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

Author

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  • Jae-Yeol Kim

    (Pohang University of Science and Technology)

  • Cheolhee Kim

    (Pohang University of Science and Technology)

  • Nam Ki Lee

    (Pohang University of Science and Technology
    School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology)

Abstract

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.

Suggested Citation

  • Jae-Yeol Kim & Cheolhee Kim & Nam Ki Lee, 2015. "Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering," Nature Communications, Nature, vol. 6(1), pages 1-9, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms7992
    DOI: 10.1038/ncomms7992
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    Cited by:

    1. Antonino Ingargiola & Eitan Lerner & SangYoon Chung & Francesco Panzeri & Angelo Gulinatti & Ivan Rech & Massimo Ghioni & Shimon Weiss & Xavier Michalet, 2017. "Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules," PLOS ONE, Public Library of Science, vol. 12(4), pages 1-27, April.

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