Convergent Biocatalytic Mediated Synthesis of siRNA.

Stephanie Paul, Darren Gray, Jill Caswell, Joshua Brooks, Wenjie Ye, Thomas S Moody, Roumen Radinov, Lubomir Nechev

ACS chemical biology 2023 Oct 20

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New technologies are required to combat the challenges faced with manufacturing commercial quantities of oligonucleotide drug substances which are required for treating large patient populations. Herein we report a convergent biocatalytic synthesis strategy for an Alnylam model siRNA. The siRNA chemical structure includes several of the unnatural modifications and conjugations typical of siRNA drug substances. Using Almac's 3-2-3-2 hybrid RNA ligase enzyme strategy that sequentially ligates short oligonucleotide fragments (blockmers), the target siRNA was produced to high purity at 1 mM concentration. Additional strategies were investigated including the use of polynucleotide kinase phosphorylation and the use of crude blockmer starting materials without chromatographic purification. These findings highlight a path toward a convergent synthesis of siRNAs for large-scale manufacture marrying both enzymatic liquid and classical solid-phase synthesis.

Citation

Stephanie Paul, Darren Gray, Jill Caswell, Joshua Brooks, Wenjie Ye, Thomas S Moody, Roumen Radinov, Lubomir Nechev. Convergent Biocatalytic Mediated Synthesis of siRNA. ACS chemical biology. 2023 Oct 20;18(10):2183-2187