In vitro selection and characterization of transition metal-dependent DNAzymes and RNAzymes.

摘要

Since the discovery that nucleic acids could perform catalytic functions other than the storage and transfer of genetic information, much research has been devoted to examining the mechanisms by which these “DNAzymes” and “RNAzymes” work. Most of the systems being examined are dependent on divalent metal ions for activity, and understanding the roles of these metal ions in the catalytic activity of DNAzymes and RNAzymes in conjunction with the nucleic acids themselves has become of paramount importance.; One of the major goals in studying DNAzymes is understanding which ligands from the DNA are binding the catalytic metal ions. Towards this goal, we have used a combinatorial technique known as in vitro selection to isolate transition-metal dependent DNAzymes that cleave RNA in a site-specific manner. By incorporating a negative selection strategy, we tailored the metal dependence of cis-cleaving DNAzymes away from a Pb 2+-dependent system towards one that showed high cleavage activity in the presence of Co2+.; The Co2+-dependent systems were fully characterized in both cis- and trans-cleaving forms to obtain a better understanding of the reaction characteristics of the systems. Three constructs were examined for activity and its dependence on pH, metal ion identity and concentration, and monovalent ion identity and concentration. All of the systems showed similar reaction properties to previously characterized RNAzyme and DNAzyme systems, most notably the hammerhead RNAzyme and the “8–17” DNAzyme. As trans-cleaving constructs, the metal binding affinity for Co2+ was tightest versus Pb 2+ and Zn2+.; These DNAzymes are dependent on Co2+ for cleavage activity. By exploiting the electronic properties of Co2+, we have used UV-Visible spectroscopy to obtain a better understanding of the metal binding properties of the selected DNAzyme systems. Through analysis of the spectroscopic and metal binding properties of these systems, in conjunction with the activity properties, a better understanding of how metals are binding in these systems has been developed. A previously selected DNAzyme system, the “8-17” motif, and a naturally-occurring RNAzyme system, the hammerhead motif, were also further examined in terms of the diversity of conditions under which RNA cleavage activity is supported.