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Monitoring multiple distances within a single molecule using switchable FRET

Abstract

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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Figure 1: Concept of switchable FRET.
Figure 2: Proof-of-principle of switchable FRET.
Figure 3: Switchable FRET with an alternative acceptor.
Figure 4: Switchable FRET monitors two distances within the CAP-DNA complex.
Figure 5: Holliday junction dynamics probed from two perspectives.

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Acknowledgements

We thank K. Gryte, J. Hohlbein, T.M.C. Rito and A. Riegert for technical and editorial assistance, and M. Leake for suggestions. S.U., S.J.H., L.L.R., J.P. and A.N.K. were supported by a UK Bionanotechnology Interdisciplinary Research Collaboration grant, Engineering and Physical Science Research Council grant EP/D058775 and European Community Seventh Framework Programme (FP7/2007-2013) grant HEALTH-F4-2008-201418 (entitled READNA). S.U. was supported by the German National Academic Foundation and the company SAP AG. M.H. and S.v.d.L. were supported by the Systems Biology Initiative (Forschungseinheiten der Systembiologie) of the German Ministry of Research and Education (0315262).

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Authors

Contributions

S.U., M.H. and A.N.K. designed research; S.U. performed experiments and simulations; S.U. and S.J.H. analyzed data; L.L.R. built experimental setup; S.U., J.P. and S.v.d.L. prepared reagents; S.U. and A.N.K. wrote manuscript; and S.U., S.J.H., L.L.R., J.P., S.v.d.L., M.H. and A.N.K. discussed results and commented on the manuscript.

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Correspondence to Achillefs N Kapanidis.

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Uphoff, S., Holden, S., Le Reste, L. et al. Monitoring multiple distances within a single molecule using switchable FRET. Nat Methods 7, 831–836 (2010). https://doi.org/10.1038/nmeth.1502

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