High-throughput electrophoretic mobility shift assays for quantitative analysis of molecular binding reactions

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Electrophoretic mobility shift assays (EMSAs) are crucial for studying molecular binding.
• Existing EMSA methods lack high-throughput capabilities for rapid screening.
• Characterizing molecular interactions, like riboswitch-ligand binding, requires efficient assays.

Purpose of the Study:

• To develop and validate a high-throughput platform for EMSAs.
• To enable rapid identification and characterization of molecular binding reactions.
• To assess the binding of the Vc2 cyclic-di-GMP riboswitch to its ligand.

Main Methods:

• A photopatterned, free-standing polyacrylamide gel array with 96 concurrent EMSAs was designed.
• Optimization focused on minimizing sample dispersion, mitigating evaporation, and controlling unit variation.
• The platform was used to analyze riboswitch-ligand interactions.

Main Results:

• The 96-plex EMSA platform achieved a throughput of ~10 data/min, significantly higher than conventional methods.
• Optimized conditions allowed for 10% mobility shift baseline resolution within 3 minutes.
• Reliable quantification of molecular binding and mobility shifts was demonstrated.

Conclusions:

• The developed free-standing polyacrylamide gel EMSA platform offers a powerful tool for high-throughput screening.
• This assay platform is suitable for riboswitches and potentially other RNA and macromolecular targets.
• The technology significantly enhances the efficiency of molecular binding characterization.