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.

逆转录酶（RTs）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的能力巩固了分子遗传学和合成遗传学中的关键方法。但是，事实证明，RT很难发现和设计，尤其是对于XNA化学种类更多的化学品。在这里，我们描述了针对任何称为模板化小珠标记的模板化学，RT功能的定向进化的一般策略，并通过2'- O-甲基RNA和己糖醇核酸的有效RT的定向进化以及对RT的发现来证明它。孤XNA化学品d-altroi​​tol核酸和2'-甲氧基乙基RNA，以前没有RTs。最后，我们描述了具有主动核酸外切法校正的XNA RT的工程设计，以及具有非常高的互补DNA合成保真度的RNA RT的定向进化，即使在没有校正的情况下。