近藤 次郎

Oligonucleotides containing polyamines are currently being evaluated as potential antigene compounds for therapeutic purposes. Among them, 5-(N-aminohexyl) carbamoyl-2'-deoxyuridine ((N)U) and 5-(N-aminohexyl) carbamoyl-2'-O-methyluridine ((N)Um) substituted oligonucleotides have higher resistance against nuclease degradation compared to native DNA. Furthermore, oligonucleotides containing (N)U stabilizes duplex formation with the complementary DNA. To elucidate the mechanisms behind these improved antigene properties, we synthesized and crystallized two Dickerson-Drew-type DNA duplexes containing (N)U and (N)Um. The 2'-O-methyl modification in (N)Um was found to induce the ribose ring to adopt the C3'-endo conformation. Electron density maps show possible interactions of the terminal ammonium ion of the aminohexyl groups with the phosphate oxygen anions.

DNA fragments containing the sequence d(GCGAAAGC) prefer to adopt a base-intercalated (zipper-like) duplex in crystalline state. To investigate effects of point mutation at the 5th residue on the structure, two crystal structures of d(GCGAGAGC) and d(GCGATAGC) have been determined by X-ray crystallography. In the respective crystals, the two octamers related by a crystallographic two-fold symmetry are aligned in an anti-parallel fashion and associated to each other to form a duplex, suggesting that the base-intercalated duplex is stable even when the 5th residue is mutated with other bases. The sheared G(3):A(6) pair formation makes the two phosphate backbones closer and facilitates formation of the A-X*-X-A* base-intercalated motif. The three duplexes are assembled around the three-fold axis, and their 3rd and 4th residues are bound to the hexamine cobalt chloride. The central 5th residues are bound to another cation.

A DNA fragment d( GCGAAAGC), postulated to adopt a stable mini- hairpin structure on the basis of its extraordinary properties, has been X- ray analyzed. Two octamers related by a crystallographic twofold symmetry are aligned in an antiparallel fashion and associate to form a duplex, which is maintained by two Watson - Crick G . C base pairs and a subsequent sheared G . A pair at both ends. The central two A residues are free from base- pair formation. The corresponding base moieties of the two strands are intercalated and stacked on each other, forming a long column of G(1)- C-2- G(3)- A(4)- A(5)*-A(5)-A*(4)- G*(3)-C*(2)-G*(1) ( asterisks indicate the counter- strand). The Watson - Crick and major- groove sites of the four stacked adenine bases are exposed to the solvent region, suggesting a functional role. Since this structural motif is similar to those found in the nonamers d(G(Br)CGAAAGCT) and d(G(I)CGAAAGCT), the base- intercalated duplex may be a stable form of the specific sequence. Electrophoresis results suggest that the octamer has two states, monomeric and dimeric, in solution depending on the Mg2+ concentration. The present duplex is preferred under the crystallization conditions, which correspond to physiologically allowed conditions.

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4(1)22 with the unit-cell dimensions a = b = 53.4 Angstrom and c = 54.0 Angstrom, and the crystal structure has been determined at 2.5 Angstrom resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C-2:C-2(+) (singly-protonated between the Watson- Crick sites), G(3):G(3) (between the minor groove sites), A(4):A(4) (between the major groove sites) and A(5):A(5) (between the Watson-Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.

To investigate the role of divalent cations in crystal packing, a Dickerson-Drew-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2'-deoxy-5-formyluridine at X, was crystallized under several conditions with Ba2+ ion instead of Mg2+ ion. The crystal structure is isomorphous with the original Dickerson-type crystal containing Mg2+ ion. In the Mg2+-free crystals, however, a five-membered ring of water molecules occupies the same position as the magnesium site found in the Mg2+-containing crystals, and connects the two duplexes similarly to the hydrated Mg2+ ion. It has been concluded that the five-membered water molecules can take the place of the hydrated magnesium cation in crystallization. The 5-formyluracil residues form the canonical Watson-Crick pair with the opposite adenine residues. (C) 2002 Elsevier Science B.V. All rights reserved.