Once considered passive messengers of genetic information, RNA molecules are now understood to play an active and dynamic role in gene regulation, modulated by a rich layer of chemical modifications collectively known as the epitranscriptome. Among these, N6-methyladenosine (m6A) is the most abundant mRNA internal modification, present on thousands of transcripts and implicated in diseases such as cancer and neurological disorders.1,2 These modifications add regulatory depth to gene expression by influencing mRNA stability, translation, trafficking, and splicing. Critically, their biological impact depends not only on where they occur, such as at splice junctions or near stop codons, but also on the proportion of transcripts that are modified at a given locus. Capturing both the genomic coordinates and relative abundance of RNA modifications requires methods that combine read mapping with a quantitative framework.
Limitations of current methodologies
Current approaches, including m6A RNA immunoprecipitation (meRIP) and chemical conversion assays such as GLORI, have advanced the field, but with key limitations: they often require high RNA input, lack internal normalization, and struggle to distinguish subtle biological differences obscured by technical variability. Direct detection by nanopore sequencing can provide quantitation, but requires large sample inputs, varies in algorithm accuracy, and cannot be used for degraded samples, thus limiting clinical applications.3,4 Epitranscriptomics lacks a comprehensive truth set, so well-designed internal controls, benchmarking strategies, and dedicated bioinformatics pipelines are essential for validating findings and ensuring scientific rigor.
Quantitation unlocks new biological insights
RNA modifications are a powerful but often overlooked layer of genetic regulation, shaping everything from aging to disease. This tutorial highlights how AlidaBio’s EpiPlexTM Platform enables accurate, quantitative measurements of m6A and inosine across the transcriptome. By applying this approach to study RNA methylation dynamics in HEK293T cells and liver tumors, we show that RNA modifications reveal a distinct regulatory landscape, one that diverges from gene expression and exposes new biological pathways that may hold untapped therapeutic potential.
EpiPlex assay and analysis
The EpiPlex assay minimizes technical noise and enables relative quantitation of multiple modifications by combining specific enrichment of modified RNA fragments with an innovative proximity barcoding chemistry. Currently optimized for m6A and inosine, this platform is inherently scalable and positioned to expand to additional modifications. With peak performance achieved at only 20 ng of polyA-enriched RNA or 250 ng of total RNA, the assay opens the door to high-resolution, quantitative modification profiling across a wide range of biological contexts.
Overall workflow
The streamlined workflow can be completed in approximately seven hours, including ~three hours of hands-on time. RNA sample and spike-in controls are fragmented and ligated to a common sequencing adapter. Magnetic beads coated with modification-specific binders (m6A and inosine non-antibody binders) and corresponding barcoded adapters capture and encode modified RNA fragments through close proximity on the bead surface during reverse transcription. The cDNA is then amplified by PCR and normalized for short-read sequencing (Figure 1A).
Relative quantitation
The EpiPlex assay’s quantitation framework includes multiple components:
- Bold Enrichment: RNA fragments bound to modification-specific beads are sequenced by NGS and mapped to their genomic location. Read pileups create peaks that provide a raw measurement of modification abundance.
- Spike-in standards: RNA standards, generated by in vitro transcription (IVT) of a lambda phage genome, are added to every sample. Negative controls (unmodified RNA) and positive controls (RNAs with known modification density) provide a standard curve for each sample (Figure 1B). Enrichment peaks are normalized to the standards to account for sample variations.
- Solution control: A parallel sample that bypasses bead capture is used to measure baseline gene expression.
The EpiScout™ software suite, a companion analysis pipeline for the EpiPlex assay, integrates these components to provide a practical solution for relative quantitation, while maintaining rigor across diverse sample sets.
Bioinformatic analysis and data interpretation
After sequencing, RNA modifications are mapped as peaks or base variants and quantified as fold enrichment over local gene expression. Because modification density on most transcripts is low and region-specific, accurate analysis requires tools that account for gene expression, sequence motifs, and spike-in controls. EpiScout software integrates all these factors to reliably detect m6A and inosine in parallel.
