Molecular modeling study of an abasic DNA undecamer duplex: d(GCGTGOGTGCG) · d(CGCACTCACGC)

Molecular mechanics calculations were performed with the JUMNA program on d(GCGTGOGTGCG) · d(CGCACTCACGC) where “O” is a modified abasic site: 3-hydroxy-2-(hydroxymethyl)tetrahydrofuran. From energy minimizations, for intrahelical or extrahelical positions of the unpaired thymine, various structures with different curvatures were obtained. Dynamical properties of this abasic sequence were also investigated through the controlled studies of DNA bending. Poisson-Boltzmann calculations were used to mimic the electrostatic effect of solvent on this sequence. The lowest energy structures show an acceptable agreement with experimental data. Received: 1 June 1998 / Accepted: 17 September 1998 / Published online: 10 December 1998     

Use of abasic site-containing DNA strands for nucleobase recognition in water

Nucleobase recognition in water is successfully achieved by the use of an abasic site (AP site) as the molecular recognition field. We intentionally construct the AP site in DNA duplex so as to orient the AP site toward a target nucleobase and examine the complexation of 2-amino-7-methylnaphthyridine (AMND) with nucleobases at the AP site. AMND is found to selectively bind to cytosine (C) base with a 1:1 binding constant of >106 M-1, accompanied by remarkable quenching of its fluorescence. In addition to hydrogen bonding, a stacking interaction with nucleobases flanking the AP site seems responsible for the binding properties of AMND at the AP site. Possible use of AMND is also presented for selective and visible detection of a single-base alternation related to the cytosine base.     

Identification of molecular reactive sites with an interactive volume rendering tool

A new molecular visualization tool is presented, EVolVis (an abbreviation of electronic volume visualizer). This software enables the user to interactively explore a molecule's electronic charge density for topological features. Using novel volume rendering techniques, the entire molecule can be probed without obstruction by opaque surfaces or preselection of specific orbitals or contours. Menus with sliding scales permit various ranges of the function of interest to be examined interactively. We have investigated the Laplacian of the electronic charge density calculated for several biomolecules that contain a variety of reactive sites: cisplatin, penamecillin and isomers of nitrogen bases from DNA. EVolVis is useful in identifying inner-shell features (cisplatin), polar, nonpolar and aromatic regions (penamecillin), hydrogen-bonding sites of varying strength (nitrogen bases and penamecillin) and other reactive sites (strained heterocycle in penamecillin). Received: 1 June 2000 / Accepted: 4 October 2000 / Published online: 28 February 2001     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

Sequence dependence of cytochrome c electrochemistry on DNA modified electrodes: Effect of hydrogen bonding of a ligand to nucleobases opposite an abasic site

The binding of a hydrogen bond-forming ligand to abasic (AP) site-containing DNA was electrochemically investigated for discrimination of a nucleotide opposite the AP site. The surface of a gold electrode was modified by AP site-containing DNA duplexes on which cytochrome c (Cyto c) was attached electrostatically as a probe. Cyto c showed quasi-reversible electrochemical behavior depending on the base opposite the AP site. When the base opposite the AP site was cytosine, much slower kinetics of Cyto c electron transfer was observed. This observation could be explained by previous reports that the base stacking was disturbed to a much greater extent because the cytosine base opposite the AP site was flipped out extra-helically. The binding of a hydrogen bond-forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), to cytosine opposite the AP site could significantly improve the electrochemical behavior of Cyto c, indicating effective base stacking due to the AMND binding. The present method demonstrates an easy way for investigating the binding of a small ligand to the AP site through DNA-mediated charge transfer.     

New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies.     

Use of vitamin B2 for fluorescence detection of thymidine-related single-nucleotide polymorphisms

In combination with abasic site (AP site)-containing DNAs, potential use of a biotic fluorescence compound, Vitamin B₂ (riboflavin), is demonstrated for the fluorescence detection of the thymine (T)-related single-nucleotide polymorphisms. Our method is based on construction of the AP site in DNA duplexes, which allows small ligands to bind to target nucleotides accompanied by fluorescence signaling: an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleotides through stacking and hydrogen-bonding interactions. In 10 mM sodium cacodylate buffer solutions (pH 7.0) containing 100 mM NaCl and 1.0 mM EDTA, Vitamin B₂ is found to selectively bind to T (K₁₁ = 1.8 × 10⁶ M⁻¹ at 5 °C) over other nucleobases, and this is accompanied by significant quenching of its fluorescence. While the sensing functions depend on the flanking sequences to the AP site, Vitamin B₂ is applicable to the detection of T/C (cytosine), T/G (guanine) and T/A (adenine) mutation sequences of the CYP2A6 gene, where the flanking nucleobases are guanines in both positions (-GXG-, X = AP site).     

Recognizing a triple bond between main group atoms

The electron pair density, in conjunction with the theory of an atom in a molecule, enables one to unambiguously determine the nature of the bonding between the gallium atoms in bent [HGa-GaH]²⁻. The Ga-Ga bonding in the dianion at the experimental bond length is found to be the result of the sharing of two electron pairs at the Hartree-Fock level of theory, the level consistent with the Lewis model of the electron pair. Received: 27 July 2000 / Accepted: 4 October 2000 / Published online: 19 January 2001     

Prototropic changes in cationic base-pair adducts. II. Guanine methylation

AM 1 calculations have been used to study the effects of CH attachment on the structures, energies, and, in some cases, proton transfer reactions of guanine cytosine base pairs. Methylation of both the guanine 3- and O⁶-positions is predicted to lead to chemically significant concentrations of intermediate base pairs arising from proton transfer from the guanine 1- to the cytosine 3-position. The possible biological implications of such intermediates in nucleic acids is discussed in relation to the formation of either doubly abasic sites or abasic sites opposite potentially miscoding DNA lesions. © 1992 John Wiley & Sons, Inc.     

Prediction of DNA far-IR absorption spectra based on normal mode analysis

 We present a computational method which couples normal mode analysis in internal coordinates of a molecule with very far IR spectroscopy. The analytical expression for the dependence of IR absorption on frequency incorporates frequencies and optical activities of each normal mode. In order to predict far-IR spectra of a molecule we evaluate the optical activity of each normal mode. This optical activity is determined by the vibration amplitude of the dipole moment produced by a normal mode. We calculated normal modes of DNA double-helical fragments (dA)₁₂ · (dT)₁₂ and (dA-dT)₆ · (dA-dT)₆ and evaluated their optical activities. These were found to be very sensitive to the DNA base-pair sequence. The positions of the resonance peaks in the calculated absorption spectrum of (dA)₁₂ · (dT)₁₂ are in a good agreement with those obtained by Fourier transform IR spectroscopy (Powell JW et al. 1987 Phys Rev A 35: 3929–3939). Received: 20 June 2000 / Accepted: 5 January 2001/ Published online: 3 May 2001     

Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy

A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a single fluorochrome-DNA complex was calibrated. The number of abasic sites on the DNA molecule was estimated by counting each fluorochrome-DNA complex. The present study directly visualized and characterized the abasic sites of single DNA molecules.     

The glycosyl C1′—N9 bond of deoxyadenosine and deoxyguanosine: response to electrophilic attacks on the purinic nitrogen atoms

