Template-independent enzymatic synthesis of RNA oligonucleotides

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Abstract

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.

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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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