Characterization of dark quencher chromophores as nonfluorescent acceptors for single-molecule FRET.
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 Förster 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 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.
Biological Physics Research Group, Department of Physics, University of Oxford, Oxford, United Kingdom. l.lereste1@physics.ox.ac.uk
Ludovic
L
Le Reste
Johannes
J
Hohlbein
Kristofer
K
Gryte
Achillefs N
AN
Kapanidis
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003624:Darkness; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012015:Reference Standards; D013997:Time Factors
D005456:Fluorescent Dyes; D004247:DNA; D004259:DNA-Directed DNA Polymerase