Reactivity of ferrocenylcarbodiimide with DNA duplex containing single-mismatched base pairs.

Ferrocenylcarbodiimide (1), which is known to react with a guanine (G) or thymine (T) base of single stranded DNA, was allowed to react with DNA duplex having a single mismatched base pair of G-T, T-T, or T-cytosine (C). Electrophoreograms of the reaction mixture showed that 1 could react with G or T base of the mismatched sites on the DNA duplex. However, 1 also reacted with the G base of the terminal site on the DNA duplex. This showed that 1 can react with an unpaired base or unstable base pair such as a terminal or mismatched base on the DNA duplex. Electrochemical mismatch detection could be achieved after hybridization of the ferrocenylated mismatched DNA duplex with a selected DNA probe-immobilized electrode. These results revealed that 1 has a potentiality of serving as a labeling reagent of mismatched bases on the DNA duplex, which is important in the search for heterozygous single nucleotide polymorphisms (SNPs).     

5-hydroxyuracil can form stable base pairs with all four bases in a DNA duplex

NMR studies, UV-monitored melting experiments, and ab initio calculations show that 5-hydroxyuracil, produced by the oxidative de-amination of cytosines by reactive oxygen species, can form stable base-pairs with dA, dG, dC and dT residues in a DNA duplex, providing a basis for the in-vivo incorporation of 5-hydroxyuracil during DNA replication.     

Theoretical study of a new DNA structure: the antiparallel Hoogsteen duplex

The structure of a new form of duplex DNA, the antiparallel Hoogsteen duplex, is studied in polyd(AT) sequences by means of state-of-the-art molecular dynamics simulations in aqueous solution. The structure, which was found to be stable in all of the simulations, has many similarities with the standard Watson-Crick duplex in terms of general structure, flexibility, and molecular recognition patterns. Accurate MM-PB/SA (and MM-GB/SA) analysis shows that the new structure has an effective energy similar to that of the B-type duplex, while it is slightly disfavored by intramolecular entropic considerations. Overall, MD simulations strongly suggest that the antiparallel Hoogsteen duplex is an accessible structure for a polyd(AT) sequence, which might compete under proper experimental conditions with normal B-DNA. MD simulations also suggest that chimeras containing Watson-Crick duplex and Hoogsteen antiparallel helices might coexist in a common structure, but with the differential characteristics of both type of structures preserved.     

Selective one-electron oxidation of duplex DNA oligomers: reaction at thymines

The one-electron oxidation of duplex DNA generates a nucleobase radical cation (electron "hole") that migrates long distances by a hopping mechanism. The radical cation reacts irreversibly with H2O or O2 to form oxidation products (damaged bases). In normal DNA (containing the four common DNA bases), reaction occurs most frequently at guanine. However, in DNA duplexes that do not contain guanine (i.e., those comprised exclusively of A/T base pairs), we discovered that reaction occurs primarily at thymine and gives products resulting from oxidation of the T-C5 methyl group and from addition to its C5-C6 double bond. This surprising result shows that it is the relative reactivity, not the stability, of a nucleobase radical cation that determines the nature of the products formed from oxidation of DNA. A mechanism for reaction is proposed whereby a thymine radical cation may either lose a proton from its methyl group or H2O/O2 may add across its double bond. In the latter case, addition may initiate a tandem reaction that converts both thymines of a TT step to oxidation products.     

Consecutive GC base pairs determine the energy barrier of DNA duplex formation under molecularly crowded conditions

The kinetics of DNA duplex formation was affected by the addition of PEGs with different masses (MW = 200-8000) to an aqueous solution; for each condition, two duplexes (5'-TAGGTTATAA-3'/5'-TTATAACCTA-3' and 5'-CAGGTCACAG-3'/5'-CTGTGACCTG-3') with different stabilities were formed after overcoming the same association activation energy barrier, suggesting that the formation of consecutive GC base pairs in the helices rather than the helix terminus is the initiation nucleus for DNA duplex formation not only in the absence, but also in the presence of PEGs.     

Halogen bonding in DNA base pairs

Halogen bonding (R-X···Y) is a qualitative analogue of hydrogen bonding that may prove useful in the rational design of artificial proteins and nucleotides. We explore halogen-bonded DNA base pairs containing modified guanine, cytosine, adenine and thymine nucleosides. The structures and stabilities of the halogenated systems are compared to the normal hydrogen bonded base pairs. In most cases, energetically stable, coplanar structures are identified. In the most favorable cases, halogenated base pair stabilities are within 2 kcal mol(-1) of the hydrogen bonded analogues. Among the halogens X = Cl, Br, and I, bromine is best suited for inclusion in these biological systems because it possesses the best combination of polarizability and steric suitability. We find that the most stable structures result from a single substitution of a hydrogen bond for a halogen bond in dA:dT and dG:dC base pairs, which allows 1 or 2 hydrogen bonds, respectively, to complement the halogen bond.     

