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

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 (Ca 2+, Cu 2+, Mg 2+, and Mn 2+) 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 Cu 2+ or Mn 2+ as the divalent metal cofactor. We found that Cu 2+- and Mn 2+-derived deoxyribozymes were strictly metal specific, while those selected by Ca 2+ and Mg 2+ were less specific. Further optimization by in vitro evolution resulted in a Mn 2+-dependent deoxyribozyme with a k cat 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.