Effect of DNA structure on the formation of collagen-DNA complex

Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms.     

Dielectric behaviour and conformational stability of collagen on interaction with DNA

Collagen-DNA interaction studies will aid in improving the stability of DNA against nucleases. In the present study, the effect of DNA on different physico-chemical properties of collagen like viscosity, conformation and dielectric behaviour has been studied. Increase of DNA concentration leads to the increment of viscosity of collagen at the pH 4 and 5, but the trend is reversed at the pH of 6 and 7 due to the formation of collagen fibrils. The temperature dependent CD spectroscopic studies for collagen-DNA conjugate showed that thermal stability of collagen is modulated with increasing molar concentration of DNA. It also shows that DNA interactions with collagen did not result in change in the triple helical structure of collagen. Impedance measurements show that the strength of ion pairs for different molar concentrations of collagen-DNA conjugates has changed. Nyquist plot for collagen-DNA conjugate posses higher Y″ at DNA concentration of 1:25 and 1:50 whereas at 1:1 and 1:10 lower Y″ than the native collagen have been observed. An understanding of this nature of the collagen-DNA interactions is helpful for gene delivery applications.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Hydrodynamic properties of DNA and DNA-lipid complex in an elongational flow field

The aim of this study was to determine the difference between hydrodynamic properties of DNA-cetyltrimethylammonium (CTA) complex and those of DNA, which may be related to the difference in fibre-forming ability of DNA-CTA from that of DNA. Responses of DNA and DNA-CTA complex to an elongational flow field were investigated. In both solution systems, results suggesting a coil-stretch transition were obtained. From a critical strain rate value, the radius of gyration of DNA-CTA molecules in ethanol-glycerol solution was revealed to be 0.3-0.5 times of that of DNA in aqueous NaCl solution. Shear viscosity of DNA-CTA solution was much smaller than that of DNA solution, also suggesting a smaller size of DNA-CTA in ethanol-glycerol solution than that of DNA in aqueous NaCl solution. The plateau birefringence value of the DNA-CTA system, a parameter that indicates the local molecular conformation and the molecular arrangement, was only about 1/10 of that of the DNA system. There is an empirically determined molecular model of DNA-CTA complex in which a DNA molecule is sheathed by a cylindrical crust made of CTA chains. This structure reduces the DNA molecular density in a pure elongational flow field region but cannot explain the observed reduction of birefringence intensity. The small plateau birefringence value of DNA-CTA compared with that of DNA was attributed to the reduced molecular polarizability by the particular conformation of DNA molecules and CTA chains in the DNA-CTA system such as that expected by the conformational models.     

Interaction of DNA with connective tissue matrix proteins reveals preferential binding to type V collagen

The interaction of DNA with type I to VI collagens and laminin was studied in vitro in systems in which the connective tissue components were immobilized, as well as when in solution. In studies on immobilized components, significant binding of DNA was observed only for type V collagen, and the binding of radiolabeled DNA to this component could be effectively inhibited in a concentration-dependent manner by the addition of unlabeled DNA. Similar results were observed in solution assays in which it was observed that DNA binding to type V collagen was dependent on the native triple-helical conformation of the collagen. The preferential binding of DNA to native type V collagen may be due to the relative basicity of type V collagen chains, as well as the unique spatial arrangement of amino acid side chains in the native molecules. The data are of potential clinical relevance in that binding of DNA to type V collagen may represent at least one component of the mechanism whereby DNA and its immune complexes are deposited in connective tissues in certain pathologic conditions.     

Preparation of insolubilized-DNA film with three-dimensional network and removal of endocrine disruptors.

A water-insolubilized film was prepared by UV irradiation on a dried DNA film. When a UV-irradiated DNA was examined using a circular dichroism spectroscopy, a double stranded structure was observed as well as that of native DNA. The UV irradiated DNA film was also accumulated intercalating reagents. These results suggested that the double stranded structure was involved in the UV irradiated DNA film with a three-dimensional network. The thymine-thymine dimer formation was suggested to be involved in the cross-linking reactions by the polymerization analysis using poly(dA)-poly(dT) and poly(dG)-poly(dC). We also demonstrate the utilization of the UV-irradiated DNA film as a functional material for the accumulation of harmful DNA intercalating pollutants in aqueous solution. These results suggested that the UV-irradiated DNA film was applicable as a functional material for medical, engineering and environmental sciences.     