Self-consistent-field computations shed light on two relevant conformations of deoxyadenosine (dA) and deoxyguanosine (dG): one with a pseudoequatorial C_1′N₉ glycosyl bond and the other, a slightly more stable one, with its C_1′N₉ bond in a bisectional orientation. In dA, both the N₃ and N₇ nitrogens are plausible sites for electrophilic attack, but only N₇ is a plausible site in dG. The addition of H⁺, CH₃ ⁺, C₂H₅ ⁺ or tert-C₄H₉ ⁺ onto N₇ does not provoke notable structural modifications and leaves the base of dA and dG in an antiperiplanar (or nearly antiperiplanar) position with respect to the sugar C_1′O_4′ bond, but N₃ additions cause the base to adopt a synperiplanar or strongly chiral position. This produces strong interactions between the purine and deoxyribose moieties, whose relief could aid the eventual cleavage of the glycosyl bond of dA. Addition of a radical cation onto N₇ reduces the dissociation energy of the glycosyl bond by an estimated 8 kcal mol⁻¹ in dA and 4 kcal mol⁻¹ in dG – a bond weakening likely to concur to a depurination of DNA induced by radical cations. Received: 13 September 1999 / Accepted: 3 February 2000 / Published online: 21 June 2000     

Quantum mechanical/molecular mechanical study of three stationary points along the deacylation step of the catalytic mechanism of elastase

A large amount of experimental as well as theoretical information is available about the mechanism of serine proteases, but many questions remain unanswered. Here we study the deacylation step of the reaction mechanism of elastase. The water molecule in the acyl-enzyme active site, the binding mode of the carbonyl oxygen in the oxyanion hole, the characteristics of the tetrahedral intermediate structure, and the mobility of the imidazole ring of His-57 were studied with quantum mechanical/molecular mechanical methods. The models are based on a recent high-resolution crystal structure of the acyl-enzyme intermediate. The nucleophilic water in the active site of the acyl-enzyme has been shown to have two minima that differ by only 2 kcalmol⁻¹ in energy. The carbonyl group of the acyl-enzyme is located in the oxyanion hole and is positioned for attack by the hydrolytic water. The tetrahedral intermediate is a weakly bonded system, which is electrostatically stabilized by short hydrogen bonds to the backbone NH groups of Gly-193 and Ser-195 in the oxyanion hole. The short distance between the N^ɛ2 of His-57 and the O^γ of Ser-195 in the tetrahedral intermediate indicates a small movement of the imidazole ring towards the product in the deacylation step. The carbonyl group of the enzyme-product complex is not held strongly in the oxyanion hole, which shows that the peptide is first released from the oxyanion hole before it leaves the active site to regenerate the native state of the enzyme. Received: 11 September 2000 / Accepted: 15 September 2000 / Published online: 21 March 2001     

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag⁺ binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag⁺ preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag⁺ binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.     

A first principles study of Pd deposition on the TiO2(1 1 0) surface

 The adsorption of isolated Pd atoms on the (1 1 0) surface of rutile TiO₂ was investigated through ab initio embedded-cluster calculations performed at the Hartree–Fock, second-order M?ller–Plesset and Becke's three parameter hybrid method with the Lee–Yang–Parr correlation functional levels. The role played by the magnitude of the surrounding charges used in the embedding procedure was carefully analyzed. The most stable site for adsorption consisted of a fourfold hollow site in which the Pd atom was coordinated to a fivefold Ti atom, two basal oxygens, and a protruding oxygen atom. However, the adsorption energies computed after basis set superposition error corrections seemed to favor a bridge site in which the Pd atom binds two protruding oxygen atoms. A periodic slab calculation using gradient-corrected functionals and plane-wave basis sets confirmed that for full coverage, the hollow site was more stable, although Pd displacement along the fivefold Ti channels was almost free. These results agree with the experimental data obtained from scanning tunneling microscopy. Finally, the adsorption energy computed from the periodic calculations was found to be 1.88 eV. Received: 14 September 1999 / Accepted: 3 February 2000 / Published online: 19 April 2000     

Tetrahydrofuran analogues with silicon and sulphur atoms

Quantum-mechanical calculations at the self-consistent-field–M?ller–Plesset level have been performed for various compounds obtained by substituting two or four CH₂ groups of the tetrahydrofuran molecule with SiH₂ groups, as well as the oxygen with a sulphur atom. Cyclic tetrasilane oxide, (SiH₂)₄O, and sulphide, (SiH₂)₄S, are suggested as possible alternatives to the carbon ring of natural sugars for the design of exobiological molecules because of certain similarities with tetrahydrofuran, (CH₂)₄O, especially the relatively small energy differences between different conformers and the subsequent ability to cross the energy barriers between them by puckering on the pseudorotation wheel. Received: 16 September 1999 / Accepted: 3 February 2000 / Published online: 21 June 2000     

Fluorescence light-up recognition of DNA nucleotide based on selective abasic site binding of an excited-state intramolecular proton transfer probe

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.     

