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Hepatitis delta virus (HDV) RNA ribozyme system which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer, Fig. 1) was designed. Effects of Mg2+ concentration on the pseudo first-order rate constant (kobs) of RNA cleavage reaction and on conformation of ribozyme complex were examined. The secondary structure of the complex was also analyzed by limited digestion with ribonucleases. The kobs and CD data were analyzed by curve-fitting analysis using equations derived for two-Mg2+ and three-Mg2+ ion binding models. The result revealed that a three-Mg2+ binding model can explain the Mg(2+)-concentration-dependent changes of both conformation and activity of the HDV ribozyme.

We have reported that d(GGAGGAA) forms a new type of quadruplex in the presence of K+. Here we report that in the absence of K+ it forms another kind of new structure composed of non-standard base pairs exclusively. The structure is neither a quadruplex nor a hairpin. The structure gives three clear imino proton signals of G at 10.0, 13.0 and 13.8 ppm, indicating the formation of G:A and/or G:G base pairs. The two signals at 13.0 and 13.8 ppm indicate that there are two base pairs in which the imino protons of G are hydrogen bonded. All residues take anti conformation, which imposes the restriction on the combination of a mutual direction of strands and types of base pairs. Phosphorus signals of the second and fifth residues appear at higher field, while that of the seventh residue at lower field, when compared with those of A and B form structures, suggesting deviations of a backbone conformation from the standard one. Replacement of the second or fifth G by I results in destabilization of the structure, which implies the involvement of amino groups of these residues in base pairing. On the basis of these results, the structure of d(GGAGGAA) is discussed.

Properties of a mutant hepatitis delta virus (HDV) ribozyme which consists of three RNA oligomer strands (Fig. 1c) are described. Effects of the MgCl2 concentration, pH and temperature on the cleavage reaction were examined. The dependence on these conditions did not resemble those of the published data for other HDV ribozymes (the parent) which consist of single-strand RNA of about 90 nt. The highest cleavage for the mutant was observed at 35-40 degrees C, while the parent ribozymes show higher activity at around 60 degrees C than at 40 degrees C. Thus we suppose that the most of the parent ribozymes are in inactive conformation at 40 degrees C and high temperature is needed to change the equilibrium between the inactive and active conformations. Since the Tm of the mutant was around 50 degrees C, these results suggest that the most of the mutant ribozyme complex molecules are in active conformation at 40 degrees C.

The mechanism of mutagenesis induced by dihydropyrimido [4,5-c][1,2]oxazin-7-one deoxyriboside, P-nucleoside, was studied. This analog is highly mutagenic toward Escherichia coli and Salmonella typhimurium. In E. coli, it induces GC-to-AT and AT-to-GC transitions specifically. No transversions are inducible. P-nucleoside was highly mutagenic to a wild-type E. coli, but little mutagenic in a strain lacking thymidine kinase. This indicates that P-nucleoside may be phosphorylated by thymidine kinase after its uptake into bacteria. The mutagenesis induced by P-nucleoside was efficiently inhibited by the addition of thymidine. This inhibition further confirmed the involvement of thymidine kinase in the first step of the metabolism of P-nucleoside in the bacterial cells. These findings indicate that P-nucleoside is a mutagen of a nucleoside-analog type, causing mutations by the erroneous incorporation and replication. The experiments to prove its ambiguous nature in DNA synthesis is now under way.

We enzymatically synthesized c-myc mRNA fragment containing the translation initiation (TI) region (36mer) and examined the structure by CD, 1H-NMR and RNaseV1 digestion. These study demonstrated that the c-myc mRNA fragment forms a hairpin structure at 37 degrees C. Six 15-mer antisense oligodeoxynucleotides(ODNs), which hybridize to the c-myc mRNA fragment were designed, and synhesized. Thermostabilities of the antisense ODNs-mRNA fragment complexes were examined. Antisense A (complementary to the c-myc mRNA from -9 to +6)-c-myc mRNA fragment complex had the highest melting temperature(Tm), and Antisense D (complementary to the c-myc mRNA from +1 to +15)-c-myc mRNA fragment complex had the most different Tm from a predicted Tm. Furthermore, Antisense D exhibited high inhibitory effect on HL60 cell proliferation, whereas Antisense A did not. The results suggest that the difference in the antisense effect is not simply due to the complex thermostability.

