An ultrasensitive fluorescent strategy based on ligation-promoted exponential amplification for site-specific...

8-oxo-7,8-dihydroguanine (8-oxoG), the primary oxidative lesion originated from reactive oxygen species (ROS) attacking guanine (G), serves as a key biomarker associated with a variety of major diseases due to inducing the mutations of G:C → T:A. Accurately identifying 8-oxoG is of great significance for initial diagnosis, therapeutic monitoring and research on pathogenesis mechanisms. Here, we present a novel ligation-promoted exponential amplification for ultra sensitively detecting 8-oxoG in cells and tissues. The exponential amplification reaction (EXPAR) template and hairpin template could be ligated in the presence of 8-oxoG in KRAS sequence based on Bsu DNA polymerase inserting adenine opposite 8-oxoG. And then EXPAR could proceed along with the ligation product to generate more amplification products than the traditional EXPAR reaction. The method showed an amplified fluorescence signal proportional to the logarithm of the 8-oxoG concentration over a range of 1 × 10^-13 to 1 × 10^-19 M, with an excellent detection limit of 7.35 × 10^-20 M (73.5 zM). Notably, the method was able to maintain accurate for the site-specific identification of 8-oxoG in genomic DNA from the complex biological matrices containing A375, DLD1, HeLa and 293 T cell lines under different oxidative stress conditions. The results showed that genomic 8-oxoG levels rose with the increase of H₂O₂ treatment concentrations. Overall, this method established a facile, cost-effective and sensitive platform for quantifying locus-specific 8-oxoG in DNA, holding promise for dynamic monitoring of oxidative damage at critical sites, early disease detection and studies of DNA damage repair.