Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity.

Nature Chemistry
Gillian HoulihanPhilipp Holliger

Abstract

The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2'-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries D-altritol nucleic acid and 2'-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.

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Citations

Jul 22, 2020·Nature Chemistry·Melanie Henkel, Andreas Marx
Sep 11, 2020·Organic & Biomolecular Chemistry·Kasper M BeckPoul Nielsen
Oct 18, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Steven Ochoa, Valeria T Milam
Oct 3, 2020·The Journal of Biological Chemistry·Zahra OuarayNigel G J Richards
Feb 4, 2021·Accounts of Chemical Research·John C Chaput
Oct 22, 2020·Chemical Society Reviews·Nils KlöckerAndrea Rentmeister
Mar 2, 2021·Chemical Society Reviews·Luke K McKenzieMarcel Hollenstein
Jan 18, 2021·The Journal of Biological Chemistry·Zahra OuarayNigel G J Richards
Mar 18, 2021·Advanced Biology·Yang ZhangShuobo Shi
May 25, 2021·ACS Synthetic Biology·Esau MedinaJohn C Chaput
Jun 15, 2021·Biochemistry·Thomas W ChristyKevin M Weeks

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Methods Mentioned

BETA
PCR
FACS
fluorescent
activated bead sorting
antisense oligonucleotides

Software Mentioned

MuLV
CBL

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