Minimal rhodamine probes for covalent protein labeling in live cells

Silja Friederike Zedlitz

Max Planck Institute of Molecular Cell Biology & Genetics, Hyman Group, Dresden, Germany, <a>zedlitz@mpi-cbg.de

Rhodamine dyes are exceptionally bright, photostable and tunable in color.[1] Paired with genetically-encoded self-labeling protein tags, like HaloTag7[2], the respective rhodamine derivatives allow selective labeling of target proteins in cultured cells. Yet, in vivo imaging of multicellular organisms with HaloTag7 remains challenging due to the limited pharmacokinetic characteristics of these fluorophore substrates.[3] Here, novel rhodamine-binding protein tags (Rho-tags)[4] present an attractive alternative because of their direct high-affinity binding to unsubstituted rhodamines. Rho-tag has been demonstrated for efficient in vivo labeling, but the non-covalent interaction remains a persistent drawback as the dye may be washed out over time. Therefore, a covalent Rho-tag (cRho-tag) modality – bearing a minimal reactive linker on the rhodamine scaffolds – was developed to address this challenge. The reactive moieties were carefully selected to ensure high labeling specificity to a residue within the binding site and favorable pharmacokinetic properties. This approach represents a promising step towards the next generation of permanent protein labeling for bioimaging.

[1] Chen, F. et al. Analysis & Sensing 2 (2022).
[2] Los, G.V. et al. ACS Chem Biol (2008).
[3] Chen, W. et al. Biocell (2022).
[4] Kompa. J. et al. bioRxiv (2025).

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