Journal
NATURE METHODS
Volume 7, Issue 10, Pages 831-U90Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1502
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Funding
- UK Bionanotechnology Interdisciplinary Research Collaboration grant
- Engineering and Physical Science Research Council [EP/D058775]
- European Community [HEALTH-F4-2008-201418]
- German National Academic Foundation
- company SAP AG
- Systems Biology Initiative (Forschungseinheiten der Systembiologie) of the German Ministry of Research and Education [0315262]
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The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET 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 FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.
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