+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

Characterization of dark quencher chromophores as nonfluorescent acceptors for single-molecule FRET

, : Characterization of dark quencher chromophores as nonfluorescent acceptors for single-molecule FRET. Biophysical Journal 102(11): 2658-2668

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.


PMID: 22713582

DOI: 10.1016/j.bpj.2012.04.028

Other references

Ying, H.; Zhang, Y.; Lin, S.; Han, Y.; Zhu, H-Zhang., 2010: Histone deacetylase inhibitor Scriptaid reactivates latent HIV-1 promoter by inducing histone modification in in vitro latency cell lines. Human immunodeficiency virus type 1 (HIV-1) latency remains a major problem for the eradication of viruses in infected individuals undergoing highly active anti-retroviral therapy. By inhibiting HIV-1 gene expression and virus production, histone...

Sanz, M.A.; Lorenzo, J.I.; Sanz, G.F.; García, S.; Amigó, V.; Vallespí, T.; Torrabadella, M.; Cañizo, M.C.; San Miguel, J., 1989: Validation of survival-predictive models in myelodysplastic syndromes. American Journal of Hematology 30(1): 44-44

Lacko, L.; Burger, M., 1964: The inhibition of transport of glucose into human erythrocytes by 2,3,4,6-tetra -d-glucopyranose. 2,3,4,6-Tetra[O-acetyl]-[beta]-D-glucopyranose penetrated into human erythrocytes following a transport mechanism different from that of glucose. However, TAG interacted with the glucose-carrier system, inhibiting the transport of glucose to a var...

Panasyuk, D.I.; Yakovlev, S.A., 1959: Phenothiazine, a highly effective drug in the control of Diayoeaulus and other strongylate infections in sheep.. Literature on the use of phenothiazine against helminths and particularly Dictyocaulus in sheep in Russia is briefly reviewed. The regular application of phenothiazine led to a six-fold reduction in dictyocauliasis between 1951 and 1958. G. I. Poz...

El-Sawaby, Ms, 1981: Reclamation of salt affected soils in Egypt. Conference on biosalinity: the problem of salinity in agriculture: a joint conference of Egyptian Israeli and American scientists Univ of California Davis September 1-4-1981 organized and edited by DW Rains: 4

Vulliamy, B.; Potts, S.G.; Willmer, P., 2006: The effects of cattle grazing on plant-pollinator communities in a fragmented Mediterranean landscape. The main aims of this study were to assess grazing impacts on bee communities in fragmented mediterranean shrubland (phrygana) and woodland habitats that also experience frequent wildfires, and to explain the mechanisms by which these impacts occu...

Saybulatov S.Z.; Kalmykova L.F.; Kapustin A.P., 1990: Structural-mechanical properties of waste masses from Ekibastuz coal mining basin. Kompleksnoye Ispol'zovaniye Mineral'nogo Syr'ya 1990(6(143)): 61-64

Koppejan, A.W.G., 1965: Agriculture in the (Dutch) central economic plan. Information is given on the setting-up of the agricultural section in the Dutch central economic plan. The estimates on future agricultural production, taking into account several specific uncertainties, form one of the bases of this plan which fo...

Bardon, K., 1992: A wintering oldsquaw in the Twin Cities. Loon, 642: 122-123

Paplinski, Z.; Sawa, A., 1964: Rare Forms Of Broncho-Abdominal Fistulae. Polski Przeglad Chirurgiczny 36: 873-879