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http://hdl.handle.net/1942/26239
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DC Field | Value | Language |
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dc.contributor.author | Vandenberk, Niels | - |
dc.contributor.author | Barth, Anders | - |
dc.contributor.author | Borrenberghs, Doortje | - |
dc.contributor.author | Hofkens, Johan | - |
dc.contributor.author | HENDRIX, Jelle | - |
dc.date.accessioned | 2018-06-29T08:11:28Z | - |
dc.date.available | 2018-06-29T08:11:28Z | - |
dc.date.issued | 2018 | - |
dc.identifier.citation | JOURNAL OF PHYSICAL CHEMISTRY B, 122(15), p. 4249-4266 | - |
dc.identifier.issn | 1520-6106 | - |
dc.identifier.uri | http://hdl.handle.net/1942/26239 | - |
dc.description.abstract | Forster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of single labeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5-9 nm), we show that none of the dye pairs stands out: distance accuracies were generally <1 nm and precision was similar to 0.5 nm. Finally, excitation of photoblinking dyes such as Alexa647 influences their fluorescence quantum yield, which has to be taken into account in distance measurements and leads to FRET dynamics. Although dye performance might differ in experiments on immobilized molecules, our combined ensemble and single-molecule approach is a robust characterization tool for all types of smFRET experiments. This is especially important when smFRET is used for atomic-scale distance measurements. | - |
dc.description.sponsorship | Olga Doroshenko (Heinrich-Heine-Universitat Dusseldorf, Germany) is thanked for kindly providing the NMR-derived theoretical DNA model structure. Peter Dedecker (KU Leuven), Don C. Lamb (Ludwig-Maximilians-Universitat Munchen, Germany), Claus Seidel (Heinrich-Heine-Universitat Dusseldorf, Germany), and co-workers are gratefully acknowledged for fruitful discussions. Research was funded through the Research Foundation Flanders (FWO Vlaanderen, grant no. G.0B49.15) and KU Leuven (grant no. C14/16/053). N.V. acknowledges the Agency for Innovation by Science and Technology in Flanders (IWT Vlaanderen) for a doctoral scholarship. A.B. thankfully acknowledges the financial support of the Deutsche Forschungsgemeinschaft (DFG) through SFB1035 (project A11). IBA technologies is acknowledged for their excellent customer service. | - |
dc.language.iso | en | - |
dc.title | Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Forster Resonance Energy Transfer Experiments | - |
dc.type | Journal Contribution | - |
local.bibliographicCitation.conferencedate | APR 16-17, 2016 | - |
local.bibliographicCitation.conferencename | International Conference on NMR Spectroscopy of Biomolecules | - |
local.bibliographicCitation.conferenceplace | Univ Michigan, Ann Arbor, MI | - |
dc.identifier.epage | 4266 | - |
dc.identifier.issue | 15 | - |
dc.identifier.spage | 4249 | - |
dc.identifier.volume | 122 | - |
local.bibliographicCitation.jcat | A1 | - |
dc.description.notes | Hendrix, J (reprint author), Katholieke Univ Leuven, Dept Chem, Div Mol Imaging & Photon, Lab Photochem & Spect, Celestijnenlaan 200F, B-3001 Leuven, Belgium. Hasselt Univ, Adv Opt Microscopy Ctr, Dynam Bioimaging Lab, Agoralaan BIOMED C, B-3590 Diepenbeek, Belgium. Hasselt Univ, Biomed Res Inst, Agoralaan BIOMED C, B-3590 Diepenbeek, Belgium. jelle.hendrix@uhasselt.be | - |
local.type.refereed | Refereed | - |
local.type.specified | Article | - |
dc.identifier.doi | 10.1021/acs.jpcb.8b00108 | - |
dc.identifier.isi | 000430784000007 | - |
item.fullcitation | Vandenberk, Niels; Barth, Anders; Borrenberghs, Doortje; Hofkens, Johan & HENDRIX, Jelle (2018) Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Forster Resonance Energy Transfer Experiments. In: JOURNAL OF PHYSICAL CHEMISTRY B, 122(15), p. 4249-4266. | - |
item.validation | ecoom 2019 | - |
item.accessRights | Restricted Access | - |
item.fulltext | With Fulltext | - |
item.contributor | Vandenberk, Niels | - |
item.contributor | Barth, Anders | - |
item.contributor | Borrenberghs, Doortje | - |
item.contributor | Hofkens, Johan | - |
item.contributor | HENDRIX, Jelle | - |
crisitem.journal.issn | 1520-6106 | - |
crisitem.journal.eissn | 1520-5207 | - |
Appears in Collections: | Research publications |
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File | Description | Size | Format | |
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vandenberk 1.pdf Restricted Access | Published version | 5.01 MB | Adobe PDF | View/Open Request a copy |
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