School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
Methods Mol Biol. 2021;2209:175-191. doi: 10.1007/978-1-0716-0935-4_12.
Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe the magnetic tweezers-based single-molecule investigation of the binding of helicases to G-quadruplex structures and their ATP-dependent unwinding activity, using DHX36 (also known as RHAU and G4R1) helicase and a DNA G-quadruplex structure for an example. We specifically emphasize on the principle and method to probe the interactions between DHX36 and the DNA G-quadruplex in different intermediate states during an ATPase cycle of DHX36, based on detecting the DHX36-induced changes in the lifetime of the DNA G-quadruplex under tension. The principle of the measurement can be broadly extended to the studies of other DNA or RNA G-quadruplex helicases.
单分子操纵方法是探测蛋白质和核酸结构相互作用的有用技术。在这里，我们以 DHX36（也称为 RHAU 和 G4R1）解旋酶和 DNA G-四链体结构为例，描述了基于磁镊的单分子研究解旋酶与 G-四链体结构的结合及其 ATP 依赖性解旋活性。我们特别强调了在 DHX36 的 ATP 酶循环过程中探测 DHX36 与 DNA G-四链体之间相互作用的原理和方法，该方法基于检测在张力下 DHX36 诱导的 DNA G-四链体寿命变化。该测量原理可以广泛扩展到其他 DNA 或 RNA G-四链体解旋酶的研究。
