Direct RNA modification mapping: Technological advances, gaps, and emerging trends

Mapping RNA modifications is essential for elucidating biological pathways and ensuring the quality and safety of therapeutic RNAs. This review critically examines current and emerging technologies for mapping RNA modifications by direct RNA sequencing. Top-down mass spectrometry (MS) is preferred for short RNAs, whereas bottom-up MS suits longer ones. Recent developments in multidimensional liquid chromatography (MD-LC-MS) and online digestion techniques have improved sample throughput while reducing sample requirements. However, the commercialization of only a few nucleases and the scarcity of suitable software packages have long impeded full sequence coverage and untargeted analysis. Nanopore sequencing excels in untargeted, long-read analysis of RNA modifications. The accuracy of nanopore sequencing is limited, and modification-aware basecallers require large data sets for training and powerful computational tools for analysis. Further research is needed to harness the full potential of the technologies in mapping RNA modifications and possibly elucidating RNA structures.