Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Specific protein labeling is crucial for studying protein function in living cells.
• Chemical probes offer a versatile method for protein imaging but can suffer from high background fluorescence.
• SNAP-tag fusion proteins are widely used for protein labeling and imaging.

Purpose of the Study:

• To design and apply novel fluorogenic probes for SNAP-tag labeling in living cells.
• To overcome the challenge of background fluorescence in chemical probe detection.
• To develop a more sensitive method for spatiotemporal investigation of protein dynamics.

Main Methods:

• Design of intramolecularly quenched fluorogenic probes incorporating a quencher on the guanine group.
• Application of probes for wash-free labeling of SNAP-tag fused to epidermal growth factor receptor (EGFR).
• Characterization of a fast-labeling SNAP-tag variant (SNAP(f)) and its reactivity with fluorogenic substrates.

Main Results:

• Developed novel fluorogenic probes that become highly fluorescent only upon SNAP-tag labeling.
• Demonstrated wash-free labeling of cell surface EGFR and quantification of β-tubulin.
• Showcased a tenfold increase in reactivity with the SNAP(f) variant, significantly reducing background fluorescence.

Conclusions:

• The combination of SNAP(f) and fluorogenic substrates offers a powerful, sensitive, and wash-free method for protein labeling and imaging.
• This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
• Reduced background fluorescence enhances the utility of SNAP-tag technology for various biological applications.