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United States Patent 19 11 Patent Number: 5,646,031 Deyoung Et Al

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United States Patent 19 11 Patent Number: 5,646,031 Deyoung Et Al US005646031A United States Patent 19 11 Patent Number: 5,646,031 DeYoung et al. 45 Date of Patent: Jul. 8, 1997 54 SARMV AND SCYMVI HAIRPIN Haseloff and Gerlach, "Simple RNA enzymes with new and RBOZYMES highly specific endoribonuclease activities” Nature, 334:585-591 (1988). 75 Inventors: Mary Beth DeYoung, Elmhurst; Haseloff and Gerlach, “Sequences required for self-cata Andrew M. Siwkowski, Sycamore; lyzed cleavage of the satellite RNA of tobacco ringspot Arnold E. Hampel, DeKalb, all of Ill. virus' Gene, 82:43-52 (1989). Kaper et al., "Nucleotide sequence predicts circularity and 73 Assignee: Northern Illinois University, DeKalb, self-cleavage of 300-ribonucleotide satellite of arabis...” I. Biochem. Biophys. Res. Com., 154:318-325 (1988). Milligan et al., “Oligoribonucleotide synthesis using T7 21 Appl. No.: 442,513 RNA polymerase and synthetic DNA templates' Nucleic Acids Res., 15:8783-8798 (1987). 22 Filed: May 16, 1995 Ojwang et al., "Inhibition of human immunodeficiency virus (51) Int. Cl. .............. C07H 21/04; C12Q 1/68; type-I (HIV-1) expression by a hairpin ribozyme” Proc. A61K 48/00; C12N 15/85 Nat. Acad. Sci. USA, 89:10802-10806 (1992). 52 U.S. Cl. .......................... 435/325; 435/6: 435/91.31; Perriman et al., “Extended target-site specificity for a ham 435/172.1; 435/1723; 435/252.3; 435/320.1; merhead ribozyme” Gene, 113:157-163 (1992). 536/23.1536/23.2:536/24.5: 514/44 Rubino et al., "Nucleotide sequence and structural analysis 58 Field of Search ....................... 435/6, 91.31, 172.1, of two satellite RNAS associated with chicory yellow mottle 435/172.3, 320.1, 240.1, 240.2, 252.3: 536/23.1, virus' J. General Virology, 7:1897-1903 (1990). 23.2, 24.5; 514/44 Santa Lucia et al., "Effects of GA mismatches on the structure and thermodynamics of RNA internal loops' Bio 56 References Cited chemistry, 29:8813-8819 (1990). Siwkowski et al., "Catalytic properties of hairpin-like U.S. PATENT DOCUMENTS ribozymes derived from CYMV . .” Presented at RNA 5,093,246 3/1992 Cech et al. ........................... 435/91.31 Processing Meeting: Cold Spring Harbor, Cold Spring Har 5,144,019 9/1992 Rossi et al. ............ ... 536/23.2 bor, NY. (Poster)(May, 1995). 5,225,337 7/1993 Robertson et al. ... 435/9.31 Sullivan, “Development of ribozymes for gene therapy" J. 5,225,347 7/1993 Goldberg et al. ... ... 435/320.1 Investigative Dermatology (Suppl), 103:95s (1994). 5,254,678 10/1993 Haseloff et al. ....................... 536,23.2 Symons, “Self-cleavage of RNA in the replication of small OTHER PUBLICATIONS pathogens of plants and animals” TIBS, 14:445-450 (1989). Symons, "Small catalytic RNAs” Annu. Rey. Biochem, Altschuler et al., "Method for generating transcripts with 61:641-671 (1992). defined 5' and 3' termini by autolytic processing” Gene, Uhlenbeck, "Small catalytic oligoribonucleotide” Nature, 122:85-90 (1992). 328:596-600 (1987). Anderson et al., “Mutagenesis of the hairpin ribozyme” Yamada et al., “Intracellular immunization of human T cells Nucleic Acids Res., 22: 1096-1100 (1994). with hairpin ribozyme against human immunodeficiency Berzal-Harranz et al., “Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme” virus type 1" Gene Therapy, 1:38-45 (1994). EMBO.J., 12:2567-2573 (1993). Yu et al., "Ahairpin ribozyme inhibits expression of diverse Buzayan et al., "Nucleotide sequence of satellite tobacco strains of human immunodeficiency virus type 1" Proc. Nat. ringspot virus RNA and its relationship to multimeric forms" Acad, Sci. USA, 90:6340-6344 (1993). Virology, 151:186-199 (1986). Wong-Staal and Nabel, "Is HTV's achilles heel hidden in its Cech, “RNA as an enzyme” Scientific American, 255:64-75 genes” HIV Advances in Res. and Therapy, 4:3-8 (1994). (1986). Cech, "Self-splicing of Group I introns" Annu. Rev. Bio Primary Examiner-John L. LeGuyader chem, 59:543-568 (1990). Attorney, Agent, or Firm-Kohn & Associates Fritsch et al., “Properties of satellite RNA of nepoviruses” 57 ABSTRACT Biochemie, 75:561-567 (1993). Gilboa et al., “Transfer and expression of clones genes using A method for the identification by modeling of synthetic retroviral vectors" BioTechniques, 4(6):504-512 (1986). catalytic RNAs and ribozymes thereof are disclosed includ Hampel and Tritz, "RNA catalytic properties of the mini ing a hairpin portion and binding sites for cleaving an RNA mum (-)sTRSV sequence” Biochemistry, 28:4929-4933 substrate at a site which has a GUA sequence following the (1989). site of cleavage. Synthetic catalytic RNA has been modeled Hampe et al., “Hairpin catalytic RNA model: evidence for from negative strands of satellite RNA from the group helices and sequence requirement for substrate RNA” consisting of chicory yellow mottle virus (sCYMVT) and Nucleic Acids Research, 18:299-304 (1990). arabis mosaic virus (sArMV). Hampel et al., "The hairpin ribozyme” Methods: A Com parison to Methods in Enzymology, 5:37-42 (1993). 6 Claims, 4 Drawing Sheets U.S. Patent Jul. 8, 1997 Sheet 1 of 4 5,646,031 STRSV l, CLEAVAGE (?) CATALYTIC RNA OOP 4. s Loop SUBSTRATE RNA U GUG UAUAUUAct4.O toUCAC "lagCUCUUU f4 U O) O O. O. b ) CAC A A AC AAC , aca 5 BASE 1 G CAAAG 2O AG LOOP3 HELIX 4 LOOP2 HELIX 3 HELIX 2 LOOP 1 HELIX 1 |Fig-1A SCYMV1 y- SITE OF CLEAVAGE CATALYTIC RNA 2 t SUBSTRATE RNA LOOP 4. LOOP5 52A f4 G U GUG40G UAUAUUA U C to2.4" A *CU''. A16 CUCUU U CAC G CGCG GACAAc 5 BASE 1 C CAAAG 20 a LOOP3 HELIX4 LOOP2 HELIX 3 HELIX2 OOP HELIX |Fig-1B SArNW T SITE OF CLEAVAGE CATALYTIC RNA S SUBSTRATE RNA LOOP 4. w OOP5 C U GUG40AUAUUA c. 6 C "e UCUU f4 U OOO O O O O ( ) CAC A GGCG GACAAc 5 BASE C CAAAG 20 A. LOOP3 HELIX 4 LOOP2 HELIX3 HELIX 2 LOOP 1 HELIX 1 |Fig-1 C U.S. Patent Jul. 8, 1997 Sheet 2 of 4 5,646,031 : 3 x : x 8 8 : x : x 3 : 8 : U.S. Patent Jul. 8, 1997 Sheet 3 of 4 5,646,031 SITE OF is . CLEAVAGE CATALYTICATALYT RNA LOOP 4 io S2 t LOOP 5 SUBSTRATE RNA U UAUAUUA A CU G GUG G U CUGGC unUCUC LJ - A CLCCAACC 5 y ASA U O ) O. O. O. O O CAC GACC GACAC GACGUUCG A A G A C s C CAAAG AG 5 BASE LOOP3 HELIX4 LOOP2 HELIX 3 HELIX 2 LOOP 1 HELIX 1 U.S. Patent Jul. 8, 1997 Sheet 4 of 4 5,646,031 5,646,031 1. 2 SARMV AND SCYMV HARPN cal testing in human AIDS patients (Wong-Staal, 1994). RBOZYMES Given the therapeutic potential of the hairpin ribozyme, as well as the general importance of a designed "molecular GRANT SUPPORT knife”, it would be useful to have additional hairpin Research in this application was supported in part by a 5 ribozymes. grant from the National Institutes of Health to AEH However, currently such ribozymes are identified empiri (A129870). The Government has certain rights in the inven cally. It would be useful to have a method whereby any tion. nucleotide sequence can be screened to identify new sequences with catalytic activity. Further, hairpin ribozymes TECHNICAL FIELD O that are currently available cleave an RNA substrate within the catalytic region at a site which has a GUC sequence The present invention is in the general area of a RNA following the site of cleavage. It would be useful to have catalyst, i.e., ribozyme, and methods of modeling new hairpin ribozymes which can cleave at additional sites. hairpin ribozymes as well as two new hairpin ribozymes. 15 SUMMARY OF THE INVENTION AND BACKGROUND OF THE INVENTION ADVANTAGES Ribozymes are RNA molecules that possess RNA cata According to the present invention, a synthetic catalytic lytic ability (see Cech. 1986, 1990, for review) that cleave RNA is disclosed including a hairpin portion and binding a specific site in a target RNA substrate in a trans reaction. sites for cleaving an RNA substrate at a site which has a The catalytic ability is shown in that the number of RNA GUA sequence following the site of cleavage. The synthetic molecules that are cleaved by a ribozyme is greater than the catalytic RNA is modeled from negative strands of satellite number predicted by stochiochemistry (Hampel and Tritz, RNA from the group consisting of chicory yellow mottle 1989; Uhlenbeck, 1987). virus (sCYMV1) and arabis mosaic virus (sArMV). Ribozymes catalyze the phosphodiester bond cleavage of 25 According to the present invention, a method of identi RNA and are emerging as a new tool for controlling the fication by modeling of a hairpin ribozyme whereby cellular RNA levels of specific genes. Several ribozyme sequence and structural homologies are identified is dis structural families have been identified including Group I closed. The method includes the steps of searching introns, RNase P, the hepatitis delta virus ribozyme, ham databases, such as GenBank, which contain genetic merhead ribozymes and the hairpin ribozyme originally sequences for short nucleotide sequences homologous to the derived from the negative strand of the tobacco ringspot STRSV hairpin ribozyme at conserved homologous regions virus satellite RNA (STRSV) (Sullivan, 1994; U.S. Pat. No. from Loop 2 through Loop 4 of STRSV. The STRSV 5,225.347, columns 4-5). The latter two families are derived ribozyme sequence is then aligned with the identified from viroids and virusoids, in which the ribozyme is sequence and structural analysis is undertaken whereby believed to separate monomers from oligomers created 35 Helices 3 and 4 are identified followed by Loop 3.
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