Characterization of Dark Quencher Chromophores as Nonfluorescent Acceptors for Single-Molecule FRET

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• 作者：Ludovic Le Reste ; ^{l.lereste1@physics.ox.ac.uk} ; Johannes Hohlbein ; Kristofer Gryte ; Achillefs  ; N. Kapanidis ; ^{a.kapanidis1@physics.ox.ac.uk}
• 刊名：Biophysical Journal
• 出版年：2012
• 出版时间：6 June, 2012
• 年：2012
• 卷：102
• 期：11
• 页码：2658-2668
• 全文大小：1206 K

文摘

Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for F?rster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 ¦ÌM or more without introducing a significant background, the use of dark quenchers should permit single-molecule F?rster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.