• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Jun 11, 1993; 21(11): 2605–2611.
PMCID: PMC309588

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cech TR. RNA as an enzyme. Sci Am. 1986 Nov;255(5):64–75. [PubMed]
  • Altman S. Ribonuclease P: an enzyme with a catalytic RNA subunit. Adv Enzymol Relat Areas Mol Biol. 1989;62:1–36. [PubMed]
  • Forster AC, Symons RH. 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. [PubMed]
  • Garriga G, Lambowitz AM. RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro. Cell. 1984 Dec;39(3 Pt 2):631–641. [PubMed]
  • Epstein LM, Gall JG. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987 Feb 13;48(3):535–543. [PubMed]
  • Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986 Mar 28;231(4745):1577–1580. [PubMed]
  • Symons RH. Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem Sci. 1989 Nov;14(11):445–450. [PubMed]
  • Uhlenbeck OC. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. [PubMed]
  • Haseloff J, Gerlach WL. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. [PubMed]
  • 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. [PubMed]
  • Koizumi M, Iwai S, Ohtsuka E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988 Nov 7;239(2):285–288. [PubMed]
  • Sheldon CC, Symons RH. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989 Jul 25;17(14):5679–5685. [PMC free article] [PubMed]
  • Ruffner DE, Dahm SC, Uhlenbeck OC. Studies on the hammerhead RNA self-cleaving domain. Gene. 1989 Oct 15;82(1):31–41. [PubMed]
  • Ruffner DE, Stormo GD, Uhlenbeck OC. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. [PubMed]
  • Perreault JP, Wu TF, Cousineau B, Ogilvie KK, Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. [PubMed]
  • Ruffner DE, Uhlenbeck OC. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. [PMC free article] [PubMed]
  • Fedor MJ, Uhlenbeck OC. Substrate sequence effects on "hammerhead" RNA catalytic efficiency. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1668–1672. [PMC free article] [PubMed]
  • Yang JH, Perreault JP, Labuda D, Usman N, Cedergren R. Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme. Biochemistry. 1990 Dec 25;29(51):11156–11160. [PubMed]
  • Pieken WA, Olsen DB, Benseler F, Aurup H, Eckstein F. Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes. Science. 1991 Jul 19;253(5017):314–317. [PubMed]
  • Olsen DB, Benseler F, Aurup H, Pieken WA, Eckstein F. Study of a hammerhead ribozyme containing 2'-modified adenosine residues. Biochemistry. 1991 Oct 8;30(40):9735–9741. [PubMed]
  • Perreault JP, Labuda D, Usman N, Yang JH, 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. [PubMed]
  • Perriman R, Delves A, Gerlach WL. Extended target-site specificity for a hammerhead ribozyme. Gene. 1992 Apr 15;113(2):157–163. [PubMed]
  • Yang JH, Usman N, Chartrand P, Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. [PubMed]
  • Williams DM, Pieken WA, 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. [PMC free article] [PubMed]
  • Fu DJ, McLaughlin LW. 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. [PMC free article] [PubMed]
  • Fu DJ, McLaughlin LW. 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. [PubMed]
  • McCall MJ, Hendry P, Jennings PA. Minimal sequence requirements for ribozyme activity. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5710–5714. [PMC free article] [PubMed]
  • Uhlenbeck OC, Dahm SC, Ruffner DE, Fedor MJ. Structure and mechanism of the hammerhead self-cleaving domain. Nucleic Acids Symp Ser. 1989;(21):95–96. [PubMed]
  • Dahm SC, Uhlenbeck OC. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. [PubMed]
  • 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. [PubMed]
  • Slim G, Gait MJ. 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. [PMC free article] [PubMed]
  • Fedor MJ, Uhlenbeck OC. Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry. 1992 Dec 8;31(48):12042–12054. [PubMed]
  • 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. [PubMed]
  • 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]
  • 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. [PubMed]
  • Piccirilli JA, Vyle JS, Caruthers MH, Cech TR. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993 Jan 7;361(6407):85–88. [PubMed]
  • 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. [PubMed]
  • 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]
  • 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]
  • 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. [PMC free article] [PubMed]
  • Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, Rossi JJ. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. [PubMed]
  • Ojwang JO, Hampel A, Looney DJ, 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. [PMC free article] [PubMed]
  • Shimayama T, Sawata S, Komiyama M, Takagi Y, Tanaka Y, Wada A, Sugimoto N, Rossi JJ, Nishikawa F, Nishikawa S, et al. 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]
  • Taylor NR, Kaplan BE, Swiderski P, Li H, Rossi JJ. 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. [PMC free article] [PubMed]
  • Hendry P, McCall MJ, Santiago FS, Jennings PA. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability. Nucleic Acids Res. 1992 Nov 11;20(21):5737–5741. [PMC free article] [PubMed]
  • Paolella G, Sproat BS, Lamond AI. Nuclease resistant ribozymes with high catalytic activity. EMBO J. 1992 May;11(5):1913–1919. [PMC free article] [PubMed]
  • Hertel KJ, Pardi A, Uhlenbeck OC, Koizumi M, Ohtsuka E, Uesugi S, Cedergren R, Eckstein F, Gerlach WL, Hodgson R, et al. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. [PMC free article] [PubMed]
  • Young B, Herschlag D, Cech TR. Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity. Cell. 1991 Nov 29;67(5):1007–1019. [PubMed]
  • 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. [PubMed]
  • Stec WJ, 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. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...