A general strategy for expanding polymerase function by droplet microfluidics

Andrew C. Larsen, Matthew R. Dunn, Andrew Hatch, Sujay P. Sau, Cody Youngbull and John C. Chaput ()
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Andrew C. Larsen: The Biodesign Institute, Arizona State University
Matthew R. Dunn: The Biodesign Institute, Arizona State University
Andrew Hatch: School of Earth and Space Exploration, Arizona State University
Sujay P. Sau: The Biodesign Institute, Arizona State University
Cody Youngbull: School of Earth and Space Exploration, Arizona State University
John C. Chaput: The Biodesign Institute, Arizona State University

Nature Communications, 2016, vol. 7, issue 1, 1-9

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms11235

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