title: Monitoring multiple distances within a single molecule using switchable FRET.
abstract: The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.
authors.affiliation: Biological Physics Research Group, Department of Physics, University of Oxford, Oxford, UK.
authors.forename: Stephan
authors.initials: S
authors.lastname: Uphoff
authors.forename: Seamus J
authors.initials: SJ
authors.lastname: Holden
authors.forename: Ludovic
authors.initials: L
authors.lastname: Le Reste
authors.forename: Javier
authors.initials: J
authors.lastname: Periz
authors.forename: Sebastian
authors.initials: S
authors.lastname: van de Linde
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D019921:Biotinylation; D003198:Computer Simulation; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
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