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

Area of Science:

• Synthetic biology
• Molecular imaging
• Biotechnology

Background:

• Fluorescent nanobodies offer sensitive detection but often suffer from background noise.
• Existing fluorescent tools lack broad spectral coverage and antigen-specific activation.
• Need for advanced imaging probes for studying dynamic biological processes in vivo.

Purpose of the Study:

• To create a versatile toolkit of fluorescent nanobodies (VIS-Fbs) with antigen-dependent fluorescence.
• To cover the entire visible spectrum (450-660 nm) for multicolor imaging applications.
• To enable background-free visualization and tracking of intracellular proteins and metabolites.

Main Methods:

• Engineering of over 20 fluorescent proteins and biosensors into 8 nanobodies.
• Development of a generalizable design for VIS-Fbs with constitutive, photoactivatable, and photoswitchable fluorescent proteins.
• Incorporation of intensiometric fluorescent protein-based biosensors for metabolite monitoring.

Main Results:

• Established VIS-Fbs spanning 450-660 nm that fluoresce brightly only upon antigen binding.
• Demonstrated simultaneous monitoring of two metabolites using VIS-Fbs with biosensors.
• Achieved ratiometric functional imaging in mouse brain and tracked endogenous β-catenin dynamics in zebrafish embryos.
• Showcased background-free visualization, multicolor detection, and selective targeting capabilities.

Conclusions:

• VIS-Fbs provide a powerful, synthetic biology-driven platform for precise biological studies.
• This toolkit enables high-specificity, low-background imaging of protein dynamics and cellular processes.
• VIS-Fbs advance the study of complex biological systems, including development and signaling pathways.