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An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Author

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  • Andreas Hartmann

    (B CUBE Center for Molecular Bioengineering, TU Dresden)

  • Koushik Sreenivasa

    (B CUBE Center for Molecular Bioengineering, TU Dresden
    Delft University of Technology)

  • Mathias Schenkel

    (B CUBE Center for Molecular Bioengineering, TU Dresden)

  • Neharika Chamachi

    (B CUBE Center for Molecular Bioengineering, TU Dresden)

  • Philipp Schake

    (B CUBE Center for Molecular Bioengineering, TU Dresden)

  • Georg Krainer

    (B CUBE Center for Molecular Bioengineering, TU Dresden
    University of Cambridge
    University of Graz)

  • Michael Schlierf

    (B CUBE Center for Molecular Bioengineering, TU Dresden
    Physics of Life, DFG Cluster of Excellence, TU Dresden
    Faculty of Physics, TU Dresden)

Abstract

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

Suggested Citation

  • Andreas Hartmann & Koushik Sreenivasa & Mathias Schenkel & Neharika Chamachi & Philipp Schake & Georg Krainer & Michael Schlierf, 2023. "An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42232-3
    DOI: 10.1038/s41467-023-42232-3
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    References listed on IDEAS

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    1. Rahul Roy & Alexander G. Kozlov & Timothy M. Lohman & Taekjip Ha, 2009. "SSB protein diffusion on single-stranded DNA stimulates RecA filament formation," Nature, Nature, vol. 461(7267), pages 1092-1097, October.
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    4. Rahul Roy & Alexander G. Kozlov & Timothy M. Lohman & Taekjip Ha, 2009. "Erratum: SSB protein diffusion on single-stranded DNA stimulates RecA filament formation," Nature, Nature, vol. 462(7275), pages 944-944, December.
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