Structural dynamics of nucleic acids by single-molecule FRET

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.