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Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

  • Mikhail Baibakov
    Mikhail Baibakov
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
  • Satyajit Patra
    Satyajit Patra
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
  • Jean-Benoît Claude
    Jean-Benoît Claude
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
  • Antonin Moreau
    Antonin Moreau
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
  • Julien Lumeau
    Julien Lumeau
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
  • , and 
  • Jérôme Wenger*
    Jérôme Wenger
    Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France
    *E-mail: [email protected]
Cite this: ACS Nano 2019, 13, 7, 8469–8480
Publication Date (Web):July 8, 2019
Copyright © 2019 American Chemical Society

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    Single-molecule Förster resonance energy transfer (smFRET) is widely used to monitor conformations and interaction dynamics at the molecular level. However, conventional smFRET measurements are ineffective at donor–acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.

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    The Supporting Information is available free of charge on the ACS Publications Web site at DOI: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b04378.

    • Orientation-dependent numerical simulations of the donor intensity inside the ZMW; single-molecule fluorescence time traces with ZMWs of different diameters; FCS analysis with ZMWs; FRET histograms recorded for the sample labeled only with the donor; influence of the crosstalk parameter α on the measured FRET efficiency; influence of the direct excitation parameter δ on the measured FRET efficiency; influence of the γ parameter on the measured FRET efficiency; influence of the binning time on the measured FRET efficiency; comparison of models to fit the smFRET histograms; fluorescence lifetime analysis, S–E plot diagrams; experimental determination of the γ correction factor; fluorescence spectra of the multiacceptor DNA sample; expression of the total energy transfer rate constant inside the ZMW (PDF)

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