Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, München 81377, Germany.
Nucleic Acids Res. 2022 Apr 8;50(6):e31. doi: 10.1093/nar/gkab1212.
DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case-specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq, and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease, and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays and showed comparable performance to commercial hot-start enzymes.
DNA 加工酶，如 DNA 聚合酶和内切核酸酶，在生物技术、分子诊断和合成生物学等领域有广泛的应用。具有可控活性的酶的开发，如热启动或光激活版本，提高了它们的应用，提高了现有酶的灵敏度和特异性。然而，目前生产可控酶的方法在实验上开发要求高，且针对特定情况。在这里，我们介绍了一种设计光启动 DNA 加工酶的简单而通用的方法。为了证明其通用性，我们将该方法应用于生物技术中常用的三种 DNA 聚合酶，包括嗜热 Phi29（mesophilic）聚合酶、Taq 聚合酶和 Pfu 聚合酶，以及一种限制酶。光启动酶在 UV 脉冲重新激活之前，表现出抑制聚合酶、外切核酸酶和内切核酸酶的活性。最后，我们将我们的酶应用于常见的分子生物学检测中，表现出与商业热启动酶相当的性能。
