Medina Esau, Yik Eric J, Herdewijn Piet, Chaput John C
KU Leuven, Rega Institute for Medical Research, Herestraat 49-bus 1041, 3000 Leuven, Belgium.
ACS Synth Biol. 2021 Jun 18;10(6):1429-1437. doi: 10.1021/acssynbio.1c00048. Epub 2021 May 24.
Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2'-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability. Bst DNA polymerase was found to have weak reverse transcriptase activity on ANA and uncontrolled reverse transcriptase activity on HNA, differing from its known recognition of FANA and TNA templates. These data benchmark the activity of current XNA polymerases and provide opportunities for generating new polymerase variants that function with greater activity and substrate specificity.
人工遗传聚合物（XNAs）作为合成生物学、生物技术和分子医学的新材料具有巨大潜力；然而，对于已开发用于合成和逆转录XNAs聚合物的XNA聚合酶的生化特性却知之甚少。在此，我们比较了通过实验室进化建立的用于合成RNA、2'-氟阿拉伯糖核酸（FANA）、阿拉伯糖核酸（ANA）、己糖醇核酸（HNA）、苏糖核酸（TNA）和膦酰甲基苏糖核酸（PMT）的聚合酶的底物特异性、热稳定性、逆转录酶活性和保真度。我们发现，为促进XNA合成而获得的突变增加了酶对糖修饰底物的耐受性，但在一定程度上牺牲了蛋白质折叠稳定性。发现Bst DNA聚合酶对ANA具有较弱的逆转录酶活性，对HNA具有不受控制的逆转录酶活性，这与其对FANA和TNA模板的已知识别不同。这些数据为当前XNA聚合酶的活性提供了基准，并为生成具有更高活性和底物特异性的新聚合酶变体提供了机会。
