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Detection of 8-oxoguanine and apurinic/apyrimidinic Sites Using a Fluorophore-Labeled Probe With Cell-Penetrating Ability

Detection of 8-oxoguanine and apurinic/apyrimidinic Sites Using a Fluorophore-Labeled Probe With Cell-Penetrating Ability

Dong Min Kang, Jong-Il Shin, Ji Beom Kim, Kyungho Lee, Ji Hyung Chung, Hye-Won Yang, Kil-Nam Kim, Ye Sun Han

BMC Mol Cell Biol. 2019 Nov 27;20(1):54.

PMID: 31775627

Abstract:

Background:
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.
Results:
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.
Conclusions:
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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP350997396	OGG1 Inhibitor O8		350997-39-6	Price

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