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Publication Abstract from PubMed

Photosensitive fluorophores, whose emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features and facilitating super-resolution microscopy techniques. Although irreversibly photoactivatable fluorophores are well established, reversible reporters that can be reactivated multiple times remain scarce, and only a few have been applied in living cells using generalizable protein labeling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labeling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show excellent performance in living cells, with large, reversible, deep-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets and SMLM compatibility. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells.,Walterspiel F, Ugarte-Uribe B, Weidenhausen J, Vincent M, Narayanasamy KK, Dimitriadi A, Khan AUM, Fritsch M, Muller CW, Zimmermann T, Deo C Angew Chem Int Ed Engl. 2025 Oct 23:e202424955. doi: 10.1002/anie.202424955. PMID:41131894^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.