Shaping Nanobodies and Intrabodies against Proteoforms

Area of Science:

• Molecular Biology
• Immunology
• Virology

Background:

• Proteoforms contribute to genomic diversity and regulate biological processes.
• While mass spectrometry advances proteoform characterization, methods for targeting specific proteoforms remain limited.
• Developing tools to bind and modulate proteoform function is crucial.

Purpose of the Study:

• To develop novel intrabodies capable of binding specific proteoforms.
• To identify nanobody binders for distinct SARS-CoV-2 receptor binding domain (RBD) proteoforms using a yeast-based system.
• To establish a framework for creating proteoform-targeting nanobodies and intrabodies.

Main Methods:

• Utilized a synthetic camelid nanobody library expressed in yeast.
• Employed positive and negative selection strategies for nanobody identification.
• Validated nanobodies against specific RBD proteoforms using yeast-2-hybrid analysis and sequence comparisons.

Main Results:

• Successfully identified nanobodies that bind to specific SARS-CoV-2 RBD proteoforms.
• Demonstrated the ability to select nanobodies that differentiate between RBD variants (e.g., Wuhan strain vs. Beta variant with E484K mutation).
• Validated nanobody specificity through yeast-2-hybrid assays.

Conclusions:

• The study provides a robust framework for developing proteoform-specific nanobodies and intrabodies.
• This approach enables the creation of molecular tools to target and potentially disrupt specific proteoform functions.
• The developed nanobodies can distinguish between SARS-CoV-2 RBD variants, offering potential applications in diagnostics or therapeutics.