Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.     

The electron affinities of deprotonated adenine, guanine, cytosine, uracil, and thymine

Electron attachment rates and gas phase acidities for the canonical tautomers of the nucleobases and electron affinities for thymine, deprotonated thymine, and cytosine are reported The latter are from a new analysis of published photoelectron spectra. The values for deprotonated thymine are (all in eV) keto-N1-H, 3.327(5); enol-N3-H, 3.250(5), enol-C2OH, 3.120(5) enol-N1-H, 3.013(5), and enol-C4OH,3.123(5). The values for deprotonated cytosine, keto-N1-H, 3.184(5); trans-NH-H, 3.008(5); cis-NH-H, 3.039(5); and enol-N1-H, 2.750(5) and enol-O-H, 2.950(5). The gas phase acidities from these values are obtained from these values using experimental or theoretical calculations of bond dissociation energies. Kinetic and thermodynamic properties for thermal electron attachment to thymine are obtained from mass spectrometric data. We report an activation energy of 0.60 eV and electron affinity of thymine, 1.0(1) eV.     

Repair effect of thymine radical anion by echinocoside using pulse radiolysis

Repair activities of thymine radical anion by echinocoside, isolated from Pedicularis plicata. were studied using pulse radiolysis technique. The thymine radical anion was produced by the reaction of hydrated electron with thymine. Echinocoside. one of the polyphenols of phenylpropanoid glycoside, was added to the thymine aqueous solution saturated with N2. Kinetic analysis by transient absorption spectrum showed that thymine radical anion was formed at first, and then after several decades of microseconds of pulse radiolysis. the spectrum of thymine radical anion was changed to that of echinocoside radical anion. The evidence indicated that thymine radical anion was repaired through one-electron-transfer between the DNA base radical anion and echinocoside. The rate constant of electron transfer by echinocoside was 1.45× 109 dm3 · mol1 · s 1.     

Stabilisation of the iminooxo tautomer of 1-methylcytosine in Pt complexes: Role of the ancillary ligands

Reaction of cis-[L₂Pt(μ-OH)]₂(NO₃)₂ (L = PPh₃) with 1-methylthymine (1-MeTy), in DMF, leads to the formation of the mononuclear neutral adduct cis-L₂Pt{1-MeTy(-H)}(ONO₂) (1) whose structure in the solid state has been obtained by single crystal X-ray diffraction. The deprotonated nucleobase is bounded at the N(3) site, with the pyrimidinic ring almost perpendicular (78.0(1)°) to the metal coordination plane. The fourth ligand is a monodentate nitrate group. Addition of 1 equiv. of 1-methylcytosine (1-MeCy) causes the immediate replacement of the nitrato ligand to form the cationic complex cis-[L₂Pt{1-MeTy(-H)}(1-MeCy,N³)]NO₃ (2) in which both the nucleobases are N(3)-platinated. In CD₂Cl₂ at −40 °C 2 exists as a mixture of two conformers (2:1 molar ratio) arising from the different orientation of the nucleobases with respect to the metal coordination plane.In solution of DMSO, DMF or chlorinated solvents, 2 slowly converts into the isomer cis-[L₂Pt{1-MeTy(-H)}(1-MeCy,N⁴)]NO₃ (3), containing the tautomeric form of the cytosine stabilised through the coordination at the N(4) atom, as a mixture of conformers whose relative abundance is dependent on the solvent and the temperature.In contrast, the analogous complex of 2 containing the phosphine PMe₃, cis-[(PMe₃)₂Pt{1-MeTy(-H)}(1-MeCy,N³)]NO₃ (4), also isolated as pure compound, in DMSO solution slowly rearranges leading to the elimination of the neutral 1-MeTy, with the formation of the dinuclear cytosinate complex cis-[(PMe₃)₂Pt{1-MeCy(-H),N³N⁴}]₂(NO₃)₂, previously characterised by us.     

Landscape of π–π and sugar–π contacts in DNA–protein interactions

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA–protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π–π (according to biological edge) or deoxyribose sugar–π (according to sugar edge). Overall, 54% of contacts are nucleobase π–π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar–π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase–cyclic amino acid contacts, sugar–π interactions are more common than nucleobase π–π contacts for the acyclic series (61% of contacts). Similar to DNA–protein π–π interactions, sugar–π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA–protein π-interactions stabilize biological systems, by up to approximately ?40 kJ mol^?1 for neutral nucleobase or sugar–amino acid interactions, but up to approximately ?95 kJ mol^?1 for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.     

