Influence of prior complex formation on the photoaddition of chlorpromazine to calf thymus deoxyribonucleic acid

Chlorpromazine (CPZ), 2-chloro-N-(3-dimethylaminopropyl) phenothiazine, causes cutaneous photosensitivity in man. The photoaddition of CPZ to deoxyribonucleic acid (DNA) may be an important mechanism for the phototoxicity. We have investigated the complexes formed between CPZ and calf thymus DNA prior to irradiation, related their formation to the photoaddition of CPZ to DNA and initiated studies to identify the photoadducts. In the presence of high concentrations of double-stranded DNA, the CPZ absorption maximum shifted from 305 to 340 nm with an isosbestic point at 323 nm. The CPZ fluorescence at 460 nm was quenched a maximum of 90%. The excitation and emission spectra for the unquenchable fluorescence are the same as those for free CPZ. These results together with those from flow dichroism measurements indicated that CPZ formed two complexes with double-stranded DNA. One complex involves intercalations, is non-fluorescent and absorbs at 345 nm. The second complex absorbs at 310 nm, fluoresces at 460 nm and has the phenothiazine ring parallel to the DNA axis. Non-covalent binding of CPZ to heat-denatured DNA did not shift the CPZ absorption spectrum but quenched 65% of the CPZ fluorescence. One complex between CPZ and denatured DNA will account for these results. CPZ photolysis was inhibited compared with that of free CPZ by binding to double-stranded DNA (more than 98%) or denatured DNA (65%). CPZ photoadded ten times more efficiently to denatured DNA than to double-stranded DNA. These results indicate that CPZ photolysis and photoaddition are quenched in the intercalation complex. The photoaddition to double-stranded DNA does not result from intercalated CPZ because the action spectrum maximized at 310 nm rather than at 340 nm.     

Interaction of asterriquinone with deoxyribonucleic acid in vitro

The interaction of asterriquinone (ARQ), a novel antitumor agent isolated from Aspergillus fungi, with deoxyribonucleic acid (DNA), has been studied. The binding of ARQ in vitro with DNA (calf thymus) was ascertained by its behavior in gel filtration using a Sephadex G-25 column at pH 5.4. Some ARQ analogs having no, or less, antitumor activity did not exhibit any evidence of interaction with DNA under the same condition. From the results obtained in this work, the pKa value of ARQs seemed to be critical between 6 and 7 for their binding to DNA and for exhibition of antitumor activity. Also, ARQ showed serious membrane deformations and an inhibitory effect on the membranous adenosine triphosphatase of Ehrlich carcinoma cells.     

Formation of metal coating on deoxyribonucleic acid molecule

The Monte Carlo method is employed to study the conditions for nanoconductor formation on a DNA molecule in aqueous solution via electrostatic interactions between negatively charged groups of the matrix molecule and positively charged functionalized gold nanoparticles. A model is developed that explicitly takes into account low-molecular-mass counterions resulting from the dissociation of DNA molecule and surface functional groups of nanoparticles. It is shown that the explicit regard to the counterions is of great importance for investigating conditions of nanoaggregate formation. The model parameters are estimated at which continuous metal coatings are formed via self-assembly.     

Singlet oxygen takes part in 8-hydroxydeoxyguanosine formation in deoxyribonucleic acid treated with the horseradish peroxidase-H2O2 system

Treatment of calf thymus deoxyribonucleic acid (DNA) with the horseradish peroxidase-H2O2 system resulted in efficient formation of 8-hydroxydeoxyguanosine (8-OH-dG) residues. It was concluded that singlet oxygen was the reactive species involved, based on experiments using active oxygen scavengers and D2O. For 8-OH-dG formation, a higher-ordered polynucleotide structure seems to be required: double stranded DNA was a better substrate for the reactive species than single stranded DNA, and monomeric deoxyguanosine underwent C8-hydroxylation to a lesser extent.     

Spectroscopic studies on the interaction of alpha‐eleostearic acid with calf thymus DNA

Interaction between alpha‐eleostearic acid (α‐ESA) and calf thymus DNA in Tris‐HCl buffer (pH = 7.4) using neutral red (NR) dye as a spectral probe was investigated using UV–Vis absorption and fluorescence spectroscopy. Spectral data matrix of the complexed reaction between α‐ESA and NR with DNA was processed with an alternative least‐squares (ALS) algorithm, the obtained concentration profiles and the corresponding pure spectra for species (NR, DNA–NR, and DNA–NR–ESA) demonstrated three kinds of reactions might occur in the system. The major groove binding between α‐ESA and DNA was further validated using circular dichroism, viscosity, DNA melting, and ionic strength effect measurements. Moreover, the calculated values of thermodynamic parameters, such as enthalpy (ΔH^θ, ?22.04 kJ/mol) and entropy change (ΔS^θ, 91.52 J K^?1 mol^?1), suggested binding between α‐ESA and DNA was mainly driven by hydrophobic interactions and hydrogen bonds without electrostatic force.     

