Optimised nanobody-based quenchbodies for enhanced protein detection

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Quenchbodies are fluorescently labeled antibodies that enhance signal upon antigen binding, showing promise for biosensor applications.
• Nanobody-based quenchbodies offer advantages like small size, stability, and engineerability, but existing designs have limited dynamic range (1.1-1.4 fold increase).

Purpose of the Study:

• To identify critical tryptophan residues in nanobody complementarity-determining regions (CDRs) for improved quenchbody performance.
• To develop an optimized nanobody scaffold for enhanced quenchbody fluorescence and dynamic range.
• To create novel quenchbodies for sensitive detection of interleukin-6 (IL-6).

Main Methods:

• Rational design and molecular dynamics simulations to identify key tryptophan residues.
• Introduction of tryptophans into an optimized nanobody scaffold.
• In vitro directed evolution against human interleukin-6 (IL-6).

Main Results:

• Identification of critical tryptophan residues in nanobody CDRs essential for quenchbody function.
• Development of an optimized nanobody scaffold enabling higher fluorescence increases.
• Engineered quenchbodies demonstrated 1.5-2.4 fold fluorescence increases, allowing IL-6 detection at 1-2 nM concentrations.

Conclusions:

• The optimized nanobody scaffold significantly enhances quenchbody performance and dynamic range.
• This platform facilitates the development of sensitive biosensors for various protein targets.
• Applications include research, diagnostics, and environmental monitoring.