title: Riboswitch structure and dynamics by smFRET microscopy.
abstract: Riboswitches are structured noncoding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single-molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy for smFRET studies of the structure, dynamics, and ligand-binding mechanisms of riboswitches.
authors.affiliation: Biophysics, University of Michigan, Ann Arbor, Michigan, USA; Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Krishna C
authors.initials: KC
authors.lastname: Suddala
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA. Electronic address: nwalter@umich.edu.
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D004867:Equipment Design; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009690:Nucleic Acid Conformation; D059370:RNA Folding; D058928:Riboswitch
chemical_list: D058928:Riboswitch
keywords: Conformation; Dynamics; Folding; Riboswitches; smFRET