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Monitoring multiple distances within a single molecule using switchable FRET
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Uphoff, Stephan, Holden, Séamus, Le Reste, Ludovic, Periz, Javier, van de Linde, Sebastian, Heilemann, Mike and Kapanidis, Achillefs N. (2010) Monitoring multiple distances within a single molecule using switchable FRET. Nature Methods, 7 (10). pp. 831-836. doi:10.1038/nmeth.1502 ISSN 1548-7091.
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Official URL: http://dx.doi.org/10.1038/nmeth.1502
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.
Item Type: | Journal Article | ||||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
Journal or Publication Title: | Nature Methods | ||||||||
Publisher: | Nature Publishing Group | ||||||||
ISSN: | 1548-7091 | ||||||||
Official Date: | October 2010 | ||||||||
Dates: |
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Volume: | 7 | ||||||||
Number: | 10 | ||||||||
Page Range: | pp. 831-836 | ||||||||
DOI: | 10.1038/nmeth.1502 | ||||||||
Status: | Peer Reviewed | ||||||||
Publication Status: | Published | ||||||||
Access rights to Published version: | Restricted or Subscription Access |
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