An Enzyme Cascade Enables Production of Therapeutic Oligonucleotides in a Single Operation

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases, however the rising number of therapies now poses a considerable manufacturing challenge. Existing synthetic methods rely on iterative coupling, capping, oxidation and deprotection to achieve stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. Here, we report a transformative biocatalytic approach to efficiently produce oligonucleotides in a single operation, where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks, aqueous conditions and can be used to produce diverse oligonucleotide sequences containing a range of pharmaceutically relevant modifications. Our methodology is showcased through the synthesis of a range of clinically relevant molecules.