Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers.
Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers.
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从多种合成遗传聚合物中定向进化人工酶（XNAzymes）。
DOI：
10.1038/nprot.2015.104
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发表时间：
2015
期刊：
影响因子：
14.8
通讯作者：
Taylor AI
中科院分区：
文献类型：
--
作者：
Taylor AI
该方案描述了人工内切酶和连接酶组成的合成遗传聚合物（XNAzymes）的定向进化，使用“交叉化学选择性富集指数扩增”（X-SELEX）。该协议类似于（脱氧）核酶选择，但它使开发完全取代的催化剂成为可能。X-SELEX是通过使用xeno核酸（XNA）聚合酶，在DNA模板上以DNA、RNA或XNA寡核苷酸作为底物引物，合成多种序列（这里有1014种不同的序列）而启动的，允许选择XNA催化的切割或连接。XNAzymes使用依赖于xna的DNA聚合酶反向转录成cDNA，然后用pcr扩增生成模板，用于后续轮次或深度测序。我们描述了针对四种XNA化学物质，阿拉伯人核酸（ANAs）， 2 ' -氟阿拉伯人核酸（FANAs），己醇核酸（hna）和环己烯核酸（CeNAs）开发的方法，每轮需要~ 1周，通常需要10-20轮；原则上，这些方法是可扩展的，适用于广泛的新型XNAzyme化学，底物和反应。
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 1014different 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.