As part of the quest for antisense compounds with relative to DNA and RNA improved nuclease-resistance and favorable RNA hybridization properties, a large variety of oligonucleotide analogues has been generated in recent years. Among these, the oligonucleotide N3' --> P5' phosphoramidate DNA (3'-NP DNA), an analogue with the 3'-oxygen in the nucleic acid sugar-phosphodiester backbone replaced by an amino group, displays several unique features. Self-pairing of 3'-NP DNA single strands is significantly favored thermodynamically over self-pairing of both DNA and RNA (Gryaznov, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5798-5802). CD measurements in solution have shown that the duplex conformation of 3'-NP DNA is very similar to the RNA A-form. Moreover, 3'-NP DNA can form stable triplexes with double stranded DNA under conditions where native DNA fails to do so. Recently, it was shown that all-phosphoramidate DNA analogues of HIV-1 RRE and TAR RNA specifically bind to the RNA-binding Rev- and Tat-related peptides (Rigl, C. T.; et al. Biochemistry 1997, 36, 650-659). We have determined the X-ray crystal structure of the all-modified 3'-NP DNA duplex [5'-d(CnpGnpCnpGnpAnpAnpTnpTnpCnpGnpCnpG at 2-Angstrom resolution. Whereas the Dickerson-Drew type phosphodiester DNA 5'-d(CGCGAATTCGCG) adopts a B-form duplex in the crystal as well as in solution, the 3'-NP DNA duplex with identical sequence displays an A-RNA conformation in the crystal. Combined with the earlier CD results in solution, our observation provides convincing evidence that the A-conformation with 3'-NP DNA is modification-and not sequence-induced. The crystal structure reveals a dramatically improved hydration of the phosphoramidate DNA relative to DNA due to the presence of the amino group in its sugar-phosphate backbone. Compared with A-DNA and A-RNA, the 3'-NP DNA duplex geometry appears more uniform, with backbone torsion angles of individual nucleotides displaying only minor variations. This is consistent with an observed repetitive pattern of coordination by either chloride anions or water molecules to the 3'-amino groups in the crystal, suggesting a strong anomeric effect between the 3'-nitrogen lone electron pair and the sigma* orbital of the P-O5' bond. Our crystal structure can qualitatively explain the exceptional thermodynamic stability of 3'-NP DNA, and helps to rationalize previously ill understood findings, such as the surprising fact that DNA with NH substituted for O5' fails to pair with either DNA or RNA. The crystal structure also establishes 3'-NP DNA as the quintessential RNA mimetic, in terms of overall duplex structure, rigidity, and level of hydration.