Abstract: The visualization of the microcosmic behavior of proteins in vivo is the key to real-time monitoring of proteins. A series of wash-free SNAP-tag probes were designed and synthesized based on the combination of SNAP-tag and small organic molecule fluorescent dyes. SNAP-tag, which specifically recognized O6-benzylguanine, could be labeled with a fluorophore (e. g., 1,8-naphthalimide) through the formation of covalent bonds. Furthermore, the change from a hydrophilic environment to the hydrophobic cavum of SNAP-tag realized a 2-13-fold enhancement in fluorescence. Through the fusion of SNAP-tag and the target protein, the probes could recognize the mitochondrial proteins (e. g., cytochrome oxidase, Cox8A) and nuclear proteins (e. g., H2B) in living cells. Besides, the fluorescent probes allowed the in-situ real-time monitoring of proteins without washing.

Key words: in-situ, organic small molecule fluorescent dyes, SNAP-tag, wash-free fluorescent probes