Conditional Control of Benzylguanine Reaction with the Self-Labeling SNAP-tag Protein

SNAP-tag, a mutant of the O⁶-alkylguanine-DNA-alkyltransferase, self-labels by reacting with benzylguanine (BG) substrates, thereby forming a thioether bond. SNAP-tag has been genetically fused to a wide range of proteins of interest in order to covalently modify them. In the context of both diagnostic and therapeutic applications, as well as use as a biological recording device, precise control in a spatial and temporal fashion over the covalent bond-forming reaction is desired to direct inputs, readouts, or therapeutic actions to specific locations, at specific time points, in cells and organisms. Here, we introduce a comprehensive suite of six caged BG molecules: one light-triggered and five others that can be activated through various chemical and biochemical stimuli, such as small molecules, transition metal catalysts, reactive oxygen species, and enzymes. These molecules are unable to react with SNAP-tag until the trigger is present, which leads to near complete SNAP-tag conjugation, as illustrated both in biochemical assays and on human cell surfaces. This approach holds promise for targeted therapeutic assembly at disease sites, offering the potential to reduce off-target effects and toxicity through precise trigger titration.