The self-association of human apolipoprotein A-IV. Evidence for an in vivo circulating dimeric form

We have investigated the self-association properties of human apolipoprotein A-IV using several complementary physical techniques. Sedimentation equilibrium analysis demonstrated that human apolipoprotein A-IV formed oligomeric species in aqueous solution at physiologic pH. Computer analysis established that the best model of self-association is a monomer-dimer-tetramer scheme, with an unusually large monomer-dimer association constant of 2.9 X 10(5) liters/mol. Fluorescence spectroscopy and electrophoretic analysis demonstrated that the rate of monomer-oligomer interconversion is sufficiently slow that a stable population of dimeric protein exists in solution, even at low total protein concentrations, and that the extent of dimerization is minimally influenced by pH. Moreover, these techniques established that the dissociation of oligomeric forms and the unfolding of the monomeric form are discrete and sequential events. In experiments where apolipoprotein A-IV was incubated with human high density lipoproteins, fractionated by gradient gel electrophoresis, and localized by immunoblotting, dimer formation occurred, but very little binding to lipoproteins was observed. Immunoblots of human serum fractionated on acrylamide gradient gels and isopycnic density gradients demonstrated an apolipoprotein A-IV band of size and density consistent with a circulating dimeric form, unassociated with lipid. We conclude that human apolipoprotein A-IV undergoes high affinity self-association in aqueous solutions, and that such self-association likely occurs in vivo. Self-association may thus be important in determining the biologic behavior of human apolipoprotein A-IV by influencing both the kinetics and distribution of its association with plasma lipoproteins.     

A vibrational spectroscopic study of the metastable form of associated polyinosinic acid

We have studied by Raman and ir spectroscopy the metastable complex formed by the self-association of polyinosinic acid in aqueous solution. The complex is easily prepared by quickly cooling to ca. 0°C a warm solution of the polyribonucleotide to which a small amount of rubidium salt has been added. Upon heating, this metastable form melts cooperatively near 13°C, well below the dissociation temperature of a stable four-stranded complex, which occurs at 47°C in the same conditions. The presence of several components in the stretching-mode region of the carbonyl groups in the vibrational spectra of the metastable complex suggests that it also has a parallel four-stranded structure. The difference in structure between the two forms is believed to be caused by the presence of fewer metal ions in the central channel of the metastable complex, in agreement with conclusions reached in previous investigations. The Raman spectra further show that the ribose units in the metastable form have a C3′-endo conformation, in contrast with the stable form, for which we have previously suggested a mixed C2′-endo/C3′-endo conformation. © 1996 John Wiley & Sons, Inc.     

DNA-collagen complex as a carrier for Ag+ delivery

The possibility of DNA-collagen complex as a drug carrier was investigated. The interaction between DNA and silver ions was proved by CD spectra. The release property of the complex of DNA-Ag+ was measured through turbidity of PBS solution to indicate that silver ions could coordinate with base pairs of DNA, and be released slowly from the complex of DNA-Ag+. Collagen film, collagen-Ag+ film, DNA-collagen film and DNA-collagen-Ag+ film were prepared, and studied through SEM. Particles were found present in DNA-collagen-Ag+ film by SEM. These show that silver ions may be enclosed inside these particles, which led to the slow release of Ag+ to the environments. Two bacteria, Escherichia coli and Staphylococcus aureus, were used to study the antibiotic properties of the complex films. The growth of E. coli and S. aureus could be inhibited by these films. It indicates that DNA-collagen may be a good drug carrier for the drug-controlled release.     

PEG-assisted DNA solubilization in organic solvents for preparing cytosol specifically degradable PEG/DNA nanogels

DNA was dissolved in selected organic solvents in the presence of poly(ethylene glycol) (PEG). Nanoscale PEG/DNA complex (approximately 100 nm) was produced in dimethylsulfoxide (DMSO) phase. Using a thiol-functionalized six-arm branched PEG for DNA solubilization, the PEG/DNA nanocomplex was cross-linked through the formation of disulfide linkages between the thiol groups, resulting in the production of stable PEG/DNA nanogels in aqueous solution. DNA release from the nanogels could be modulated by changing the concentration of an external reducing agent. The released plasmid DNA from the nanogels maintained intact structural integrity and exhibited appreciable gene transfection efficiency. The PEG/DNA nanogels could be potentially applied for gene therapy including DNA vaccination.     

