DNA In Aqueous Solution Research Articles

Netropsin, a Minor Groove Binding Ligand: A Potential Radioprotective Agent

Minimizing radiation-induced damages in DNA is an important aspect in the development of chemical radioprotectors. The aim of this study was to evaluate the possible radioprotective ability of the DNA minor groove binding ligand netropsin in an aqueous solution of plasmid DNA (pBR322) and to compare its efficacy with that of Hoechst 33258, a known radioprotector. The radiochemical parameters D(0), G(SSB) and DMF were calculated in pBR322 DNA. Based on a comparison of the DMFs of netropsin and Hoechst 33258, netropsin appeared to be the better radioprotector. The ligand binding site accessibility of the restriction enzyme EcoRI at the ligand-pBR322 complex was assessed using a restriction-digestion assay in irradiated solutions. A distinct ligand-bound site protection in netropsin-DNA was observed in irradiated solutions. However, no site protection was observed in the presence of Hoechst 33258. The possible role of ligand-induced structural stabilization in irradiated aqueous solutions was also investigated using netropsin-calf thymus DNA melting temperature measurements. The greater radioprotective ability of netropsin in solutions of DNA was suggested to be due to its higher binding affinity and its ability to provide higher structural stabilization. EcoRI digestion revealed that hydroxyl radical (OH*) generated by ionizing radiation is not able to radiolyse the netropsin-DNA complex. These results will help in developing better radioprotectors.

Conversion of ortho-para H2O isomers in water and a jump in erythrocyte fluidity through a microcapillary at a temperature of 36.6±0.3°C

A mechanism is proposed for the previously observed [1] jump in erythrocyte fluidity through a microcapillary 1.3 µm in diameter at a temperature of 36.6±0.3°C. Our interpretation is based on the experimental evidence both for existence of ortho and para H2O isomers in water and on spin-selective interaction of proteins with para H2O isomers as hydration shells of biomolecules are being formed [2]. It is important that the formation of hydration shells of proteins and DNA in aqueous solutions is accompanied by an increase in the Brillouin shift to 0.4 cm−1 (≃0.25 cm−1 in water), which points to the formation of icelike structures. We believe that the coincidence of the translational energy kT of the Brownian motion and the energy of the rotational quanta for the 313–202 transition of para H2O isomers at the temperature 36.6°C increases the probability for excitation of para H2O isomers in collisions. Collisions mix quantum states of closely spaced levels in para H2O (313, 285.2 cm−1) and ortho H2O (330, 285.4 cm−1) and induce conversion of para isomers to ortho H2O. It is assumed that this conversion in the icelike hydration shell of hemoglobin (Hb) is accelerated under the catalyzing effect of oxygen and iron present in Hb and triggers a chain reaction: release of ortho H2O isomers through the erythrocyte membrane→compaction of Hb molecules and increase in concentration of catalysts→acceleration of conversion→structural gel-sol transition. It is the sequence of these processes that provides a jump in fluidity of erythrocytes through a microcapillary and the anomalous increase in fluidity of the aqueous solution of hemoglobin by almost an order of magnitude at temperatures close to 36.6°C and an increase in the solution concentration by a factor of 1.7.

Conformational changes of DNA γ-irradiated in the presence of aliphatic alcohols in solution

The conformational changes of DNA in aqueous solutions containing ethanol and iso- and n-propanol having concentrations of 0–23 wt.% and ionic strength μ = 0.003 NaCl and exposed to γ radiation at doses from 0 Gy to 30 Gy were studied by low-gradient viscosimetry and circular dichroism. The dependence of the specific volume (Vsp) of DNA on the radiation dose changed at different contents of alcohol. There is a critical concentration (Ccr) at which the structure of the mixed solvent is rearranged. The Vsp of DNA decreased as the radiation dose increased at Calc = 12 wt.% Ccr. In both cases, the largest Vsp of DNA was observed in water ethanol solutions. The secondary structure of the macromolecule in the systems corresponds to the form. It was shown that the observed conformational changes in irradiated DNA at Calc Ccr could not be explained only by the ability of alcohols to intercept the radicals formed in radiolysis of water.