Molecular dynamics simulations of the effects of ring-saturated thymine lesions on DNA structure

The effect of thymine lesions produced by radiation or oxidative damage on DNA structure was studied by molecular dynamics simulations of native and damaged DNA. Thymine in position 7 of native dodecamer d(CGCGAATTCGCG)₂ was replaced by one of the four thymine lesions 5-hydroxy-5,6-dihydrothymine, 6-hydroxy-5,6-dihydrothymine (thymine photohydrate), 5,6-dihydmxy-5,6-dihydro-thymine (thymine glycol), and 5,6-dihydmthymine. Simulations were performed with Assisted Model Building with Energy Refinement force field. Solvent was represented by a rectangular box of water with periodic boundary conditions applied. A constant temperature and constant volume protocol was used, the observed level of distortions of DNA structure depends on the specific nature of the lesion. The 5,6-dihydrothymine does not cause distinguishable perturbations to DNA. Other lesions produce a dramatic increase in the rise parameter between the lesion and the 5′ adjacent adenine. These changes are accompanied by weakening of Watson–Crick hydrogen bonds in the A6-T19 base pair on the 5′ side of the lesion. The lesioned bases also show negative values of inclination relative to the helical axis. No changes in the pattern of backbone torsional angles are observed with any of the lesions incorporated into DNA. The structural distortions in DNA correlate well with known biological effects of 5,6-dihydrothymine and thymine glycol on such processes as polymerase action or recognition by repair enzymes. © 1995 John Wiley & Sons, Inc.     

Interconversion of thymine and thymidine in a thymine requiring strain of Bacillus subtilis

In a $\text{B. subtilis}$ Thy^? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr$\text{e}$ sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis.     

DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands

DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase–polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase–polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.     

Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

S_N1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O⁶ position of guanine (O⁶meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O⁶meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S_N1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S_N1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.     

Silver(I)-mediated Hoogsteen-type base pairs

Metal-mediated Hoogsteen-type base pairs are useful for the construction of DNA duplexes containing contiguous stretches of metal ions along the helical axis. To fine-tune the stability of such base pairs and the selectivity toward different metal ions, the availability of a selection of artificial nucleobases is highly desirable. In this study, we follow a theoretical approach utilizing dispersion-corrected density functional methods to evaluate a variety of artificial nucleobases as candidates for metal-mediated Hoogsteen-type base pairs. We focus on silver(I)-mediated Hoogsteen- and reverse Hoogsteen-type base pairs formed between 1-deaza- and 1,3-dideazapurine-derived nucleobases, respectively, and cytosine. Apart from two coordinative bonds, these base pairs are stabilized by a hydrogen bond. We elucidate the impact of different substituents at the C6 position and the presence or absence of an endocyclic N3 nitrogen atom on the overall stability of a base pair and concomitantly on the strength of the hydrogen and coordinative bonds. All artificial base pairs investigated in this study are less stable than the experimentally established benchmark base pair C–Ag⁺–G. The base pair formed from 1,3-dideaza-6-methoxypurine is isoenergetic to the experimentally observed C–Ag⁺–C base pair. This makes 1,3-dideaza-6-methoxypurine a promising candidate for the use as an artificial nucleobase in DNA.     

Effective syntheses of 2′,4′-BNANC monomers bearing adenine,guanine, thymine,and 5-methylcytosine,and the properties of oligonucleotides fully modified with 2′,4′-BNANC

We efficiently synthesized 2′-O,4′-C-aminomethylene-bridged nucleic acid (2′,4′-BNA^NC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2′-O-substituted 5-methyluridines to evaluate the effects of 2′-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2′,4′-BNA^NC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2′,4′-BNA^NC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2′,4′-BNA^NC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2′,4′-BNA^NC monomers in both or either strands were found to possess excellent thermal stabilities.     

Antiproliferative activity of Pt(II) and Pd(II) phosphine complexes with thymine and thymidine

Oxidative addition reactions between [M(PPh(3))(4)] (M=Pt and Pd) and N1-methylthymine (t)/3',5'-di-O-acetylthymidine (T) were carried out to give [M(II)(PPh(3))(2)Cl t (or T)] complexes, in which the metal is coordinated to the N3 of the base. All complexes were characterized by spectroscopic analyses (IR, NMR) and Fast Atom Bombardment mass spectrometry (FAB-MS); X-ray data for the thymine complexes and elemental analysis for the thymidine complexes are reported. The antiproliferative activity of the complexes was tested on human chronic myelogenous leukaemia K562 cells. Arrested polymerase-chain reaction analysis was carried on to correlate antiproliferative activity and inhibition of DNA replication. All Pd and Pt complexes exhibit antiproliferative activity, Pd complexes resulting always more active than Pt complexes. Arrested PCR data are strongly in agreement with the effects on cell growth, suggesting that inhibition of the DNA replication by the synthesized compounds is the major basis for their in vitro antiproliferative activity.     