The CD study of r(GGAGGAA) has indicated that it forms a higher order structure upon either increase of the strand concentration or addition of K+. Gel electrophoresis showed that a high molecular weight species is formed in the presence of K+. The NMR study revealed a nearly 1:1 equilibrium of two kinds of higher order structures at the strand concentration of 2.1 mM and 100 mM Na+. This equilibrium shifted drastically upon addition of K+, and only one structure remained at 40 mM K+. It is suggested that the structure formed by r(GGAGGAA) in the presence of K+ is a new type of RNA quadruplex, where an A-quartet stabilized by mutual hydrogen bonding among the four adenine bases is sandwiched by conventional G-quartets.

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)2 and d(GGACGAGTCC)2, both of which have been demonstrated to form two consecutive 'sheared' A:G base pairs at the center by our previous studies. The parameters were determined also for r(GGACUAGUCC)2 and d(GGACTAGTCC)2, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes.

Base pairing between A and G residues has been suggested in some ribozymes and the structure derived from this non-standard base pairing could play a crucial role in enzymatic actions of the ribozymes. We have studied the structures of three DNAs and an RNA that base sequences were modeled after the ribozymes by NMR. It was found that the A and G residues can form a basepair in a very unique fashion both in the DNAs and RNA. "Side" instead of "head to head" alignment of the two bases was found, where an amino proton instead of an imino proton of the G residue is involved in the hydrogen bonding. As a result of the unique basepairing, certain bases stack over the bases of the opposite strand instead of over the ones of the same strand. It was also shown that the formation and thermal stability of this unique AG basepair depend on the neighbouring base sequences. Moreover existence of the unique AG basepairs was suggested in a real small metalloribozyme itself.

Structural features and DNA binding properties of bacterially expressed c-Myc related oncoproteins were examined. The circular dichroism spectra demonstrated highly alpha-helical nature of these proteins. Dimerization and the DNA binding of these proteins were examined by electrophoretic mobility shift assay.

The solution structure of the DNA-binding domain (DBD) of the human retinoic acid receptor-beta (hRAR-beta) has been determined by nuclear magnetic resonance (NMR) spectroscopy and distance geometry (DG). The assignments of 1H and 15N resonances were carried out by the use of 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional NMR experiments. The structure of RAR DBD has been obtained on the basis of distance constrains derived from NMR experiments. The structure shows that two "zinc-finger" domains of the protein are followed by two perpendicular alpha-helices and a short beta-sheet near the N-terminus. Apolar residues in both helices form a hydrophobic core. Binding models of RAR DBD to its inverted and direct repeat response elements have been constructed based on this three-dimensional structure.

The conformation of the chromomycin-d(GGGGCCCC)2 complex in aqueous solution was studied by NMR spectroscopy. The NMR spectrum of the complex indicated that the chromomycin binds as a symmetry-related dimer to the minor groove of the central four residues of d(GGGGCCCC)2. The drastic conformational change in the central four residues of d(GGGGCCCC)2 from the B form family to the A-form was demonstrated by the characteristic NOEs and coupling patterns. The change seems to be indispensable for accommodation of the bulky chromomycin dimer in the minor groove. On the basis of the intermolecular NOEs between chromomycin and d(GGGGCCCC)2, the structure of the complex has been constructed and refined by energy minimization.

Eighteen kinds of oligodeoxyribonucleic acids have been examined to reveal their structures in aqueous solutions at different ionic strengths by Raman spectroscopy. The structures in solutions were found to be very polymorphic depending on their sequences as well as on the salt concentrations. At a low salt condition a DNA oligomer assumes a unique B form within a B family, for examples Ba, Bh, B', or Bn form. Amongst these DNA oligomers, d(CGCG)2 showed a salt induced Ba-Z transition, while d(GGGGCCCC)2 showed a salt induced Bh-A transition. DNA oligomers with AA/TT sequences were found to prefer B' form even at high salt condition. From comparing the structures of DNA oligomers in solutions with their crystal structures, it is safe to say that the crystal structure of a DNA oligomer is very similar to the structure in the high salt solution.

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.

The conformations of double stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) and d(GCATTTTGAAACG).d(CGTTTCAAAATGC) have been studied by means of NMR spectroscopy. Analyses of cross peaks in NOESY spectra between H2 of an adenine and H1' of the deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' along the tract in four oligonucleotides. A new model is proposed as to DNA bending based on the evidence of gradual compression of the minor groove. This model can explain why d(GGAAATTTCC) n x 2 and d(GAAAATTTTC) n x 2 are bent, and d(GGTTTAAACC) n x 2 and d(GTTTTAAAAC) n x 2 are not bent. The bending of d(AjN10-j) n x 2 sequences can also be explained.