FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)

IF 11.3 1区化学 Q1 CHEMISTRY, PHYSICAL

ACS Catalysis Pub Date : 2025-03-18 DOI:10.1021/acscatal.4c07614

Xiao Li, Qiongwei Tang, Jingjie Jiang, Yuepeng Shang, Zelin Lu, Mingli Chen, Jiajia He, Feng Liu, Sisi Zhu, Zengping Zhang, Hui Han, Xixi Yu, Qiuxian Li, Yuansong Xiu, Yuhong Yang, Ping Gui, Xuefeng Wang, Feng Lu, Wei Jing, Langping Xu, Yanna Lin, Xinglong Wang, Shu Quan, Xiang Liu, Huancai Yin, Fuqiang Ma

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Abstract

Bst DNA polymerase is a key enzyme used in both molecular diagnosis and scientific research. Employing the fluorescence-activated droplet sorting (FADS) technique, we successfully evolved a suboptimal wild-type Bst DNA polymerase into practically valuable mutants through directed evolution. The mutants exhibited significantly improved thermostability and strand displacement capability, enabling much better loop-mediated isothermal amplification (LAMP) performance, with a faster reaction speed (reduced from 40 to 10 min) and a highly stable solid reagent that remained stable for 2 months at 50 °C. Moreover, these robust mutants facilitated high-temperature LAMP assays at 70 °C, thereby eliminating the common issue of false positives in LAMP assays. To better understand the molecular mechanism behind the strand displacement capability, we proposed the strand displacement index (SDI) as a parameter to quantify this property. We also proposed the “hydrophobic blade” hypothesis, providing insights into the mechanism underlying enhanced strand displacement capability. This work serves as a successful example of molecular engineering and LAMP applications of the Bst DNA polymerase.

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来源期刊

ACS Catalysis CHEMISTRY, PHYSICAL-

CiteScore

20.80

自引率

6.20%

发文量

1253

审稿时长

1.5 months

期刊介绍： ACS Catalysis is an esteemed journal that publishes original research in the fields of heterogeneous catalysis, molecular catalysis, and biocatalysis. It offers broad coverage across diverse areas such as life sciences, organometallics and synthesis, photochemistry and electrochemistry, drug discovery and synthesis, materials science, environmental protection, polymer discovery and synthesis, and energy and fuels. The scope of the journal is to showcase innovative work in various aspects of catalysis. This includes new reactions and novel synthetic approaches utilizing known catalysts, the discovery or modification of new catalysts, elucidation of catalytic mechanisms through cutting-edge investigations, practical enhancements of existing processes, as well as conceptual advances in the field. Contributions to ACS Catalysis can encompass both experimental and theoretical research focused on catalytic molecules, macromolecules, and materials that exhibit catalytic turnover.