Howard Hughes Medical Institute, Cornell University, Ithaca, NY, USA.
Laboratory of Atomic and Solid State Physics (LASSP), Department of Physics, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2022;2478:37-73. doi: 10.1007/978-1-0716-2229-2_4.
Angular optical trapping (AOT) is a powerful technique that permits direct angular manipulation of a trapped particle with simultaneous measurement of torque and rotation, while also retaining the capabilities of position and force detection. This technique provides unique approaches to investigate the torsional properties of nucleic acids and DNA-protein complexes, as well as impacts of torsional stress on fundamental biological processes, such as transcription and replication. Here we describe the principle, construction, and calibration of the AOT in detail and provide a guide to the performance of single-molecule torque measurements on DNA molecules. We include the constant-force method and, notably, a new constant-extension method that enables measurement of the twist persistence length of both extended DNA, under an extremely low force, and plectonemic DNA. This chapter can assist in the implementation and application of this technique for general researchers in the single-molecule field.
角向光镊（AOT）是一种强大的技术，它允许直接对角向捕获的粒子进行操纵，同时测量扭矩和旋转，同时保留位置和力检测的能力。这项技术为研究核酸和 DNA-蛋白质复合物的扭转特性以及扭转应力对转录和复制等基本生物过程的影响提供了独特的方法。在这里，我们详细描述了 AOT 的原理、结构和校准，并提供了在 DNA 分子上进行单分子扭矩测量的性能指南。我们包括恒力法，特别是一种新的恒伸长法，该方法可在极低的力下测量伸展 DNA 的扭转持久长度，以及扭结 DNA 的扭转持久长度。本章可以帮助单细胞领域的一般研究人员实施和应用这项技术。
