Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.
Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.
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DOI：
10.1038/nchem.2493
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发表时间：
2016-06
期刊：
影响因子：
21.8
通讯作者：
Romesberg FE
中科院分区：
文献类型：
--
作者：
Chen T;Hongdilokkul N;Liu Z;Adhikary R;Tsuen SS;Romesberg FE
寡核苷酸的PCR扩增使得能够进化称为适体的序列，所述适体以抗体样亲和力结合特异性靶标。然而，这些适体的使用在许多应用中受到核酸酶介导的降解的限制。相反，在其糖C2'位置用甲氧基或氟取代基修饰的寡核苷酸对核酸酶稳定，但不能通过天然聚合酶合成。在这里，我们报告了聚合酶进化系统的开发及其用于进化热稳定聚合酶的用途，所述热稳定聚合酶通过“转录”和“逆转录”有效地相互转化C2 '-OMe修饰的寡核苷酸及其DNA对应物，或者更重要的是，PCR扩增部分C2'-OMe或C2 '-F修饰的寡核苷酸。一个机械分析表明，扩增的修饰的寡核苷酸的能力是通过优化结构域间的相互作用，稳定的催化活性的聚合酶的封闭构象的演变。进化的聚合酶应该找到实际的应用和开发的进化系统应该是一个强大的工具，聚合酶的剪裁有其他类型的新功能。DNA聚合酶可以有效地PCR扩增天然DNA，但不能利用C2 '修饰的底物。通过选择和筛选的组合，斯托费尔片段DNA聚合酶进化为从DNA模板“转录”C2 '修饰的DNA，将C2'修饰的DNA“逆转录”回DNA，并PCR扩增C2 '修饰的DNA。
The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, the use of these aptamers is limited in many applications by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases but cannot be synthesized by natural polymerases. Here, we report the development of a polymerase evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe modified oligonucleotides and their DNA counterparts via “transcription” and “reverse transcription,” or more importantly, PCR amplify partially C2'-OMe or C2'-F modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides was evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for the tailoring of polymerases to have other types of novel function. DNA polymerase can PCR amplify natural DNA efficiently, but cannot utilize C2’-modified substrates. Via a combination of selection and screening, Stoffel fragment DNA polymerase was evolved to “transcribe” C2’-modified-DNA from a DNA template, “reverse transcribe” C2’-modified-DNA back into DNA, and PCR amplify C2’-modified DNA.