Journal of Molecular Evolution

, Volume 61, Issue 2, pp 216–225 | Cite as

In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes



In vitro selection of Zn2+-dependent RNA-cleaving DNAzymes with activity at 90°C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn2+ over Pb2+, Ca2+, Cd2+, Co2+, Hg2+, and Mg2+. The Zn2+-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn2+-binding dissociation constant of 234 μM and that its activity increases with increasing temperatures from 50–90°C. A fit of the Arrhenius plot data gave Ea = 15.3 kcal mol−1. Surprisingly, the selected Zn2+-dependent DNAzymes showed only a modest (∼3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn2+ is present. Since this effect is highly specific for Zn2+, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.


DNAzymes Deoxyribozymes Catalytic DNA Ribozymes High temperature RNA hydrolysis In vitro selection Metal ions 


  1. Achenback JC, Chiuman W, Cruz RPG, Li Y (2004) DNAzymes: From creation in vitro to application in vivo. Curr Pharmaceut Bioctechnol 5:312–336CrossRefGoogle Scholar
  2. Adamidi C, Fedorova O, Pyle AM (2003) A group II intron inserted into a bacterial heat-shock operon shows autocatalytc activity and unusual thermostability. Biochemistry 42:3408–3418Google Scholar
  3. Baidya N, Uhlenbeck OC (1997) Ax-Kinetic and thermodynamic analysis of cleavage site mutations in the hammerhead ribozyme. Biochemistry 36:1108–1114CrossRefPubMedGoogle Scholar
  4. Banerjee AR, Jaeger JA, Turner DH (1993) Thermal unfolding of a group I ribozyme: the low-temperature transition is primarily disruption of tertiary structure. Biochemistry 32:153–163CrossRefPubMedGoogle Scholar
  5. Bonaccio M, Credali A, Peracchi A (2004) Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme. Nucleic Acids Res 32:916–925CrossRefPubMedGoogle Scholar
  6. Breaker RR (1997a) DNA enzymes. Nat Bioteclinol 15:427–431CrossRefGoogle Scholar
  7. Breaker RR (1997b) In vitro selection of catalytic polynucleotides. Chem Rev 97:371–390CrossRefGoogle Scholar
  8. Breaker RR (2000) Making catalytic DNAs. Science 290:2095–2096Google Scholar
  9. Breaker RR, Joyce GF (1994) A DNA enzyme that cleaves RNA. Chem Biol 1:223–229CrossRefPubMedGoogle Scholar
  10. Breaker RR, Joyce GF (1995) A DNA enzyme with Mg2+-dependent RNA phosphoesterase activity. Chem Biol 2:655–660CrossRefPubMedGoogle Scholar
  11. Brown AK, Li J, Pavot CMB, Lu Y (2003) A lead-dependent DNAzyme with a step mechanism. Biochemistry 42:7152–7161CrossRefPubMedGoogle Scholar
  12. Brown JW, Haas ES, Pace NR (1993) Characterization of ribonuclease P RNAs from thermophilic bacteria, Nucleic Acids Res 1:671–679PubMedGoogle Scholar
  13. Bruesehoff PJ, Li J, Augustine AJ, Lu Y (2002) Improving metal ion specificity during in vitro selection of catalytic DNA. Combin Chem High T Scr 5:327–353Google Scholar
  14. Butzow JJ, Eichhorn GL (1971) Interaction of metal ions with nucleic acids and related compounds, XVII. Mechanism of degradation of polyribonucleotides and oligoribonucleotides by zinc(II) ions. Biochemistry 10:2019–2027Google Scholar
  15. Canny MD, Jucker FM, Kellogg E, Khvorova A, Jayasena SD, Pardi A (2004) Fast Cleavage kinetics of natural hammerhead ribozyme. J Am Chem Soc 126:10848–10849CrossRefPubMedGoogle Scholar
