解旋酶与 G-四链体结合和解旋的动力学测量。
Kinetics measurements of G-quadruplex binding and unfolding by helicases.
机构信息
Department of Molecular Biosciences, University of Texas at Austin, Austin TX 78712, United States.
Department of Molecular Biosciences, University of Texas at Austin, Austin TX 78712, United States.
出版信息
Methods. 2022 Aug;204:1-13. doi: 10.1016/j.ymeth.2022.04.012. Epub 2022 Apr 25.
G-quadruplex structures (G4s) form readily in DNA and RNA and play diverse roles in gene expression and other processes, and their inappropriate formation and stabilization are linked to human diseases. G4s are inherently long-lived, such that their timely unfolding depends on a suite of DNA and RNA helicase proteins. Biochemical analysis of G4 binding and unfolding by individual helicase proteins is important for establishing their levels of activity, affinity, and specificity for G4s, including individual G4s of varying sequence and structure. Here we describe a set of simple, accessible methods in which electrophoretic mobility shift assays (EMSA) are used to measure the kinetics of G4 binding, dissociation, and unfolding by helicase proteins. We focus on practical considerations and the pitfalls that are most likely to arise when these methods are used to study the activities of helicases on G4s.
G-四链体结构（G4s）在 DNA 和 RNA 中易于形成，并在基因表达和其他过程中发挥着多样化的作用，其形成和稳定的不当与人类疾病有关。G4s 本质上是长寿命的，因此它们的适时展开取决于一系列 DNA 和 RNA 解旋酶蛋白。对单个解旋酶蛋白与 G4 结合和解折叠的生化分析对于确定其 G4 结合的活性、亲和力和特异性水平很重要，包括具有不同序列和结构的单个 G4。在这里，我们描述了一组简单、可访问的方法，其中电泳迁移率变动分析（EMSA）用于测量解旋酶蛋白对 G4 的结合、解离和展开的动力学。我们重点介绍了在使用这些方法研究解旋酶在 G4 上的活性时最有可能出现的实际考虑因素和陷阱。
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