XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases

Abstract

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. © 2018 by John Wiley & Sons, Inc.