RNA 和 XNA 逆转录酶功能和保真度的发现和进化
Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity
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.
逆转录酶 (RT) 能够从天然 RNA 和一系列非天然异种核酸 (XNA) 模板化学中合成互补 DNA，这是分子和合成遗传学中关键方法的基础。然而，RT 的发现和设计已证明具有挑战性，尤其是对于更多样化的 XNA 化学。
在这里，我们描述了一种用于定向进化任何模板化学的 RT 功能的通用策略，称为区室化珠子标记，并通过定向进化 2'- O-甲基 RNA 和己糖醇核酸的有效 RT 以及发现孤儿 XNA 化学d -altritol 核酸和 2'-甲氧基乙基 RNA 的 RT 来证明这一点，而之前没有 RT。最后，我们描述了具有主动核酸外切校对的 XNA RT 的设计，以及即使在没有校对的情况下也具有非常高的互补 DNA 合成保真度的 RNA RT 的定向进化。
