期刊
NATURE CHEMISTRY
卷 12, 期 8, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41557-020-0502-8
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资金
- Medical Research Council (MRC) [MC_U105178804]
- UK Biotechnology and Biological Sciences Research Council [09-EuroSYNBIO-OP-013]
- AstraZeneca UK
- Medical Research Council
- MRC [MC_U105178804] Funding Source: UKRI
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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