Used in a wide range of experimental assays, DNA is a valuable biotechnological tool because it is easily amplified without the loss of information. 2’ modifications of DNA impart nuclease resistance, thereby increasing the biological application of DNA. However, DNA polymerases are highly evolved to recognize only natural nucleotides, and therefore cannot effectively synthesize modified DNA. Current efforts are focused on further engineering the most active mutants of Thermus aquaticus DNA polymerase I (Taq) to enzymatically synthesize long polymers of modified DNA (M-DNA).<br/>In order to apply or utilize M-DNA or apply secondary amplification applications, M-DNA dependent DNA polymerases must be able to competently reverse transcribe M-DNA into natural DNA. A library of rationally designed polymerases mutants were generated by site directed mutagenesis and the proteins were assessed for their reverse transcription activity. In this study, four mutants showed significantly improved reverse transcription activity and give potential for further engineering and analysis on the specific mutations that impart unnatural function. This research will inform future M-DNA polymerase characterization, and improve the prospect of developing improved M-DNA dependent DNA polymerases.

Engineering Taq polymerases for the reverse transcription of 2' modified DNA

253rd National Meeting (2017)

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