title: Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments.
abstract: Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor-acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.|Institute of Biophysics and Biophysical Chemistry, Universität Regensburg , 95053 Regensburg, Germany.
authors.forename: Atieh Aminian
authors.initials: AA
authors.lastname: Jazi
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Ploetz
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Muhamad
authors.initials: M
authors.lastname: Arizki
authors.affiliation: Institute of Biophysics and Biophysical Chemistry, Universität Regensburg , 95053 Regensburg, Germany.
authors.forename: Balasubramaniam
authors.initials: B
authors.lastname: Dhandayuthapani
authors.affiliation: Institute of Biophysics and Biophysical Chemistry, Universität Regensburg , 95053 Regensburg, Germany.
authors.forename: Izabela
authors.initials: I
authors.lastname: Waclawska
authors.affiliation: Institute for Biochemistry, Universität Köln , 50674 Köln, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Krämer
authors.affiliation: Institute of Biophysics and Biophysical Chemistry, Universität Regensburg , 95053 Regensburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Ziegler
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Thorben
authors.identifier: 0000-0002-8598-5499
authors.initials: T
authors.lastname: Cordes
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry*; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D007700:Kinetics; D008027:Light; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D055668:Photochemical Processes; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry*; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D010720:Phosphines; D011994:Recombinant Proteins; D012235:Rhodamines; D027981:Symporters; C101652:BetP protein, Corynebacterium glutamicum; C080938:tris(2-carboxyethyl)phosphine