Sanger Sequencing of Xeno‐Nucleic Acid

Xeno-nucleic acids (XNAs) offer biostable genetic polymers for biotechnology and information technology, but are incompatible with prevailing DNA sequencing methodologies. Here we report a Sanger sequencing approach tailored for XNA, enabling direct readout of XNA strands up to 50 bases. This new method relies on a Bst DNA polymerase mutant, which not only recognizes XNA as a template but also efficiently incorporates dideoxyribonucleoside triphosphate (ddNTP) substrates, facilitating sequencing of XNA by synthesis of complementary DNA. The Bst F710Y mutant exhibits at least two orders of magnitude higher activity in catalyzing ddNTP incorporation. Our method demonstrates accurate sequence determination of diverse XNA strands of distinct backbone chemistries such as TNA and FANA. Lastly, we showcase proof-of-concept automated XNA sequencing on a genetic analyzer instrument, by leveraging Bst mutant's ability to stochastically incorporate BigDye-labeled ddNTP substrates in a single reaction. This work establishes a direct sequencing platform for XNA employing a chain termination strategy, and lays a foundation for future de novo identification of functional XNA and development of XNA-based data storage system.