2618 Biochemistry, Vol. 49, No. 12, 2010 Shanmugam et al. FIGURE 1: Temperature-dependent 1H NMR spectra (imino proton region) for the 1-BD (A) and unmodified 1-BD (B) duplex. was increased to 47 C, the G2, T5, and T10 imino proton resonances at 13.1, 13.5, and 12.7 ppm, respectively, were the first to disappear from the spectrum. However, the T16, T17, T20, G21/G14, and G7 imino proton resonances remained visible (Figure 1A). In contrast, only three sharp resonances were observed for guanines from the unmodified 1-BD duplex at 7 C (Figure 1B). The peak assigned as G7, next to the modifica- tion site in the 30 direction, was broadened in the unmodified 1-BD duplex (Figure 1B). (i) Nonexchangeable DNA Protons. The assignments were made using standard protocols (46, 47). The NOESY spectra for the 1-BD duplex exhibited resolved cross-peaks (Figure 2A,B). The anticipated pattern of sequential NOE connectivity between base aromatic and deoxyribose anomeric protons was identified for both the modified and complementary strands from C1 to C12 and from G13 to G23, respectively (Figure 2A,B). There were no interruptions to the sequential NOEs at or adjacent to the adduct site. However, the T5 H10 f X6 H2 NOE (the imidazole proton of 1,N2-εdG; note the modified numbering scheme for this nucleotide) was weak compared to the corresponding NOEs (Figure 2A,B). Likewise, the connectivity between C18 H10 and A19 H8 in the complementary strand was weak. The T17 H10 f C18 H6 and C18 H6 f C18 H10 NOE peaks overlapped. For T5, X6, G7, C18, and A19, the NOEs between the base protons and the sugar H10 protons were similar in intensity to NOEs between other base and sugar H10 protons. All of the intranucleotide NOE peaks between adenine H2 and H10 protons of the attached deoxyribose moieties were observed. Expected sequential NOE peaks between the adenine H2 proton and the H10 proton of the 30 neighbor were also observed (Figure 2A,B). Likewise, the sequential NOE peaks between a base proton (H8 or H6) and the H5 proton of cytosine from the 30 neighbor were observed (Figure 2A,B). The pattern of NOEs between the base aromatic protons and the deoxyribose H20 and H200 protons was similar to that observed for the NOEs between the aromatic protons and the deoxyribose H10 protons, in both the modified and complementary stands. Sequential NOEs from C18 H20 to A19 H8 and from C18 H200 to A19 H8 were weak compared to other corresponding NOEs. The assignments of the nonex- changeable protons of the 1-BD and unmodified 1-BD duplexes are listed in Tables S1 and S2, respectively, of the Supporting Information. FIGURE 2: Expanded plot of a phase sensitive NOESY spectrum (250 ms) in D2O buffer for the 1-BD oligodeoxynucleotide duplex showing the sequential NOEs from the aromatic to H10 protons. The spectrum was recorded at 25 C. (A) Connectivities are traced for the 1,N2-εdG-modified strand: Additional cross-peaks, (A) A4 H2 f A4 H10, (B) A4 H2 f T5 H10, (C) A8 H2 f A8 H10, (D) A8 H2 f A9 H10, (E) A9 H2 f A9 H10, (F) A9 H2 f T10 H10, (G) G2 H8 f C3 H5, (H) T10 H6 f C11 H5, and (I) C11 H6 f C12 H5. (B) Connectivities are traced for the complementary strand: Additional cross-peaks, (J) A15 H2 f A15 H10, (K) A15 H2 f T16 H10, (L) A19 H2 f A19 H10, (M) A19 H2 f T20 H10, (N) T17 H6 f C18 H5, and (O) G21 H8 f C22 H5. (ii) Exchangeable DNA Protons. Seven well-resolved and two partially resolved peaks between 11.5 and 13.9 ppm ac- counted for all of the imino protons of the 1-BD duplex, except