biostudies-literatureKuhn SDeutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft (German Research Foundation)École Polytechnique Fédérale de LausanneMax-Planck-Gesellschaft1754-1761https://www.ebi.ac.uk/biostudies/studies/S-EPMC12568630biostudies-literatureUnknown21(11)SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 10⁷ s^-1 M^-1, a 100-fold improvement over the corresponding SNAP-tag-substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a fivefold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translatNature chemical biologySNAP-tag2 for faster and brighter protein labeling.PMC12568630TRR 186Johnsson Kde Lange EMFEgoldt CLennoi ATarnawski MLin YHVlijm RKompa JReinstein JNasufovic VHiblot JWilhelm JFischer JKuhn SfalseSNAP-tag2 for faster and brighter protein labeling.SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 10⁷ s^-1 M^-1, a 100-fold improvement over the corresponding SNAP-tag-substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a fivefold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translat2025-01-01T00:00:00Z2025 Nov2026-06-05T07:12:01.551Z2026-05-14T03:08:46.152ZS-EPMC125686304061072010.1038/s41589-025-01942-z