XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases.

The B-family polymerases of hyperthermophilic archaea have proven an exceptional platform for engineering polymerases with extended substrate spectra, despite multiple mechanisms for detecting and avoiding incorporation of non-cognate substrates. These polymerases can efficiently synthesize and reverse-transcribe a number of xenonucleic acids (XNAs) that differ significantly from the canonical B-form of DNA. We present here a protocol for hexitol nucleic acid (HNA) synthesis by an engineered Thermococcus gorgonarius polymerase variant, including adaptation for large-scale synthesis and purification, and for other XNAs. We describe XNA purification and reverse transcription (with a previously reported XNA RT also based on Thermococcus gorgonarius), as well as key considerations for the characterization and optimization of XNA reactions.