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Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

Author

Listed:
  • Antonino Ingargiola
  • Eitan Lerner
  • SangYoon Chung
  • Francesco Panzeri
  • Angelo Gulinatti
  • Ivan Rech
  • Massimo Ghioni
  • Shimon Weiss
  • Xavier Michalet

Abstract

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Suggested Citation

  • 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.
  • Handle: RePEc:plo:pone00:0175766
    DOI: 10.1371/journal.pone.0175766
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    References listed on IDEAS

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    1. Elio A. Abbondanzieri & William J. Greenleaf & Joshua W. Shaevitz & Robert Landick & Steven M. Block, 2005. "Direct observation of base-pair stepping by RNA polymerase," Nature, Nature, vol. 438(7067), pages 460-465, November.
    2. Antonino Ingargiola & Eitan Lerner & SangYoon Chung & Shimon Weiss & Xavier Michalet, 2016. "FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET," PLOS ONE, Public Library of Science, vol. 11(8), pages 1-27, August.
    3. 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.
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    Cited by:

    1. Paul David Harris & Alessandra Narducci & Christian Gebhardt & Thorben Cordes & Shimon Weiss & Eitan Lerner, 2022. "Multi-parameter photon-by-photon hidden Markov modeling," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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