Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

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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.

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Source data for Fig. 2-5 are included in an accompanying zip file.

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Thomas George Wade Graham

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
Thomas George Wade Graham, is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..

"This ORCID iD identifies the author of this article:" 0000-0001-5189-4313
John Joseph Ferrie III

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Gina M Dailey

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-8988-963X
Robert Tjian

Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States

For correspondence
tijcal@berkeley.edu

Competing interests
Robert Tjian, is a member of eLife's Board of Directors.Is a co-founder of Eikon Therapeutics, Inc.Is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..
Xavier Darzacq

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

For correspondence
darzacq@berkeley.edu

Competing interests
Xavier Darzacq, is a co-founder of Eikon Therapeutics, Inc.Is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..

"This ORCID iD identifies the author of this article:" 0000-0003-2537-8395

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.

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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.