Accurate epitranscriptome mapping
METTL3 is the core methyltransferase (MTase) of the m6A writer complex, and it specifically recognizes a 5-base RNA motif. EIF4A3, a component of the exon junction complex (EJC), protects splice junctions from methylation by METTL3.5,6 The EpiPlex assay was used to examine the regulation of m6A by METTL3 and EIF4A3 in HEK293T cells and to profile paired tumor/normal samples. These studies reveal how the m6A landscape is incredibly dynamic and that spike-in controls must account for this wide
dynamic range.
METTL3 and EIF4A3 inhibition
Pharmacological inhibition of the m6A writer METTL3 using STM2457 revealed a steep, dose-dependent decline in m6A peak counts. Residual peaks persist at maximal inhibition, but fall below the threshold of statistical significance, reflecting a substantial loss of modification abundance rather than complete site erasure. Importantly, inosine peaks remain stable across the titration series, highlighting how synthetic spike-in controls calibrate for relative read differences between samples and allow confident, relative quantitation across samples (Figure 2A). These controls also ensured high reproducibility: DMSO-treated samples processed on three separate days exhibited only 0.8% variation in total m6A peaks.
In a complementary experiment, inhibition of the EJC component, EIF4A3, reveals the flip side of methylation dynamics (Figure 2B, left).7 Inhibition of EIF4A3 helicase activity leads to m6A peaks nearly doubling, and a closer look at these peaks shows they populate new regions. These peaks are predominantly localized to short exons, regions typically protected by the EJC, consistent with a model in which the complex blocks methylation at these regions (Figure 2B, red arrow). Together, these findings show how robust internal controls and quantitative analysis enable detection of both global and region-specific changes in RNA methylation, underscoring the EpiPlex assay’s capacity to resolve subtle effects.8
Liver tumor global hypomethylation
To explore the potential of RNA modifications in uncovering novel therapeutic targets, RNA methylation was profiled in a matched tumor-normal liver pair from a patient with hepatocellular carcinoma (HCC), the most common form of liver cancer and the third leading cause of cancer-related death worldwide.
Strikingly, the tumor sample exhibits global hypomethylation, as the volcano plot displays a large leftward shift. At the gene level, both hypo- and hyper-methylation of specific transcripts can be observed (Figure 3, left). Therefore, care should be taken to deconvolute these global effects from gene-specific changes. As is common in cancer, there are many differentially expressed genes, however, differential methylation is often tied to a unique set of genes. REACTOME pathway analysis reveals that RNA modifications and gene expression primarily regulate distinct processes, reflecting their roles as independent but occasionally overlapping layers of control.
Conclusion
Accurately and quantitatively mapping RNA modifications remains a fundamental challenge in epitranscriptomics. By integrating spike-in and solution controls directly into the workflow and bioinformatic analysis, the EpiPlex assay can enable robust, reproducible, and quantitative location and abundance mapping of m6A and inosine, even with minimal RNA input.
References
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2. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):201-206. doi:10.1038/nature11112
3. Spangenberg J, Mündnich S, Busch A, et al. The RMaP challenge of predicting RNA modifications by nanopore sequencing. Commun Chem. 2025;8(1):115. doi:10.1038/s42004-025-01507-0
4. Prawer YDJ, Gleeson J, De Paoli-Iseppi R, Clark MB. Pervasive effects of RNA degradation on Nanopore direct RNA sequencing. NAR Genomics Bioinforma. 2023;5(2):lqad060. doi:10.1093/nargab/lqad060
5. Yankova E, Blackaby W, Albertella M, et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593(7860):597-601. doi:10.1038/s41586-021-03536-w
6. Bensaude O, Barbosa I, Morillo L, Dikstein R, Le Hir H. Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly. Nat Commun. 2024;15(1):4209. doi:10.1038/s41467-024-48371-5
7. Ito M, Tanaka T, Cary DR, et al. Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors. J Med Chem. 2017;60(8):3335-3351. doi:10.1021/acs.jmedchem.6b01904
8. Sendinc E, Yu H, Hwang Fu YH, et al. Mapping multiple RNA modifications simultaneously by proximity barcode sequencing. bioRxiv [Preprint]. 2024 doi:10.1101/2024.10.09.617509
Ananya Anmangandla, PhD, is a Scientist II at Alida Biosciences.