  16. Carmi N, Shultz LA, Breaker RR (1996) In vitro selection of self-cleaving DNAs. Chem Biol 3:1039–1046CrossRefPubMedGoogle Scholar
  17. Cesjolka J, Yarus M (1996) Small RNA-divalent domains. RNA 2:785–793 PubMedGoogle Scholar
  18. Ciesiolka J, Gorski J, Yarus M (1995) Selection of an RNA domain that binds Zn2+, RNA 1:538–550PubMedGoogle Scholar
  19. Cuenoud B, Szostak JW (1995) A DNA metalloenzyme with DNA ligase activity. Nature 375:611–614CrossRefPubMedGoogle Scholar
  20. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–22CrossRefPubMedGoogle Scholar
  21. Fang XW, Golden BL, Littrell K, Shelton V, Thiyagarajan P, Pan T, Sosnick TR (2001) The thermodynamic origin of the stability of a thermophilic ribozyme. Proc Nat Acad Sci USA 98:4355–4360CrossRefPubMedGoogle Scholar
  22. Fang XW, Srividya N, Golden BL, Sosnick TR, Pan T (2003) Stepwise conversion of a mesophilic to a thermophilic ribozyme. J Mol Biol 330:177–183Google Scholar
  23. Galtier N, Lobry JR (1997) Relationships between genomic G + C content, RNA secondary structures, and optimal growth temperature in prokaryotes. Mol Evol 44:632–636PubMedGoogle Scholar
  24. Gold L, Polisky B, Uhlenbeck O, Yarus M (1995) Diversity of oligonucleotide functions. Annu Rev Biochem 64:763–797CrossRefPubMedGoogle Scholar
  25. Guo F, Cech TR (2002) Evolution of Tetrahymena ribozyme mutants with increased structural stability. Nature Struct Biol 9:855–861PubMedGoogle Scholar
  26. Hammann C, Hormes R, Sczakiel G, Tabler M (1997) A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics. Nucleic Acids Res 5:4715–4722CrossRefGoogle Scholar
  27. Hirao I, Nishimura Y, Naraoka T, Watanabe K, Arata Y, Miura K (1989) Extraordinary stable structure of short single-stranded DNA fragments containing a specific base sequence: d(GCGAAAGC). Nucleic Acids Res 17:2223–2231PubMedGoogle Scholar
  28. Hirao I, Nishimura Y, Tagawa Y, Watanabe K, Miura K (1992) Extraordinarily stable mini-hairpins; electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing. Nucleic Acids Res 20:3891–3896PubMedGoogle Scholar
  29. Hirao I, Kawai G, Yoshizawa S, Nishimura Y, Ishido Y, Watanabe K, Miura K (1994) Most compact hairpin-turn structure exerted by a short DNA fragment. d(GCGAAGC) in solution: an extraordinarily stable structure resistant to nucleases and heat. Nucleic Acids Res 22:576–582PubMedGoogle Scholar
  30. Ikenaga H, Inoue Y (1974) Metal(II) ion catalyzed transphosphorylation of four homodinucleotides and five pairs of dinucleotide sequence isomers. Biochemistry 13:577–582CrossRefPubMedGoogle Scholar
  31. Jankowsky E, Schwenzer B (1996) Efficient improvement of hammerhead ribozyme mediated cleavage of long substrates by oligonucleotide facilitators. Biochemistry 35:15313–15321CrossRefPubMedGoogle Scholar
  32. Jencks WP(1969) Catalysis in chemistry and enzymology. Dover, New York, pp 605–611Google Scholar
  33. Joyce GF (1999) Reactions Catalyzed by RNA and DNA enzymes. In: Gesteland RF, Cech TR, Atkins JF (eds) The RNA world. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p 687–689 Google Scholar
  34. Joyce GF (2004) Directed evolution of nucleic acid enzymes. Annu Rev Biochem 73:791–836CrossRefPubMedGoogle Scholar
  35. Juneau K, Cech TR (1999) In vitro selection of RNAs with increased teriary structural stability. RNA 5:1119–1129CrossRefPubMedGoogle Scholar
