Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity
Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity
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DOI：
10.1038/s41557-020-0502-8
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发表时间：
2020-07-20
期刊：
影响因子：
21.8
通讯作者：
Holliger, Philipp
中科院分区：
文献类型：
--
作者：
Houlihan, Gillian;Arangundy-Franklin, Sebastian;Holliger, Philipp
逆转录酶(RT)从天然RNA和一系列非天然异种核酸(XNA)模板化学合成互补DNA的能力，是分子和合成遗传学的关键方法。然而，事实证明，RTS的发现和工程具有挑战性，特别是对于更具分歧的XNA化学。在这里，我们描述了一种用于任何模板化学的RT功能的定向进化的一般策略，称为区隔微珠标记，并通过2‘-O-甲基RNA和己醇核酸的有效RT的定向进化以及对于以前没有RT S的孤立的XNA化学物质d-altritol核酸和2’-甲氧基乙基RNA的RT的发现来展示它。最后，我们描述了具有主动外切校对的XNA RT S的工程，以及即使在没有校对的情况下，具有非常高互补DNA合成保真度的RNA RTS的定向进化。
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.