Studies on human serum high-density lipoproteins. Self-association of human serum apolipoprotein A-II in aqueous solutions.

Some of the solution properties of pure preparations of human serum high-density apolipoprotein A-II were studied by sedimentation equilibrium ultracentrifugation, conducted at different apoprotein concentrations and at several speeds. The concentration dependence of the apparent weight average molecular weight indicated that apolipoprotein A-II, when dissolved in 0.02 MEDTA (pH 8.6), undergoes self-association. Over a protein concentration range between 0.8 and 1.5 mg/ml, the self-association could best be described by a monomer-dimer-trimer step association, although indefinite self-association could not be ruled out. The equilibrium constants obtained were sufficient to describe the system over the concentration range investigated.     

Formation of Au(III)-DNA coordinate complex by laser ablation of Au nanoparticles in solution

We discovered that an Au(III)-DNA coordinate complex, Au(III)(DNA-base)2(amine)L, are formed by laser ablation of Au nanoparticles in an aqueous solution containing DNA molecules in the presence of amines and multi-valent cations, where L represents an unknown ligand (either amine or water). Optical absorption spectrum of the solution after laser ablation exhibited a 360 nm absorption peak assined to ligand-->Au(III) charge transfer (LMCT) band of the coordinate complex. The complex is considered to be formed as follows: (1) the DNA molecules are neutralized by binding the multi-valent cations to their negatively charged phosphate groups, and adsorbed on the surface of the Au nanoparticles by a hydrophobic interaction, (2) Au(III) ions are liberated from the Au nanoparticles by laser ablation, and (3) an Au(III) ion reacts with amine and two DNA bases of a DNA molecule into an Au(III)(DNA-base)2(amine)L.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

Nucleosome Interactions and Stability in an Ordered Nucleosome Array Model System

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.     

Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence

A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.     

The self-organization of adenosine 5'-triphosphate and adenosine 5'-diphosphate in aqueous solution as determined from ultraviolet hypochromic effects

The self-association of adenosine 5'-triphosphate (ATP) and of adenosine 5'-diphosphate (ADP) was studied in aqueous solution at different pH values, over the concentration range from 5 x 10(-6) to 5 x 10(-2) M, by ultraviolet spectroscopy. Measures of the molar absorptivity of the ultraviolet bands of these compounds with increasing concentration have shown two hypochromic effects, at concentrations below 10(-3) and above 10(-3) M, respectively. These results can be interpreted in terms of self-association processes involving the formation of dimers and of polymers. From the fitting of the experimental curves of hypochromic effects, self-association constants for dimerization and polymerization were calculated. The results obtained are discussed in relation to the values reported in the literature and indicate the influence of the concentration range not only on the numerical value but also on the order of magnitude of the association constants. Comparison of ATP with ADP shows that the length of the phosphate chain may be a relevant feature in the nature of the self-organization processes in these adenine nucleotides in aqueous solution.     

The self-association of ATP: thermodynamics and geometry.

The concentration and temperature dependence of the self-association of ademosin-5'-triphosphate (ATP) in aqueous solution was studied by means of ultraviolet absorption spectroscopy and circular dichroism (CD). Of several possible models, a model was indefinite linear self-association, in which each step has the same equilibrium constant, describes the data best. The two different methods lead within experimental error to the same thermodynamic parameters. At pH 8.7, IN 1 M Tris and 0.5 M 7gCl-2, we find deltaH-0 equals -5.1 kcal/mole and deltaS-0 equals -13.0 e.u. These values do not differ much from those found for the self-association of uncharged bases and nucleosides in aqueous solution. The CD spectrum that results from the self-association is conservative and quite similar in shape to that observed for some stacked dinucleotides: it is interpreted as a first approximation within the framework of the exciton model.     

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 the 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 energy transfer efficiency 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.     

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.