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

  1. Thomas George Wade Graham
  2. John Joseph Ferrie III
  3. Gina M Dailey
  4. Robert Tjian  Is a corresponding author
  5. Xavier Darzacq  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States

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

  1. 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..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5189-4313
  2. John Joseph Ferrie III

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Gina M Dailey

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8988-963X
  4. 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..
  5. 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..
    ORCID icon "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.

Reviewing Editor

  1. Jie Xiao, Johns Hopkins University, United States

Version history

  1. Preprint posted: December 15, 2021 (view preprint)
  2. Received: January 7, 2022
  3. Accepted: August 16, 2022
  4. Accepted Manuscript published: August 17, 2022 (version 1)
  5. Version of Record published: October 4, 2022 (version 2)
  6. 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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  1. Thomas George Wade Graham
  2. John Joseph Ferrie III
  3. Gina M Dailey
  4. Robert Tjian
  5. Xavier Darzacq
(2022)
Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
eLife 11:e76870.
https://doi.org/10.7554/eLife.76870

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https://doi.org/10.7554/eLife.76870

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