  36. Kaukinen U, Lyytikainen S, Mikkola S, Loennberg H (2002) The reactivity of phosphodiester bonds within linear single-stranded oligoribonucleotides is strongly dependent on the base sequence. Nucleic Acids Res 30:468–474CrossRefPubMedGoogle Scholar
  37. Kawakami J, Imanaka H, Yokota Y, Sugimoto N (2000) In vitro selection of aptamers that act with Zn2+. J Inorg Biochem 82:197–206CrossRefPubMedGoogle Scholar
  38. Khvorova A, Lescoute A, Westhof E, Jayasena SD (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nature Struct Biol 10:708–712CrossRefPubMedGoogle Scholar
  39. Kowalak JA, Dalluge JJ, McCloskey JA, Stetter KO (1994) The role of modification in stabilization of transfer RNA from hyerthermophiles. Biochemistry 33:7869–7876CrossRefPubMedGoogle Scholar
  40. Kuusela S, Lonnberg H (1993) Metal ions that promote the hydrolysis of nucleoside phosphoesters do not enhance intramolecular phosphate migration. J Phys Org Chem 6:347–356CrossRefGoogle Scholar
  41. Li J, Lu Y (2000) A highly Sensitive and selective catalytic DNA biosensor for lead ions. J Am Chem Soc 122:10466–10467CrossRefGoogle Scholar
  42. Li J, Zheng W, Kwon AH, Lu Y (2000) In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme. Nucleic Acids Res. 28:481–488CrossRefPubMedGoogle Scholar
  43. Li Y, Breaker RR (1999a) Deoxyribozymes: new players in the ancient game of biocatalysis. Curr Opin Struct Biol 9:315–323CrossRefGoogle Scholar
  44. Li Y, Breaker RR (1999b) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group. J Am Chem Soc 121:5364–5372CrossRefGoogle Scholar
  45. Liu J, Lu Y (2003a) A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J Am Chem Soc 125:6642–6643CrossRefGoogle Scholar
  46. Liu J, Lu Y (2003b) Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers. Anal Chem 75:6666–6672CrossRefGoogle Scholar
  47. Liu J, Lu Y (2004a) Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor. Analytic 76:1627–1632CrossRefGoogle Scholar
  48. Liu J, Lu Y (2004b) Colorimetric biosensor based on DNAzyme-assembled gold nanoparticles. J Fluoresc 14:343–354CrossRefGoogle Scholar
  49. Lu Y (2002) New transition metal-dependent DNAzymes as Efficient endonucleases and as selective metal biosensors. Chem Eur J 8:4588–4596CrossRefGoogle Scholar
  50. Lu Y, Liu J, Li J, Bruesehoff PJ, Pavot CMB, Brown AK (2003) New highly sensitive and selective catalytic DNA biosensors for metal ions. Biosens Bioelectr 18:529–540CrossRefPubMedGoogle Scholar
  51. Martin RB (1985) Nucleoside sites for transition metal ion binding. Accounts Chem Res 18:32–38CrossRefGoogle Scholar
  52. McCloskey JA, Graham DE, Zhou S, Grain PF, Ibba M, Konisky J, Soll D, Olsen GJ (2001) Post-transcriptional modification in archaeal tRNAs; identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales. Nucleic Acids Res 729:4699–4706CrossRefPubMedGoogle Scholar
  53. Mei SHJ, Liu Z, Brennan JD, Li Y (2003) An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with flurorescence signaling. J Am Chem soc 125:412–420CrossRefPubMedGoogle Scholar
  54. Mikkola S, Zagorowska I, Lonnberg H (1999) Zn2+ promoted hydrolysis of RNA. Nucleosides Nucleotides 18:1267–1268Google Scholar
  55. Moody EM, Bevilacqua PC (2003a) Folding of stable DNA motif involves a highly cooperative network interactions. J Am Chem Sol 125:16285–16293CrossRefGoogle Scholar