Investigation of the interaction between DNA and cobalt ferrite nanoparticles by FTIR spectroscopy

The interaction of DNA with nanoparticles of cobalt ferrite powder prepared by the mechano-chemical method was studied. It was shown that CoFe(2)O(4) nanoparticles efficiently bind DNA in aqueous solutions (Tris-HCl), forming a bionanocomposite. The adsorption capacity of CoFe(2)O(4) nanoparticles for DNA was evaluated to be 5.25 x 10(-3) mol/m(2). The desorption of DNA from the surface of the particles was analyzed while changing the pH, the ionic strength, and the chemical content of the medium. The DNA-CoFe(2)O(4) nanocomposite was investigated by FTIR spectroscopy. The block of the data allowed one to consider the mechanism of the interaction between a polynucleotide and CoFe(2)O(4) nanoparticles and to make the assumption that the binding occurred due to the coordination interaction of the phosphate groups and heterocyclic bases of DNA (oxygen atoms of thymine and guanine) with metal ions on the particle surface. The analysis of the IR spectra showed that binding can lead to the partial destabilization of the DNA structure, with the B conformation of a polynucleotide being preserved.

Spectral-luminescent study of the interaction of some styrylcyanine dyes with bovine serum albumin and DNA in aqueous solutions

Spectral-luminescent characteristics of the several new styrylcyanine dyes, synthesized on the base of F dye ((E)-4-(4-(dimethylamino)styryl)-1-methylpyridinium iodide), were studied in aqueous solutions in the presence and absence of bovine serum albumin and deoxyribonucleic acid. It was shown, that with the rise of concentration of bovine serum albumin and deoxyribonucleic acid in aqueous solution, the bathochromic shift of electronic absorption spectra band and sharp intensity increase of fluorescence band intensity are observed for studied dyes, while the shape of absorption and fluorescence bands remains intact. Biophysical parameters (the binding constant and quantity of the binding sites) of interaction of studied dyes with BSA and DNA were determined. It is shown, that homodimer styrylcyanine dyes with a pentyl – or longer linking chain have larger binding affinity to specified biomolecules than dyes with shorter link-chain.

Photophysical Properties of Quinolizinium Salts and Their Interactions with DNA in Aqueous Solution

The photophysical properties of three quinolizinium salts (naphto[2,1-b]quinolizinium bromide (Q2), naphto[1,2-b]quinolizinium bromide (Q3), and indolo[2,3-b]quinolizinium tetrafluoroborate (HI)) in fluid media and their interactions with DNA were investigated by steady-state and by nanosecond and femtosecond time-resolved techniques. The main decay pathways of the excited singlet state S(1), fluorescence, intersystem crossing, and internal conversion, were characterized in terms of quantum yields and rate constants. The lowest triplet state of the quinolizinium salts is able to sensitize singlet oxygen in rather high efficiency (phi(Delta) = 0.4 to 0.5 in MeCN). The complexes between quinolizinium salts and DNA formed in the ground state were characterized in terms of lifetimes and decay channels to give more details of the mechanism of photoinduced DNA strand break.

Selective accumulation of harmful compounds by the DNA-inorganic hybrid-immobilized glass bead

Previously, we reported the DNA-inorganic hybrid material including double-stranded DNA by mixing the aqueous DNA solution and silane coupling reagents. Here, we immobilized the DNA-inorganic hybrid material onto the glass bead and prepared the DNA-immobilized glass bead column. The DNA-immobilized glass beads were stable in water and the amount of eluted DNA from the DNA-glass beads did not change for more than 1 week. Additionally, this DNA-immobilized column selectively accumulated the harmful compounds with the planar structure, such as dioxin- and polychlorinated biphenyl (PCB)-derivatives, and these accumulation percentages were 50–70%. Furthermore, the DNA-immobilized glass bead was recycled nine times by the application of ethanol solution and the accumulative ratio was maintained at more than 60% and did not appear to be decreasing. Therefore, these DNA-columns might have a potential for the selective removal and separation of DNA-intercalating molecules and harmful compounds with the planar structure from experimental or industrial drainages.

Light scattering study of natural DNAs over a wide range of molecular weights: Evidence for compaction of the large molecules

This work reports light scattering measurements on DNA in aqueous solutions (100 mM NaCl, 1 mM EDTA and 10 mM Tris–HCl buffer, pH 7.8) over a wide range of molecular weights (10 2–10 5 base pairs) and shows that, in the above standard solvent, shorter chains (<10 4 base pairs) behave as a “wormlike chain” and their diffusion coefficients as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains (>10 4 base pairs) behave in a different manner. Dynamic and static light scattering and SEM analysis indicate that DNA molecules 10 5 base pairs long, condense into compact structures in our solvent conditions. Calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature.

