Issue 8, 2022

Mutant polymerases capable of 2′ fluoro-modified nucleic acid synthesis and amplification with improved accuracy

Abstract

Nonnatural nucleic acids (xeno nucleic acids, XNA) can possess several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize XNA, so, in recent years mutant XNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approach natural error rates and make XNA polymerases useful for applications that require high fidelity. Here, we systematically evaluate leading Taq DNA polymerase mutants for their fidelity during synthesis of 2′F XNA. To further improve their accuracy, we add mutations that have been shown to increase the fidelity of wild-type Taq polymerases, to some of the best current XNA polymerases (SFM4–3, SFM4–6, and SFP1). The resulting polymerases show significant improvements in synthesis accuracy. In addition to generating more accurate XNA polymerases, this study also informs future polymerase engineering efforts by demonstrating that mutations that improve the accuracy of DNA synthesis may also have utility in improving the accuracy of XNA synthesis.

Graphical abstract: Mutant polymerases capable of 2′ fluoro-modified nucleic acid synthesis and amplification with improved accuracy

Supplementary files

Article information

Article type
Paper
Submitted
28 Feb 2022
Accepted
16 Jun 2022
First published
17 Jun 2022
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2022,3, 1044-1051

Mutant polymerases capable of 2′ fluoro-modified nucleic acid synthesis and amplification with improved accuracy

T. A. Christensen, K. Y. Lee, S. Z. P. Gottlieb, M. B. Carrier and A. M. Leconte, RSC Chem. Biol., 2022, 3, 1044 DOI: 10.1039/D2CB00064D

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