Reactive oxygen species (ROS) produce different lesions in DNA by ROS-induced DNA damage. Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for study. Human ribosomal protein S3 (hRpS3) has a high binding affinity to 8-oxoG. In this study, we developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are known to have cell-penetrating properties. Therefore, we developed a TAT-S3 probe by attaching a TAT peptide to our imaging probe. A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and apurinic/apyrimidinic (AP) sites. We confirmed that the TAT-S3 probe localized in the mitochondria, without permeabilization, and fluoresced in H2O2-treated HeLa cells and zebrafish embryos. Treatment with Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted with an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm binding of TAT-S3 to the AP sites. The TAT-S3 probe showed competitive binding to AP sites with ARP and MX. 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.

中文翻译：

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使用具有细胞穿透能力的荧光团标记探针检测8-氧代鸟嘌呤和嘌呤/嘧啶类化合物位点

活性氧（ROS）通过ROS诱导的DNA损伤在DNA中产生不同的损伤。细胞内8-氧代-7,8-二氢鸟嘌呤（8-oxoG）的检测和定量对于研究很重要。人核糖体蛋白S3（hRpS3）对8-oxoG具有高结合亲和力。在这项研究中，我们开发了一种成像探针，可使用hRpS3的特定肽来检测8-oxoG。已知反式激活蛋白（TAT）具有穿透细胞的特性。因此，我们通过将TAT肽连接到我们的成像探针上来开发TAT-S3探针。进行了DNA结合测定，以确认我们的探针与8-oxoG和嘌呤/嘧啶（AP）位点结合。我们证实，TAT-S3探针位于线粒体中，没有通透性，并在H2O2处理的HeLa细胞和斑马鱼胚胎中发出荧光。用Mitoquinone（MitoQ）进行治疗，一种针对线粒体的抗氧化剂，可减少TAT-S3探针的荧光。此外，用O8（OGG1的抑制剂）治疗可增加探针荧光。用醛反应探针（ARP）和甲氧基胺（MX）进行竞争测定，以确认TAT-S3与AP位点的结合。TAT-S3探针显示通过ARP和MX与AP站点竞争结合。这些结果表明，TAT-S3探针成功检测到受损细胞中存在8-oxoG和AP位点。TAT-S3探针可用于检测由活性氧引起的疾病。用醛反应探针（ARP）和甲氧基胺（MX）进行竞争测定，以确认TAT-S3与AP位点的结合。TAT-S3探针显示通过ARP和MX与AP站点竞争结合。这些结果表明，TAT-S3探针成功检测到受损细胞中存在8-oxoG和AP位点。TAT-S3探针可用于检测由活性氧引起的疾病。用醛反应探针（ARP）和甲氧基胺（MX）进行竞争测定，以确认TAT-S3与AP位点的结合。TAT-S3探针显示通过ARP和MX与AP站点竞争结合。这些结果表明，TAT-S3探针成功检测到受损细胞中存在8-oxoG和AP位点。TAT-S3探针可用于检测由活性氧引起的疾病。