Single-molecule imaging at high fluorophore concentrations by local activation of dye

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; Spenkelink, Lisanne M.; McGrath, William J.; Morrone, Seamus R.; Sohn, Jungsan; Robinson, Andrew; van Oijen, Antoine M.

doi:10.1016/j.bpj.2014.12.019

Title: Single-molecule imaging at high fluorophore concentrations by local activation of dye

Journal Article · Tue Feb 17 00:00:00 EST 2015 · Biophysical Journal

DOI:https://doi.org/10.1016/j.bpj.2014.12.019· OSTI ID:1184512

Geertsema, Hylkje J. ^[1]; Mangel, Walter F. ^[2]; Schulte, Aartje C. ^[1]; Spenkelink, Lisanne M. ^[1]; McGrath, William J. ^[2]; Morrone, Seamus R. ^[3]; Sohn, Jungsan ^[3]; Robinson, Andrew ^[1]; van Oijen, Antoine M. ^[1]

Zernike Inst. of Advanced Materials, Groningen (The Netherlands)
Brookhaven National Lab. (BNL), Upton, NY (United States)
John Hopkins School of Medicine, Baltimore, MD (United States)

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use of short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: Netherlands Organization for Scientific Research (NWO); European Research Council (ERC); National Institutes of Health (NIH)

Grant/Contract Number:: SC00112704

OSTI ID:: 1184512

Report Number(s):: BNL-107889-2015-JA; 400412000

Journal Information:: Biophysical Journal, Vol. 108, Issue 4; ISSN 0006-3495

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 11 works

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Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments Jazi, Atieh Aminian; Ploetz, Evelyn; Arizki, Muhamad Biochemistry, Vol. 56, Issue 14 https://doi.org/10.1021/acs.biochem.6b00916	journal	March 2017
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