Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase,ACS Catalysis

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Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase
ACS Catalysis ( IF 11.3 ) Pub Date : 2022-02-18 , DOI: 10.1021/acscatal.1c04879
Xiaoyun Lu _{1,

2,

3,

4} , Jinlong Li _{1,

2} , Congyu Li _{1,

5} , Qianqian Lou _{1,

2} , Kai Peng _{1,

2} , Bijun Cai _{1,

2} , Ying Liu _{1,

2} , Yonghong Yao _{1,

2} , Lina Lu _{1,

2} , Zhenyang Tian _{1,

2} , Hongwu Ma _{1,

2} , Wen Wang ₃ , Jian Cheng _{1,

2} , Xiaoxian Guo _{1,

2} , Huifeng Jiang _{1,

2} , Yanhe Ma _{1,

2}

Affiliation

Template-free enzymatic approaches are considered the most promising solution for next-generation artificial DNA synthesis. However, the development of these technologies has been hampered by the lack of efficient enzymes specialized for stepwise nucleotide addition. By combining evolutionary analysis, high-throughput mutagenesis scanning, and rational design, we identified a terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3′-ONH₂-modified nucleotides. The catalytic activity of the engineered ZaTdT for 3′-ONH₂-dNTPs is 3 orders of magnitude higher than that of the commonly used mammalian TdT. The engineered ZaTdT enables highly efficient single-nucleotide extension of the growing oligonucleotide chain with an average stepwise yield of 98.7%, which makes it practical for de novo enzymatic DNA synthesis.

中文翻译：

工程末端脱氧核苷酸转移酶酶促 DNA 合成

无模板酶法被认为是下一代人工 DNA 合成最有希望的解决方案。然而，由于缺乏专门用于逐步添加核苷酸的有效酶，这些技术的发展受到了阻碍。通过结合进化分析、高通量诱变扫描和合理设计，我们从Zonotrichia albicollis (ZaTdT) 中鉴定出一种末端脱氧核苷酸转移酶，并重塑其催化腔以更好地适应 3'-ONH ₂修饰的核苷酸。工程化 ZaTdT 对 3'-ONH _{2的催化活性}-dNTPs 比常用的哺乳动物 TdT 高 3 个数量级。工程化的 ZaTdT 能够以 98.7% 的平均逐步产率对生长中的寡核苷酸链进行高效的单核苷酸延伸，这使其可用于从头酶促 DNA 合成。

更新日期：2022-02-18

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