Advances in Detecting RNA Modifications Using Direct RNA Nanopore Sequencing

RNA modifications add a dynamic and versatile regulatory layer to gene expression, influencing RNA stability, splicing, translation, and cellular responses. Despite their importance, traditional detection methods-such as antibody-based enrichment, chemical labeling, or indirect sequencing approaches-often suffer from limited resolution, biases, and an inability to capture modifications in their native RNA context. Oxford Nanopore Technologies (ONT) direct RNA sequencing (DRS) overcomes many of these limitations by enabling amplification-free, single-molecule, and single-nucleotide detection of diverse RNA modifications directly from native RNA molecules. In this review, recent advances in applying ONT DRS to characterize modifications beyond the extensively studied N⁶-methyladenosine (m⁶A), including 2'-O-methylation (Nm), N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C), N⁴-acetylcytidine (ac⁴C), N⁷-methylguanosine (m⁷G), pseudouridine (Ψ), and adenosine-to-inosine (A-to-I) editing are summarized. Computational frameworks and basecalling innovations are highlighted that improve modification calling, with particular emphasis on approaches that detect co-occurring modifications and reveal their potential regulatory cross-talk within individual transcripts. Finally, emerging applications across synthetic systems, non-model organisms, and disease contexts are discussed, and offer a forward-looking perspective on integrating nanopore-based epitranscriptomics with multi-omics platforms to achieve a deeper and more comprehensive understanding of RNA regulation.