Article Biochemistry, Vol. 49, No. 12, 2010 2619 FIGURE 3: (A) Downfield region of the 1H NMR spectrum for the 1-BD duplex showing the imino proton resonances. (B) Expanded plot of a NOESY spectrum with a mixing time of 250 ms for the 1-BD duplex showing the sequential NOEs between the imino protons and the amino and base protons: (A and A0) G2 N1H f C22 N4H, h/ n; (B and B0) G21 N1H f C3 N4H, h/n; (C and C0) G7 N1H f C18 N4H, h/n; (D and D0) G14 N1H f C11 N4H, h/n; (E) T20 N3H f A4 H2; (F) T5 N3H f A19 H2; (G) T17 N3H f A8 H2; (H) T16 N3H f A9 H2; and (I) T10 N3H f A15 H2. The symbols h and n denote hydrogen-bonded and non-hydrogen-bonded amino protons of cyto- sine, respectively. (C). Expanded plot of a NOESY spectrum showing sequential NOE connectivity for the imino protons of the G2 3 C23 f G14 3 C11 base pairs for the 1-BD duplex. The labels designate the imino proton of the indicated nucleotide. The spectra were recorded at 7 C. for the two terminal imino protons that were not observable, presumably because of exchange with solvent (Figure 3). Five sharp resonances between 13.2 and 13.9 ppm were assigned to thymine protons (Figure 3A), as evidenced by NOEs to the corresponding adenine H2 protons (Figure 3B). Four other sharp resonances between 11.5 and 13.2 ppm were assigned to guanine imino protons (Figure 3A), as evidenced by NOEs to the cor- responding cytosine amino protons (Figure 3B). The resonance at 11.8 ppm was assigned to G7 N1H, next to the modified lesion, on the basis of the NOEs to the complementary C18 amino protons (Figure 3B) and sequential NOE connectivity from G7 FIGURE 4: Expanded region of the 1H COSY spectrum showing a cross-peak between etheno H7 and H6 protons. The spectrum was recorded at 25 C. N1H to T17 N3H protons (Figure 3C). From Figure 3C, the expected pattern of NOE cross-peaks was observed for the G2 3 C22, C3 3 G21, A4 3 T20, and T5 3 A19 base pairs, located in the 50 direction from the 1,N2-εdG adduct. Likewise, NOE connec- tivity was observed between the G7 3 C18, A8 3 T17, A9 3 T16, T10 3 A15, and C11 3 G14 base pairs, located in the 30 direction from the 1,N2-εdG adduct. The sequential NOE connectivity between imino protons of neighboring base pairs was interrupted between the T5 3 A19 and G7 3 C18 base pairs. The amino proton of the 1,N2- εdG nucleotide (Scheme 1) was not observed, suggesting rapid exchange with solvent. The resonance assignments for the imino and amino protons of the unmodified 1-BD and modified 1-BD duplexes are listed in Table S3 of the Supporting Information. (iii) 1,N2-εdG Etheno Protons. The COSY spectrum showed a cross-peak between 5.84 and 5.55 ppm (Figure 4). This signal was assigned as arising between the 1,N2-εdG H6 and H7 etheno protons. Hydrogen-deuterium exchange at 50 C estab- lished the assignments of those two protons (19) (Figure 5). The intensity of the peak at 5.84 ppm decreased over time (Figure 5A, curves b-d), whereas that of the peak at 5.55 ppm did not (Figure 5B). The peak at 5.55 ppm became a singlet due to H-D exchange of the proton resonating at 5.84 ppm, which is assigned as the H7 etheno proton. Accordingly, the H6 proton was assigned to 5.55 ppm. (iv) NOEs between Etheno Protons and DNA Protons. Figure 6 contains a tile plot of the NOESY spectrum recorded with a mixing time of 250 ms and shows the NOE between the two etheno protons and NOEs between the etheno protons and the nonexchangeable DNA protons. The H6 and H7 protons showed only cross-strand NOE cross-peaks. The H6 proton showed more NOE cross-peaks than did the H7 proton. A total of 17 cross-peaks were assigned between the etheno protons and nonexchangeable protons (Figure 6). Ten cross-peaks represent NOEs between X6 H6 and nonexchangeable protons, and seven cross-peaks represent NOEs between X6 H7 and nonexchange- able protons. The X6 H7 proton had medium-intensity cross- peaks to C18 H20, C18 H200, C18 H5, and C18 H6, and the X6 H6 proton had medium-intensity cross-peaks to C18 H20, C18 H200, and C18 H10. Weak cross-peaks were observed between the X6 H6 proton and the A19 H50, A19 H500, A19 H40, A19 H10, A19 H2, C18 H6, and A19 H8 protons. The X6 H7 proton had weak cross-peaks to A19 H50/H500, C18 H10, and A19 H2. In addition, three weak NOEs were observed between X6 H7 and H6 and