The structure of the dodecamer d(CGCGAATXCGCG)(2), in which X = Z3dU, 5-(3-aminopropyl)-2'-deoxyuridine, was determined. At neutral pH, Z3dU introduced a positive charge into the major groove. NMR spectroscopy revealed that the Z3dU omega-aminopropyl moiety oriented in the 3'-direction from the site of modification. Watson-Crick base pairing remained intact throughout the dodecamer. The presence of the charged amino group in the major groove resulted in a 0.24 ppm upfield shift of one P-31 NMR resonance in the 3'-direction at the phosphodiester linkage between nucleotides C-9 and G(10). Molecular dynamics calculations restrained by distances obtained from H-1 NOE data and torsion angles obtained from H-1 NMR (3)J coupling data, and in which the omega-amino group was constrained to be proximate to G(10)O(6), predicted from the P-31 NMR data and molecular modeling (Dande, P.; Liang, G.; Chen, F.-X.; Roberts, C.; Nelson, M. G.; Hashimoto, H.; Switzer, C.; Gold, B. Biochemistry 1997, 36, 6024-6032), were consistent with experimental NOES. These refined structures exhibited bending. The distance from the amino group to the 5'-phosphate oxygen of Z3dU was >5 Angstrom, which indicated that in this dodecamer the Z3dU amino group did not participate in a salt bridge to its 5'-phosphate.