Wang, Lu, et al. Nature Chemistry 12.2 (2020): 165-172.
MaP555-SNAP, a novel N,N-dimethylsulfamide-based 6-TAMRA probe, is designed for high-efficiency, no-wash live-cell imaging of SNAP-tagged fusion proteins. Traditional SNAP-tag probes often suffer from limited cell permeability and high background signals, necessitating high concentrations and multiple wash steps. MaP555-SNAP addresses these limitations through rational fluorophore design that optimizes the spirolactam-zwitterion equilibrium to enhance fluorogenicity and cellular uptake.
In vitro analyses demonstrated a 15-fold increase in absorbance at 555 nm and a 21-fold increase in fluorescence emission at 580 nm upon binding to SNAP-tag, confirming MaP555-SNAP's robust activation mechanism. In live-cell imaging experiments using U2OS cells expressing nuclear SNAP-Halo fusion proteins, MaP555-SNAP achieved strong nuclear labeling at concentrations as low as 250 nM without requiring wash steps. It produced an impressive nuclei-to-cytosol fluorescence ratio (Fnuc/Fcyt = 15), significantly outperforming conventional probe 6 and commercial alternatives like TMR-Star.
Additionally, MaP555-SNAP exhibited faster intracellular labeling kinetics compared to control probes, while still maintaining low background fluorescence due to its spirolactam-based quenched state in the unbound form. These properties make MaP555-SNAP particularly well-suited for dynamic, live-cell imaging applications where rapid and specific labeling is essential.
In conclusion, MaP555-SNAP represents a powerful advancement in the toolkit for live-cell protein imaging, enabling fast, low-background, and wash-free visualization of SNAP-tagged proteins.
