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. 1993 Jun 11;21(11):2605–2611. doi: 10.1093/nar/21.11.2605

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

T Shimayama 1, F Nishikawa 1, S Nishikawa 1, K Taira 1
PMCID: PMC309588  PMID: 8332458

Abstract

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altman S. Ribonuclease P: an enzyme with a catalytic RNA subunit. Adv Enzymol Relat Areas Mol Biol. 1989;62:1–36. doi: 10.1002/9780470123089.ch1. [DOI] [PubMed] [Google Scholar]
  2. Cech T. R. RNA as an enzyme. Sci Am. 1986 Nov;255(5):64–75. doi: 10.1038/scientificamerican1186-64. [DOI] [PubMed] [Google Scholar]
  3. Dahm S. C., Uhlenbeck O. C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. doi: 10.1021/bi00103a011. [DOI] [PubMed] [Google Scholar]
  4. Epstein L. M., Gall J. G. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987 Feb 13;48(3):535–543. doi: 10.1016/0092-8674(87)90204-2. [DOI] [PubMed] [Google Scholar]
  5. Fedor M. J., Uhlenbeck O. C. Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry. 1992 Dec 8;31(48):12042–12054. doi: 10.1021/bi00163a012. [DOI] [PubMed] [Google Scholar]
  6. Fedor M. J., Uhlenbeck O. C. Substrate sequence effects on "hammerhead" RNA catalytic efficiency. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1668–1672. doi: 10.1073/pnas.87.5.1668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  8. Fu D. J., McLaughlin L. W. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry. 1992 Nov 17;31(45):10941–10949. doi: 10.1021/bi00160a001. [DOI] [PubMed] [Google Scholar]
  9. Fu D. J., McLaughlin L. W. Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3985–3989. doi: 10.1073/pnas.89.9.3985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garriga G., Lambowitz A. M. RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro. Cell. 1984 Dec;39(3 Pt 2):631–641. doi: 10.1016/0092-8674(84)90470-7. [DOI] [PubMed] [Google Scholar]
  11. Goodchild J., Kohli V. Ribozymes that cleave an RNA sequence from human immunodeficiency virus: the effect of flanking sequence on rate. Arch Biochem Biophys. 1991 Feb 1;284(2):386–391. doi: 10.1016/0003-9861(91)90313-8. [DOI] [PubMed] [Google Scholar]
  12. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  13. Heidenreich O., Eckstein F. Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem. 1992 Jan 25;267(3):1904–1909. [PubMed] [Google Scholar]
  14. Hendry P., McCall M. J., Santiago F. S., Jennings P. A. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability. Nucleic Acids Res. 1992 Nov 11;20(21):5737–5741. doi: 10.1093/nar/20.21.5737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Herschlag D. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry. 1992 Feb 11;31(5):1386–1399. doi: 10.1021/bi00120a015. [DOI] [PubMed] [Google Scholar]
  16. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Koizumi M., Iwai S., Ohtsuka E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988 Nov 7;239(2):285–288. doi: 10.1016/0014-5793(88)80935-9. [DOI] [PubMed] [Google Scholar]
  18. Koizumi M., Iwai S., Ohtsuka E. Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mers. FEBS Lett. 1988 Feb 15;228(2):228–230. doi: 10.1016/0014-5793(88)80004-8. [DOI] [PubMed] [Google Scholar]
  19. Koizumi M., Ohtsuka E. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry. 1991 May 28;30(21):5145–5150. doi: 10.1021/bi00235a005. [DOI] [PubMed] [Google Scholar]
  20. McCall M. J., Hendry P., Jennings P. A. Minimal sequence requirements for ribozyme activity. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5710–5714. doi: 10.1073/pnas.89.13.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohkawa J., Yuyama N., Taira K. Activities of HIV-RNA targeted ribozymes transcribed from a 'shot-gun' type ribozyme-trimming plasmid. Nucleic Acids Symp Ser. 1992;(27):15–16. [PubMed] [Google Scholar]
  22. Ojwang J. O., Hampel A., Looney D. J., Wong-Staal F., Rappaport J. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10802–10806. doi: 10.1073/pnas.89.22.10802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olsen D. B., Benseler F., Aurup H., Pieken W. A., Eckstein F. Study of a hammerhead ribozyme containing 2'-modified adenosine residues. Biochemistry. 1991 Oct 8;30(40):9735–9741. doi: 10.1021/bi00104a024. [DOI] [PubMed] [Google Scholar]
  24. Paolella G., Sproat B. S., Lamond A. I. Nuclease resistant ribozymes with high catalytic activity. EMBO J. 1992 May;11(5):1913–1919. doi: 10.1002/j.1460-2075.1992.tb05244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perreault J. P., Labuda D., Usman N., Yang J. H., Cedergren R. Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry. 1991 Apr 23;30(16):4020–4025. doi: 10.1021/bi00230a029. [DOI] [PubMed] [Google Scholar]
