Department of Chemistry, University of Zurich, Zurich, Switzerland.
Laserinstitut Hochschule Mittweida, University of Applied Sciences Mittweida, Mittweida, Germany.
Methods Mol Biol. 2022;2439:173-190. doi: 10.1007/978-1-0716-2047-2_12.
Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).
单分子显微镜常用于观察和描述核酸（NA）的构象动力学。由于 NA 结构种类繁多，且单分子观察技术具有独特的挑战，因此此类实验中记录的数据必须经过多次统计处理，最终才能得到 NA 动力学的可靠机制观点。在本章中，我们提出了一种综合方案，用于分析单分子Förster 共振能量转移（FRET）显微镜范围内的单分子轨迹。该方案可生成研究样本中所有分子共有的构象状态，以及相关的构象转变动力学。所提出的模型可解析通过观察 FRET 信号无法区分的状态，并使用 FRET 状态和转变速率常数的误差计算以 95%置信度进行估计。最后，提供了分步用户指南，以使用多功能分析软件处理单分子 FRET 数据（MASH-FRET）重现该方案。
