Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Genetic code expansion is a powerful technique for protein manipulation.
• Site-specific protein labeling is crucial for studying protein function.
• Click chemistry offers efficient and specific bioconjugation methods.

Purpose of the Study:

• To detail the application of genetic code expansion with click chemistry for protein labeling.
• To highlight the advantages of this method for advanced biological research.

Main Methods:

• Utilizing orthogonal tRNA/amino-acyl tRNA synthetase pairs for cotranslational incorporation of noncanonical amino acids (ncAAs).
• Employing click chemistry for subsequent labeling of incorporated ncAAs with small organic fluorophores.
• Applying the method for in vitro and in vivo protein manipulation and imaging.

Main Results:

• Achieved site-specific labeling of target proteins with small organic fluorophores.
• Demonstrated the utility of genetically encoded click chemistry tools for high-resolution studies.
• Enabled precise control over fluorophore choice and labeling site within proteins.

Conclusions:

• Genetic code expansion combined with click chemistry provides a versatile platform for protein labeling.
• This approach significantly advances capabilities in single molecule science, superresolution microscopy, and live-cell imaging.
• Facilitates improved design of antibody-drug conjugates and viral imaging applications.