Strand-specific quantification of 8-oxo-dG and apurinic sites via Ligation-Dependent Probe Amplification (LPA)

Abstract

Oxidative DNA damage, characterized by the prominent lesion 8-oxo-7,8-dihydroguanine (8-oxo-dG), is linked to mutagenesis and genome instability. Accurately mapping these lesions with strand specificity and high resolution remains a major challenge, limiting our understanding of damage dynamics during transcription and repair. Here, we introduce a novel, highly sensitive ligation-dependent probe amplification (LPA) method that enables quantitative, strand-specific analysis of 8-oxo-dG and apurinic (AP) sites at single-nucleotide resolution. Our technique uses enzymatic digestion, highly selective ligation, and quantitative PCR to distinguish damaged from intact DNA strands, offering detailed insight into lesion localization and repair kinetics. When applied to estrogen-stimulated breast cancer cells, LPA reveals asymmetric, strand-specific guanine oxidation during transcriptional activation, characterized by rapid repair of the transcribed strand and more persistent damage on the non-transcribed strand. Our findings show that oxidative lesions are dynamically regulated by biological stimuli, reflecting a finely tuned balance between repair and damage buildup. This LPA approach is a powerful tool for exploring the complex relationship among redox signaling, DNA damage, and transcription regulation, furthering our understanding of redox-driven genome modulation in health and disease.