title: Structural dynamics of nucleic acids by single-molecule FRET.
abstract: 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.
authors: Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.
authors: Asger Christian
authors: AC
authors: Krüger
authors: Lasse Lava
authors: LL
authors: Hildebrandt
authors: Sofie Louise
authors: SL
authors: Kragh
authors: Victoria
authors: V
authors: Birkedal
mesh_terms: D002021:Buffers; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D061925:Immobilized Nucleic Acids / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D012996:Solutions
chemical_list: D002021:Buffers; D005456:Fluorescent Dyes; D061925:Immobilized Nucleic Acids; D012996:Solutions; D012313:RNA; D004247:DNA