Discrimination between streptavidin and avidin with fluorescent affinity-based probes

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Imaging

Background:

• Streptavidin (SA) and avidin (AV) are structurally similar proteins with distinct properties.
• Differentiating between SA and AV is crucial for various biological and diagnostic applications.
• Development of selective probes is needed for accurate identification of these proteins.

Purpose of the Study:

• To design and synthesize novel fluorescent probes for distinguishing between streptavidin and avidin.
• To investigate the mechanism of fluorescence modulation in the presence of SA and AV.
• To evaluate the potential of these probes in bioimaging applications.

Main Methods:

• Design and synthesis of two coumarin-based fluorescent probes, SPS3 and RC3, incorporating biotin.
• Utilizing fluorescence spectroscopy to study probe-protein interactions.
• Investigating the role of twist intramolecular charge transfer (TICT) in probe behavior.
• Testing probe performance in fluorescence imaging of Hela cells.

Main Results:

• SPS3 exhibits fluorescence turn-on upon binding to SA, attributed to restrained TICT.
• RC3 shows significant fluorescence enhancement when binding to AV, due to its fluorophore entering a less polar pocket.
• Both probes successfully differentiate between SA and AV.
• SPS3 and RC3 demonstrate utility in fluorescence imaging of biotin receptor-overexpressed Hela cells.

Conclusions:

• The developed fluorescent probes SPS3 and RC3 enable selective differentiation of streptavidin and avidin.
• The probes' fluorescence modulation mechanisms are linked to their specific binding interactions.
• These probes hold promise for applications in protein analysis and cellular imaging.