Milligan John N, Shroff Raghav, Garry Daniel J, Ellington Andrew D
Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences , University of Texas , 2500 Speedway , Austin , Texas 78712 , United States.
Biochemistry. 2018 Aug 7;57(31):4607-4619. doi: 10.1021/acs.biochem.8b00200. Epub 2018 Apr 18.
Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.
链置换聚合酶是等温扩增（IA）反应的关键组成部分，在等温扩增反应中，无需热循环可减少设备需求并缩短检测时间，这在即时检测应用中尤为重要。为了改善链置换聚合酶的功能，我们开发了一种基于乳液的定向进化方案——高温等温分隔式自我复制（HTI-CSR），该方案不依赖热循环。我们从嗜热栖热放线菌（Bst LF）和嗜热水生栖热菌（Klentaq）的核酸外切酶缺陷型A家族聚合酶的算法优化改组文库出发，应用HTI-CSR生成了一种更耐热的链置换聚合酶变体，该变体在环介导等温扩增和滚环扩增中表现良好，即使在高达95°C的热激处理后仍能实现更好的引物退火。这种新酶（v5.9）还能够进行多种新反应，包括等温超分支滚环扩增。现在，HTI-CSR方法应被证明有助于在链置换聚合酶中进化出更多有益的表型。