Stereoselective excision of thymine glycol from oxidatively damaged DNA

DNA damage created by reactive oxygen species includes the prototypic oxidized pyrimidine, thymine glycol (Tg), which exists in oxidatively damaged DNA as two diastereoisomeric pairs. In Escherichia coli, Saccharomyces cerevesiae and mice, Tg is preferentially excised by endonuclease III (Endo III) and endonuclease VIII (Endo VIII), yNTG1 and yNTG2, and mNTH and mNEIL1, respectively. We have explored the ability of these DNA glycosylases to discriminate between Tg stereoisomers. Oligonucleotides containing a single, chromatographically pure (5S,6R) or (5R,6S) stereoisomer of Tg were prepared by oxidation with osmium tetroxide. Steady-state kinetic analyses of the excision process revealed that Endo III, Endo VIII, yNTG1, mNTH and mNEIL1, but not yNTG2, excise Tg isomers from DNA in a stereoselective manner, as reflected in the parameter of catalytic efficiency (k_cat/K_m). When DNA glycosylases occur as complementary pairs, failure of one or both enzymes to excise their cognate Tg stereoisomer from oxidatively damaged DNA could have deleterious consequences for the cell.     

Exploring the roles of nucleobase desolvation and shape complementarity during the misreplication of O(6)-methylguanine

O⁶-methylguanine (O⁶-MeG) is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles of hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and nonnatural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O⁶-MeG. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase's active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O⁶-MeG observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of nonnatural nucleotides identified an analog that displays high selectivity for incorporation opposite O⁶-MeG compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis.     

Cation—π and Amino-Acceptor Interactions Between Hydrated Metal Cations and DNA Bases. A Quantum-Chemical View

Abstract

Cation—π interactions between cytosine and hexahydrated cations have been characterized using ab initio method with inclusion of electron correlation effects, assuming idealized and crystal geometries of the interacting species. Hydrated metal cations can interact with nucleobases in a cation—π manner. The stabilization energy of such complexes would be large and comparable to the one for cation—π complex with benzene. Further, polarized water molecules belonging to the hydration shell of the cation are capable to form a strong hydrogen bond interaction with the nitrogen lone electron pair of the amino groups of bases and enforce a pronounced sp³ pyramidalization of the nucleobase amino groups. However, in contrast to the benzene—cation complexes, the cation—π configurations are highly unstable for a nucleobase since the conventional in plane binding of hydrated cations to the acceptor sites on the nucleobase is strongly preferred. Thus, a cation—π interaction with a nucle-obase can occur only if the position of the cation is locked above the nucleobase plane by another strong interaction. This indeed can occur in biopolymers and may have an effect on the local DNA architecture. Nevertheless, nucleobases have no intrinsic propensity to form cation—π interactions.     

The protonation of the 5-thymyl radical in single crystals of thymine derivatives: e.s.r. and INDO evidence.

For the 5-thymyl radical, minor couplings additional to beta-protons bonded to C6 and C7 are observed in single crystals of 1-methylthymine. Hyperfine coupling tensors are given of the N3 nitrogen (+3.5/0.0/0.0 G) and of the exchangeable proton (-4.1/-2.5/-0.9 G) bonded to it. In single crystals of thymine . anhydrate, thymine . monohydrate and 5,6-dihydrothymine these additional couplings are unresolved, but are also present. INDO-calculations were performed to reproduce consistently both beta-coupling constants and additional couplings from N3 and H(N3). Comparison of experimental and calculated values to the conclusion that the 5-thymyl radical is protonated at 04 in all single crystals of thymine derivatives investigated.     

Selective base excision repair of DNA damage by the non‐base‐flipping DNA glycosylase AlkC

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.     

Derivatization of thymine and thymine photodimers with 4-bromomethyl-7-methoxycoumarin for fluorescence detection in high-performance liquid chromatography

Exposure of DNA to ultraviolet radiation results in the formation of a number of photoproducts, including thymine photodimers. A sensitive and selective analytical method based on high-performance liquid chromatography (HPLC) and fluorescent labeling with 4-bromomethyl-7-methoxycoumarin has been developed to quantify both thymine and thymine photodimers. The identity of the thymine and thymine dimer derivatives were determined by HPLC–electrospray ionization mass spectrometry. The derivatization reaction yield was maximized by optimizing several reaction variables. The limit of detection for HPLC method was 1.0 pmol thymine and 0.4 pmol thymine dimer for S/N=3.     

The Electron Affinities of Deprotonated Adenine,Guanine, Cytosine,Uracil, and Thymine

Electron attachment rates and gas phase acidities for the canonical tautomers of the nucleobases and electron affinities for thymine, deprotonated thymine, and cytosine are reported The latter are from a new analysis of published photoelectron spectra. The values for deprotonated thymine are (all in eV) keto-N1-H, 3.327(5); enol-N3-H, 3.250(5), enol-C2OH, 3.120(5) enol-N1-H, 3.013(5), and enol-C4OH,3.123(5). The values for deprotonated cytosine, keto-N1-H, 3.184(5); trans-NH-H, 3.008(5); cis-NH-H, 3.039(5); and enol-N1-H, 2.750(5) and enol-O-H, 2.950(5). The gas phase acidities from these values are obtained from these values using experimental or theoretical calculations of bond dissociation energies. Kinetic and thermodynamic properties for thermal electron attachment to thymine are obtained from mass spectrometric data. We report an activation energy of 0.60 eV and electron affinity of thymine, 1.0(1) eV.