A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

Area of Science:

• Biotechnology
• Molecular Biology
• Microfluidics

Background:

• Drop-based microfluidics offers stable genotype-phenotype linkage for high-throughput screening.
• Screening high-affinity peptide binders using microfluidics is limited by the lack of sensitive assays for single DNA molecules in drops.

Purpose of the Study:

• To develop a sensitive functional assay for screening high-affinity peptide binders using drop-based microfluidics.
• To introduce the in vitro two-hybrid system (IVT2H) into microfluidic drops for a streamlined mix-and-read workflow.

Main Methods:

• Encapsulation of a random DNA library encoding potential peptide binders with IVT2H in microfluidic drops.
• Utilizing the correlation between protein-protein interaction affinity and fluorescent reporter activation within IVT2H.
• Validation through screening a library derived from a known high-affinity MDM2 inhibitor (PMI).

Main Results:

• Demonstrated the feasibility of screening peptide binders using drop-IVT2H.
• Achieved high-throughput screening of DNA libraries within a single day.
• Obtained hits from a small-to-medium-sized library (10^3-10^6) with minimal reagent consumption.

Conclusions:

• Drop-IVT2H is a sensitive and efficient method for screening peptide binders.
• This platform simplifies and accelerates microfluidic workflows for DNA library screening.
• Represents a novel approach for protein engineering and in vitro directed protein evolution.