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

Abstract

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

Microfluidic approaches for the analysis of protein–protein interactions in solution

“…d, (upper) Identities of high-binding peptides selected by droplet-enrichment relative to (lower) those transcribed from a random library. Figures taken with permission from (Cui et al 2016). e Schematic showing principle for protein aggregation inside micro-droplet environments.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…However, two-hybrid and three-hybrid systems have been developed in PURExpress supplemented with E. coli core RNAP enzyme . Cui et al (2016) used such an in vitro two-hybrid system encapsulated in single-emulsion droplets to screen a library of 105 peptide binders in a single day.…”

Microfluidic single-cell analysis—Toward integration and total on-chip analysis

“…Due to the aforementioned complications to incubate on-chip, the majority of droplet microfluidics protocols to date have opted for incubation off-chip to ensure continuous operation of upstream processes, thereby preserving the inherently high throughput of dropletmediated systems. 12,42,43 In valve-based microfluidics, having at least two layers consisting of a flow layer for the reagent and cell flow and a control layer for valve control increases the complexity of chip design. Additional peripherals for controlling valves add another layer of operational complexity as control software and electronics could be a barrier for biologists.…”