Synthesis and investigation on the interaction with calf thymus deoxyribonucleic acid of a novel fluorescent probe 7-oxobenzo[b][1,10]phenanthroline-12(7H)-sulfonic acid

In this paper, the synthetic route of a potential antitumor reagent, benzo[b][1,10] phenanthrolin-7(12H)-one (BPO), was improved. A sulfonic group was introduced to BPO to form a new compound, 7-oxobenzo[b][1,10]phenan-throline-12(7H)-sulfonic acid (OPSA), in order to enhance its water-solubility. The molecular structure of OPSA has been confirmed by IR, UV, MS, ¹H NMR and elements analysis. It was proved in our experiments that DNA could quench the fluorescence of OPSA and the maximum quenched intensity appeared at 408 nm (λ_ex = 284 nm). The quenched fluorescence intensity was proportional to the concentration of DNA. Based on this phenomenon, OPSA had been used as the fluorescent probe for detection of calf thymus DNA (ct-DNA) and the corresponding linear response range was from 1.0 to 150.0 μg mL⁻¹ and the limit of detection (LOD) was 3.8 ng mL⁻¹. Its interaction with ct-DNA was investigated by fluorescence, absorption and viscosity measurements. When binding to ct-DNA, OPSA showed obvious fluorescence quenching and the quenched intensity was stable with the presence and absence of NaCl. The absorption spectra of OPSA had no evidence of increasing or decreasing when ct-DNA was added. The viscosity of OPSA and ct-DNA mixture showed no obvious change comparing with the viscosity of ct-DNA along. The results suggested that the interaction between OPSA and ct-DNA was groove binding in nature. Scatchard plots constructed from fluorescence titration data gave a binding constant of 8.9 × 10⁵ L mol⁻¹ and a binding site size of 0.35 base pairs per bound drug molecule.     

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.     

Electrochemical investigation of nucleic acid behaviour: Part I. Native calf thymus DNA

The polarographic behaviour of DNA extracted from the calf thymus has been studied using phase sensitive a.c. polarography. The conditions of adsorption on the dropping mercury electrode have been studied in terms of the conditions of medium (ionic strength, pH) and of the molecular weight of DNA. The ionic strength plays a deciding role in the conditions of adsorption. The adsorption also depends on the form of the macromolecule. When it is in rod form (low molecular weight) the adsorption occurs parallel to the charged surface. When it adopts a wormlike form, loops are formed which extend into the solution. The adsorption does not depend on the nature of the monovalent counterion thus the adsorption is identical in Na⁺ and NH₄⁺ media.The polarograms of native and denatured DNA present an initial rounded peak situated at the limit of the adsorption zone. The native DNA is characterised by a second peak which appears, whatever the medium and the molecular weight of the DNA may be, on the recordings of the in-phase component of the current. This peak is situated at a more positive potential than that which is normally characteristic of the denatured DNA.The characteristics of the peaks of the native DNA have been specified. The first peak corresponds to the desorption of a certain number of adsorbed elements of the macromolecule. The second peak changes its properties according to the pH. In a slightly acid medium, it corresponds to the reduction of the adsorbed bases without desorption of the reduced products. The zones of reduction are the locally destabilized, i.e. the A-T rich regions of the DNA. In alkaline medium it corresponds to the desorption of the macromolecule. A general schema of the behaviour of native DNA on the dropping mercury electrode is proposed.     

温度对中性红与小牛胸腺DNA相互作用的影响

用光谱法和电化学法研究了温度对中性红 (NR)与小牛胸腺DNA (CTDNA)相互作用的影响 ,在低温下和在低浓度下NR嵌入到CTDNA碱基内部 ,NR的显色团嵌入G -C碱基对并与核酸双螺旋的对称轴垂直 (γ =90°) ,当温度升高时 ,部分NR从DNA双螺旋中脱出 ,嵌入的NR发色团的长轴转变为与DNA双螺旋的对称轴成 45°角 (γ =45°)。     

Spectroscopic studies on the interaction of pazufloxacin with calf thymus DNA

Fluorescence spectra, absorption spectra, DNA viscosity titrations, competition experiment, and iodide quenching experiment were used to study the interaction of DNA with pazufloxacin. DNA quenches the fluorescence of pazufloxacin significantly. No red shift and isobestic points are observed in UV titration experiment. DNA viscosity and iodide quenching results suggest that pazufloxacin does not intercalate into DNA. SsDNA has a stronger quenching effect on pazufloxacin than dsDNA has. Pazufloxacin interacts with DNA in a different mode from ethidium bromide, which is a typical intercalator of DNA. All these results indicate that pazufloxacin interacts with calf thymus DNA in the mode of groove binding. The quenching constant and thermodynamic constants have also been determined.     

