title: Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.
abstract: Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, California 94305-4090, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Albert
authors.initials: A
authors.lastname: Tsai
authors.forename: Alexey
authors.initials: A
authors.lastname: Petrov
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008968:Molecular Conformation; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry / Q000235:genetics
chemical_list: D002232:Carbocyanines; C085321:cyanine dye 5; D012343:RNA, Transfer