Dissolved oxygen alteration of the spectrophotometric analysis and quantification of nucleic acid solutions

Nucleic acids are routinely and readily analysed using the A(260)/A(280) ratio, although this method is known to be prone to erroneous results owing to contaminants in solution that absorb at similar wavelengths. The aim of the present review, while highlighting the problems and alternatives of using UV spectrophotometry for nucleic acid measurements, is to bring forth an observational result from our recent studies, namely that DO (dissolved oxygen) and nitrogen can alter the A(260) of aqueous DNA solutions. Our finding is of importance because DO is highly variable between protocols and storage conditions of DNA preparations. The physicochemical nature of the oxygen-DNA interactions is briefly discussed.

Effect of high power ultrasound on aqueous solution of DNA

The effect of (20 kHz continuous wave) ultrasound (power from 17 to 77 W) on the damage of nitrogen bases, the loss of double-helical structure, strand breaks and fragmentation of Calf spleen DNA were studied in vitro. The relative ultrasound sensitivity of the hydrogen bonded base pairs A-T and G-C were also studied. The techniques used in this investigation were ultraviolet absorption spectroscopy and constant-field gel electrophoresis (CFGE). The results showed that ultrasound decreased the hydrogen-bonded nucleotides. The ultrasound sensitivity was greater for the A-T base pairs than for the G-C base pairs. It was also shown that high power ultrasound damaged the nitrogen bases. The loss of the double helical structure by the ultrasound was mainly due to the separation of certain sections of the DNA by single strand breaks (SSB) and also to a small extent due to base damage. The size of the DNA fragments decreased as the ultrasound power increased and finally a plateau effect was seen at the highest powers. Key words: DNA damage, ultrasound, spectroscopy.

Study of DNA Interactions with Steroidal and Nonsteroidal Estrogen–Platinum (II)–Based Anticancer Drugs

The interactions of the steroidal and nonsteroidal estrogen-platinum (Pt) (II)-based anticancer drugs 16beta-hydroxymethyl-16alpha-[8-(2-pyridin-2-yl-ethylamino)-3,6-dioxaoctyl]-1,3,5(10)-estratrien-3,17betadiol dichloroplatinum (II) (JPM-39), 4-[6-(2'-pyridylethylamino)-butyloxy)-phenyl]-7-methoxy-2,2-dimethyl-3-phenyl-chroman dichloroplatinum (II) (ATG-99), and 1-[(2-aminoethyl)amino]-9,10,10-tris(4-hydroxyphenyl)-9-decene dichloroplatinum (II) (GEB-28) with calf-thymus DNA in vitro using constant DNA concentration and various drug levels were studied. Fourier transform infrared (FTIR) and circular dichroism (CD) were studied with calf-thymus DNA in vitro using constant DNA concentration and various drug levels. FTIR, UV-visible, and CD spectroscopic methods were used to characterize the drug binding mode, the binding constant, and structural variations of DNA in aqueous solution. Spectroscopic evidence showed that the various Pt-based drugs bind indirectly to the major and minor grooves of DNA duplex with some degree of drug-phosphate interaction. The overall binding constants for JPM-39, GEB-28, and ATG-99 are K(JPM-39) = 4.2 (+/-0.75) x 10(3) M(-1), K(GEB-28) = 3.4 (+/-0.65) x 10(3) M(-1), and K(ATG-99) = 2.1 (+/-0.45) x 10(3) M(-1). DNA aggregation occurs at high drug concentration, while DNA remains in the B-family structure.

Interaction of ethidium bromide and caffeine with DNA in aqueous solution

Two component (ethidium bromide–caffeine, ethidium bromide–DNA) and three component (ethidium bromide–caffeine–DNA) systems in aqueous saline (0.01 M NaCl) phosphate buffer solutions (pH 6.86, T = 298 K) are studied spectrophotometrically. The equilibrium constants for dimerization of caffeine, K D = 1.22 ± 2 M−1, and for heteroassociation of ethidium bromide with caffeine, K = 71 ± 8 M−1, in ethidium bromide–caffeine systems are determined. When the concentration of caffeine is increased, the dynamic equilibrium of the solution shifts toward formation of heterocomplexes which are, presumably, stabilized by dispersive and hydrophobic interactions of chromophores. The equilibrium parameters for ethidium bromide complex formation with DNA are calculated: the coupling constant for the dye with the biopolymer, K 1 = (232 ± 16)⋅103 M−1, and the number of base pairs of the biopolymer participating in bonding with the ligand, n 1 = 3.6 ± 0.2, are calculated. Given these values, it is suggested that under these experimental conditions there are two types of bonding between ethidium bromide and the nucleic acid — intercalation and “external” bonds. A McGhee–von Hippel model for a three component system and the numerical values of the parameters for molecular complex formation in two component systems are used to calculate the bonding constant for caffeine with DNA, K 2 = 127 ± 30 M−1, and the number of base pairs of DNA which bond with caffeine, n 2 = 1.7 ± 0.2. The concentrations of ethidium bromide and caffeine in the composition of two and three component complexes are calculated as functions of the nucleic acid content in the solution. An analysis of the concentration dependences shows that heteroassociation of ligands has a significant effect on the reduction in the concentration of ethidium bromide–DNA complexes in a three component system for low DNA concentrations, while at high DNA concentrations the bonding of caffeine with the biopolymer has this effect.

