Journal
BIOPHYSICAL JOURNAL
Volume 102, Issue 11, Pages 2658-2668Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2012.04.028
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Funding
- European Commission [HEALTH-F4-2008-201418]
- Biotechnology and Biological Research Council [BB/H01795X/1]
- European Research Council [261227]
- European Research Council (ERC) [261227] Funding Source: European Research Council (ERC)
- Biotechnology and Biological Sciences Research Council [BB/H01795X/1] Funding Source: researchfish
- BBSRC [BB/H01795X/1] Funding Source: UKRI
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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 Forster 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 mu M or more without introducing a significant background, the use of dark quenchers should permit single-molecule Forster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.
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