Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and...

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Nanobodies are valuable tools for protein manipulation and visualization in vivo.
• Controlling nanobody/antigen interactions with light offers precise spatiotemporal control over protein function.

Purpose of the Study:

• To engineer photo-activatable nanobodies using photocaged amino acids for light-inducible protein binding.
• To demonstrate the application of these engineered nanobodies for controlling protein localization in vivo.

Main Methods:

• Genetic code expansion to introduce photocaged amino acids into nanobody binding interfaces.
• Computational alanine scanning and molecular dynamics simulations to guide affinity tuning.
• In vivo expression of engineered nanobodies and their antigens in Caenorhabditis elegans.

Main Results:

• Developed photo-activatable nanobodies with light-tunable binding affinity.
• Demonstrated robust function of engineered nanobodies in complex intracellular environments.
• Successfully controlled subcellular protein localization in a living nematode worm.

Conclusions:

• Engineered photocaged nanobodies provide a generalizable method for light-controlled protein interactions.
• Computational modeling predictions can be effectively applied in vivo.
• In vivo effects on protein-protein interactions must be considered for nanobody design.