Structural ultrafast dynamics of macromolecules: diffraction of free DNA and effect of hydration

Of special interest in molecular biology is the study of structural and conformational changes which are free of the additional effects of the environment. In the present contribution, we report on the ultrafast unfolding dynamics of a large DNA macromolecular ensemble in vacuo for a number of temperature jumps, and make a comparison with the unfolding dynamics of the DNA in aqueous solution. A number of coarse-graining approaches, such as kinetic intermediate structure (KIS) model and ensemble-averaged radial distribution functions, are used to account for the transitional dynamics of the DNA without sacrificing the structural resolution. The studied ensembles of DNA macromolecules were generated using distributed molecular dynamics (MD) simulations, and the ensemble convergence was ensured by monitoring the ensemble-averaged radial distribution functions and KIS unfolding trajectories. Because the order-disorder transition in free DNA implies unzipping, coiling, and strand-separation processes which occur consecutively or competitively depending on the initial and final temperature of the ensemble, DNA order-disorder transition in vacuo cannot be described as a two-state (un)folding process.

Advantage of Being a Dimer for Serratia marcescens Endonuclease

The monomer and dimer of the bacterium Serratia marcescens endonuclease (SMnase) are each catalytically active, and the two subunits of the dimer function independently of each other. Nature, however, chooses the dimer form instead of the monomer. In order to explain this, we performed molecular dynamics (MD) simulations of both model-built complexes of a subunit of SMnase and the dimer with DNA in aqueous solution. We estimated the electrostatic binding energy, analyzed the distribution and dynamics of water around the complexes, identified water clusters in the protein, and related the dynamics of water to the protein's function. We find that the dimer form has an electrostatic advantage over the monomer to associate with DNA. Although Mg(2+) remains hexa-coordinated during the simulation, the binding pathway of DNA to Mg(2+) changes from inner-sphere binding in the monomer to outer-sphere in the dimer, which may be more energetically favorable. In addition, two water clusters in the active site of each monomer and in the dimer complex were identified and localized in two regions, named the "stabilizing" and "working" regions. Water in the "working" region in the dimer complex has larger fluctuations than that in the monomer.

A Fluorinated Ruthenium Porphyrin as a Potential Photodynamic Therapy Agent: Synthesis, Characterization, DNA Binding, and Melanoma Cell Studies

When the new porphyrin 5,10-(4-pyridyl)-15,20-(pentafluorophenyl)porphyrin is reacted with 2 equiv of Ru(bipy)(2)Cl(2) (where bipy = 2,2'-bipyridine) formation of the target ruthenated porphyrin is achieved with 40% yield. Strong electronic transitions are observed in the visible region of the spectrum associated with the porphyrin Soret and four Q-bands. A shoulder at slightly higher energy than the Soret band is attributed to the Ru(dpi) to bipy(pi*) metal to ligand charge transfer (MLCT) band. The bipyridyl pi to pi* transition occurs at 295 nm. Cyclic voltammetry experiments reveal two single-electron redox couples in the cathodic region at E(1/2) = -0.80 and -1.18 V vs Ag/AgCl associated with the porphyrin. Two overlapping redox couples at E(1/2) = 0.83 V vs Ag/AgCl due to the Ru(III/II) centers is also observed. DNA titrations using calf thymus (CT) DNA and the ruthenium porphyrin give a K(b) = 7.6 x 10(5) M(-1) indicating a strong interaction between complex and DNA. When aqueous solutions of supercoiled DNA and ruthenium porphyrin are irradiated with visible light (energy lower than 400 nm), complete nicking of the DNA is observed. Cell studies show that the ruthenated porphyrin is more toxic to melanoma skin cells than to normal fibroblast cells. When irradiated with a 60 W tungsten lamp, the ruthenium porphyrin preferentially leads to apoptosis of the melanoma cells over the normal skin cells.

AFM for analysis of structure and dynamics of DNA and protein–DNA complexes

This paper describes protocols for studies of structure and dynamics of DNA and protein–DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein–DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein–DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI–DNA complexes. When necessary, additional specifics are added to the corresponding example.

