CONSEQUENCES OF REPLACING THE DNA 3'-OXYGEN BY AN AMINO GROUP - HIGH-RESOLUTION CRYSTAL-STRUCTURE OF A FULLY MODIFIED N3'-]P5' PHOSPHORAMIDATE DNA DODECAMER DUPLEX

As part of the quest for antisense compounds with relative to DNA and RNA improved nuclease-resistance and favorable RNA hybridization prope rties, a large variety of oligonucleotide analogues has been generated in recent years. Among these, the oligonucleotide N3' --> P5' phospho ramidate DNA (3'-NP DNA), an analogue with the 3'-oxygen in the nuclei c acid sugar-phosphodiester backbone replaced by an amino group, displ ays several unique features. Self-pairing of 3'-NP DNA single strands is significantly favored thermodynamically over self-pairing of both D NA 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 dupl ex conformation of 3'-NP DNA is very similar to the RNA A-form. Moreov er, 3'-NP DNA can form stable triplexes with double stranded DNA under conditions where native DNA fails to do so. Recently, it was shown th at all-phosphoramidate DNA analogues of HIV-1 RRE and TAR RNA specific ally 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-ra y crystal structure of the all-modified 3'-NP DNA duplex [5'-d(CnpGnpC npGnpAnpAnpTnpTnpCnpGnpCnpG at 2-Angstrom resolution. Whereas the Dick erson-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 wit h identical sequence displays an A-RNA conformation in the crystal. Co mbined with the earlier CD results in solution, our observation provid es convincing evidence that the A-conformation with 3'-NP DNA is modif ication-and not sequence-induced. The crystal structure reveals a dram atically improved hydration of the phosphoramidate DNA relative to DNA due to the presence of the amino group in its sugar-phosphate backbon e. Compared with A-DNA and A-RNA, the 3'-NP DNA duplex geometry appear s 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 sig ma orbital of the P-O5' bond. Our crystal structure can qualitatively explain the exceptional thermodynamic stability of 3'-NP DNA, and hel ps to rationalize previously ill understood findings, such as the surp rising fact that DNA with NH substituted for O5' fails to pair with ei ther DNA or RNA. The crystal structure also establishes 3'-NP DNA as t he quintessential RNA mimetic, in terms of overall duplex structure, r igidity, and level of hydration.