Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Nat Protoc. 2015 Oct;10(10):1625-42. doi: 10.1038/nprot.2015.104. Epub 2015 Sep 24.
This protocol describes the directed evolution of artificial endonuclease and ligase enzymes composed of synthetic genetic polymers (XNAzymes), using 'cross-chemistry selective enrichment by exponential amplification' (X-SELEX). The protocol is analogous to (deoxy)ribozyme selections, but it enables the development of fully substituted catalysts. X-SELEX is initiated by the synthesis of diverse repertoires (here 10(14) different sequences), using xeno nucleic acid (XNA) polymerases, on DNA templates primed with DNA, RNA or XNA oligonucleotides that double as substrates, allowing selection for XNA-catalyzed cleavage or ligation. XNAzymes are reverse-transcribed into cDNA using XNA-dependent DNA polymerases, and then PCR-amplified to generate templates for subsequent rounds or deep sequencing. We describe methods developed for four XNA chemistries, arabino nucleic acids (ANAs), 2'-fluoroarabino nucleic acids (FANAs), hexitol nucleic acids (HNAs) and cyclohexene nucleic acids (CeNAs), which require ∼1 week per round, and typically 10-20 rounds; in principle, these methods are scalable and applicable to a wide range of novel XNAzyme chemistries, substrates and reactions.
本方案描述了使用“交叉化学选择富集指数扩增（X-SELEX）”对人工内切酶和连接酶酶进行定向进化的方法，这些酶由合成遗传聚合物（XNAzymes）组成。该方案类似于（脱氧）核酶选择，但它能够开发出完全取代的催化剂。X-SELEX 通过使用异源核酸（XNA）聚合酶在 DNA 模板上合成各种文库（此处为 10^14 种不同序列）开始，这些模板由 DNA、RNA 或 XNA 寡核苷酸引发，这些寡核苷酸既可以作为底物，也可以作为选择 XNA 催化切割或连接的试剂。XNAzymes 使用 XNA 依赖性 DNA 聚合酶逆转录成 cDNA，然后进行 PCR 扩增，以生成模板，用于后续的循环或深度测序。我们描述了为四种 XNA 化学物质（阿拉伯糖核酸（ANAs）、2'-氟阿拉伯糖核酸（FANAs）、己糖醇核酸（HNAs）和环己烯核酸（CeNAs））开发的方法，每个循环大约需要 1 周，通常需要 10-20 个循环；原则上，这些方法是可扩展的，并适用于广泛的新型 XNAzyme 化学物质、底物和反应。
