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

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.