Efficient incorporation of a copper hydroxypyridone base pair in DNA

Recently, we reported the first artificial nucleoside for alternative DNA base pairing through metal complexation (J. Org. Chem. 1999, 64, 5002-5003). In this regard, we report here the synthesis of a hydroxypyridone-bearing nucleoside and the incorporation of a neutral Cu(2+)-mediated base pair of hydroxypyridone nucleobases (H-Cu-H) in a DNA duplex. When the hydroxypyridone bases are incorporated into the middle of a 15 nucleotide duplex, the duplex displays high thermal stabilization in the presence of equimolar Cu(2+) ions in comparison with a duplex containing an A-T pair in place of the H-H pair. Monitoring temperature dependence of UV-absorption changes verified that a Cu(2+)-mediated base pair is stoichiometrically formed inside the duplex and dissociates upon thermal denaturation at elevated temperature. In addition, EPR and CD studies suggested that the radical site of a Cu(2+) center is formed within the right-handed double-strand structure of the oligonucleotide. The present strategy could be developed for controlled and periodic spacing of neutral metallobase pairs along the helix axis of DNA.     

Drug-induced stabilisation of a mismatched C-T base pair in a DNA hairpin

We have shown that a key feature of drug binding, namely specific G-C base pair recognition at a 5'-TG step, can induce a number of novel structural features when an extrahelical base is inserted in close proximity to the drug binding site; we have clearly demonstrated the formation of a stabilised C-T mismatched base pair at a non-terminal site.     

DNA deformability at the base pair level

A complete set of harmonic force constants describing the DNA deformation energetics at the base pair level was obtained using unrestrained atomic-resolution molecular dynamics simulations of selected duplex oligonucleotides and subsequent analysis of structural fluctuations from the simulated trajectories. The deformation was described by the six base pair conformational parameters (buckle, propeller, opening, shear, stretch, stagger). The results for 13 AT pairs and 11 GC pairs in different sequence contexts suggest that buckle and propeller are very flexible (more than roll in TA dinucleotide steps), while stretch is exceptionally stiff. Only stretch and opening stiffness were found to depend unambiguously on the base pair identity (AT vs GC). The relationship of the results to a simple plates-and-springs model of base-base interactions is discussed.     

Ultrafast deactivation of an excited cytosine-guanine base pair in DNA

Multiconfigurational ab initio calculations and QM/MM molecular dynamics simulations of a photoexcited cytosine-guanine base pair in both gas phase and embedded in the DNA provide detailed structural and dynamical insights into the ultrafast radiationless deactivation mechanism. Photon absorption promotes transfer of a proton from the guanine to the cytosine. This proton transfer is followed by an efficient radiationless decay of the excited state via an extended conical intersection seam. The optimization of the conical intersection revealed that it has an unusual topology, in that there is only one degeneracy-lifting coordinate. This is the central mechanistic feature for the decay both in vacuo and in the DNA. Radiationless decay occurs along an extended hyperline nearly parallel to the proton-transfer coordinate, indicating the proton transfer itself is not directly responsible for the deactivation. The seam is displaced from the minimum energy proton-transfer path along a skeletal deformation of the bases. Decay can thus occur anywhere along the single proton-transfer coordinate, accounting for the remarkably short excited-state lifetime of the Watson-Crick base pair. In vacuo, decay occurs after a complete proton transfer, whereas in DNA, decay can also occur much earlier. The origin of this effect lies in the temporal electrostatic stabilization of dipole in the charge-transfer state in DNA.     

The elusive atomic rationale for DNA base pair stability

A systematic analysis of the electrostatic interaction between 27 natural DNA base pairs was carried out, based on ab initio correlated wave functions and the topology of the electron density. Using high rank multipole moments we show that the atomic partitioning of the interaction energy contains many substantial contributions between distant atoms. Profiles of cumulative energy versus internuclear distance show large fluctuations and provide an electrostatic fingerprint of the partitioning of interaction energy in a complex. A quantified comparison between each pair of energy profiles, one for each base pair, makes clear that there is no correlation between the total base pair interaction energy and the shape of the profile. In other words, base pairs with similar interaction energy are not stable for the same reasons in terms of atomic partitioning. In summary, simple rules to rationalize the pattern of energetic stability of naturally occurring base pairs in terms of subsets of atoms are elusive. Our work cautions against inappropriate use of Jorgensen's secondary interaction hypothesis.     

A pyrimidine-like nickel(II) DNA base pair

4-(2'-Pyridyl)-pyrimidinone deoxyriboside is synthesized and characterized as a DNA metallo base-pair; this novel nucleoside forms a self-pair in the presence of Ni(II) and stabilizes double helical DNA to the same extent as a G.C pair.     

An extremely stable and orthogonal DNA base pair with a simplified three-carbon backbone

A nucleotide C3HQ with a minimal three-carbon backbone displays unprecedented pairing strength and orthogonality in a homopair C3HQ:C3HQ in the presence of one equivalent of Cu2+. The pairing stability in DNA even exceeds the related base pair having the regular 2'-deoxyribose backbone. This discovery of a synergy between an artificial backbone and base-pairing scheme opens new avenues for the economical design of modified oligonucleotides with tailored properties.     

A novel silver(i)-mediated DNA base pair

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base pairs dA:dT and dC:dG. UV-melting experiments revealed that the dSPy:dSPy self-pair can replace natural base pairs at multiple sites and still form stable DNA duplexes.     

Recognition of DNA base pair mismatches by a cyclometalated Rh(III) intercalator

Two cyclometalated complexes of Rh(III), rac-[Rh(ppy)2chrysi]+ and rac-[Rh(ppy)2 phi]+, have been synthesized and characterized with respect to their binding to DNA. The structure of rac-[Rh(ppy)2 phi]Cl.H2O.CH2Cl2 has been determined by X-ray diffraction (monoclinic, P2(1)/c, Z = 4, a = 18.447(3) A, b = 9.770(1) A, c = 17.661(3) A, beta = 94.821(11) degrees, V = 3172.0(8) A3) and reveals that the complex is a distorted octahedron with nearly planar ligands, similar in structure to the DNA mismatch recognition agent [Rh(bpy)2chrysi]3+. The 2-phenylpyridyl nitrogen atoms are shown to be in the axial positions, as a result of trans-directing effects. This tendency simplifies the synthesis and purification of such complexes by limiting the number of possible isomers generated. The abilities of [Rh(ppy)2chrysi]+ and [Rh(ppy)2 phi]+ to bind and, with photoactivation, to cleave DNA have been demonstrated in assays on duplex DNA in the absence and presence of a single CC mismatch. [Rh(ppy)2chrysi]+ was shown upon photoactivation to cleave DNA selectively at the base pair mismatch whereas [Rh(ppy)2 phi]+ cleaves B-DNA nonspecifically. The reactivity of [Rh(ppy)2chrysi]+ was also compared to that of the known mismatch recognition agent [Rh(bpy)2chrysi]3+. Competitive photocleavage studies revealed that a 14-fold excess of [Rh(ppy)2chrysi]+ was required to achieve the same level of binding as that of [Rh(bpy)2chrysi]3+. However, the ratio of damage induced by [Rh(bpy)2chrysi]3+ to that induced by [Rh(ppy)2chrysi]+ is considerably greater than this value, indicating that decreased photoefficiency for the cyclometalated complex must contribute to its significantly attenuated photoreactivity. These cyclometalated intercalators provide the starting points for the design of a new family of metal complexes targeted to DNA.     

Electronic coupling between base pair dimers of LNA:DNA oligomers

We calculated ab initio electronic coupling elements between neighboring base-pair dimers in a set of LNA:DNA oligomers with different numbers of locked nucleotides and compared them by averaging the values over ensembles of snapshots from molecular dynamics trajectories. Averaging was based on coupling elements for various ensembles comprising of 33,000 structures. The known pronounced variations of coupling elements on the nanosecond timescale due to thermal fluctuations of the DNA structure were confirmed. We found significant differences in electronic coupling at the dimer level between a non-modified DNA:DNA duplex and the corresponding duplex containing one fully LNA-substituted strand. We rationalized these differences by very dissimilar overlap in the pi-stack as a consequence of the LNA-modified system approximating an A-DNA-type helix. The calculated coupling elements for the non-modified reference duplex were similar to those of standard B-DNA and those for the fully modified oligomer resembled the matrix elements estimated for standard A-DNA.     

Targeting DNA base pair mismatch with artificial nucleobases. Advances and perspectives in triple helix strategy

This review, divided into three sections, describes the contribution of the chemists' community to the development and application of triple helix strategy by using artificial nucleic acids, particularly for the recognition of DNA sequences incorporating base pair inversions. Firstly, the development of nucleobases that recognise CG inversion is surveyed followed secondly by specific recognition of TA inverted base pair. Finally, we point out in the last section recent perspectives and applications, driven from knowledge in nucleic acids interactions, in the growing field of nanotechnology and supramolecular chemistry at the border area of physics, chemistry and molecular biology.     

Tuning urea-phenanthridinium conjugates for DNA/RNA and base pair recognition

A series of novel urea-phenanthridine conjugates was prepared. The variation of linker length connecting two urea-phenanthridinium conjugates regulated their binding mode toward double stranded polynucleotides, consequently controlling selectivity of compounds toward ds-RNA over ds-DNA stabilization as well as selective fluorescence response toward addition of G-C base pair and A-U(T) base pair containing polynucleotides.     

Comparative studies between hydrated hydrogen bonded and stacked DNA base pair

Influence of hydration on the Watson–Crick guanine–cytosine hydrogen bonded (h-bonded) base pair (GC) and stacked pair (G/C) was investigated in their first hydration shell. An electrostatic based approach has been used to identify the potential binding sites for water molecules around GC and G/C pairs. Several geometries of the complexes, GC…(H₂O)_n and G/C…(H₂O)_n (n=1–6) were investigated using HF/6-31G** and HF/6-31G++** methods. Further minimization calculations were performed at both B3P86/6-31G** and MP2/6-31G** levels to assess the role of electron correlation contribution in the hydration process. It can be concluded from the present findings that the stacked base-pair hydrate better than the corresponding h-bonded base pair, and DNA base pairs can accommodate up to 4–5 water molecules whereas stacked pair do accommodate 5–6 water molecules.     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.