@article {cui2016mix, title = {A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders}, journal = {Scientific reports}, volume = {6}, year = {2016}, pages = {22575}, publisher = {Nature Publishing Group}, 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{\textendash}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. }, url = {https://www.nature.com/articles/srep22575}, author = {Cui, Naiwen and Zhang, Huidan and Schneider, Nils and Tao, Ye and Asahara, Haruichi and Sun, Zhiyi and Cai, Yamei and Koehler, Stephan A. and de Greef, Tom FA and Abbaspourrad, Alireza and others} }