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 intermed iate oxidant derived from the oxidation of bicarbonate anions and nitrosope roxocarboxylate anions (formed in the reaction of CO2 with ONOO-) in cellul ar environments. Employing nanosecond laser flash photolysis methods, we sh ow that the CO3.- anion can selectively oxidize guanines in the self-comple mentary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilib rated aqueous buffer solution (pH 7.5), In these time-resolved transient ab sorbance experiments, the CO3.- radicals are generated by one-electron oxid ation of the bicarbonate anions (HCO3-) with sulfate radical anions (SO4.-) that, in turn, are derived from the photodissociation of persulfate anions (S2O82-) initiated by 308-nm XeCl excimer laser pulse excitation. The kine tics of the CO3.- anion and neutral guanine radicals, G(-H)(.), arising fro m the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxims 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 10(7) M-1 s(-1). The decay of the CO3.- anions and th e formation of G(-H)(.) radicals are correlated with one another on the mil lisecond time scale, whereas the neutral guanine radicals decay on time sca les 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 guan ine sites only, The biological implications of these oxidative processes ar e discussed.