Engineering TNA polymerases through iterative cycles of directed evolution
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Updated: Jul 12, 2025

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Published on: October 6, 2017
Eric J Yik1, Victoria A Maola1, John C Chaput2
1Department of Pharmaceutical Sciences, University of California, Irvine, CA, United States.
Abstract:
DNA polymerases are important tools for biotechnology, synthetic biology, and chemical biology as they are routinely used to amplify and edit genetic information. However, natural polymerases do not recognize artificial genetic polymers (also known as xeno-nucleic acids or XNAs) with unique sugar-phosphate backbone structures. Directed evolution offers a possible solution to this problem by facilitating the discovery of engineered versions of natural polymerases that can copy genetic information back and forth between DNA and XNA. Here we report a directed evolution strategy for discovering polymerases that can synthesize threose nucleic acid (TNA) on DNA templates. The workflow involves library generation and expression in E. coli, high-throughput microfluidics-based screening of uniform water-in-oil droplets, plasmid recovery, secondary screening, and library regeneration. This technique is sufficiently general that it could be applied to a wide range of problems involving DNA modifying enzymes.
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