Immunoelectroanalytical multiplexing of RNA methylation signatures to decode oncogenic point mutations in cancer cells.

Epigenomic and epitranscriptomic alterations-particularly RNA methylations such as N6-methyladenosine (m6A), N5-methylcytosine (m5C), N1-methyladenosine (m1A), and N7-methylguanosine (m7G)-are increasingly recognized as pivotal regulators in the pathobiology of colorectal cancer (CRC) and other highly prevalent disorders. Despite their growing clinical and biological relevance, rapid and robust electrochemical strategies enabling the single (m1A, m7G) or multiplexed quantification of these RNA modifications remain unavailable. Herein, we report an indirect competitive, four-analyte amperometric immunoassay that enables the simultaneous determination of m6A, m5C, m1A, and m7G. BSA-conjugated methylated synthetic nucleotides covalently attached to magnetic carboxylated microsized-beads (HOOC-MμBs) competed with freely methylated nucleotides in the target sample for specific anti-epimark detection antibodies. Thereafter, labeling with horseradish peroxidase (HRP)-conjugated secondary antibodies (Ab2-HRP) enabled amperometric readout at screen-printed carbon electrodes (SPCEs) for single or multiplexed transduction. The developed bioplatforms exhibited high selectivity, negligible cross-interference, and a significantly reduced assay time (30 min), supporting their integration into a robust multiplexed format. Application in CRC cellular scenarios, using only nanograms of total RNA, allowed differential profiling of basal and pathway-activated states across five cell lines, revealing distinct RNA methylation patterns associated with diverse biological and genetic backgrounds. Moreover, as proof of concept, an octuple-detection configuration was implemented for parallel analysis of the four methylations in two cell types, wild-type and harboring the clinically relevant KRAS G12V mutation, allowing the evaluation of associations between the target epimark expression levels and this oncogenic point mutation.