The varying levels of a polymerase’s fidelity affect what biological applications it’s most suitable for. A panel of polymerases has been recently identified – P4, P6, P7, SFM4-3, SFM4-6, and SFM4-9 – that are able to synthesize 2’F M-DNA. This expands the uses of M-DNA as its utility is limited by the difficulty of polymerases to synthesize it. A side-by-side fidelity assay for the six promising mutant polymerases was performed where HT-Seq gave the errors per base pair for each of the six enzymes. It was found that P6 was the most accurate enzyme and position encoding cytosine had the highest error rate for all six polymerases. Mutations that increase the accuracy can be highly beneficial, but it is also important to assess whether other properties have been affected by the modifications. Thus, to begin to understand the relationship between efficiency and fidelity, we adapted and developed a kinetic profiling efficiency. The approach monitors 2’F M-DNA synthesis on a priming template using high-resolution melting (HRM) fluorescent dyes that are incorporated into the growing strand of M-DNA. Preliminary results using this developed method for a few enzymes have been collected. The findings of these experiments will help uncover the relationship between efficiency and fidelity as well as give insight into how certain mutations contribute to enhanced enzymatic function.

Evaluation of 2’fluoro M-DNA polymerase’s fidelity and efficiency

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.