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DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2'-deoxyadenosine and 2'-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO4 and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2'-deoxycytidine lesions form the base pair with guanine while the 2'-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.

Base stacking is important for the base pair interaction of a DNA duplex, DNA replication by polymerases, and single-stranded nucleotide overhangs. To study the mechanisms responsible for DNA stacking interactions, we measured the thermal stability of DNA duplexes containing a non-natural nucleotide tethered to a simple aromatic hydrocarbon group devoid of dipole moments and hydrogen bonding sites. The duplexes containing tetrahydrofuran were paired with a deoxyadenosine derivative (A/T base pair analog) or a deoxycytidine derivative (C/G base pair analog) and showed a lower stability than Watson-Crick base pairing, partly due to the loss of interbase hydrogen bonds. Conversely, non-natural nucleotides present at a dangling end yielded an interaction energy as high as that observed with base pairing. Importantly, the non-natural nucleotides yielded an interaction energy with a linear correlation similar to that of the analogous Watson-Crick base pairs both in the middle and at the end of a DNA duplex, although a different stacking mechanism between the middle and the end was suggested. Moreover, a positive cooperativity was observed in dangling end stacking of the nucleotide base moiety and aromatic hydrocarbon group. These observations are useful to understand nucleic acid interactions and to design new non-natural nucleotides.

A base flipping conformation is found in many biological processes, including DNA repair and DNA and RNA modification processes. To investigate the dynamics and energetics of this unusual conformation in a double helix, base flipping induced by the base pair analogues of deoxyadenosine and deoxycytidine derivatives tethering a phenyl or naphthyl group was investigated. DNA strands bearing the base pair analogues stabilized the base flipping conformation of a complementary RNA, resulting in a site-specific hydrolysis by specific base catalysis. Measurements of the hydrolysis rate and the thermal stability of DNA/RNA duplexes suggested an unconstrained flexibility of the flipped-out ribonucleotide. As established in the base flipping by DNA repair and DNA and RNA modification enzymes, the results suggested that base flipping occurred in competition with base pair formation. In addition, the deoxycytidine derivatives discriminated G from I (inosine), with respect to the base pair interaction energy, as observed for a damaged base or a weakened base pair search by DNA repair proteins. The base pair mimic nucleosides would be useful for investigating the base flipping conformation under the equilibrium with base pairing.

We developed the base pair-mimic nucleosides tethering the phenyl group and the naphtyl group, synthesized from deoxyadenosine (Aphe and Anaph) and deoxycytidine (Cphe and Cnaph). Structure and the thermal stability of the DNA duplexes containing the deoxyadenosine derivatives or the deoxycytidine ones in the middle of a DNA strand were obtained by the CD spectra and the UV melting curves. The DeltaGo37 values among DNA duplexes containing Aphe-A, Aphe-G, Aphe-C and Aphe-T pair differed only by 1.9 kcal mol(-1). The DNA duplexes containing Cphe also showed the DeltaGo37 values differed by 1.0 kcal mol-1 despite opposite base nucleotide, except the duplex containing the Cphe-G pair. Interestingly, the duplex consisting of the DNA containing the base pair-mimic nucleosides and the complementary RNA strand caused the site-selective RNA hydrolysis except the Cphe-G pair. This data suggests that the Cphe has an ability of forming the base pair with guanosine.

We previously reported that the deoxyadenosine derivative tethering the phenyl group at the N6 position of deoxyadenosine, A(phe) stacked efficiently with the adjacent nucleotide bases at a DNA duplex terminus and in the middle of a DNA duplex. In contrast with the observations for A(phe), this study revealed that the phenyl group of the deoxycytidine derivative, C(phe) located outside the helix and allowed the base pair formation with guanine in a DNA duplex, although its phenyl group could stack with the adjacent DNA bases as efficient as A(phe). Klenow fragment of DNA polymerase I and T7 DNA polymerase selectively incorporated dTTP and dGTP opposite A(phe) and C(phe) in the template DNA, respectively, implying that the conformation of A(phe) differs between in the DNA polymerase and in solution.

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.

On the basis of the non-covalent bond interactions in nucleic acids, we have synthesized a novel adenosine derivative tethering a phenyl group. Structure and thermal stability of the DNA duplexes bearing the adenosine derivative in the middle of a strand were investigated by CD spectra and the UV melting curves. We found that the conformation and the interactions of the duplexes were similar regardless of the nucleotide facing with the adenosine derivative in a duplex. This observation suggests the base pair-mimic geometry of the adenosine derivative accompanied with a conformational change of the nucleotide in the complementary strand.

Novel deoxyadenosine derivatives tethering a phenyl or naphthyl group by means of an amido linker have been synthesized, and these derivatives, stacking on the 5' end of a DNA duplex, provide free energy contributions equal to or greater than that of the Watson-Crick A/T base pair.

We synthesized novel adenosine derivatives tethering an aromatic hydrocarbon group by an amido linker. The single adenosine derivatives at 5' dangling end stabilized the DNA duplex of 5'-ATGCGCAT-3' more or equally than Watson-Crick base pair. When the number of the dangling residues increased from one to three, the duplex stability became larger by 1.8-3.6 kcal/mol. When the adenosine derivative was opposite to an abasic site in a DNA duplex, the destabilization by the abasic site was significantly reduced. These observations suggest that the adenosine derivatives developed in this study can stack with a DNA base pair by forming a base pair-mimic geometry.