While DNA is one of the most versatile and valuable molecules in biotechnology due to its ability to encode information, it is limited by its susceptibility to nuclease degradation. The development of modified DNA is a rapidly growing field in biotechnology which aims to develop modified-DNA (M-DNA) biopolymers that can encode information but which are not easily degraded by nucleases. Here, we describe our efforts to engineer and discover enzymes capable of accurate synthesis of 2'F M-DNA under mild conditions. Our most promising enzyme, SFP1, can synthesize M-DNA at substoichiometric enzyme concentrations, in less than one hour, without manganese or other additives. We also describe our efforts to identify commercially available DNA polymerases which are capable of reverse transcribing 2'F M-DNA accurately and efficiently to enable study and applications of 2'F M-DNA. Our most promising enzyme, Q5, can, in a one-pot reaction taking less than one hour, reverse transcribe and amplify 2'F M-DNA with minimal errors. Collectively, we have developed a system that is robust, requires mild conditions, and largely relies on commercially available materials, which should broadly enable M-DNA applications.

Efficient, robust, and accurate synthesis and reverse transcription of 2'F modified DNA

ACS Spring Meeting 2021

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