Design of Antigen-Targeting Fluorogenic Probes Utilizing Intramolecular Addition Reaction of Protein-Dye Hybrids.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Fluorogenic probes are crucial for medical diagnostics and imaging.
• A key challenge is achieving rapid and substantial fluorescence enhancement upon target binding.

Purpose of the Study:

• To develop a new class of fluorogenic probes for antigens with a large fluorescence turn-on response.
• To utilize antibody-mimetic DARPins conjugated with silicon-pyronine (SiP) for antigen detection.

Main Methods:

• Engineered DARPins with site-specific cysteine residues were conjugated to SiP.
• A library-screening approach identified SiP fluorescence quenching via cysteine-SiP reaction.
• Antigen binding was shown to shift the equilibrium, restoring SiP fluorescence.

Main Results:

• Developed probes targeting Green Fluorescent Protein (GFP) and Epithelial Cell Adhesion Molecule (EpCAM).
• Achieved significant fluorescence increases of 25-fold for GFP and 12-fold for EpCAM upon binding.
• Demonstrated wash-free cancer cell imaging with low background using the EpCAM probe.

Conclusions:

• The novel DARPin-SiP conjugate system provides a robust platform for highly sensitive fluorogenic probes.
• This approach overcomes limitations of existing probes by enabling large, rapid fluorescence increases.
• The probes show promise for advanced diagnostic and imaging applications, including in vivo imaging.