In vitro selection and metal specificity of transition metal-dependent DNAzymes

Since the discovery of DNA/RNAzymes, nucleic acid systems with catalytic potential have been identified in increasing number. In order to develop the potential therapeutic and biotechnology applications of DNA/RNAzymes, increased knowledge of the metal specificity, catalytic activity and structure of nucleic acids is necessary. In vitro selection was employed under varied conditions in attempts to isolate sequences with more complex transition metal ion binding sites that are likely to be infrequent in sequence space. Metal ion cofactors, random pool design and other selection parameters are vital in the selection of Fe3+- and Hg2+-dependent DNAzymes.;To investigate the contribution of divalent metal ions to DNAzyme structural stability, in vitro selection of RNA-cleaving DNAzymes was carried out at 90°C with Mg2+ or Zn2+. A family of high temperature Zn2+-dependent DNAzmes with a modest rate enhancement and high Zn2+-specificity was isolated. Detailed characterization showed that the most active sequence was specific for high temperature, although little secondary structure was predicted. The ability of Zn2+ to enhance the RNA-cleavage activity over a high background rate suggests a special role for this metal ion in the activation and stabilization of DNAzymes at high temperatures.;The characterization of two families of Co2+-dependent DNAzymes further demonstrates the interplay between DNAzyme structure and metal specificity. The clone 11 and 18 DNAzymes were selected for study because the primary sequences differ by only four bases, producing vast differences in Co2+ specificity. Phylogenetic and mutational analyses were used to identify the sequence and secondary structure responsible for Co 2+ specificity and catalysis. Sequence elements within the random domain and conserved primer binding regions are important in stabilizing structures with high specificity and activity. Truncation of peripheral sequences leads to an increased propensity for formation of inactive, self-complementary or metastable structures. The Co2+ ion likely stabilizes a conformation with high activity and specificity in the clone 11 DNAzyme system. The aspects of DNA/RNAzyme function and metal specificity discussed will likely aid in the rational design or in vitro selection of DNA/RNAzyme systems well suited for the spectroscopic characterization of metal binding sites and showing greater potential for therapeutic and biotechnology applications.