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. Here, we establish a single-nucleotide resolution method to identify 8-oxoguanine in RNA based on its ability to hinder ligation.
