Mass spectrometry-based mRNA sequence mapping via complementary RNase digests and bespoke visualisation tools

mRNA technology has significantly changed the timeline for developing and delivering a new vaccine from years to months, as demonstrated by the development and approval of two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. Analytical methods are required to characterise mRNA therapeutics and underpin manufacturing development. In this study, we have developed and utilised partial RNase digests of mRNA using RNase T1 and RNase U2 in conjunction with an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of mRNA therapeutics. In conjunction with this, we have developed novel software engineered to optimise and streamline the visualisation and analysis of sequence mapping of mRNA using LC-MS/MS. We show that increased mRNA sequence coverage is obtained by combining multiple partial RNase T1 digests-44% and 37% individually, 64% together-or RNase T1 and U2 partial digests-73% and 52% individually, 88% combined. The developed software automates the process of combining digests, ensuring faster and more accurate analysis. Furthermore, the software provides additional information on sequence coverage by taking into account multiple overlapping oligoribonucleotide fragments to increase the confidence of the sequence mapping. Finally, the software enables powerful and accessible visualisation capabilities by generating spiral plots to quickly analyse the sequence maps in a single output from combined multiple partial RNase digests.