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Formation of a catalytically active dimer by tRNAVal -driven short ribozymes

Nature Biotechnology volume 16, pages 961–965 (1998)Cite this article

Abstract

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNAVal promoter. The tRNA1Val portion of the transcript did not hinder dimerization as the tRNA-embed-ded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNAVal-embedded conventional hammerhead ribozyme. The tRNAVal-driven minizyme inhibited reporter gene activity (95%) whereas the tRNAVal-driven hammerhead ribozyme resulted in approximately 55% inhibition

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References

  1. Symons,R.H. 1989. Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem. Sci. 14: 445–450.

    Article  CAS  PubMed  Google Scholar 

  2. Kazakov,S. and Altman,S. . 1992. A trinucleotide can promote metal ion-dependent specific cleavage of RNA. Proc. Natl. Acad. Sci. USA 89: 7939–7943.

    Article  CAS  Google Scholar 

  3. Picirilli,J.A., Vyle,J.S., Caruthers,M.H.,and Cech,T.R. 1993. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature 361: 85–88.

    Article  Google Scholar 

  4. Dahm,S.C., Derrick,W.B., and Uhlenbeck,O.C. . 1993. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry 30: 9464–9469.

    Article  Google Scholar 

  5. Steitz,T.A. and Steitz,J.A. 1993. A general twometal-ion mechanism for catalytic RNA. Proc. Watt Acad. Sci. USA 90: 6498–6502.

    Article  CAS  Google Scholar 

  6. Eckstein,R. and Lilley,D.M.J. (eds.). (eds). 1996. Catalytic RNA nucleic acids and molecular biology. Vol. 10. Springer-Verlag Press, Berlin, Germany

  7. Weinstein,L.B., Jones,B.C., Cosstick,R., and Cech,T.R. 1997. A second catalytic metal ion on group I ribozyme. Nature 388: 805–808.

    Article  CAS  PubMed  Google Scholar 

  8. Sawata,S., Komiyama,M., and Taira,K. 1995. Kinetic evidence based on solvent isotope effects for the nonexistence of a proton-transfer process in reactions catalyzed by a hammerhead ribozyme: implication to the double-metal-ion mechanism of catalysis. J. Am. Chem. Soc. 117: 2357–2358.

    Article  CAS  Google Scholar 

  9. Zhou,D.-M., Zhang,L.-H.,and Taira,K. 1997. Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion-effects on the cleavage rates of 5′-oxy and 5-thio substrates by a hammerhead ribozyme. Proc. Watt Acad. Sci. USA 94: 14343–14348.

    Article  CAS  Google Scholar 

  10. Lott,W.B., Pontius,B.W., and von Hippel,P.H. 1998. A two metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate. Proc. Watt Acad. Sci. USA 95: 542–547.

    Article  CAS  Google Scholar 

  11. Zhou,D.-M., and Taira,K. 1998. The hydrolysis of RNA: from theoretical calculations to the hammerhead ribozyme-mediated cleavage of RNA. Chem. Rev. 98: 991–1026.

    Article  CAS  PubMed  Google Scholar 

  12. Pley,H.W., Flaherty,K.M., and Mackay,D.B. 1994. Three-dimensional structure of a hammerhead ribozyme. Wafure 372: 68–74.

    CAS  Google Scholar 

  13. Scott,W.G., Finch,J.T., and Klug,A. 1995. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81: 991–1002.

    Article  CAS  PubMed  Google Scholar 

  14. Scott,W.G., Murray,J.B., Arnold,J.R.P., Stoddard,B.L., and Klug,A. 1996. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 274: 2065–2069.

    Article  CAS  PubMed  Google Scholar 

  15. McCall,M.J., Hendry,P., and Jennings,PA. 1992. Minimal sequence requirements for ribozyme activity. Proc. Natl.Acad. Sci. USA 89: 5710–5714.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tuschl,T. and Eckstein,F. 1993. Hammerhead ribozymes:importance of stem-loop II activity. Proc. Watt Acad. Sci. USA 90: 6991–6994.

    Article  CAS  Google Scholar 

  17. Fu,D.J., Benseler,F. and Mclaughlin,L.W. 1994. Hammerhead ribozymes containing non-nucleoside linkers are active RNA catalysts. J. Am. Chem. Soc. 116: 4591–4598.

    Article  CAS  Google Scholar 

  18. Long,D.M. and Uhlenbeck,O.C. 1994. Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. Proc. Natl. Acad. Sci. USA 91: 6977–6981.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Amontov,S. and Taira,K. 1996. Hammerhead minizymes with high cleavage activity a dimeric structure as the active conformation of minizymes. J. Am. Chem. Soc. 118: 1624–1628.

    Article  CAS  Google Scholar 

  20. Kuwabara,T., Amontov,S., Warashina,M., Ohkawa,J., and Taira,K. 1996. Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes. Nucleic Acids Res. 24: 2302–2310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Geiduschek,E.P. and Tocchini-Valentini,G.P. 1988. Transcription by RNA poly-merase III. Annu. Rev. Biochem. 57: 873–914.

    Article  CAS  PubMed  Google Scholar 

  22. Perriman,R. and de Feyter,R. 1988. tRNA-delivery systems for ribozymes, pp.393–402 in Methods in molecular biology, ribozyme protocols. Turner, PC. (ed.). Humana Press, Totowa, NJ.

    Google Scholar 

  23. Orita,M., Vinayak,R., Andrus,A., Warashina,M., Chiba,A., Kaniwa,H. et al 1996. Magnesium-mediated conversion of an inactive form of a hammerhead ribozyme to an active complex with its substrate. An investigation by NMR spec-troscopy. J. Biol. Chem. 271: 9447–9454.

