Ultrasensitive and Specific Detection of 8-Oxoguanine DNA Glycosylase via Locus-Specific Rolling Circle Amplification

8-Oxoguanine DNA glycosylase 1 (OGG1) is a key enzyme for maintaining genomic integrity, as it specifically recognizes and excises 8-oxoguanine (OG), a major oxidative DNA lesion, to initiate base excision repair. Dysregulation of OGG1 activity is closely associated with genomic instability, apoptosis, and tumorigenesis. However, conventional methods for detecting OGG1 activity often lack the sensitivity required for trace-level analysis, limiting their application in early diagnostics and mechanistic studies. In this study, we report an innovative assay termed OG-specific rolling circle amplification (OG-RCA), which for the first time integrates OGG1-mediated OG excision with RCA and subsequent G-triplex formation for signal readout. This novel approach establishes a unique conversion strategy that translates enzymatic activity into quantifiable amplification signals, significantly enhancing detection sensitivity and specificity. After systematic optimization of key reaction conditions, including dsOG substrate concentration, enzyme amounts, and DNA probe concentrations, the OG-RCA assay achieved an exceptionally low limit of detection (LOD) of 3 × 10–8 mg/mL (equivalent to ∼1.6 × 10–5 U/mL) for OGG1 activity, surpassing existing methods. The assay also exhibited high specificity, showing minimal cross-reactivity with other DNA repair glycosylases. Moreover, spike-recovery experiments using HeLa cell protein extracts and 293T cell lysates confirmed its robustness in complex biological samples. The OG-RCA method not only provides a powerful tool for the ultrasensitive detection of DNA base damage biomarkers but also offers a novel platform for investigating DNA damage and repair mechanisms. It holds significant promise for applications involving limited biological samples and mechanistic studies of DNA repair.