DNA is a useful tool for biological applications as it can be amplified by DNA polymerases very quickly and without the loss of information. The effectiveness of DNA in these applications, however, is limited by the fact that DNA can be degraded by nucleases. Modifications at the 2’-position of the sugar can prevent the mechanism of the cleavage of the phosphate backbone. Unfortunately, native DNA polymerases cannot synthesize any length of DNA with 2’-modified nucleotide triphosphates. Mutant DNA polymerases have been evolved for their ability to synthesize DNA with 2’-modified nucleotide triphosphates, but cannot perform full-length synthesis, are highly inefficient, or have low fidelity. SFM19 is the best-characterized and most promising Taq pol mutant for further engineering to date, but cannot incorporate more than 6 to 8 modified nucleotides. In an effort to improve the ability of SFM19 to extend 2’-modified DNA, we cloned a library of ten novel SFM19 mutants that encoded additional mutations found in the literature to increase recognition of a modified DNA duplex, improve DNA binding affinity or processivity. From the library of novel SFM19 mutants, three novel polymerases with additional mutations, SFM19 x KR (E742K and A743R), SFM19 x RR (E742R and A743R), and SFM19 x SFM1 (K314R, E520G, F598L, A608V, and E742G), are more active than SFM19 when tested for their ability to incorporate both ribonucleotides (rNTPs) and 2’-fluoro nucleotide triphosphates (2’-FNTPs). The novel SFM19 mutants generated in this study demonstrate significantly improved activity compared to SFM19 with both rNTP and 2’-FNTP substrates above.

Rationally designing DNA polymerases for 2’-modified DNA synthesis

253rd National Meeting (2017)

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