Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
Abstract
Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.
Data availability
Source data for Fig. 2-5 are included in an accompanying zip file.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Robert Tjian
Jane Coffin Childs Memorial Fund for Medical Research
- Thomas George Wade Graham
Life Sciences Research Foundation
- John Joseph Ferrie III
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jie Xiao, Johns Hopkins University, United States
Version history
- Preprint posted: December 15, 2021 (view preprint)
- Received: January 7, 2022
- Accepted: August 16, 2022
- Accepted Manuscript published: August 17, 2022 (version 1)
- Version of Record published: October 4, 2022 (version 2)
- Version of Record updated: February 20, 2024 (version 3)
Copyright
© 2022, Graham et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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