LAUSR

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’ capping 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 or a DNAzyme, 5’ cleavage products enrichment, and LC-MS intact mass analysis. In this communication, we describe 1) our results on the sequence preference of RNase H cleavage and a framework to design site-specific RNA cleavage; 2) a method to fluorescently label the RNase H cleavage fragments for readouts such as easily accessible urea-PAGE 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.