title: A general strategy for expanding polymerase function by droplet microfluidics.
abstract: Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.
authors.affiliation: The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Larsen
authors.affiliation: The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301, USA.|School of Life Sciences, Arizona State University, Tempe, Arizona 85287-5301, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Dunn
authors.affiliation: School of Earth and Space Exploration, Arizona State University, Tempe, Arizona 85287-5301, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hatch
authors.affiliation: The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301, USA.
authors.forename: Sujay P
authors.initials: SP
authors.lastname: Sau
authors.affiliation: School of Earth and Space Exploration, Arizona State University, Tempe, Arizona 85287-5301, USA.
authors.forename: Cody
authors.initials: C
authors.lastname: Youngbull
authors.affiliation: The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301, USA.|Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-5301, USA.|Department of Pharmaceutical Sciences, University of California, 147 Bison Modular, Building 515, Irvine, California 92697.
authors.forename: John C
authors.initials: JC
authors.lastname: Chaput
mesh_terms: D020029:Base Pairing; D001681:Biological Assay; D040761:Biomimetic Materials / Q000737:chemistry*; D018914:Cells, Immobilized / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D009005:Monosaccharides / Q000737:chemistry; D009696:Nucleic Acids / Q000737:chemistry*; D055096:Optical Devices; D015202:Protein Engineering / Q000379:methods
chemical_list: D009005:Monosaccharides; D009696:Nucleic Acids; D004259:DNA-Directed DNA Polymerase