Reading between the bases: technological advances in RNA modification detection

Despite the rapid expansion of epitranscriptomics, accurately detecting and mapping RNA modifications remains a major technical challenge that limits our understanding of their functional and therapeutic relevance. More than 170 chemical RNA modifications have been identified, collectively shaping the epitranscriptome through dynamic regulation of RNA processing, stability, localization, and translation. Among these, m 6 A, m 5 C, pseudouridine (Ψ), m 1 A, m7G, 2′-O-Me and A-to-I editing are among the best characterized marks and have emerged as crucial regulators of RNA functions affecting a broad range of cellular functions, whose dysregulation is broadly implicated in disease. The growing therapeutic interest in targeting epitranscriptomic pathways, particularly in cancer, has intensified the need for robust, quantitative, and modification-specific detection strategies. In this mini-review, we critically examine the principal technologies currently used for epitranscriptomic profiling, including immunoprecipitation-based sequencing, chemical and enzymatic probing, RNA mass spectrometry, and direct RNA sequencing. Rather than treating these approaches as isolated methodologies, we emphasize the methodological complementarities required for rigorous epitranscriptomic mapping across distinct RNA modification classes. While every approach involves trade-offs among resolution, throughput, and systematic bias, hybrid strategies integrating native multimodal sequencing, orthogonal chemical reactivity, and genetic validation are improving detection accuracy and specificity. Persistent challenges in stoichiometric quantification, rare mark detection, and causal inference remain, but emerging advances in AI-driven de novo detection may overcome these limitations. Together with advances in single-cell spatial transcriptomics and large-scale CRISPR-epitranscriptomic screening, these technologies are poised to accelerate the functional decoding of RNA modifications and their translation into therapeutic applications.