title: 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 (10(3)-10(6)). 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.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Naiwen
authors.initials: N
authors.lastname: Cui
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.|Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.
authors.forename: Huidan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400, Illkirch, France.|New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Nils
authors.initials: N
authors.lastname: Schneider
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.|School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tao
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Haruichi
authors.initials: H
authors.lastname: Asahara
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Sun
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yamei
authors.initials: Y
authors.lastname: Cai
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Koehler
authors.affiliation: Institute for Complex Molecular Systems, Department of Biomedical Engineering Eindhoven University of Technology Den Dolech 2, 5600 MB Eindhoven, The Netherlands.
authors.forename: Tom F A
authors.initials: TF
authors.lastname: de Greef
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Alireza
authors.initials: A
authors.lastname: Abbaspourrad
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.|Department of Physics, Harvard University, Cambridge, MA02138, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Shaorong
authors.initials: S
authors.lastname: Chong
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D044085:Microfluidics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D010455:Peptides; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2