A General Strategy for Expanding Polymerase Function by Droplet Microfluidics

Overview

Journal Nat Commun

Specialty Biology

Date 2016 Apr 6

PMID 27044725

Citations 56

Authors

Andrew C Larsen

Matthew R Dunn

Andrew Hatch

Sujay P Sau

Cody Youngbull

John C Chaput

Affiliations

Soon will be listed here.

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.

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