Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N-2-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N-2-dG:N-2-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc. 2003, 12S, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12)). 3'.5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X-7 is the dG adjacent to the C6 carbon of the cross-link or the alpha-carbon of the (6S,8R,11S) 1,N-2-dG adduct, and Y-19 is the dG adjacent to the C8 carbon of the cross-link or the gamma-carbon of the HNE-derived (6S,8R,11S) 1,N-2-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of C-13 at the C8 carbon of the cross-link revealed one (13)C8 -> H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y-19 H1', C-20 H1', and C-20 H4', orienting it toward the complementary strand, consistent with the (65,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y-19 H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y-19 (NH)-H-2 and X-7 N1H protons, respectively. A strong H8 -> H11 NOE and no (3)J(C-13 -> H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N-2-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X-7 N-2 and Y-19 N-2 atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C-20 O-2 of the 5'-neighbor base pair G(5)center dot C-20 and O11H with (CO2)-O-18 of X-7 center dot C-18. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.