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Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s –1  between two conformers of a Holliday junction. The rapid diffusional migration of  Deinococcus radiodurans  single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of  Escherichia coli  SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.

Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering