Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology
Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology
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DOI：
10.1021/acssynbio.1c00048
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发表时间：
2021-05-24
影响因子：
4.7
通讯作者：
Chaput, John C.
中科院分区：
文献类型：
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作者：
Medina, Esau;Yik, Eric J.;Chaput, John C.
人工遗传聚合物（XNA）作为合成生物学、生物技术和分子医学的新材料具有巨大的潜力;然而，对于已经开发用于合成和逆转录XNA聚合物的XNA聚合酶的生物化学性质知之甚少。在这里，我们比较了底物特异性，热稳定性，逆转录酶活性，和保真度的实验室进化的聚合酶，建立了合成RNA，2 '-氟阿拉伯核酸（FANA），阿拉伯核酸（ANA），己糖醇核酸（HNA），苏糖核酸（TNA），和膦酰基甲基苏糖核酸（PMT）。我们发现，获得促进XNA合成的突变增加了酶对糖修饰底物的耐受性，同时牺牲了蛋白质折叠的稳定性。发现Bst DNA聚合酶对ANA具有弱的逆转录酶活性，而对HNA具有不受控制的逆转录酶活性，这与其已知的对FANA和TNA模板的识别不同。这些数据对当前XNA聚合酶的活性进行了基准测试，并为产生具有更大活性和底物特异性的新聚合酶变体提供了机会。
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.