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

Andreas Hartmann (), Koushik Sreenivasa, Mathias Schenkel, Neharika Chamachi, Philipp Schake, Georg Krainer and Michael Schlierf ()
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Andreas Hartmann: B CUBE Center for Molecular Bioengineering, TU Dresden
Koushik Sreenivasa: B CUBE Center for Molecular Bioengineering, TU Dresden
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
Michael Schlierf: B CUBE Center for Molecular Bioengineering, TU Dresden

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-42232-3

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