Energy transfer photoproximity labelling in live cells using an organic cofactor

Photocatalytic proximity labelling has recently emerged as a powerful tool to resolve a wide variety of biomolecular and cellular interactions. While the use of high-resolution probe species, such as diazirines, enables cell-surface protein labelling with nanometre precision by generating highly reactive intermediates, intracellular applications are limited either by the intrinsic toxicity of frequently employed photocatalysts or lower resolution when long-lived reactive intermediates are used. In this work, we describe the discovery and application of an organic flavin cofactor derivative, deazaflavin, capable of diazirine activation to form carbenes through triplet energy transfer and offers unparalleled biocompatibility. We demonstrate deazaflavin-diazirine energy transfer labelling (DarT-labelling) not only allows for targeted extracellular scenarios using antibody conjugates but, most importantly, for intracellular interactome mapping of cellpenetrating peptides (CPPs). We successfully mapped the localisation of two popular polyarginine CPPs and identified potential key membrane interactors. Furthermore, we showed the applicability of DarT-labelling over extended time by mapping the intracellular trafficking of a stable cyclic derivative to reveal its eventual exocytosis from the cell. We envision DarT-labelling has the unmet potential to enable detailed profiling of intracellular dynamics across diverse biological systems with unprecedented spatiotemporal control.