Sequence Diversity, Metal Specificity, and Catalytic Proficiency of Metal-Dependent Phosphorylating DNA Enzymes Original Research Article

Although DNA has not been found responsible for biological catalysis, many artificial DNA enzymes have been created by “in vitro selection.” Here we describe a new selection approach to assess the influence of four common divalent metal ions (Ca2+, Cu2+, Mg2+, and Mn2+) on sequence diversity, metal specificity, and catalytic proficiency of self-phosphorylating deoxyribozymes. Numerous autocatalytic DNA sequences were isolated, a majority of which were selected using Cu2+ or Mn2+ as the divalent metal cofactor. We found that Cu2+- and Mn2+-derived deoxyribozymes were strictly metal specific, while those selected by Ca2+ and Mg2+ were less specific. Further optimization by in vitro evolution resulted in a Mn2+-dependent deoxyribozyme with a kcat of 2.8 min−1. Our findings suggest that DNA has sufficient structural diversity to facilitate efficient catalysis using a broad scope of metal cofactor utilizing mechanisms.