NMR Solution Structure of the N3‘ → P5‘ Phosphoramidate Duplex d(CGCGAATTCGCG)₂ by the Iterative Relaxation Matrix Approach

Abstract

High-resolution 2D NMR spectra of the duplex CGCGAATTCGCG with deoxyribose sugars but with the normal phosphodiester linker replaced by an N3' --> P5' phosphoramidate (NP) group have been used to establish a solution structure for the duplex. Distance, angle, and base pair hydrogen-bonding constraints were used to refine the structure by use of the iterative relaxation matrix approach (IRMA). The phosphoramidate NH proton signal could be observed in DMSO at low temperature but not in H2O and D2O. For this reason, the structure was refined with the -NH in each of the two possible low-energy configurations. The structure with the nitrogen lone pair located between the nonbridging oxygen atoms of the 5'-phosphate group consistently had the lowest energy and RMSD values, consistent with an X-ray analysis of the same duplex [Tereshko, V., Gryaznov, S. , and Egli, M. (1998) J. Am. Chem. Soc. 120, 269-283]. In the refined structure, the sugars are in the C3'-endo conformation with the change from the normal C2'-endo conformation of deoxyribose apparently being driven by the gauche effect and the change in electronegativity from the 3'O to the 3'NH group. In agreement with preliminary studies [Ding, D., Gryaznov, S. M., Lloyd, D. H., Chandrasekaran, S., Yao, S., Ratmeyer, L., Pan, Y., and Wilson, W. D. (1996) Nucleic Acids Res. 24, 354-360], the backbone conformation in the NP duplex is very close to classical A-form values. Comparison of phosphodiester and phosphoramidate structures suggests that their backbones have global conformations that are primarily a function of the low-energy state of the sugar ring. A somewhat more complex situation arises when base pair conformation is analyzed with many of the base pairs having a conformation between those of classical A- and B-form helices. The effects of the 2' substituent are obviously important in specifying the final conformation of the stacked bases in either an A-form or B-form helix. It is clear, however, that conversion of the normal phosphodiester of DNA into a phosphoramidate linkage yields a nucleic acid that behaves much more like RNA than DNA, and it has been shown that NP sequences can bind to RNA-directed proteins [Rigl, C. T., Lloyd, D. H., Tsou, D. S., Gryaznov, S. M., and Wilson, W. D. (1997) Biochemistry 36, 650-659].

Loop–loop interaction of HIV-1 TAR RNA with N3′ → P5′ deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription

“…This conclusion is further supported by CD experiments on both complexes (data not shown). Only slight differences in CD spectra are observed, which likely originate from those reported in the structure of an A-type helix NP-DNA duplex (22).…”

“…Synthesis of N3Ј 3 P5Ј phosphoramidate oligodeoxynucleotides (NP-DNA), which are resistant to digestion by snake venom phosphodiesterase and by nucleases in HeLa cell nuclear extract, has been described (20,21). NMR experiments showed that a NP-DNA duplex d(CGCGAATTCGCG) 2 adopts an A-type helix conformation in solution (22), suggesting that NP-DNA could be used as RNA mimetics. To ascertain this possibility, a NP-DNA version of the anti-TAR RNA hairpin aptamer was synthesized.…”

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

“…P5' phosphoramidates [19] [20]. In contrast, the 3'-hydroxy-2'-deoxy nucleosides prefer C2'-endo, or S-type, conformations under similar experimental conditions.…”

“…P5' internucleoside phosphoramidate linkages [17]. This phosphoramidate duplex adopts a RNA-like A-type double helix [17] [20]. Additionally, X-ray analysis revealed a more extensive hydration pattern of the oligonucleotide phosphoramidate sugar-phosphate backbone compared to that for the phosphodiester counterpart.…”

“…Oligonucleotide N3' ! P5' phosphoramidates in single-and in double-stranded forms are structurally similar to their natural phosphodiester RNA counterparts [8] [17] [20]. Consequently, it may be plausible to prepare functionally important natural RNA structural elements from the oligonucleotide phosphoramidates and to use them as synthetic decoys for the RNAbinding proteins.…”