Construction of a damage site-specific fluorescent biosensor for single-molecule detection of DNA damage

The 8-oxoguanine (8-oxoG) represents the most common DNA damage type, and it has been regarded as the oxidative stress biomarker, but the reported 8-oxoguanine assays are limited by poor specificity and low sensitivity. Herein, we demonstrate the construction of damage site-specific fluorescent biosensor for 8-oxoG assay by integrating single-molecule detection with hyperbranched signal amplification. In this assay, the 8-oxoG damages in DNA can generate free 3' OH with the assistance of formamidopyrimidine DNA glycosylase (Fpg) and polynucleotide kinase (PNK), which subsequently triggers the incorporation of abundant Cy5-labeled dUTPs via terminal deoxynucleotidyl transferase (TDT)-mediated site-specific hyperbranched nucleic acid amplification. After digestion of amplification products with nuclease treatment, abundant mononucleotide Cy5-dUTPs are produced, which will be easily monitored via single-molecule imaging and detection. The introduction of hyperbranched nucleic acid amplification and single-molecule detection can greatly improve the sensitivity to achieve a detection limit of 7.62 × 10^-18 M. This biosensor is highly specific with the capability of discriminating 0.001% 8-oxoG target from the DNA mixture. Moreover, it can be applied for quantitative detection of 8-oxoG damage in genomic DNAs with a detection limit of 0.0017 ng, and even accurately quantifies the absolute number (7025 - 8506) of 8-oxoG damage base in single HeLa cell treated with 150 μM H₂O₂. Importantly, this biosensor can measure the 8-oxoG damage level in different cancer cell lines, facilitating the oxidative damage-associated biomedical researches and clinical diagnosis.