posted on 2021-05-24, 21:29authored byEsau Medina, Eric J. Yik, Piet Herdewijn, John C. Chaput
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.