Formation of DNA complexes with actinomycins in aqueous solutions and films

The mechanisms of DNA interaction with actinomycin D (AMD), 7-amino-actinomycin D (7-AAMD), and ethidium bromide (EtBr) were studied in aqueous solutions and in condensed state (films coating plates). The use of the methods of absorption (UV, IR, and visible spectral ranges) and fluorescence (steady-state, polarization, and phase-modulation) spectroscopy revealed that (1) the formation of DNA complexes with 7-AAMD in solution was not accompanied by energy transfer from photoexcited nucleotides to phenoxazone chromophore and (2) the mechanism of ligand incorporation was distinct from stacking. In the film of the DNA-7-AAMD complex, which simulated the native state in a biological cell, the efficiency of the energy transfer was high. This indicates that a stacking-type mechanism underlies actinomycin intercalation into DNA. In the presence of high concentrations of 7-AAMD in the film, DNA denatured and its double-helical structure, degraded. In the DNA-AMD complex, the native B-form of DNA molecule was conserved both in films and in solution.     

Nonstacking Binding of 7-Aminoactinomycin D and Actinomycin D to DNA and Model Nucleotide Systems in Solutions

The mechanism of actinomycin D (AMD) and 7-aminoactinomycin D (7AAMD) interaction with DNA and model nucleotide compounds was studied by absorption and fluorescence spectroscopy (steady-state, phase-modulation, and polarization). It was shown that complex formation does not result in energy transfer from photoexcited nucleotides to the phenoxazone chromophore of 7AAMD, which indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. A basic difference was revealed in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them; 7AAMD forms no complexes with either guanine micelles or polyguanylic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, strong competition is observed between AMD and 7AAMD for the binding site in oligonucleotide HP1 used as a DNA hairpin model. The effective diameters of 7AAMD–HP1 complex and free 7AAMD were determined using the Levshin–Perren equation.     

Non stacking binding of 7-amino-actinomycin D and actinomycin D to DNA and model nucleotide systems in solutions

Mechanism of actinomycin D (AMD) and 7-aminoactinomycin D (7AAMD) interaction with DNA and model nucleotide compounds was studied by absorption and fluorescence spectroscopy (steady-state, phase-modulation, and polarization). It was shown that complex formation does not result in energy transfer from photoexcited nucleotides to phenoxazone chromophore of 7AAMD that indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. It was revealed a fundamental difference in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them. 7AAMD forms complexes neither guanine micelles nor polyguanilic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, a strong competition is observed between AMD and 7AAMD for binding site in oligonucleotide HP1 used as DNA hairpin model. The efficient diameters of 7AAMD-HP1 complex and free 7AAMD were determined using the Levshin-Perren equation.     

Prompt non-stacking binding of actinomycin D to hairpin oligonucleotide HP1 and slow redistribution from HP1 to DNA

Complexes of actinomycin D (AMD) and 7-amino-actinomycin D (7AAMD) with model hairpin oligonucleotide HP1 and various types of DNA in aqueous solutions were investigated by steady-state, polarized, time-resolved and stopped-flow fluorimetry, and photometry. Prompt non-stacking binding of the actinomycins inside HP1 was observed. No energy transfer from nucleotides to 7AAMD in the complex was detected, most likely because of the absence of stacking intercalation. Complex formation of AMD or 7AAMD and HP1 was followed by the transition from a random flexible conformation of the hairpin to a more compact rigid structure, and subsequently to hypochromism. Strong competition between AMD and 7AAMD for a cavity in HP1 was observed. The decrease in the 7AAMD emission after addition of DNA to the 7AAMD/HP1 complex indicates that actinomycins can be redistributed from HP1 to DNA, i.e. hairpin oligonucleotides can serve as molecular carriers of actinomycins.     

