Figure 5.

Interrogating precision RES signaling by T-REX. A protein of interest (POI) is expressed as a functional Halo-fusion POI in live cells, worms, or fish. Following non-invasive treatment of these living systems with a HaloTag-targetable bioinert cell/organism-permeable photocaged precursor to a given RES, a 1:1 covalent complex of Halo:photocaged-RES is achieved. Photoactivation following washing away of excess probe liberates the RES (in an amount stoichiometric to POI) in the immediate vicinity of the POI, resulting in the formation of an “encounter complex” (blue shell). The POI is given first refusal of the RES and competition between native reactivity of the POI to a specific RES and native diffusivity of the RES is allowed for. Provided the POI is a bona fide sensor able to react rapidly with the RES before its diffusion, fractional target occupancy is achieved between a specific RES-chemotype and a specific POI. If this occupancy fulfills biological sufficiency, T-REX enables on-target functional redox response to be measured in real time in living systems.