In vitro selection of high temperature Zn2+-dependent DNAzymes

Kevin E. Nelson, Peter J. Bruesehoff, Yi Lu

Research output: Contribution to journalArticlepeer-review

Abstract

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+, Hg 2+, 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 E a ∈=∈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.

Original languageEnglish (US)
Pages (from-to)216-225
Number of pages10
JournalJournal of Molecular Evolution
Volume61
Issue number2
DOIs
StatePublished - Aug 1 2005

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Genetics(clinical)
  • Ecology, Evolution, Behavior and Systematics
  • Molecular Biology
  • Agricultural and Biological Sciences(all)
  • Agricultural and Biological Sciences (miscellaneous)

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