title: Conformational flexibility of viral RNA switches studied by FRET.
abstract: The function of RNA switches involved in the regulation of transcription and translation relies on their ability to adopt different, structurally well-defined states. A new class of ligand-responsive RNA switches, which we recently discovered in positive strand RNA viruses, are distinct from conventional riboswitches. The viral switches undergo large conformational changes in response to ligand binding while retaining the same secondary structure in their free and ligand-bound forms. Here, we describe FRET experiments to study folding and ligand binding of the viral RNA switches. In addition to reviewing previous approaches involving RNA model constructs which were directly conjugated with fluorescent dyes, we outline the design and application of new modular constructs for FRET experiments, in which dye labeling is achieved by hybridization of a core RNA switch module with universal DNA fluorescent probes. As an example, folding and ligand binding of the RNA switch from the internal ribosome entry site of hepatitis C virus is studied comparatively with conventional and modular FRET constructs.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Boerneke
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States; Center for Drug Discovery Innovation, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States. Electronic address: tch@ucsd.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hermann
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D058928:Riboswitch
chemical_list: D012367:RNA, Viral; D058928:Riboswitch
keywords: FRET; Hepatitis C virus; IRES; RNA switch; RNA virus