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.
机构信息
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.
Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK.
出版信息
Nat Chem. 2020 Aug;12(8):683-690. doi: 10.1038/s41557-020-0502-8. Epub 2020 Jul 20.
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）能够将天然 RNA 和多种非天然的异源核酸（XNA）模板化学物质合成互补 DNA，这为分子和合成遗传学的关键方法提供了基础。然而，RTs 的发现和工程改造一直具有挑战性，特别是对于更具差异的 XNA 化学物质。在这里，我们描述了一种针对任何模板化学物质的 RT 功能定向进化的通用策略，称为隔室化珠标记，并通过定向进化用于 2'-O-甲基 RNA 和己糖醇核酸的高效 RT 以及用于孤儿 XNA 化学物质 D-阿糖醇核酸和 2'-甲氧基乙基 RNA 的 RT 的发现证明了这一策略，对于后者，以前不存在 RT。最后，我们描述了具有活性外切核酸酶校对功能的 XNA RT 的工程改造，以及在没有校对的情况下，具有非常高的互补 DNA 合成保真度的 RNA RT 的定向进化。
Zotero 插件