Rapid photo-crosslinking in living cells reveals protein–nucleic acid dynamics on a timescale of minutes

The activation of chemical reactions in living cells using ultraviolet (UV) light enables the interrogation of biomolecules in their native environment with photoreactive probes or crosslinking reagents. Although numerous photo-crosslinking approaches have been successfully employed, they often suffer from common limitations, including low reaction yields, the need for long exposure times, and irradiation-induced cellular damage from heat, desiccation, or side reactions. We recently showed that 365 nm light-emitting diodes enable rapid, biorthogonal protein-DNA crosslinking in living cells, incurring minimal photodamage. Here, we generalize this approach and demonstrate that high-intensity, longwave UV light reduces the irradiation time for in-cell photo-crosslinking reactions by up to 1000-fold, allowing protein-drug, protein-protein, protein-DNA, and protein-RNA interactions to be fixed within seconds. Benchmarking this rapid photo-activation for the analysis of RNA-interacting proteomes responding to RNA-binding drugs or UV-induced RNA damage, we demonstrate both qualitative and quantitative advantages of controlled, high-intensity UV irradiation, uncovering emergent experimental opportunities that were previously inaccessible to light-activated chemistry in intact cells and tissues.