In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin

The low therapeutic index of digoxin necessitates careful monitoring of its serum levels. Most of digoxin immunoassays suffer from interferences with digoxin-like immunoreactive substances. Since aptamers have been shown to be highly specific for their targets, the aim of this study was to develop DNA aptamers for this widely used cardiac glycoside. Digoxin was coated onto the surface of streptavidin magnetic beads. DNA aptamers against digoxin were designed using Systematic Evolution of Ligands by Exponential enrichment method (SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR amplification. The PCR product was cloned and sequenced. Binding affinity was determined using digoxin–BSA conjugate, coated onto ELISA plate. Inhibitory effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. Three aptamers (D1, D2 and D3) were identified. Binding studies of fluorescein-labeled truncated (without primer binding region) D1 and D2 and full length D1 anti-digoxin aptamers were performed and their corresponding dissociation constants values were 8.2 × 10⁻⁹, 44.0 × 10⁻⁹ and 17.8 × 10⁻⁹ M, respectively. This is comparable to what other workers have obtained for interaction of monoclonal antibodies raised against digoxin. There was little difference in binding affinity between full length and truncated anti-digoxin D1 aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between digoxin and ouabain in both tissue study and binding experiments. Our finding indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further studies might show its suitability for use in digoxin assays and as a therapeutic agent in life-threatening digoxin toxicity.     

中性红分光光度法测定脱氧核糖核酸

研究了有机染料中性红与脱氧核糖核酸(DNA)的结合反应, 选择了实验的最佳条件: pH 5.0 的B-R缓冲溶液2.5 mL, 1.0×10-3 mol/L中性红溶液0.6 mL, 反应20 min后体系的吸光度很稳定, 在λ=530 nm处有最大吸收峰, 并且随着DNA的加入, 中性红的吸收峰显著下降. 因此以中性红为标记物, 根据其在波长530 nm处吸收峰下降的程度, 可用于定量测定DNA. 测量DNA的线性范围为0～10 μg/mL, 相关系数为0.998, 该方法具有较高的灵敏度和选择性, 已用于合成试样分析.     

Interactions of tannic acid and its derivatives (ellagic and gallic acid) with calf thymus DNA and bovine serum albumin using spectroscopic method

In the present investigation, an attempt has been made to study the interaction of chosen polyphenols (tannic, ellagic and gallic acids) with calf thymus DNA and bovine serum albumin (BSA) employing spectrofluorimetric technique. The fluorescence quenching of DNA-bound ethidium bromide (EB) and BSA-bound 1-anilinonaphthalene-8-sulfonic acid (ANS) by phenolic acids has been examined. As BSA contains two tryptophan residues, the polyphenols influence on protein by measuring the changes in the fluorescence of BSA in the presence of phenolic acids was also evaluated. Our experiments prove that there is a direct interaction between phenols and DNA or BSA. The obtained data suggest that used acids can intercalate to DNA and interact strongly with BSA. The strongest interactions were observed between DNA and ellagic acid and between BSA and tannic acid. The conformational changes were revealed in DNA and BSA after incubation with tested phenolic acids and the extent depended on the phenol structure and the used concentration.     

光谱法和电化学法研究中性红与小牛胸腺DNA的相互作用

利用紫外 可见和圆二色光谱(CD)法和伏安方法,研究了小分子染料中性红(NR)与小牛胸腺DNA(CTDNA)的相互作用。实验表明在NR低浓度下,NR能嵌入至核酸双螺旋的碱基内部在G C处与核酸结合,而在较高浓度情况下,嵌入的NR分子与后来的在核酸双螺旋外部的NR分子相互作用发生聚集,从而堆积在DNA双螺旋的表面,同时使核酸的构象由B型转变为Z型。用光谱滴定的方法获得NR与CTDNA作用内部结合常数,分别为:Ka1=2 4×104mol·L-1·cm-1和Ka2=2 1×10-2mol·L-1·cm-1。     

Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.     

Inhibition of deoxyribonucleic acid synthesis in vitro by anticancer platinum pyrimidine greens against Daudi cells.

Platinum pyrimidine greens inhibited the deoxyribonucleic acid (DNA) synthesis of tumor cells in the S phase of the cell cycle and exerted antitumor activity. Clear differences were observed in the activity between the samples prepared at 40 degrees C and at 75 degrees C. Using 3H-thymidine incorporation assay and cell cycle analysis we confirmed that the former had much stronger and more specific inhibitory activity against DNA synthesis than the latter. Reactivity of the 40 degrees C sample with deoxyguanosine monophosphate (dGMP) and deoxyadenosine monophosphate (dAMP) was, respectively, two and three times larger than that of the 75 degrees C sample.