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation

Summary Cortex cells of the root meristem of Cucurbita pepo (0.0–0.5 mm from the cap junction), in the 3–4, 5–6 and 7–8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7–8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5–6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (³H thymidine uptake), template activity of DNA (³H actinomycin D(³H AMD)-binding), RNA synthesis (³H uridine incorporation), and protein synthesis (³H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3–4 mm segment reach 4C ploidy state, but in the 7–8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of ³H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5–6 and 7–8 mm segments, while in 3–4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, ³H uridine uptake, ³H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3–4 and 5–6 mm segments). ³H leucine is still incorporated intensely into 7–8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of ³H thymidine uptake into nuclear DNA with ³H AMD binding and ³H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3–4 and 5–6 mm segments; in the 7–8 mm segment binding of ³H AMD slightly decreases, while ³H uridine incorporation is considerably reduced. Divergence between the ploidy state, ³H AMD binding and ³H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3–4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.This work was partly supported by Polish Academy of Sciences, Botanical Committee, Grant 217/II     

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Fluorescence microscopic and autoradiographic analyses of the differential reaction of various regions of polytene chromosomes in Chironomus to prolonged exposure to 7-amino-actinomycin D and 3H-actinomycin D

Dynamics of binding of a fluorescent analogue of actinomycin D -- 7-amino-actinomycin D -- and 3H-actinomycin D with polytene chromosomes of Ch. thummi was studied. Biological effects of AMD, 7-amino-AMD, and 3H-AMD on polytene chromosomes were found to be similar. These ligands provoke the reduction of the nucleolus and Balbiani rings and the appearance of giant pseudo-puffs in heterochromatic centromere regions of polytene chromosomes. There was no intermediate binding of 7-amino-AMD to DNA in vivo both after a longterm treatment of larvae with fluorochrome and in chase experiments. It was found that a loosening of chromatin in centromere regions accompanied by a weakening of its fluorescence took place in the formation of pseudo-puffs. Possible mechanisms of pseudo-puff formation under the influence of AMD and 7-amino-AMD are discussed. Essential factors may be peculiarities of DNA nucleotide composition in centromere regions, DNA packing, alteration of physico-chemical properties of DNA in the complex with AMD (despiralizations and elongation), and an inhibition of RNA synthesis necessary for the maintenance of normal structure of polytene chromosomes.     

Receptor-Mediated Transfer of DNA–Galactosylated Poly-L-lysine Complexes into Mammalian Cells in vitro and in vivo

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA–poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA–poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA–poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.     

Energy of interaction in actinomycin-nucleotide complexes

Complexes of the natural heterocyclic antibiotic actinomycin D (AMD) with its putative carriers: purine and pyrimidine nucleotides, as well as with fragmented DNA and phospholipid liposomes have been studied by high-sensitivity spectrophotometry. The antibiotic is not only adsorbed onto the surface of purine clusters but also is incorporated into them. It is especially readily incorporated into unwound DNA regions. The incorporation is accompanied by a long-wavelength shift of the absorption spectrum. From the magnitude of the shift, the energy of interaction was calculated. In the case of AMD in the complex with caffeine and adenosine, it is 2.4 and 2.7 kcal/mol, and in the complex with guanosine and fragmented DNA it is considerably higher, 3.3 and 3.7 kcal/mol. It is assumed that guanosine, adenosine, caffeine and fragmented DNA may serve as carriers of the antibiotic.     

Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 10⁵ M^–1 at 20°C, in a solution containing 18 mM Na⁺. The DNA–drug interaction was enthalpy driven, with a ΔH_b of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔG_obs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.     

Biological, proton-magnetic-resonance- and ultraviolet-spectroscopic evidence for a molecular complex of actinomycin D and 10,11-dihydro-3H-naphth[1,2-g]indazol-7-ol (Short Communication)

A proton-magnetic-resonance study of the complex formed between actinomycin D and 10,11-dihydro-3H-naphth[1,2-g]indazol-7-ol strongly suggests that the indazole lies above the phenoxazine ring of actinomycin D. The complex can be destroyed by addition of dimethyl sulphoxide or dimethylformamide. The actinomycin D–indazole complex inhibits growth of Pseudomonas fluorescens and raises the thermal denaturation response of DNA. These data support the hypothesis that a molecular complex is formed which readily inhibits cell growth and interacts with DNA.     

