Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO(3)(·−) radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO(3)(·−) radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpC(n)pT) and 5′-d(TpC(n)pG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO(3)(·−) radicals and enzymatic excision of the 5′-d(G(*)pT(*)) product.