A density functional theory study of the chemoselectivity and regioselectivity of the domino cycloaddition reactions of nitroalkenes with substituted alkenes

The chemoselectivity and regioselectivity of the domino intermolecular [4 + 2]/[3 + 2] cycloaddition reactions of nitroalkenes with substituted alkenes, vinyl ethers as electron-rich alkenes and vinyl ketones as electron-poor alkenes, have been studied using density functional theory (DFT) methods with the B3LYP functional and the 6-31G^* basis set. These domino processes comprise two consecutive cycloaddition reactions: the first one is an intermolecular [4 + 2] cycloaddition of the vinyl ether to the nitroalkene to give a nitronate intermediate, which then affords the final nitroso acetal adduct through an intermolecular [3 + 2] cycloaddition reaction with the vinyl ketone. The two consecutive cycloadditions present total chemoselectivity and ortho regioselectivity. While first [4 + 2] cycloaddition reaction takes place along the attack of the electron-rich alkene to nitroalkene, the [3 + 2] one takes place along the attack of the electron-poor alkene to the corresponding nitronate intermediate. This DFT study is in complete agreement with the experimental results. Received: 16 September 1999 / Accepted: 3 February 2000 / Published online: 2 May 2000     

Synthesis and analysis of oligonucleotides containing abasic site analogues

DNA damage results in the formation of abasic sites from the formal hydrolysis of the glycosidic bond (AP) and several oxidized abasic lesions. Previous studies on AP sites revealed that DNA polymerases preferentially incorporated dA opposite them in approximately 80% of the replication events in Escherichia coli. These results were consistent with the hypothesis that the AP sites are noninstructive lesions due to the absence of a Watson-Crick base whose bypass adheres to the "A-rule." Recent replication studies of the oxidized abasic lesion, 2-deoxyribonolactone (L), revealed that DNA polymerase(s) does not apply the A-rule when bypassing it and incorporates large amounts of dG opposite L. These studies suggested that abasic sites such as L do direct polymerases to selectively incorporate nucleotides opposite them. However, it was not possible to determine the structural basis for this molecular recognition from these experiments. A group of oligonucleotides containing analogues of the AP and L lesions were synthesized and characterized as probes to gain insight into the structural basis for the distinct effect of 2-deoxyribonolactone on replication. These molecules will be useful tools for studying replication in cells and in vitro.     

Sequence dependence of DNA radioprotection by the thiols WR-1065 and WR-151326

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage. Thiols reduce breakage by scavenging damaging radiolytic OH ^. and repairing sugar radicals. As shown by sequencing gel electrophoresis, WR-1065 radioprotection is modulated by sequence, whereas that of WR-151326, a larger thiol, is more evenly distributed. Molecular modelling was performed on complexes of a 53 bp oligonucleotide (belonging to a natural restriction fragment) with one molecule of WR-1065 or WR-151326. Energy minimised structures exhibit a broadening of the minor groove of an AAATT motif upon WR-1065 binding, and a narrowing of the groove upon WR-151326 binding. Consequently, the accessibility to OH^˙ of H4′ (whose abstraction leads to strand breakage) increases near WR-1065, whereas it decreases near WR-151326. This modifies locally the otherwise homogeneous radioprotection. The effect of WR-151326 strengthens the protection at all tested binding sites, whereas that of WR-1065 diminishes it in some regions, in good agreement with the observed radioprotection distribution. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 11 November 1998