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A general strategy for expanding polymerase function by droplet microfluidics.

Andrew C Larsen1, Matthew R Dunn1,2, Andrew Hatch3

  • 1The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301, USA.

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|April 6, 2016
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Summary

Droplet-based optical polymerase sorting (DrOPS) enables engineering polymerases for artificial genetic polymers. This method achieved a 1,200-fold enrichment and evolved a high-fidelity TNA polymerase.

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Area of Science:

  • Synthetic biology
  • Biochemistry
  • Molecular biology

Background:

  • Engineering natural polymerases to synthesize artificial genetic polymers is crucial for synthetic biology.
  • Developing novel methods to expand polymerase function is a significant challenge.

Purpose of the Study:

  • To present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function.
  • To demonstrate the efficacy of DrOPS in evolving polymerases for unnatural genetic polymers.

Main Methods:

  • Utilized microfluidics to generate uniform synthetic compartments for polymerase activity monitoring.
  • Employed an optical sensor to detect polymerase activity within microfluidic droplets.
  • Performed a complete cycle of encapsulation, sorting, and recovery on a doped polymerase library.

Main Results:

  • Achieved a ~1,200-fold enrichment of a model engineered polymerase using DrOPS.
  • Successfully evolved a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase.
  • The evolved TNA polymerase demonstrated >99% template-copying fidelity.

Conclusions:

  • DrOPS is a versatile tool for evolving polymerase functions, particularly where optical detection via Watson-Crick base pairing is feasible.
  • This method significantly advances the engineering of polymerases for synthetic genetic polymers.
  • DrOPS offers a powerful platform for directed evolution of enzyme function.