In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme

Abstract

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the '8-17' deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribo-nucleotide base and G can be either ribo- or deoxy-ribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn(2+), the cleavage rate is 1.35 min(-1)at pH 6.0; based on pH profile this rate is estimated to be at least approximately 30 times faster at pH 7.5, where most assays of Mg(2+)-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn(2+)with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg(2+)-dependent nucleic acid enzymes under similar conditions.