Thermally Stable Sites for Electron Capture in Directly Ionized DNA:  Free Radicals Produced by the Net Gain of Hydrogen at C5/C6 of Cytosine and Thymine in Crystalline Oligodeoxynucleotides

Electron paramagnetic resonance (EPR) spectroscopy is used to study radical trapping in crystalline oligodeoxynucleotides exposed to 70 keV x-irradiation at 4 K and annealed to 240 K. The four oligomers studied were the Z form d(CGCACG:GCGTGC), two A forms, d(CCCTAGGG) 2 and d(GTGCGCAC) 2, and the B form d(CGCGAATTCGCG) 2 . In each of these oligomers, evidence was found for trapping of a cytosine radical formed by the net gain of a hydrogen at C 6 and a proton at N 3 (the Cyt(C 6 +H, N 3 +H + ) +• radical). The data are consistent with the trapping of another cytosine radical formed by the net gain of hydrogen at C5 (the Cyt(C 5 +H, N 3 +H) +• or Cyt(C 5 +H) • radical). The well-known thymine radical formed by the net gain of hydrogen at C5 (Thy(C 6 +H) • radical) was observed in the Z- and B-form duplexes but not in the A-form duplexes. The relative yields of these three reduction species indicate that cytosine is comparable to, or better than, thymine as a stable trapping site for reductive damage. These three radicals, Cyt(C 6 +H, N 3 +H + ) +•, Cyt(C 5 +H, N 3 +H) +•, and Thy(C 6 +H) •, account for ∼85% of the total irreversibly trapped electrons in samples irradiated at 4 K and annealed to 240 K. Extrapolation of these results to B-form DNA hydrated to 9 waters per nucleotide, x-irradiated at 4 K, and warmed to room temperature predicts end product yields of 0.04−0.06 μmol/J for 5,6-dihydrouracil and 0.03−0.05 μmol/J for 5,6-dihydrothymine.