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Can DyeCycling break the photobleaching limit in single-molecule FRET?

View ORCID ProfileBenjamin Vermeer, View ORCID ProfileSonja Schmid
doi: https://doi.org/10.1101/2022.02.08.479542
Benjamin Vermeer
NanoDynamicsLab, Laboratory of Biophysics, Wageningen University, Stippeneng 4, 6708WE Wageningen, The Netherlands
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Sonja Schmid
NanoDynamicsLab, Laboratory of Biophysics, Wageningen University, Stippeneng 4, 6708WE Wageningen, The Netherlands
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  • For correspondence: schmid@nanodynlab.org
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Abstract

Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ‘DyeCycling’, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://doi.org/10.5281/zenodo.5972932

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 08, 2022.
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Can DyeCycling break the photobleaching limit in single-molecule FRET?
Benjamin Vermeer, Sonja Schmid
bioRxiv 2022.02.08.479542; doi: https://doi.org/10.1101/2022.02.08.479542
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Can DyeCycling break the photobleaching limit in single-molecule FRET?
Benjamin Vermeer, Sonja Schmid
bioRxiv 2022.02.08.479542; doi: https://doi.org/10.1101/2022.02.08.479542

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