In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes |
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Authors: | Kevin E Nelson Peter J Bruesehoff Yi Lu |
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Institution: | (1) Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA |
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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+, 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. |
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Keywords: | DNAzymes Deoxyribozymes Catalytic DNA Ribozymes High temperature RNA hydrolysis In vitro selection Metal ions |
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