Amplified electrochemical aptasensor for thrombin based on bio-barcode method

In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was developed based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI was immobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with barcode binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcode DNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the concentration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Under optimum conditions, a detection limit of 6.2 × 10⁻¹⁵ mol L⁻¹ (M) thrombin was achieved. In addition, the sensor exhibited excellent selectivity against other proteins.     

Halomonas campisalis,an Obligatorily Alkaliphilic,Nitrous Oxide–Reducing Denitrifier with a Molybdenum Cofactor–Lacking Nitrate Reductase

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1–68.1 mol %) and DNA–DNA homology (75–92%) and represent different morphotypes of the same species. On the basis of results of partial 16S rRNA sequencing, strain Z-7398-2 was found to be most closely related to the Halomonas campisalis isolate from the Alkali Lake (USA). The DNA–DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide–reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a molybdenum cofactor.     

Formation of Liquid-Crystalline Dispersions of Double-Stranded DNA–Chitosan Complexes

Right-handed helical double-stranded DNA molecules were shown to interact with chitosans to form under certain conditions (chitosan molecular weight, content of amino groups, distance between amino groups, ionic strength and pH of solution) cholesteric liquid-crystalline dispersions characterized by abnormal positive band in CD spectrum in the absorption region of DNA nitrogen bases. Conditions were found for the appearance of intense negative band in CD spectrum upon dispersion formation. In some cases, no intense band appeared in CD spectrum in spite of dispersion formation. These results indicate not only the multiple forms of liquid-crystalline dispersions of DNA–chitosan complexes but also a possibility to control the spatial properties of these complexes. The multiplicity of liquid-crystalline forms of DNA–chitosan complexes was attempted to explain by the effect of character of dipoles distribution over the surface of DNA molecules on the sense of spatial twist of cholesteric liquid crystals resulting from molecules of the complexes.     

Modification specifique des bandes Q et R par le 5-bromodeoxyuridine et l'actinomycine D

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.     

The interaction of 7-substituted actinomycin D analogs with DNA

The interaction of actinomycin D and three new 7-substituted analogs with calf thymus DNA has been studied by a number of physical techniques. The methods utilized in this investigation include visible absorption spectrometry and ultrafiltration methodology for the determination of equilibrium binding constants; viscometry; and circular dichroism. The studies show that the 7-substituted actinomycin D analogs retain the G . C base pair specific DNA binding demonstrated by actinomycin D. The mode of binding to native DNA, despite substitution at position 7, is practically unaltered. The retention of this binding specificity by these analogs seems to be unaffected by changes in the electon properties of the chromophore.     

Calculations of DNA Condensation Caused by Ligand Adsorption

A method for calculating the curves of DNA transition from linear to condensed state upon binding of condensing ligands has been developed. The character of the transition and ligand concentration necessary for condensation have been shown to be governed by the length of DNA molecule, energy and stoichiometry parameters of the DNA–ligand complex (equilibrium constant between linear and condensed form in the absence of ligands, constants for ligand binding to linear and condensed forms, the number of base pairs covered by one ligand, etc.). The results of the calculations indicate that a slight difference in the free energies of these DNA states (less than 6 cal/mol(bp) for a DNA of 500 bp) is sufficient for the existence of a stable linear state in the absence of ligands (in free DNA) and the formation of stable condensed state upon complexation.     

Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

Actinomycin D binding to DNA and chromatin: A colorimetric procedure suitable for the analysis of turbid preparations and for simultaneous processing of several samples

A simple, sensitive, and inexpensive method is presented for measuring the binding of actinomycin D (AMD) to either chromatin or DNA. The procedure gives results which are comparable with those obtained by the optical titration procedure, but is superior to that method in that it does not require continuous attention, can be used with turbid chromatin preparations, and allows simultaneous processing of several samples. Chromatin or DNA that has been allowed to react with AMD is subjected to sucrose density gradient centrifugation to remove unbound antibiotic. The AMD-DNA or AMD-chromatin complex is then dissociated by incubation with 5m urea, and the released AMD is extracted with ether and estimated spectrophotometrically.