Photobleaching of fluorescent probes limits the observation span of a typical single molecule fluorescence measurement and hinders simultaneous observation of dynamics across timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes throughout the experiment via transient binding of fluorescently labelled single-stranded DNAs to complementary target DNA strands attached to the target molecule. We show our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the photobleaching time under our conditions. We also show our method is adaptable to FRET and study the conformational dynamics of DNA Holliday Junctions for extended periods. By adjusting the temporal resolution and observation span, we envision to capture the conformational dynamics of proteins and nucleic acids over wide range of timescales.

Photobleaching of fluorescent probes limits the observation span of typical single‐molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near‐continuous single‐molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

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