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bioRxiv · 10.1101/2022.02.02.478748

RNase H-based analysis of synthetic mRNA 5-prime cap incorporation

Chan, S.-H.·Whipple, J. M.·Dai, N.·Kelley, T. M.·Withers, K.·Tzertzinis, G.·Correa, I. R.·Robb, G. B.
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Abstract

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5 cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5 cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5 end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5 cleavage products, and LC-MS intact mass analysis. In this communication, we describe 1) a framework to design site-specific RNA cleavage using RNase H; 2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5 cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

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Chan, S.-H., Whipple, J. M., Dai, N., Kelley, T. M., Withers, K., Tzertzinis, G., Correa, I. R., Robb, G. B.. 2022-02-02. RNase H-based analysis of synthetic mRNA 5-prime cap incorporation. https://doi.org/10.1101/2022.02.02.478748

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