Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogues.
8-Oxodeoxyguanosine is present in DNA from many tissues. The direct demonstration of 8-oxodeoxyguanosine as a potential biomarker of oxidative DNA damage has implications for the study of mutagenesis, carcinogenesis, and free radical toxicity. Avidin is shown here to bind with high specificity to this potentially mutagenic oxidized nucleoside, 8-oxodeoxyguanosine, and to the oxidatively modified base, 8-oxoguanine. The serendipitous finding that avidin bound to the nuclei of UVA-irradiated cells has led to the development of a technique which allows detection of the damage product in a manner analogous to that of immunological techniques. The technique has been shown to be applicable to isolated DNA and to DNA in fixed cellular material and postmortem tissue. Statistically different levels of damage can be demonstrated in both isolated DNA and cultured cells exposed to free radical generating systems using a 96-well plate-based methodology. The sensitivity of this method allows the detection of 10(-19) mol of 8-oxodeoxyguanosine. This novel usage of avidin conjugates applies also to its bacterial analogue, streptavidin, and to a lesser extent to the monoclonal antibody to biotin (the ligand bound by the parent compound). This finding has tremendous potential as a simple method analogous to immunotechniques for the direct detection of 8-oxodeoxyguanosine. From structural considerations we speculate that avidin would also bind to 8-oxodeoxyadenosine.
Struthers
L
L
Department of Pathology, University of Leicester, United Kingdom.
Patel
R
R
Clark
J
J
Thomas
S
S
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000368:Aged; D000468:Alkalies / Q000494:pharmacology; D000818:Animals; D001360:Avidin / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001710:Biotin / Q000494:pharmacology; D019921:Biotinylation; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D004247:DNA / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000008:administration & dosage / Q000494:pharmacology; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology; D016472:Motor Neuron Disease / Q000378:metabolism / Q000503:physiopathology; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000468:Alkalies; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D001360:Avidin; C024829:8-hydroxyguanine; D006147:Guanine; D001710:Biotin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine; D016650:Fluorescein-5-isothiocyanate; D008751:Methylene Blue