  56. Moody EM, Bevilacqua PC (2003b) Thermodynamic coupling of the loop and stem in unusually stable DNA hairpins closed by CG base pairs. J Am Chem Soc 125:2032–2033CrossRefGoogle Scholar
  57. Nakano M, Moody EM, Liang J, Bevilacqua PC (2002) Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc). Biochemistry 41:14281–14292CrossRefPubMedGoogle Scholar
  58. Nesbø CL, Doolittle WF (2003) Active self-splicing group I introns in 23S rRNA genes of hyperthermophilic bacteria, derived from introns in eukaryotic organelles. Proc Natl Acad Sci USA 100:10806–10811CrossRefPubMedGoogle Scholar
  59. Nutiu R, Li Y (2004) Structure-switching signaling aptamers: Transducing molecular recognition into fluorescence signaling. Chem Eur J 10:1868–1876Google Scholar
  60. Osborne EM, Schaak JE, Derose VJ (2005) Characterization of a native hammerhead ribozyme derived from schistosomes. RNA 11:187–196CrossRefPubMedGoogle Scholar
  61. Osborne SE, Ellington AD (1997) Nucleic Acid selection and the challenge of combmatorial chemistry. Chem Rev 97:349–370CrossRefPubMedGoogle Scholar
  62. Paul R, Lazarev D, Altman S (2001) Characterization of RNase P from Thermotoga maritima. Nucleic Acids Res 29:880–885CrossRefPubMedGoogle Scholar
  63. Peracchi A (1999) Origins of the temperature dependence of hammerhead ribozyme catalysis. Nucleic Acids Res 27:2875–2882CrossRefPubMedGoogle Scholar
  64. Saksmerprome V, Roychowdhury-saha M, Jayasena S, Khvorova A, Burke DH (2004) Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures. RNA 10:1916–1924Google Scholar
  65. Santoro SW, Joyce GF, Sakthivel K, Gramatikova S, Barbas CF III (2000) RNA Cleavage by a DNA enzyme with extended chemical functionality. J Am Chem Soc 122:2433–2439CrossRefPubMedGoogle Scholar
  66. Scarabino D, Tocchini-Valentini GP (1996) Influence of substrate structure on cleavage by hammerhead ribozyme. FEES Lett 383:185–190CrossRefPubMedGoogle Scholar
  67. Sen D, Geyer CR (1998) DNA enzymes. Curr Opin Chem Biol 2:680–687CrossRefPubMedGoogle Scholar
  68. Soukup GA, Breaker RR (1999) Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5:1308–1325CrossRefPubMedGoogle Scholar
  69. Sun LQ, Cairns MJ, Saravolac EG, Baker A, Gerlach WL (2000) Catalytic nucleic acids: From lab to applications. Pharmacol Rev 52:325–347PubMedGoogle Scholar
  70. Takagi Y, Taira K (1995) Temperatures-dependent change in the rate-determining step in a reaction catalyzed a hammerhead ribozyme. FEBS Lett 361:273–6CrossRefPubMedGoogle Scholar
  71. Tanner MA, Cech TR (1996) Activity and thermostability of the smal self-splicing group I intron in the pre-tRNAIle of the purple bacterium Azoarcus. RNA 2:72–83Google Scholar
  72. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: Sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43Google Scholar
  73. Wang W, Billen LP, Li Y (2002) Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes. Chem Biol 9:507–517CrossRefPubMedGoogle Scholar
  74. Williams KP, Bartel DP (1995) PCR product with strands of unequal length. Nucleic Acids Res 23:4220–1PubMedGoogle Scholar
  75. Wilson DS, Szostak JW (1999) In vitro selection of functional nucleic acids. Annu Rev Biochem 68:611–647CrossRefPubMedGoogle Scholar
  76. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res 31:3406–3415CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations