Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labelling and functional imaging

Area of Science:

• Synthetic biology
• Molecular imaging
• Biotechnology

Background:

• Fluorescent nanobodies offer high specificity for biological imaging.
• Existing nanobody tools often suffer from background fluorescence and limited spectral range.
• Need for advanced imaging probes to study dynamic biological processes with high precision.

Purpose of the Study:

• To develop a versatile toolkit of antigen-stabilizable fluorescent nanobodies (VIS-Fbs) covering the visible spectrum.
• To engineer VIS-Fbs that fluoresce brightly only upon antigen binding, enabling background-free imaging.
• To demonstrate the utility of VIS-Fbs for tracking protein dynamics and metabolite monitoring in vivo.

Main Methods:

• Engineering of over twenty fluorescent proteins and biosensors into eight nanobodies.
• Development of constitutive, photoactivatable, and photoswitchable fluorescent proteins.
• Incorporation of intensiometric fluorescent protein-based biosensors.
• Application of VIS-Fbs for imaging in the mouse brain and tracking protein dynamics in zebrafish embryos.

Main Results:

• Established a generalizable design for VIS-Fbs spanning 450 nm to 660 nm.
• VIS-Fbs demonstrated bright, antigen-specific fluorescence with minimal background.
• Enabled simultaneous monitoring of two metabolites using biosensor-carrying VIS-Fbs.
• Facilitated ratiometric functional imaging in the mouse brain and tracking of β-catenin dynamics in zebrafish.

Conclusions:

• VIS-Fbs provide a powerful platform for background-free visualization of intracellular proteins.
• The toolkit allows multicolor detection of multiple antigens and selective targeting.
• This synthetic biology approach enables precise studies of protein dynamics and complex biological systems.