Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition Hayley J. Schultz, Andrea M. Gochi, Hannah E. Chia, Alexie L. Ogonowsky, Sharon Chiang, Nedim Filipovic, Aurora G. Weiden, Emma E. Hadley, Sara E. Gabriel, and Aaron M. Leconte* W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, California 91711, United States * S Supporting Information ABSTRACT: Chemical modifications to DNA, such as 2′ mod- ifications, 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 mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2′-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide synthesis. These studies yield an improved understanding of the mutational origins of 2′-modified substrate recognition as well as identify SFM19 as the best candidate for further engineering, whether via rational design or directed evolution. I n addition to encoding all known life, DNA is a valuable biotechnological tool because of its ability to be easily amplified without a loss of information. DNA polymerases perform this enzymatic amplification with high efficiency and fidelity, allowing a number of foundational biological technologies, including the polymerase chain reaction (PCR) 1 and Sanger sequencing, 2 as well as a number of emerging biological technologies, such as high-throughput DNA sequencing, 3 DNA-encoded libraries, 4 and many DNA nano- technologies. 5 While the remarkable biochemical properties of DNA polymerases permit these technologies, they also restrict them; DNA polymerases recognize very few modi fied substrates, limiting many emerging applications. 6 Thus, researchers have spent significant time and effort to broaden the substrate repertoire of these enzymes through directed evolution. 7 Oligonucleotides possessing altered sugar structures, such as 2′ modifications, are often refractory to nuclease digestion, improving the utility of these nucleic acids in biological settings, both in vivo and ex vivo. 7-9 For instance, RNAi and other short, therapeutic oligonucleotides bearing 2′ modifications have been chemically synthesized and shown to possess improved properties relative to those of unmodified DNA or RNA. Unfortunately, there are no known native DNA polymerases that recognize these modified nucleotides, limiting the use of modified nucleotides in nucleic acids longer than those amenable to chemical synthesis in an array of applications, including, but not limited to, the evolution of modified DNA. 10 Considering the utility of these modified nucleotides, as well as the limited success of rational design of enzymes in this area, several research groups have used directed evolution to identify mutant DNA polymerases capable of using 2′-modified nucleotides. To date, directed evolution efforts have largely focused on DNA polymerase I from Thermus aquaticus (Taq) 11-13 and, more recently, B family polymerases such as Therminator 14 and the replicative DNA polymerase from Thermococcus gorgonarius (Tgo). 15 Taq has been evolved to improve recognition of 2′- modified nucleotides on three different occasions; 11-13 in each case, a mutant enzyme with multiple mutations (Table 1) and significantly improved properties was identified. Each of these three enzymes (AA40, SFR3, and SFM19) is marked by a remarkable ability to use 2′-modified nucleotides; both AA40 and SFR3 were evolved to recognize ribonucleotides, and SFM19 was evolved to recognize 2′ -methoxy-modi fied nucleotides. However, all three enzymes possess a common inability to synthesize 2′-modified DNA beyond approximately six to eight modified nucleotides. While these results are promising, to date, these Taq variants have not been further engineered or characterized beyond the initial reports. Received: June 19, 2015 Revised: September 2, 2015 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/acs.biochem.5b00689 Biochemistry XXXX, XXX, XXX-XXX