Ligation-Triggered Assay for the Signal-Amplified Genotyping of Cancer-Associated Single Nucleotide Polymorphisms in MicroRNAs

Methods for detecting microRNA (miRNA) are well-established, yet strategies for identifying miRNA-related single-nucleotide polymorphisms (miR-SNPs) remain limited. Despite their strong association with cancer development, the clinical application of miR-SNPs has been hindered by the lack of practical detection methods. To bridge this gap, we developed a method termed ligation-triggered hybridization chain reaction for miR-SNP testing (LIGHT). LIGHT utilizes SplintR-triggered ligation for SNP discrimination in miR-196a2, combined with hybridization chain reaction for signal amplification. This approach enables precise allele discrimination with a detection limit of 0.75 aM, offering specificity by distinguishing mutations in miR-196a2 when they constitute only 0.0002% of the wild-type population. LIGHT-based miR-SNP genotyping revealed significantly elevated miR-196a2T expression in non-small cell lung cancer (NSCLC) samples, highlighting the potential of LIGHT for NSCLC diagnostics. Furthermore, by leveraging the isothermal workflow of LIGHT, we developed a 3D-printed visualizer for portable miR-SNP genotyping and demonstrated its potential to facilitate cancer diagnosis in resource-limited settings.