A Chemiluminescence Assay for Differentiating MicroRNA Isoforms with 3'-Terminal Single-Nucleotide Resolution

MicroRNAs (miRNAs) are small (19-25 nt) endogenous noncoding RNAs that regulate diverse genetic pathways and biological processes. During biogenesis, imprecise processing generates terminal length variants known as isomiRs, which harbor single-nucleotide variations at their 5'- or 3'-ends. These subtle alterations can modify target recognition and downstream signaling, positioning isomiRs as promising biomarkers for disease. Nevertheless, discriminating single-nucleotide terminal isomiRs with high fidelity remains a nontrivial task, owing to the inherent limitations in the discriminatory power of conventional techniques. Here, we report a chemiluminescent strategy for the highly sensitive detection of 3'-terminal single-nucleotide isomiRs, employing T4 RNA ligase 2 and melting temperature-based discrimination. A stem-loop hairpin probe selectively ligates perfectly matched miRNAs, while isomiRs with 3'-end additions or deletions exhibit significantly diminished ligation efficiency. The ligation products are captured and recognized by a biotinylated reporter probe conjugated with streptavidin-horseradish peroxidase (SA-HRP), yielding a quantifiable chemiluminescent signal. This method achieves precise, single-nucleotide resolution in discriminating 3' isomiRs, with superior sensitivity and specificity.