title: Analysis of RNA folding and ribonucleoprotein assembly by single-molecule fluorescence spectroscopy.
abstract: To execute their diverse range of biological functions, RNA molecules must fold into specific tertiary structures and/or associate with one or more proteins to form ribonucleoprotein (RNP) complexes. Single-molecule fluorescence spectroscopy is a powerful tool for the study of RNA folding and RNP assembly processes, directly revealing different conformational subpopulations that are hidden in conventional ensemble measurements. Moreover, kinetic processes can be observed without the need to synchronize a population of molecules. In this chapter, we describe the fluorescence spectroscopic methods used for single-molecule measurements of freely diffusing or immobilized RNA molecules or RNA-protein complexes. We also provide practical protocols to prepare the fluorescently labeled RNA and protein molecules required for such studies. Finally, we provide two examples of how these various preparative and spectroscopic methods are employed in the study of RNA folding and RNP assembly processes.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Goran
authors.initials: G
authors.lastname: Pljevaljčić
authors.forename: Rae
authors.initials: R
authors.lastname: Robertson-Anderson
authors.forename: Edwin
authors.initials: E
authors.lastname: van der Schans
authors.forename: David
authors.initials: D
authors.lastname: Millar
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D015752:Genes, env; D006801:Humans; D055029:Inverted Repeat Sequences; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059370:RNA Folding; D016337:RNA, Catalytic / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; D012261:Ribonucleoproteins; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1; D012313:RNA