Abstract
The carbonate radical anion (CO(3)) 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 CO(3) 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 CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), 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 CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) 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.
Highlights
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. In these time-resolved transient absorbance experiments, the CO3. radicals are generated by oneelectron oxidation of the bicarbonate with sulfate radical anions (SO4.) that, anions in turn, (HCO3Ϫ) are derived from the photodissociation of persulfate anionsieoinrna.idtTiiahctaeeldsk, ibGnye(؊t3iHc0s)81⁄7,onafmrtihsXienegCClOfr3e.oxmacnitmihoeenrraalnapdsiednrdepeuputrrlsaoeltonation of the guanine radical cation, are monitored via their transient absorption spectra on time scales of microseconds to seconds
Duplex Design—The duplex formed by the self-complementary 16-mer d(AACG1CG2AATTCG3CG4TT) sequence contains the 12-mer d(CG1CG2AATTCG3CG4) core. The latter is the first oligonucleotide duplex containing at least one turn of helix for which structural information was obtained by single-crystal x-ray diffraction methods [39, 40] and high resolution NMR
Summary
Ref. 33 This work Ref. 33 This work Ref. 31 Ref. 32 This work Ref. 45 This work This work This work This work This work This work This work a The product reactions radicals, were monitored spectroscopically via the G(ϪH)1⁄7 (315 nm) and 8-oxo-G(ϪH)1⁄7 (320 decay nm). (445 nm) radicals and the concomitant formation of the b The reactions were monitored by the accelerated decay of CO3. Stranded DNA and do not react significantly with any of the other nucleic acid bases A, C, or T. The oxidation of the different guanines in the self-complementary DNA duplex results in alkali-labile guanine lesions; these are revealed by the site-selective DNA strand cleavage induced by a standard hot piperidine treatment and polyacrylamide gel electrophoresis assay
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