Detection of single nucleotide polymorphisms by the specific interaction between transition metal ions and mismatched base pairs in duplex DNA

Single nucleotide polymorphisms (SNPs) are base differences in the human genome. These differences are favorable markers for genetic factors including those associated with risks of complex diseases and individual responses to drugs. When two duplex DNAs with different types of SNPs are mixed and reannealed, the two novel heteroduplexes containing mismatched base pairs are formed in addition to the two initial perfectly matched homoduplexes. Heteroduplex analysis recognizing the newly formed mismatched base pairs is useful for SNP detection. Various strategies to detect the mismatched base pairs were devised due to the potential applications of SNPs. However, they were not always convenient and accurate. Here, we propose a novel strategy to detect the mismatched base pairs by the specific interaction between the Hg²⁺ ion and a T:T mismatched base pair and that between the Ag⁺ ion and a C:C mismatched base pair. UV melting indicated that the melting temperature of only the heteroduplexes with the T:T and C:C mismatched base pair specifically increased on adding the Hg²⁺ and Ag⁺ ion, respectively. Fluorescence resonance energy transfer analyses indicated that the intensity of fluorophore emission of only the fluorophore and quencher-labeled heteroduplexes with the T:T and C:C mismatched base pair specifically decreased on adding the Hg²⁺ and Ag⁺ ion, respectively. We propose that the addition of the metal ion could be a convenient and accurate strategy to detect the mismatched base pair in the heteroduplex. This novel strategy might make the heteroduplex analysis easy and eventually lead to better SNP detection.     

Electronic properties of metal-modified DNA base pairs

The electronic properties of several metal-modified Watson-Crick guanine-cytosine base pairs are investigated by means of first-principle density functional theory calculations. Focus is placed on a new structure recently proposed as a plausible model for building an antiparallel duplex with Zn-guanine-cytosine pairs, but we also inspect several other conformations and the incorporation of Ag and Cu ions. We analyze the effects induced by the incorporation of one metal cation per base pair by comparing the structures and the electronic properties of the metalated pairs to those of the natural guanine-cytosine pair, particularly for what concerns the modifications of energy levels and charge density distributions of the frontier orbitals. Our results reveal the establishment of covalent bonding between the metal cation and the nucleobases, identified in the presence of hybrid metal-guanine and metal-cytosine orbitals. Attachment of the cation can occur either at the N1 or the N7 site of guanine and is compatible with altering or not altering the H-bond pattern of the natural pair. Cu(II) strongly contributes to the hybridization of the orbitals around the band gap, whereas Ag(I) and Zn(II) give hybrid states farther from the band gap. Most metalated pairs have smaller band gaps than the natural guanine-cytosine pair. The band gap shrinking along with the metal-base coupling suggests interesting consequences for electron transfer through DNA double helices.     

Thermodynamic stability of Hoogsteen and Watson-Crick base pairs in the presence of histone H3-mimicking peptide

We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.     

Symmetry selection in artificial DNA base pairs

We report the results of density functional theory (DFT) studies on the formation of the complex H1--Cu2+-H1- consisting of two deprotonated hydroxypyridone ligands (H1-) and a Cu2+ ion. We compare the total energies of three possible structures with different symmetries and show that the structure with plane reflection symmetry has the lowest energy. The electronic structure of the periodic extended DNA-like double helix consisting of stacked H1--Cu2+-H1- units is then calculated within the density functional method, and the double helix is found to be an insulating ferromagnet.     

