Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition

Abstract

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 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.

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

“… DNAP, DNA polymerase; RNAP, RNA polymerase; NTP, nucleoside triphosphate; CeNA, cyclohexenyl nucleic acid; LNA, locked nucleic acid; ANA, arabino nucleic acid; FANA, 2′-F-arabino nucleic acid; HNA, 1,5-anhydrohexitol nucleic acid; d5NI, 5-nitroindole; d5NIC, 5-nitroindole-3-carboxamide; ICS, isocarbostyril; 7AI, 7-azaindole. Refrences: 1 (Padilla and Sousa, 1999 ); 2 (Raines and Gottlieb, 1998 ); 3 (Padilla and Sousa, 2002 ); 4 (Burmeister et al, 2006 ); 5 (Chelliserrykattil and Ellington, 2004 ); 6 (Patel and Loeb, 2000 ); 7 (Patel et al, 2001 ); 8 (Suzuki et al, 1996 ); 9 (Ogawa et al, 2001 ); 10 (Ong et al, 2006 ); 11 (Xia et al, 2002 ); 12 (Fa et al, 2004 ); 13 (Schultz et al, 2015 ); 14 (Shinkai et al, 2001 ); 15 (Pinheiro et al, 2012 ); 16 (Ghadessy et al, 2004 ); 17 (Laos et al, 2013 ); 18 (Loakes et al, 2009 ); 19 (Leconte et al, 2005 ); 20 (Ramsay et al, 2010 ); 21 (Hansen et al, 2011 ). …”

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

“…In previous studies, we evaluated the ability of three previously evolved mutant enzymes (each evolved to recognize 2′‐modified nucleoside triphosphates) to synthesize five different types of 2′‐modified DNA . SFM19 is significantly more effective at synthesizing all five types of modified DNA than two other previously evolved enzymes, AA40 and SFR3 .…”

“…Here, we observe M‐DNA synthesis well beyond the eight‐nucleotide limit common for 2′ M‐DNA. All three first‐generation Taq mutants and early Tgo mutants showed significant pausing after the addition of six modified nucleotides; this was hypothesized to be caused by a conformational shift in the duplex following synthesis of this length . As the polymerase only contacts approximately eight nucleotides of the newly synthesized duplex at a time, as well as the fact that polymerases synthesizing beyond the six nucleotide barrier can routinely synthesize M‐DNA as long as 50–100 nt, we did not test for synthesis beyond 22 nucleotides.…”

“…However, these mutant enzymes cannot synthesize M‐DNA longer than eight nucleotides, thus severely limiting their applicability. Recently, we evaluated the catalytically active Taq C‐terminal Stoffel fragment (Sf) of these M‐DNA pols by comparative biochemical characterization . These studies revealed that one, SFM19, is capable of recognizing a wide range of M‐NTP substrates with relatively high efficiency, thus making it the most promising for further development.…”

G-quadruplex-based aptamers against protein targets in therapy and diagnostics

“…Among the methods developed for expanding the repertoire of nucleobase-modified aptamers, recently Mayer et al introduced click chemistry protocols to generate a novel class of modified aptamers, termed "clickmers", fully compatible with the common steps of established in vitro selection procedures (click-SELEX) [23]. Many enzymes that can "read" or "write" aptamer sequences containing unnatural nucleotides are available [20][21][22][23][24][25][26] and methods that identify the precise location of these nucleotides within an aptamer sequence are known as well [27][28][29].…”