Detection of 8-oxoguanine and Apurinic/Apyrimidinic Sites Using a Fluorophore-Labeled Probe With Cell-penetrating Ability

Abstract Background Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for studying the molecular mechanisms of cancer. Human ribosomal protein S3 (hRpS3), which is involved in DNA repair, has a high binding affinity to 8-oxoG. We developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are commonly known to have cell-penetrating properties. Therefore, we developed the TAT-S3 probe via attaching a TAT peptide to our imaging probe. Result A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and AP sites. We confirmed that the TAT-S3 probe is present in the mitochondria, without permeabilization, and the TAT-S3 probe also showed fluorescence in H2O2-treated HeLa cells and zebrafish embryos. Treatment with MitoQ, a mitochondria-targeted anti-oxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted to utilize an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm the binding of the TAT-S3 to the AP sites. The TAT-S3 probe showed binding to AP sites competitive with ARP and MX. Conclusion These results revealed that the TAT-S3 probe successfully detected the presence of 8-oxoG and AP sites in damaged cells. The TAT-S3 probe may have applications for the detection of diseases caused by reactive oxygen species (ROS).