In vitro selection of high temperature Zn(2+)-dependent DNAzymes.
In vitro selection of Zn(2+)-dependent RNA-cleaving DNAzymes with activity at 90 degrees C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn(2+) over Pb(2+), Ca(2+), Cd(2+), Co(2+), Hg(2+), and Mg(2+). The Zn(2+)-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn(2+)-binding dissociation constant of 234 muM and that its activity increases with increasing temperatures from 50-90 degrees C. A fit of the Arrhenius plot data gave E(a) = 15.3 kcal mol(-1). Surprisingly, the selected Zn(2+)-dependent DNAzymes showed only a modest (approximately 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 Zn(2+) is present. Since this effect is highly specific for Zn(2+), this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.
Nelson
Kevin E
KE
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Bruesehoff
Peter J
PJ
Lu
Yi
Y
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D012641:Selection, Genetic; D016415:Sequence Alignment; D015032:Zinc / Q000494:pharmacology*
D002413:Cations, Divalent; D021881:DNA, Catalytic; D015032:Zinc