Kompa, J.; Dornfeld, L. J.; Porzberg, N.; Jang, S.; Lilje, S. H.; Catapano, C.; Jocher, D.; Merk, L.; Zedlitz, S.; Mao, R.; Wilhelm, J.; Dietz, M. S.; Tarnawski, M.; Hiblot, J.; Heilemann, M.; Johnsson, K.

We present Rho-tag and SiR-tag, engineered protein tags derived from bacterial multidrug-resistance proteins that bind unsubstituted (silicon-) rhodamines with nanomolar affinity, enabling fast, reversible, and fluorogenic protein labeling. In live cells, Rho-tag labeling occurs within seconds - faster than HaloTag7 - and the tags are compatible with super-resolution methods like STED, SMLM, and MINFLUX. The high specificity of Rho-tag and SiR-tag for unsubstituted rhodamines allows their use alongside HaloTag7 and SNAP-tag. In vivo applications are demonstrated by efficient neuronal labeling in zebrafish larvae.