Krüger Asger Christian, Hildebrandt Lasse Lava, Kragh Sofie Louise, Birkedal Victoria
Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.
Methods Cell Biol. 2013;113:1-37. doi: 10.1016/B978-0-12-407239-8.00001-X.
Single-molecule Förster Resonance Energy Transfer (smFRET) has emerged as a powerful technique to study biological processes at the molecular level. It provides detailed information on the structural dynamics of nucleic acids and proteins on the nanometer scale under a wide variety of conditions. Studying each molecule individually allows going beyond traditional ensemble averaging experiments. It permits to reveal the molecular conformational heterogeneity, to sort out molecules that have a defined conformation and to identify distinct dynamical events and relaxation pathways. This chapter outlines two protocols for performing smFRET measurements on immobilized nucleic acids using wide-field fluorescence microscopy. The first explains the steps needed for performing smFRET in static sample chambers, while the second describes the additional requirements for performing the measurements in a flow cell. Static smFRET measurements are easier to setup but are limited to one set of measurement conditions at a time; in contrast, flow measurements provide superior control of buffer conditions and the possibility to flow in ligands. Here, we provide detailed steps for DNA/RNA immobilization, sample chamber and buffer preparation.
单分子荧光共振能量转移（smFRET）已成为一种在分子水平上研究生物过程的强大技术。它能在各种条件下，在纳米尺度上提供有关核酸和蛋白质结构动力学的详细信息。对每个分子进行单独研究，超越了传统的总体平均实验。它能够揭示分子构象的异质性，筛选出具有特定构象的分子，并识别不同的动力学事件和弛豫途径。本章概述了两种使用宽场荧光显微镜对固定化核酸进行smFRET测量的方法。第一种方法解释了在静态样品池中进行smFRET所需的步骤，而第二种方法描述了在流动池中进行测量的额外要求。静态smFRET测量更容易设置，但一次仅限于一组测量条件；相比之下，流动测量能更好地控制缓冲条件，并有可能引入配体。在此，我们提供了DNA/RNA固定化、样品池和缓冲液制备的详细步骤。
