Bright Molecular Strain Probe Templates for Reporting Protein-Protein Interactions

Area of Science:

• Molecular Medicine
• Biotechnology
• Biophysics

Background:

• Imaging protein-protein interactions (PPIs) is crucial in molecular medicine.
• Existing methods for PPI detection have limitations in sensitivity and scope.
• The post-genomic era demands advanced tools for studying molecular interactions.

Purpose of the Study:

• To develop novel single-chain molecular strain probe templates for sensitive PPI imaging.
• To engineer luciferase reporters (RLuc86SG, ALuc49) for enhanced signal detection.
• To investigate the mechanism of rapamycin-triggered intramolecular PPIs using these probes.

Main Methods:

• Constructed molecular strain probes by sandwiching luciferase reporters between FKBP-rapamycin binding domain (FRB) and FKBP.
• Optimized probe sensitivity through interface truncations and substrate modulation.
• Employed computational modeling to elucidate substrate-binding chemistry and probe mechanisms.

Main Results:

• Achieved high fold intensities of 9.4 (RLuc86SG) and 16.6 (ALuc49) for PPI detection.
• Demonstrated tunable emission spectra (blue, green, yellow) based on substrate selection.
• Validated the working mechanism of the molecular strain probes computationally.

Conclusions:

• The developed molecular strain probes offer bright and highly sensitive detection of PPIs.
• This approach expands the bioassay toolbox for molecular imaging applications.
• The tunable spectral properties and high sensitivity position these probes for broad utility in biological research.