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In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes

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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 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.

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Acknowledgments

We thank Andrea K. Brown and Dewain K. Garner for helpful technical discussions. This material is based upon work supported by the U.S. Department of Energy (NABIR program, DEFG02-01-ER63179).

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Correspondence to Yi Lu.

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Nelson, K.E., Bruesehoff, P.J. & Lu, Y. In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes. J Mol Evol 61, 216–225 (2005). https://doi.org/10.1007/s00239-004-0374-3

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