    Article  CAS  PubMed  Google Scholar 

  24. Sarver,N., Cantin,E., Chang,P., Ladne,P., Stephens,D., Zaia,J. et al. Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247: 1222–1225.

    Article  CAS  PubMed  Google Scholar 

  25. Altman,S. 1993. RNA enzyme-directed gene therapy. Proc. Natl. Acad. Sci. USA 90: 10898–10900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Marschall,P., Thomson,J.B., and Eckstein,F. 1994. Inhibition of gene expression with ribozymes. Cell Mol. Neurobiol. 14: 523–538.

    Article  CAS  PubMed  Google Scholar 

  27. Sullenger,B.A. and Cech,T.R. 1994. Ribozyme-mediated repair of defective mRNA by tppargeted trans-splicing. Nature 371: 619–622.

    Article  CAS  PubMed  Google Scholar 

  28. Leavitt,M.C., Yu,M., Wong-Staal,F. and Looney,D.J. 1996. Ex vivo transduction and expansion of CD4* lymphocytes from HIV* donors: prelude to a ribozyme gene therapy trial. Gene Ther. 3: 599–606.

    CAS  PubMed  Google Scholar 

  29. Good,P.J., Krikos,A.J., Li,S.X., Bertrand,E., Lee,N.S., Giver,L. et al. 1997. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4: 45–54.

    Article  CAS  PubMed  Google Scholar 

  30. Sun,L.-Q., Gerlach,W.L., and Symonds,G. 1996. The use of ribozymes to inhibit HIV replication, pp. 329-342, in Catalytic RNA. nucleic acids and molecular biology. Vol. 10. Eckstein,F. and Lilley,D.M.J. (eds.). Springer-Verlag Press, Berlin, Germany.

    Google Scholar 

  31. Yamada,0., Yu,M., Yee,J.-K., Kraus,G., Looney,D., and Wong-Staal,F. 1994. Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1. Gene Ther. 1: 38–45.

    CAS  PubMed  Google Scholar 

  32. Baier,G., Coggeshall,K.M., Baier-Bitterlich,G., Giampa,L., Telford,D., Herbert,E. et al. 1994. Construction and characterization of Ick- and fyn-specific tRNA:ribozyme chimeras. Mol. Immunol. 31: 923–932.

    Article  CAS  PubMed  Google Scholar 

  33. Yu,M., Leavitt,M.C., Maruyama,M., Yamada,O., Young,D., Ho,A.D. et al. 1995. Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 92: 699–703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kawasaki,H., Ohkawa,J., Tanishige,N., Yoshinari,K., Murata,T., Yokoyama,K.K. et al. 1996. Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase. Nucleic Acids Res. 24: 3010–3016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bertrand,E., Castanotto,D., Zhou,C, Carbonnele,C, Lee,N.S., Good,R et al. 1997. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3: 75–88.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Kawasaki,H., Eckner,R., Yao,T.-P,, Taira,K., Chiu,R., Livingston,D.M. et al. 1998. Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation. Nature 393: 284–289.

    Article  CAS  PubMed  Google Scholar 

  37. Heidenreich,O. and Eckstein,F. 1992. Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J. Biol. Chem. 267: 1904–1909.

    CAS  PubMed  Google Scholar 

  38. Ohkawa,J., Yuyama,N., Takebe,Y, Nishikawa,S., and Taira,K. 1993. Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 90: 11302–11306.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sussman,J.L., Holbrook,S.R., Warrant,R.W., Church,G.M.,and Kim,S.H. 1978. Crystal structure of yeast phenylalanine transfer RNA. I. crystallographic refinement. J. Mol. Biol. 123: 607–630.

    Article  CAS  PubMed  Google Scholar 

  40. Hendry,P., McCall,M.J. >, Santiago,F.S., and Jennings,PA. 1995. n vitro activity of minimized hammerhead ribozymes. Nucleic Acids Res. 23: 3922–3927.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Leontis,N.B., Piotto,M.E., Hills,M.T., Malhotra,A., Ouporov,I.V., Nussbaum,J.N. et al. 1995. Structural studies of DNA three-way junctions. Methods Enzymol. 261: 183–207.

    Article  CAS  PubMed  Google Scholar 

  42. Koseki,S., Ohkawa,J., Yamamoto,R., Takebe,Y. and Taira,K. 1998. A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase. J. Controlled Release 53: 159–173.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. National Institute for Advanced Interdisciplinary Research, Tsukuba Science City, 305–8562, Japan

    Tomoko Kuwabara, Masaki Warashina, Shiori Koseki, Jun Ohkawa & Kazunari Taira

  2. National Institute ofBioscience and Human Technology, Agencyof Industrial Science and Technology, MITI, Tsukuba Science City, 305–8566, Japan

    Tomoko Kuwabara, Masaki Warashina, Shiori Koseki, Jun Ohkawa & Kazunari Taira

  3. Yamanouchi Pharmaceutical Co., Tsukuba Science City, 305–0841, Japan

    Masaya Orita

  4. Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, 305–8572, Japan

    Tomoko Kuwabara, Masaki Warashina, Shiori Koseki, Jun Ohkawa & Kazunari Taira

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  1. Tomoko Kuwabara
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Correspondence to Kazunari Taira.

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Kuwabara, T., Warashina, M., Orita, M. et al. Formation of a catalytically active dimer by tRNAVal -driven short ribozymes. Nat Biotechnol 16, 961–965 (1998). https://doi.org/10.1038/nbt1098-961

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  • DOI: https://doi.org/10.1038/nbt1098-961

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