Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide...

The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m⁶A). While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m⁶A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs.