Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection

Sisamakis, Evangelos; Valeri, Alessandro; Kalinin, Stanislav; Rothwell, Paul J.; Seidel, Claus A. M.

doi:10.1016/s0076-6879(10)75018-7

Cited by 250 publications

(378 citation statements)

References 167 publications

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“…[25] This ratio is sensitive to the alignment of the system and is usually determined by calibrating with free dyes that have relatively short rotation correlation times and a residual anisotropy of zero. [32] Mathematical Details…”

Section: Accurate Spfret Experimentsmentioning

confidence: 99%

“…[32] Thus, a species-selective determination of the donor quantum yield can be performed. Acceptor quenching affects the uncorrected FRET efficiency E PR but is corrected by using the g factor determined for the quenched acceptor.…”

Section: Bce Vs T D(a)mentioning

confidence: 99%

“…Single-molecule experiments avoid difficulties with ensemble averaging and incomplete labeling of the sample, but many other factors need to be properly accounted for. These difficulties include donor and acceptor photophysics, [31,32] optical mismatch of the detection volumes, [31] and the unknown orientation of the donor and acceptor dipoles (the kappa squared problem). [33,34] Thus, at least six control measurements are required for a quantitative FRET analysis in addition to measurement of the sample of interest.…”

Section: Introductionmentioning

confidence: 99%

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Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

et al. 2012

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Section: Accurate Spfret Experimentsmentioning

confidence: 99%

Section: Bce Vs T D(a)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

et al. 2012

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“…intensity, lifetime, anisotropy) from individual molecules simultaneously, smMFD can reach a high precision and accuracy in the FRET measurements, identify unspecific quenching effects and distinguish between D-and/or A-labeled specimen. [7][8][9] An alternative method to detect low FRET efficiencies in cell-free systems is luminescence resonance energy transfer (LRET), which uses lanthanides as donors. 10,11 Due to the long lifetime (ms) of lanthanides, the large Stokes shift and Förster radii up to 9 nm, LRET efficiencies can be determined over a wide range of distances (1-14 nm).…”

Section: Introductionmentioning

confidence: 99%

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

et al. 2011

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Inter-or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleedthrough, thereby tolerating a high excess (up to 100 fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50 % in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in-vitro and in-vivo FRET-based spectroscopy and imaging.

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“…In a smFRET experiment, a biomolecule of interest is labeled with a donor and an acceptor fluorophore at specific sites. Because the FRET efficiency (E FRET ) depends on the distance between the fluorophores according to E FRET ¼ 1/(1 þ (R/R 0 ) 6 ), changes of the distance translate into changes of measurable spectroscopic quantities, such as the ratio of acceptor and donor intensities (6). R 0 (the För-ster radius), depending on the combination of the donor and acceptor labels, is 5-7 nm (7), implying that the interval of interdye distances that can be probed by FRET is in the range from a few to~10 nm.…”

Section: Introductionmentioning

confidence: 99%

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Tutkus

Marčiulionis

Sasnauskas

et al. 2017

Biophysical Journal

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The single-molecule Fö rster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field. In combination with FRET it allowed us to reveal and characterize the dynamics of what had been inaccessible conformations of the DNA-protein complex. Our model system, restriction enzyme Ecl18kI, interacts with a FRET pair-labeled DNA fragment to form two different DNA loop conformations. The DNA-protein interaction geometry is such that the efficient FRET is expected for one of these conformations-''antiparallel'' loop. In the alternative ''parallel'' loop, the expected distance between the dyes is outside the range accessible by FRET. Therefore, ''antiparallel'' looping is observed in a single-molecule time trajectory as discrete transitions to a state of high FRET efficiency. At the same time, transitions to a high-intensity state of the directly excited acceptor fluorophore on a DNA tether are due to a change of its average position in the evanescent field of excitation and can be associated with a loop of either ''parallel'' or ''antiparallel'' configuration. Simultaneous analysis of FRET and acceptor intensity trajectories then allows us to discriminate different DNA loop conformations and access the average lifetimes of different states.

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Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection

Cited by 250 publications

References 167 publications

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

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