Mauker, P.; Dessen-Weissenhorn, L.; Zecha, C.; Veprek, N.; Brandmeier, J.; Beckmann, D.; Kitowski, A.; Kernmayr, T.; Thorn-Seshold, J.; Kerschensteiner, M.; Thorn-Seshold, O.

Here, we develop a general design for high-quality fluorogenic activity probes to quantify biological processes in live cells, by creating a scaffold that efficiently generates cell-retained bright fluorescent soluble products upon reaction with biochemical targets. Live cell probes must be designed to be membrane-permeable; but that often means that their fluorophore products are similarly permeable, resulting in rapid signal loss from the activating cell: which limits their cell-by-cell resolution as well as their sensitivity for quantifying low-turnover processes. Current strategies to retain fluorescent products within cells usually disrupt native biology: e.g. by non-specific alkylation or solid precipitation. Here, scanning charge- and polarity-based approaches to trigger cell retention, we developed a bright fluorogenic rhodol scaffold Trappable Green (TraG) that balances all key requirements for signal integration (rapid probe entry, but effective product retention, across a variety of cell lines) and is easily adaptable to quantify many target types (shown here with probes for GSH, TrxR, and H2O2). The simple and rugged TraG scaffold can now permit straightforward elaboration to a range of cell-retained enzyme activity probes, that enable more accurate cell-resolved imaging as well as higher-sensitivity integration of low-turnover processes, without the drawbacks of alkylation or precipitation-based strategies.