifically cleaves RNA encoded by an aryl hydrocarbon nuclear transporter (ARNT) gene.

2. An enzymatic nucleic acid molecule with RNA
cleaving activity, wherein said enzymatic nucleic acid molecules specifically cleaves RNA encoded by an integrin subunit beta 3 ((.beta.3) gene.

3. An enzymatic nucleic acid molecule with RNA
cleaving activity, wherein said enzymatic nucleic acid molecules cleaves RNA encoded by a integrin subunit alpha 6 (.alpha.6) gene.

4. An enzymatic nucleic acid molecule with RNA
cleaving activity, wherein said enzymatic nucleic acid molecules cleaves RNA encoded by a Tie-2 gene.

5. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.

6. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises a stem II region of length greater than or equal to 2 base pairs.

7. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is in a hairpin configuration.

8. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid is in a hepatitis delta virus, group I intron, group II intron, VS
nucleic acid or RNase P nucleic acid configuration.

9. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic molecule is a DNAzyme.

10. The enzymatic nucleic acid of claim 7, wherein said enzymatic nucleic acid molecule comprises a stem II
region of length between three and seven base-pairs.

11. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to said RNA.

12. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to said mRNA.

13. The enzymatic nucleic acid molecule of claim 5 wherein said enzymatic nucleic acid molecule consists essentially of any sequence defined as Seq. I.D. Nos 1-393, 911-1611, 2449-3587, and 4915-5701.

14 The enzymatic nucleic acid molecule of claim 7, wherein said enzymatic nucleic acid molecule consists essentially of any sequence defined as Seq. ID 787-848, 2313-2380, 4727-4820 and 6489-6568.

15. A mammalian cell including an enzymatic nucleic acid molecule of any of claims 1-4.

16. The mammalian cell of claim 15, wherein said mammalian cell is a human cell.

17. An expression vector comprising nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of any of claims 1-4 in a manner which allows expression of that enzymatic nucleic acid molecule.

18. A mammalian cell including an expression vector of claim 17.

19. The mammalian cell of claim 18, wherein said mammalian cell is a human cell.

20. A method for treatment of cancer, diabetic retinopathy, age related macular degeneration (ARMD), inflammation, and arthritis comprising the step of administering to a patient an enzymatic nucleic acid molecule of any of claims 1-4.

21. A method for treatment of cancer comprising the step of administering to a patient, an expression vector of claim 17.

22. A method for the treatment of cancer, diabetic retinopathy, age related macular degeneration (ARMD), inflammation, and arthritis comprising the step of administering to a patient an expression vector of claim 17.

23. A method for treatment of cancer comprising the steps of: a) isolating cells from a patient; b) administering to said cells an enzymatic nucleic acid molecule of any of claims 1-4; and c) introducing said cells back into said patient.

282 29. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of any of claims 1-4.

25. A method of treatment of a patient having a condition associated with an elevated level of aryl hydrocarbon nuclear transporter (ARNT), comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 1.

26. A method of treatment of a patient having a condition associated with the level of Tie-2 comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 2.

27. A method of treatment of a patient having a condition associated with the level of integrin subunit alpha 6, comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 3.

28. A method of treatment of a patient having a condition associated with the level of integrin subunit beta 3 comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 4.

29. A method of treatment of a patient having a condition associated with the level of aryl hydrocarbon nuclear transporter (ARNT), comprising the steps of:
(a)contacting cells of said patient with an enzymatic nucleic acid molecule of claim 1; and (b) administering to said patient one or more additional drugs.

30. A method of treatment of a patient having a condition associated with the level of Tie-2, comprising the steps of : ( a ) contacting cells of said patient with an enzymatic nucleic acid molecule of claim 2; and (b) administering to said patient one or more additional drugs.

31. A method of treatment of a patient having a condition associated with the level of integrin subunit alpha 6, comprising the steps of: (a)contacting cells of said patient with an enzymatic nucleic acid molecule of claim 3; and (b) administering to said patient one or more additional drugs.

32. A method of treatment of a patient having a condition associated with the level of integrin subunit beta 3, comprising the steps of: (a)contacting cells of said patient with an enzymatic nucleic acid molecule of claim 4; and (b) administering to said patient one or more additional drugs.

33. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues, phosphorothioate linkages in at least three of the 5' terminal nucleotides, a 2'-C-allyl modification at position No. 4 of said nucleic acid, at least ten 2'-O-methyl modifications, and a 3'- end modification.

34. The enzymatic nucleic acid of claim 33, wherein said enzymatic nucleic acid comprises a 3'-3' linked inverted ribose moiety at said 3' end.

35. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues; phosphorothioate linkages at least three of the 5' terminal nucleotides 2'-amino modification at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2'-O-methyl modifications; and a 3'- end modification.

36. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues; phosphorothioate linkages at least three of the 5' terminal nucleotides, abasic substitution at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2;-O-methyl modifications; comprises a 3'-end modification.

37. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises of at least five ribose residues; phosphorothioate linkages at least three of the 5' terminal nucleotides; a 6-methyl uridine substitution at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2'-O-methyl modifications; and comprises a 3' end modification.

38. A method for modulating expression of ARNT gene in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 1.

39. A method for modulating expression of integrin subunit beta 3 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 2.

40. A, method for modulating expression of integrin subunit alpha 6 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 3.

41. A method for modulating expression of Tie-2 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 4.

42. A method of cleaving an ARNT RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 1 with said ARNT RNA molecule under conditions suitable for the cleavage of said ARNT RNA
molecule.

43. A method of cleaving a integrin subunit beta 3 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 2 with said integrin subunit beta 3 RNA molecule under conditions suitable for the cleavage of said integrin subunit beta 3 RNA molecule.

44. A method of cleaving a integrin subunit alpha 6 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 3 with said integrin subunit alpha 6 RNA molecule under conditions suitable for the cleavage of said integrin subunit alpha 6 RNA molecule.

45. A method of cleaving a Tie-2 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 4 with said Tie-2 RNA molecule under conditions suitable for the cleavage of said Tie-2 RNA molecule.

46. The method of any of claims 42-45, wherein said cleavage is carried out in the presence of a divalent cation.

47. The method of claim 46, wherein said divalent cation is Mg2+.

48. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is chemically synthesized.

49. The expression vector of claim 17, 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 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.

50. The expression vector of claim 17, 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 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.

51. The expression vector of claim 17, 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 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.

52. The expression vector of claim 18, 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.

53. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 394-786 and 849-910.

54. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 5702-6488 and 6569-6648.

55. The enzymatic nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 3588-4726 and 4821-4914.

56. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 1612-2312 and 2381-2448.

57. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid comprises at least one 2'-sugar modification.

58. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid comprises at least one nucleic acid base modification.

59. The enzymatic nucleic acid molecule of any of claims 1-9, wherein said enzymatic nucleic acid comprises at least one phosphorothioate modification.