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bioRxiv · 10.1101/2021.12.13.472508

Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging

Graham, T. G. W.·Ferrie, J. J.·Dailey, G. M.·Tjian, R.·Darzacq, X.
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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 Forster 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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Graham, T. G. W., Ferrie, J. J., Dailey, G. M., Tjian, R., Darzacq, X.. 2021-12-15. Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging. https://doi.org/10.1101/2021.12.13.472508

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