Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments

Jazi, Atieh Aminian; Plötz, Evelyn; Arizki, Muhamad; Dhandayuthapani, Balasubramaniam; Waclawska, Izabela; Kramer, Reinhard; Ziegler, Christine und Cordes, Thorben (2017): Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments. In: Biochemistry, Bd. 56, Nr. 14: S. 2031-2041

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 Forster 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.

• Dokumententyp: Zeitschriftenartikel
• EU Funded Grant Agreement Number: 638536
• EU-Projekte: Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638536: SM-IMPORT - Substrate import at work: single-molecule studies of ABC transporters
• Fakultät: Chemie und Pharmazie > Department Chemie
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie ; 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 0006-2960
• Sprache: Englisch