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 poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a 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 and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.