Template-independent enzymatic synthesis of RNA oligonucleotides

• Published in: Nature biotechnology
• Main Authors: Wiegand, Daniel J, Rittichier, Jonathan, Meyer, Ella, Lee, Howon, Conway, Nicholas J, Ahlstedt, Daniel, Yurtsever, Zeynep, Rainone, Dominic, Kuru, Erkin, Church, George M
• Format: Journal Article
• Language: English
• Published: United States 12.07.2024
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-024-02244-w

RNA oligonucleotides have emerged as a powerful therapeutic modality to treat disease, yet current manufacturing methods may not be able to deliver on anticipated future demand. Here, we report the development and optimization of an aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methods. The enzymatic synthesis of RNA oligonucleotides is made possible by controlled incorporation of reversible terminator nucleotides with a common 3'-O-allyl ether blocking group using new CID1 poly(U) polymerase mutant variants. We achieved an average coupling efficiency of 95% and demonstrated ten full cycles of liquid phase synthesis to produce natural and therapeutically relevant modified sequences. We then qualitatively assessed the platform on a solid phase, performing enzymatic synthesis of several N + 5 oligonucleotides on a controlled-pore glass support. Adoption of an aqueous-based process will offer key advantages including the reduction of solvent use and sustainable therapeutic oligonucleotide manufacturing.