Engineering TNA polymerases through iterative cycles of directed evolution.

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.

Synthesis and Polymerase Recognition of Threose Nucleic Acid Triphosphates Equipped with Diverse Chemical Functionalities.

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.