5 years ago

A Photoactivatable Probe for Super-Resolution Imaging of Enzymatic Activity in Live Cells

A Photoactivatable Probe for Super-Resolution Imaging of Enzymatic Activity in Live Cells
Pablo Rivera-Fuentes, Dorothea Pinotsi, Zacharias Thiel, Elias A. Halabi, Nils Trapp
A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron-poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is nonemissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich, and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules. Single-molecule localization microscopy was used to reconstruct a super-resolved image of esterase activity. These single-molecule images of enzymatic activity changed significantly upon treatment of the cells with inhibitors of human carboxylesterase I and II, both in terms of total number of signals and intracellular distribution. This proof-of-principle study introduces a sensing mechanism for single-molecule detection of enzymatic activity that could be applied to many other biologically relevant targets.

Publisher URL: http://dx.doi.org/10.1021/jacs.7b07748

DOI: 10.1021/jacs.7b07748

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