A Near-Infrared-Triggered Luminescence-Activated System for In Vivo Biomacromolecular Tagging and Photocatalytic Crosslinking for Large-Scale Investigation of RNA-Protein Complexes in Living Mice

The ability to investigate biomolecular interactions and screen for their therapeutic interventions holds great potential for elucidating biological mechanisms and enabling disease treatment. While photocatalytic cross-linking and tagging systems have emerged as attractive strategies for interrogating biomolecular interactions in cellular contexts, their application in living animals remains challenging due to the limited tissue penetration of UV–vis light. Here, we established a near-infrared (NIR)-triggered biomacromolecule in vivo cross-linking and tagging (IVCT) system based on luminescence-activation-induced photocatalysis using lanthanide-doped upconversion nanoparticles (UCNPs) as energy donors (IVCT-LAUC). IVCT-LAUC converts the deep-tissue penetration 808 nm laser into a 254 nm UV for cross-linking of RNA-protein complexes (RPCs) and simultaneously into 450/650 nm visible light for singlet oxygen ( 1 O 2 ) generation to tag the cross-linked RPCs. This enables second-scale interrogation of dynamic RPCs in living mouse organs using a single excitation source. Enrichment using the tagged handles, followed by sequencing and mass spectrometry analysis, facilitates in vivo capture of RPCs and identification of 1709 and 563 RNA-binding proteins in the mouse kidney and spleen. Our method also supports in vivo off-target evaluation of RPC stabilizers and inhibitors, an application still rarely achieved in the field of drug screening.