Molecular Simulation Guided Optimization of Multi-Epitope Nanobody Affinity for Enhanced Förster Resonance Energy...

Area of Science:

• Biotechnology
• Nanotechnology
• Immunotechnology

Background:

• Förster resonance energy transfer (FRET) detection systems are crucial for pathogen screening due to speed, specificity, and simplicity.
• Existing FRET probes face limitations in sensitivity, environmental adaptability, and epitope interference, hindering performance.
• Development of advanced probes is needed to overcome current limitations in FRET-based pathogen detection.

Purpose of the Study:

• To develop novel multiepitope probes (MEPs) for enhanced FRET-based pathogen detection.
• To improve sensitivity, robustness, and epitope recognition in FRET detection systems.
• To create a rapid, sensitive, and stable FRET system for pathogen screening.

Main Methods:

• Designed and constructed novel multiepitope probes (MEPs) using a nanobody panel.
• Utilized molecular docking to identify an optimal tetra-epitope nanobody complex for rotavirus detection.
• Conjugated nanobodies to luminescent microspheres and integrated them into a homogeneous FRET system.
• Employed site-directed saturation mutagenesis to optimize MEPs for enhanced FRET sensitivity.

Main Results:

• Developed MEPs integrated into a homogeneous FRET detection system for rotavirus.
• Achieved a 10.18-fold enhancement in FRET sensitivity through site-directed mutagenesis.
• Demonstrated a linear FRET response to rotavirus VP6 protein from 1.56 to 50 pg/mL within 50 minutes.
• Established a detection limit of 0.83 pg/mL, a 1566-fold improvement over conventional methods.

Conclusions:

• The novel MEPs-based FRET system exhibits exceptional sensitivity, specificity, stability, and accuracy.
• This technology holds significant potential for real-world applications in pathogen detection and public safety.
• Presents a novel strategy for high-performance FRET systems applicable to large-protein detection.