Accurate identification of 8-oxoguanine in RNA with single-nucleotide resolution using ligase-dependent qPCR

8-oxoguanine (o⁸G), a prevalent oxidative modification in RNA induced by reactive oxygen species (ROS), plays a pivotal role in regulating RNA functions. Accurate detection and quantification of o⁸G modifications is critical to understanding their biological significance and potential as disease biomarkers, but effective detection methods remain limited. Here, we have developed a highly specific T3 DNA ligase-dependent qPCR assay that exploits the enzyme's ability to discriminate o⁸G from guanine (G) with single-nucleotide resolution. This method can detect o⁸G in RNA at levels as low as 500 fM, with an up to 18-fold higher selectivity for discriminating o⁸G from G. By simulating oxidative stress conditions in SH-SY5Y and HS683 cell lines treated with rotenone, we successfully identified site-specific o⁸G modifications in key miRNAs associated with neuroprotective responses, including miR-124, let-7a and miR-29a. The developed assay holds significant promise for the practical identification of o⁸G, facilitating its potential for detailed studies of o⁸G dynamics in various biological contexts and diseases.