An Immunochemical Probe for 8,5′-Cycloadenosine-5′-Monophosphate and Its Deoxy Analog in Irradiated Nucleic Acids

Abstract

Polyclonal antisera specific for 8,5''-cycloadenosine-5''-monophosphate (8,5''-cyclo-AMP) or its deoxy analog (8,5''-cyclo-dAMP) were elicited by immunizing rabbits wtih a conjugate prepared by the method of Johnston et al. [Biochemistry 22, 3453-3460 (1983)]. A competitive enzyme-linked immunosorbent assay (ELISA) was developed and used to detect the formation of these products in irradiated solutions of polyadenylic acid [poly(A)] or DNA which were saturated with nitrous oxide, nitrogen, or oxygen. The 8,5''-cyclo-AMP or 8,5''-cyclo-dAMP moieties could be detected in poly(A) at 1.0 krad and in native DNA at 10 krad, respectively. The yield of 8,5''-cyclo-dAMP was found to be two to three times higher in irradiated double-stranded DNA than in single-stranded DNA. The hydroxyl radical appears to initiate 8,5''-cyclonucleotide formation in irradiated nucleic acids, as demonstrated by the inhibition of 8,5''-cyclo-AMP formation in irradiated poly(A) by dimethyl sulfoxide. However, irradiation under nitrous oxide, particularly at low doses, does not lead to the expected increases in the yield of the 8,5''-cyclonucleotide.