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. 2022 May 13;15(11):9818–9830. doi: 10.1007/s12274-022-4420-5

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

Benjamin Vermeer 1, Sonja Schmid 1,✉
PMCID: PMC9101981  PMID: 35582137

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 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 smFRET. 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.

graphic file with name 12274_2022_4420_Fig1_HTML.jpg

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s12274-022-4420-5 and is accessible for authorized users.

Keywords: biomolecular dynamics, single-molecule fluorescence resonance energy transfer (smFRET), photobleaching, conformational changes, single-molecule kinetics

Electronic Supplementary Material

12274_2022_4420_MOESM1_ESM.pdf (346.5KB, pdf)

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

Acknowledgements

We thank Chirlmin Joo, Johannes Hohlbein, Mattia Fontana, and Abbas Jabermoradi for helpful discussions before and during this project. We thank John Philippi for support with electronic triggering. We thank Johannes Hohlbein, Mattia Fontana, Katarzyna Tych, Mahipal Ganji, and David Dulin for helpful comments on the manuscript. Parts of the analysis code used herein were co-developed in Thorsten Hugel’s lab by previous lab members (including SS). We are thankful for this contribution.

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