核酸
核糖核酸
校对
化学
逆转录酶
定向进化
计算生物学
核酸结构
聚合酶
DNA
生物化学
生物
基因
突变体
作者
Gillian Houlihan,Sebastian Arangundy‐Franklin,Benjamin T. Porebski,Nithya Subramanian,Alexander I. Taylor,Philipp Holliger
出处
期刊:Nature Chemistry
[Springer Nature]
日期:2020-07-20
卷期号:12 (8): 683-690
被引量:48
标识
DOI:10.1038/s41557-020-0502-8
摘要
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. Engineering reverse transcriptases for modified or unnatural nucleic acids is challenging, but now a versatile method has been developed that enables the discovery of efficient reverse transcriptases. The method works with a wide range of template structures, including xeno-nucleic acids and can also be used to produce high-fidelity reverse transcriptases for RNA.
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