  26. Perreault J. P., Wu T. F., Cousineau B., Ogilvie K. K., Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. doi: 10.1038/344565a0. [DOI] [PubMed] [Google Scholar]
  27. Perriman R., Delves A., Gerlach W. L. Extended target-site specificity for a hammerhead ribozyme. Gene. 1992 Apr 15;113(2):157–163. doi: 10.1016/0378-1119(92)90391-2. [DOI] [PubMed] [Google Scholar]
  28. Piccirilli J. A., Vyle J. S., Caruthers M. H., Cech T. R. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993 Jan 7;361(6407):85–88. doi: 10.1038/361085a0. [DOI] [PubMed] [Google Scholar]
  29. Pieken W. A., Olsen D. B., Benseler F., Aurup H., Eckstein F. Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes. Science. 1991 Jul 19;253(5017):314–317. doi: 10.1126/science.1857967. [DOI] [PubMed] [Google Scholar]
  30. Prody G. A., Bakos J. T., Buzayan J. M., Schneider I. R., Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986 Mar 28;231(4745):1577–1580. doi: 10.1126/science.231.4745.1577. [DOI] [PubMed] [Google Scholar]
  31. Ruffner D. E., Dahm S. C., Uhlenbeck O. C. Studies on the hammerhead RNA self-cleaving domain. Gene. 1989 Oct 15;82(1):31–41. doi: 10.1016/0378-1119(89)90027-9. [DOI] [PubMed] [Google Scholar]
  32. Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
  33. Ruffner D. E., Uhlenbeck O. C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. doi: 10.1093/nar/18.20.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  35. Sheldon C. C., Symons R. H. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989 Jul 25;17(14):5679–5685. doi: 10.1093/nar/17.14.5679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shimayama T., Sawata S., Komiyama M., Takagi Y., Tanaka Y., Wada A., Sugimoto N., Rossi J. J., Nishikawa F., Nishikawa S. Substitution of non-catalytic stem and loop regions of hammerhead ribozyme with DNA counterparts only increases KM without sacrificing the catalytic step (kcat): a way to improve substrate-specificity. Nucleic Acids Symp Ser. 1992;(27):17–18. [PubMed] [Google Scholar]
  37. Sioud M., Drlica K. Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7303–7307. doi: 10.1073/pnas.88.16.7303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Slim G., Gait M. J. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 1991 Mar 25;19(6):1183–1188. doi: 10.1093/nar/19.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stec W. J., Grajkowski A., Koziolkiewicz M., Uznanski B. Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates). Nucleic Acids Res. 1991 Nov 11;19(21):5883–5888. doi: 10.1093/nar/19.21.5883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Symons R. H. Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem Sci. 1989 Nov;14(11):445–450. doi: 10.1016/0968-0004(89)90103-5. [DOI] [PubMed] [Google Scholar]
  41. Taira K., Uebayasi M., Maeda H., Furukawa K. Energetics of RNA cleavage: implications for the mechanism of action of ribozymes. Protein Eng. 1990 Aug;3(8):691–701. doi: 10.1093/protein/3.8.691. [DOI] [PubMed] [Google Scholar]
  42. Taylor N. R., Kaplan B. E., Swiderski P., Li H., Rossi J. J. Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo. Nucleic Acids Res. 1992 Sep 11;20(17):4559–4565. doi: 10.1093/nar/20.17.4559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Uchimaru T., Uebayasi M., Tanabe K., Taira K. Theoretical analyses on the role of Mg2+ ions in ribozyme reactions. FASEB J. 1993 Jan;7(1):137–142. doi: 10.1096/fasebj.7.1.8422960. [DOI] [PubMed] [Google Scholar]
  44. Uebayasi M., Uchimaru T., Tanabe K., Nishikawa S., Taira K. Preferential chelation of cationic ligands to axial-equatorial oxygens over equatorial-equatorial dianionic oxygens: implication to the mechanism of action of ribozymes. Nucleic Acids Symp Ser. 1991;(25):107–108. [PubMed] [Google Scholar]
  45. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  46. Uhlenbeck O. C., Dahm S. C., Ruffner D. E., Fedor M. J. Structure and mechanism of the hammerhead self-cleaving domain. Nucleic Acids Symp Ser. 1989;(21):95–96. [PubMed] [Google Scholar]
  47. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang J. H., Perreault J. P., Labuda D., Usman N., Cedergren R. Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme. Biochemistry. 1990 Dec 25;29(51):11156–11160. doi: 10.1021/bi00503a002. [DOI] [PubMed] [Google Scholar]
  49. Yang J. H., Usman N., Chartrand P., Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. doi: 10.1021/bi00136a013. [DOI] [PubMed] [Google Scholar]
  50. Young B., Herschlag D., Cech T. R. Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity. Cell. 1991 Nov 29;67(5):1007–1019. doi: 10.1016/0092-8674(91)90373-7. [DOI] [PubMed] [Google Scholar]

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