Comparative characterization of Taq DNA polymerase mutants capable of recognizing 2' modified nucleotides (967.3)

Abstract

In addition to encoding all known life, DNA is also an invaluable biotechnological tool. Chemical modifications to DNA, such as 2’ modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2’ modified DNA nucleotides. While these enzymes recognize these modified nucleotides, they are not capable of synthesizing DNA containing more than six to eight modified nucleotides. Here, we describe comparative biochemical studies that seek to better understand these previously evolved enzymes through quantitative characterization. We describe the identification of specific amino acids and combinations of amino acids that contribute most to unnatural activity. Notably, we have also found that these enzymes are capable of using substrates beyond the originally selected substrates, introducing the possibility of new evolved enzyme‐modified substrate pairs. Collectively, through quantitative characterization of mutations, substrate specificity, extension efficiency and structure, we hope to elucidate key features of previously evolved DNA polymerases. These studies should inform future DNA polymerase engineering efforts, whether via rational design or directed evolution.