In nature, DNA polymerases synthesize DNA by incorporating nucleotides along a template strand. Although these enzymes are both accurate and efficient, they are limited because they only recognize natural nucleotides, which in turn limits the therapeutic and biotech applications of the resulting DNA. As a result, DNA polymerases have been engineered further to increase their promiscuity. This research assessed the ability of mutant Thermus aquaticus (Taq) DNA polymerases to recognize a 3’ modified nucleotide,<br/>3’O-Methyl-CTP. It was found that the most active Taq mutants could perform a single nucleotide incorporation of 3’-O-Methyl-CTP, while the WT could not. The enzymatic efficiency of Taq mutants to incorporate a single 3’-O-Methyl-CTP was also determined. This study aims to examine possible full template extension by more evolved Taq mutants of a ribonucleotide mix that includes a 3’-O-Methyl-NTP. Synthesis of DNA under these conditions would suggest that there are site-specific 2’-5’ linkages between nucleotides in the nucleic acid sequence, which would increase binding possibilities of nucleic acid ligands and enzymes.

Incorporation of 3’OMe-NTP by mutant DNA polymerases

253rd National Meeting (2017)

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