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.
CITATION STYLE
Wiegand, D. J., Rittichier, J., Meyer, E., Lee, H., Conway, N. J., Ahlstedt, D., … Church, G. M. (2024). Template-independent enzymatic synthesis of RNA oligonucleotides. Nature Biotechnology. https://doi.org/10.1038/s41587-024-02244-w
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