A ribonucleic acid mapping brainteaser: solving a ribonuclease 4 / ribonuclease A crossword using pre- and...

Various modifications occur on both in vivo and in vitro produced messenger ribonucleic acid (mRNA), many of them defying identification by next generation sequencing (NGS). With the promising developments of therapeutic/prophylactic mRNA, the urge to characterize all modifications arising during synthesis and storage becomes prevailing. Whereas sequencing of long nucleic acids by mass spectrometry (MS) is still in its infancy, unknown modifications are dealt with by MS rather than NGS. This necessitates the development of new MS-based RNA mapping strategies where oligonucleotides derived from a ribonuclease (RNase) digestion are identified via MS/MS spectral elucidation in a liquid chromatography-MS (LC-MS) set up. Here, a complementary LC-MS platform has been developed in which oligonucleotide identification is raised to full MS level by post-column RNase digestion of the oligonucleotides. A custom-built low-volume RNase A-bearing cartridge enabled successful oligonucleotide digestion without compromising separation efficiency. Combined with the in-house developed MSsenger software, chromatogram-aligned analysis of the LC-MS data from RNase 4-digested firefly luciferase mRNA using either the RNase A-bearing or a non-functionalized cartridge, was achieved. This allowed reconstitution of the mRNA from its RNase 4-derived oligonucleotides which were identified based on their RNase A digest signature. Exploration of the complex LC-MS data unveiled (i) the complementarity between conventional MS/MS-based sequencing and the post-column digestion approach, and (ii) the high sequence coverage reached by combining both methods. Being complementary to default RNA mapping methods, incorporation of post-column RNase digestion boosts sequence coverage and provides a new method for LC-MS-based localization of unknown modifications.