Quantitative and in situ detection of oxidatively generated DNA damage 8,5'-cyclo-2'-deoxyadenosine using an immunoassay with a novel monoclonal antibody.

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix-distorting types of DNA damage including sunlight-induced pyrimidine dimers. In addition to skin disease, approximately 30% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of a particular type of oxidatively generated DNA damage called purine 8,5'-cyclo-2'-deoxynucleosides (purine cyclonucleosides). However, there are no currently available methods to detect purine cyclonucleosides in DNA without the need for DNA hydrolysis. In this study, we generated a novel monoclonal antibody (CdA-1) specific for purine cyclonucleosides in single-stranded DNA that recognizes 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA). An immunoassay using CdA-1 revealed a linear dose response between known amounts of cyclo-dA in oligonucleotides and the antibody binding to them. The quantitative immunoassay revealed that treatment with Fenton-type reagents (CuCl2/H2O2/ascorbate) efficiently produces cyclo-dA in DNA in a dose-dependent manner. Moreover, immunofluorescent analysis using CdA-1 enabled the visualization of cyclo-dA in human osteosarcoma cells, which had been transfected with oligonucleotides containing cyclo-dA. Thus, the CdA-1 antibody is a valuable tool for the detection and quantification of cyclo-dA in DNA, and may be useful for characterizing the mechanism(s) underlying the development of XP neurological disease.