Quantitative and in situ Detection of Oxidatively Generated DNA Damage 8,5′-Cyclo-2′-Deoxyadenosine Using an Immunoassay with a Novel Monoclonal Antibody

• Published in: Photochemistry and photobiology Vol. 90; no. 4; pp. 829 - 836
• Main Authors: Iwamoto, Takaaki, Brooks, Philip J., Nishiwaki, Tomohisa, Nishimura, Kazuki, Kobayashi, Nobuhiko, Sugiura, Shigeki, Mori, Toshio
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.07.2014
• Subjects: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Deoxyadenosines - chemistry; DNA Damage; Gene Expression Regulation; Humans; Hybridomas; Immunoassay; Mice; Molecular Structure; Monoclonal antibodies; Oxidation; Visualization
• ISSN: 0031-8655; 1751-1097
• DOI: 10.1111/php.12239

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. Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix‐distorting 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 oxidatively generated DNA damage called purine cyclonucleosides. In this study, we generated a novel monoclonal antibody (CdA‐1) specific for purine cyclonucleosides in DNA that recognizes 8,5′‐cyclo‐2′‐deoxyadenosine (cyclo‐dA). An immunofluorescent analysis using CdA‐1 enabled the visualization of cyclo‐dA (green dots) in human osteosarcoma cells, which had been transfected with oligonucleotides containing 5′S‐cyclo‐dA.