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

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes ha s been obtained through in vitro selection, They share a common motif with the '8-17' deoxyribozyme isolated under different conditions, including dif ferent design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimer ic substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can be either ribo- or deoxyribo-G), The pH profi le and reaction products of this deoxyribozyme are similar to those reporte d for hammerhead ribozyme, This deoxyribozyme has higher activity in the pr esence of transition metal ions compared to alkaline earth metal ions. At s aturating concentrations of Zn2+, the cleavage rate is 1.35 min(-1) at pH 6 .0; based on pH profile this rate is estimated to be at least similar to 30 times faster at pH 7.5, where most assays of Mg2+-dependent DNA and RNA en zymes are carried out. This work represents a comprehensive characterizatio n of a nucleic acid-based endonuclease that prefers transition metal ions t o alkaline earth metal ions. The results demonstrate that nucleic acid enzy mes are capable of binding transition metal ions such as Zn2+ with high aff inity, and the resulting enzymes are more efficient at RNA cleavage than mo st Mg2+-dependent nucleic acid enzymes under similar conditions.