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Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology.

Esau MedinaEric J YikPiet HerdwijnJohn C Chaput
Published in: ACS synthetic biology (2021)
Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2'-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability. Bst DNA polymerase was found to have weak reverse transcriptase activity on ANA and uncontrolled reverse transcriptase activity on HNA, differing from its known recognition of FANA and TNA templates. These data benchmark the activity of current XNA polymerases and provide opportunities for generating new polymerase variants that function with greater activity and substrate specificity.
Keyphrases
  • nucleic acid
  • structural basis
  • single molecule
  • amino acid
  • electronic health record
  • risk assessment
  • machine learning
  • artificial intelligence
  • big data
  • human health
  • circulating tumor cells