Similia similibus: Pairing of homologous chromosomes driven by the physicochemical properties of DNA

Genetic recombination in eukaryotes requires the pairing of homologous chromosomes to allow precise molecular exchanges between chromosome pairs at intertwined structures called Holliday junctions, the formation of which requires the action of the RecA protein. The mechanism behind the precise pairing of structures as long as chromosomes remains mysterious. In yeast, during the initial phases of meiosis, chromosomes are paired at approximately 65 kilobase intervals via paranemic interactions that do not involve strand breakage nor the intervention of analogs of the RecA protein. It has been proposed that these paranemic interactions could occur between G-rich chromosomal regions, but putting in register stretches of homologous sequences hundreds of kb long remains challenging. Recent developments on the theory of the physicochemical properties of DNA in aqueous solutions, in presence of di- or multivalent counterions, leads to the prediction that molecules with the same sequence tend to pair spontaneously by paranemic interactions depending on the electrostatic properties of DNA. Experimental support for this prediction has now been provided in vitro with naked DNA. This newly discovered property of DNA duplexes may thus provide a clue to solve the puzzle of the premeiotic pairing.

Surfactant–cobalt(III) complexes: Synthesis, critical micelle concentration (CMC) determination, DNA binding, antimicrobial and cytotoxicity studies

A new class of surfactant–cobalt(III) complexes, cis-[Co(bpy) 2(C 11H 23NH 2)Cl] 2+ ( 1) and cis-[Co(phen) 2(C 11H 23NH 2)Cl] 2+ ( 2) (bpy = 2,2′-bipyridyl, phen = 1,10-phenanthroline), have been synthesized and characterized. The critical micelle concentration (CMC) values of these complexes in aqueous solution were obtained from conductance measurements. The specific conductivity data (at 298, 308, 318 and 328 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization ( Δ G m 0 , Δ H m 0 and Δ S m 0 ) . The interaction between these complexes and calf thymus DNA in aqueous solution was investigated adopting electronic absorption spectroscopy, emission spectroscopy and viscosity measurements. Results suggest that the two complexes can bind to DNA via groove binding, van der Waals interactions and/or electrostatic interactions. The complexes showed moderate antibacterial and antifungal activities against certain selected microorganisms. The cytotoxic activity of the complexes on HBL-100 human breast cancer cells was determined adopting MTT assay and specific staining techniques, which revealed that the viability of the cells thus treated was significantly decreased and the cells succumbed to apoptosis as seen in the changes in the nuclear morphology and cytoplasmic features. Furthermore, the influence of complexes on normal cell lines from green monkey kidney was also determined and the results indicate that the effect is small on inhibition of viability.

DNA Adducts with Antioxidant Flavonoids: Morin, Apigenin, and Naringin

Flavonoids have recently attracted a great interest as potential therapeutic drugs against a wide range of free-radical-mediated diseases. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. While the antioxidant activity of these natural polyphenolic compounds is well known, their bindings to DNA are not fully investigated. This study was designed to examine the interactions of morin (Mor), naringin (Nar), and apigenin (Api) with calf thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) ratios of 1/40 to 1. FTIR and UV-Vis spectroscopic methods were used to determine the ligand binding modes, the binding constant, and the stability of DNA in flavonoid-DNA complexes in aqueous solution. Spectroscopic evidence shows both intercalation and external binding of flavonoids to DNA duplex with overall binding constants of K(morin) = 5.99 x 10(3) M(-1), K(apigenin) = 7.10 x 10(4) M(-1), and K(naringin) = 3.10 x 10(3) M(-1). The affinity of ligand-DNA binding is in the order of apigenin > morin > naringin. DNA aggregation and a partial B- to A-DNA transition occurs upon morin, apigenin, and naringin complexation.

Tripod-like Cationic Lipids as Novel Gene Carriers

An innovative family of tridentate-cationic "single-chained lipids" designed to enhance DNA compaction and to promote endosomal escape was synthesized by coupling various lipids to a multibranched scaffold. DNA retardation assays confirmed the ability of the most members of the library to complex DNA. Classical molecular dynamics simulations performed on the lauryl derivative, bound to a short strand of DNA in aqueous solution supported these observations. These showed that two "arms" of the tripodal molecule are ideally suited to forming strong Coulombic interactions with two contiguous phosphate groups from the DNA backbone while the lipophilic tail stays perpendicular to the DNA helix. Gene transfer abilities of the library were assessed in multiple cell lines (CHO, Cos7, and 16HBE14o-) with some library members giving excellent transfection abilities and low cytotoxicity, supporting the use of this tripodal approach for the development of efficient gene delivery agents.