The Carbonate Radical Is a Site-selective Oxidizing Agent of Guanine in Double-stranded Oligonucleotides

The carbonate radical anion (CO₃^.-) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO ₂ with ONOO^-) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO₃ ^.- anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO₃^.- radicals are generated by one-electron oxidation of the bicarbonate anions (HCO₃^-) with sulfate radical anions (SO₄^.-) that, in turn, are derived from the photodissociation of persulfate anions (S₂O ₈^2-) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO₃^.- anion and neutral guanine radicals, G(-H)^., arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO₃^.- is (1.9 ± 0.2) x 10⁷ M^-1 S^-1. The decay of the CO₃^.- anions and the formation of G(-H)^. radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.