Enzymatic Synthesis of Mixed XNA Polymers Containing 2′Fluoro and 2′Azide Modifications

Xeno Nucleic Acids (XNA) are nucleic acids not found in nature; they are valuable biotechnological and biomedical tools due to their increased nuclease stability and ability to incorporate new nonnatural functional groups. Nucleic acids containing 2'-azide (2'Az) substitutions are of particular interest due to the capacity of the azide group to participate in the highly selective biorthogonal click chemistry reaction, allowing the conjugation of interesting molecules, dyes, or probes. Laboratory-evolved XNA polymerases have been shown to incorporate 2'Az nucleotides during DNA synthesis, but it is unclear if 2'Az nucleotides can be incorporated during XNA synthesis. Here, we demonstrate that leading XNA polymerases can synthesize fully substituted mixed polymers containing a mixture of 2'Az and 2'-fluoro (2'F) modifications. We also describe a simple, one-pot reaction to convert 2'Az/2'F mixed polymers back to DNA using commercially available DNA polymerases. These XNA polymerases have improved fidelity synthesizing 2'Az/2'F polymers relative to previous systems and can be made with high accuracy. This research provides a toolkit for the accurate and efficient synthesis and reverse transcription of mixed XNA polymers containing azides.