Intramolecular Cyclization in Irradiated Nucleic Acids: Correlation between High-Performance Liquid Chromatography and an Immunochemical Assay for 8,5′-Cycloadenosine in Irradiated Poly(A)
A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5''-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5''-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5''-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5''-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5''-AMP or adenosine irradiated under similar conditions.