Eliminating Spurious Zero-Efficiency FRET States in Diffusion-Based Single-Molecule Confocal Microscopy

Abstract

Single-molecule Förster resonance energy transfer (smFRET) of freely diffusing biomolecules using confocal microscopy is a simple and powerful technique for measuring conformation and dynamics. However, a spurious zero-FRET population can significantly distort the measured histograms and lead to incorrect results, particularly in measurements of intrinsically low-FRET systems. Using a model system consisting of duplex DNAs, we show that there are two important contributions to the zero-FRET state: (1) formation of a dark triplet state of the acceptor dye and (2) the presence of donor-only strands due to incomplete hybridization between donor- and acceptor-labeled strands. The combined strategy of using Trolox as a triplet-state quencher and labeling the same DNA strand with donor and acceptor dyes effectively eliminates the zero-FRET population, even for constructs with intrinsically low FRET efficiencies. This strategy allows us to perform smFRET experiments using a simple confocal microscope with improved accuracy.

Direct Single-Molecule Observation of Sequential DNA Bending Transitions by the Sox2 HMG Box

“…These fluorescence intensities were converted to FRET efficiency ( E FRET ) histograms, providing a scheme for direct visualization of DNA conformational distributions. Without Sox2 HMG , the dual-labeled DNA showed a single-peak in its E FRET histogram with histogram width typical of smFRET studies of freely diffusing dsDNA molecules [29,30] (Figure 2a; top panel). An NLS fit of the histogram to a Gaussian function yielded E FRET value of 0.39 (±0.04).…”

“…Often, histograms of data collected in diffusion-based smFRET experiments show an additional peak at zero E FRET that arise from molecules with active donor(s) and either inactive or absent acceptor [29,30,32,33,34,35]. These zero E FRET peaks tend to significantly overlap with low E FRET peak populations and hamper direct estimation of the position of the non-zero peak(s) [36,37,38,39].…”

Selective Cell–Cell Adhesion Regulation via Cyclic Mechanical Deformation Induced by Ultrafast Nanovibrations

“…To verify the vibrational motion between the AuNPs and the substrate, we used the optical ruler method. − First, a cover glass was coated with PDMS containing the fluorescent dye BODIPY (λ = 520 nm). Then, the AuNPs were attached to the substrate by using PEG linkers.…”

Exploring Intra- and Inter-Regional Interactions in the IDP α-Synuclein Using smFRET and MD Simulations

“…For the experiments, the FRET-labeled αS variants were diluted in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4, 1× PBS), saturated with Trolox to quench the triplet state and to suppress the dark states of the fluorophores. 30 To investigate the effect of solvent quality on the αS conformation, experiments were performed in a low-salt buffer (0.1× PBS), 5 M urea, and 40% methanol. To obtain fluorescence time traces, samples were diluted to concentrations of approximately 100 pM, resulting in 1 to 10 bursts per second on average.…”