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

Naiwen Cui,Zhiyi Sun,Nils Schneider,Stephan A Koehler,Ye Tao,Tom F A De Greef,Yamei Cai,Huidan Zhang,Haruichi Asahara,Shaorong Chong,David A Weitz,Alireza Abbaspourrad

doi:10.1038/srep22575

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.

Highlights

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening
High throughput screening of individual binders using IVT2H in plate-based assay format would lead to prohibitive costs in the experimental reagents and other consumables
The major advantage of drop-IVT2H is that the expression of the binder and the target is coupled to the detection of the binding interaction in a continuous and streamlined drop-based microfluidic workflow

Summary

Introduction

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. 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. Drop-based microfluidics has become a novel tool for high throughput screening in recent years It provides a stable linkage between phenotype and genotype by partitioning single cells into picoliter drops and allows fluorescence-activated drop sorting (FADS)[18,19]. The use of such drop-based microfluidic method to screen high-affinity binders from a random DNA library has never been shown, largely due to the fact that there is no functional assay sensitive enough to detect single DNA molecules for protein binding in drops. We demonstrate that this mix-and-read drop-IVT2H has allowed successful enrichment of high-affinity peptide binders in a p53-MDM2 binding model

Methods

Results

Conclusion