Measuring Multiple Distances within a Single Molecule using Switchable FRET

t he analysis of structure and dynamics of biomolecules is important for understanding their function. t oward this aim, we introduce a method called ‘switchable F ret ’, which combines single-molecule fluorescence resonance energy transfer (F ret ) with reversible photoswitching of fluorophores. t ypically, single-molecule F ret is measured within a single donor-acceptor pair and reports on only one distance. Although multipair F ret approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. in contrast, switchable F ret sequentially probes F ret between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. o ur experiments on dnA molecules, a protein- dnA complex and dynamic holliday junctions demonstrate the potential of switchable F ret for studying dynamic, multicomponent biomolecules.