Recognition of ten base pairs of DNA by head-to-head hairpin dimers

Hairpin polyamides coupled head-to head with alkyl linkers of varying lengths were synthesized, and their DNA binding properties were determined. The DNA binding affinities of six-ring hairpin dimers Im-Im-Py-(R)[Im-Im-Py-(R)(HNCO(CH))(n)(CO)gamma-Py-Py-Py-beta-Dp](NH)gamma-Im-Py-Py-beta-Dp (1-4) (where n = 1-4) for their 10-bp, 11-bp, and 12-bp match sites 5'-TGGCATACCA-3', 5'-TGGCATTACCA-3', and 5'-TGGCATATACCA-3' were determined by quantitative DNase I footprint titrations. The most selective dimer Im-Im-Py-(R)[Im-Im-Py-(R)(HNCO(CH)(2))(2)(CO)gamma-Py-Py-Py-beta-Dp](NH)gamma-Im-Py-Py-beta-Dp (2) binds the 10-bp site match site with an equilibrium association constant of K(a) = 7.5 x 10(10) M(-1) and displays 25- and 140-fold selectivity over the 11-bp and 12-bp match sites, respectively. The affinity toward single base pair mismatched sequences is 4- to 8-fold lower if one hairpin module of the dimer is affected, but close to 200-fold lower if both hairpin modules face a single mismatch base pair. The head-to-head hairpin dimer motif expands the binding site size of DNA sequences targetable with polyamides.     

Chimeric GNA/DNA metal-mediated base pairs

DNA double helices comprising chimeric GNA/DNA metal-mediated base pairs have been synthesized and characterized (GNA = glycol nucleic acid). The possibility to combine different nucleic acid backbones within one metal-mediated base pair expands the applicability of metal-functionalized nucleic acids.     

Binding of metal ions by pyrimidine base pairs in DNA duplexes

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).     

Probing transient Hoogsteen hydrogen bonds in canonical duplex DNA using NMR relaxation dispersion and single-atom substitution

Nucleic acids transiently morph into alternative conformations that can be difficult to characterize at the atomic level by conventional methods because they exist for too little time and in too little abundance. We recently reported evidence for transient Hoogsteen (HG) base pairs in canonical B-DNA based on NMR carbon relaxation dispersion. While the carbon chemical shifts measured for the transient state were consistent with a syn orientation for the purine base, as expected for A(syn)?T(anti) and G(syn)?C(+)(anti) HG base pairing, HG type hydrogen bonding could only be inferred indirectly. Here, we develop two independent approaches for directly probing transient changes in N-H···N hydrogen bonds and apply them to the characterization of transient Hoogsteen type hydrogen bonds in canonical duplex DNA. The first approach takes advantage of the strong dependence of the imino nitrogen chemical shift on hydrogen bonding and involves measurement of R(1ρ) relaxation dispersion for the hydrogen-bond donor imino nitrogens in G and T residues. In the second approach, we assess the consequence of substituting the hydrogen-bond acceptor nitrogen (N7) with a carbon (C7H7) on both carbon and nitrogen relaxation dispersion data. Together, these data allow us to obtain direct evidence for transient Hoogsteen base pairs that are stabilized by N-H···N type hydrogen bonds in canonical duplex DNA. The methods introduced here greatly expand the utility of NMR in the structural characterization of transient states in nucleic acids.     

Multiple proton-transfer reactions in DNA base pairs by coordination of pt complex

The possibility of multiple proton-transfer reactions in DNA base pairs because of coordination of cisplatin is theoretically elucidated by density functional theory (DFT) and by quantum mechanics/molecular mechanics (QM/MM) methods with an ONIOM method. From the energetics of two base pairs with the cisplatin, it is theoretically confirmed that the Pt complex is likely to bind in the order cis-(CG)-Pt-(GC), cis-(CG)-Pt-(AT), cis-(TA)-Pt-(AT), where G, C, A, and T are guanine, cytosine, adenine, and thymine, respectively, and the Pt atom bonds to the N7 site of G and A. This result supports the experimental evidence, where the structure cis-A-Pt-A is seldom observed at room temperature. The single proton-transfer reaction occurs in one of the two GC pairs. No simultaneous single proton-transfer reaction can occur in both base pairs. Two different single proton-transferred structures (cis-(CG*)(d)-Pt-(GC)(p) and cis-(CG)(d)-Pt-(G*C)(p), where the asterisk means a proton donor of G) are as stable as the original structures (CG)(d)-Pt-(GC)(p). The same tendency was observed with cis-(CG*)-Pt-(AT). In contrast to cisplatin, multiple single proton-transfer reactions occur in the system consisting of two base pairs with transplatin. The optimized structure agrees with the experimental data for Pt-G coordination except for the hydrogen-bonding length.     

MercuryII-mediated formation of thymine-HgII-thymine base pairs in DNA duplexes

The very specific binding of the HgII ion unexpectedly and significantly stabilizes naturally occurring thymine-thymine base mispairing in DNA duplexes. Following this finding, we prepared DNA duplexes containing metal-mediated base pairs at the desired sites, as well as novel double helical architectures consisting only of thymine-HgII-thymine pairs.