Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Förster Resonance Energy Transfer Experiments

Förster 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 ∼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.