Calf Thymus DNA In Solution Research Articles

Multi-spectroscopic, calorimetric and molecular dynamics evaluation on non-classical intercalation of antiviral drug Molnupiravir with DNA

The interaction of an antiviral drug Molnupiravir (MOL) with calf thymus DNA (CT-DNA) was investigated using a series of biophysical techniques. A significant hyperchromism with a blue shift nm in the UV-Vis spectra indicated a high binding affinity of MOL for CT-DNA with binding constants in the order of 105 M−1. Competitive fluorescent dye displacement assays with ethidium bromide (EB) and Hoechst 33258 suggested an intercalative mode of binding of MOL with CT-DNA. Thermodynamic profiles determined using fluorescence titration and isothermal titration calorimetric (ITC) analysis matched well with each other. The negative free energy change revealed that the MOL/CT-DNA complexation is a spontaneous process. The negative values of enthalpy and entropy changes indicated that H-bonding and van der Walls interactions play dominant roles in stabilizing the complex. A decrease in viscosity of CT-DNA solution upon adding MOL indicated a partial intercalation mode of binding which was well supported by circular dichroism (CD) spectral and effect of KI and denaturation studies. Molecular docking and metadynamics simulation studies clearly showed the partial intercalation of the pyrimidine ring of MOL into the base pairs of DNA. Free energy surface (FES) contour indicated that the drug/DNA complex is stabilized by H-bonding and pi-pi/pi-cation interactions. Communicated by Ramaswamy H. Sarma

Elucidating the interaction of antidepressant drug paroxetine with ct-dsDNA: A comparative study by electrochemical, spectroscopic, and molecular docking approaches

This study is designed to investigate the interaction of phenylpiperidine derivative drug paroxetine, which is an effective serotonin reuptake inhibitor and biomolecules through electrochemical, fluorescence spectroscopy, and molecular docking methods. The interaction between paroxetine and biomolecules was investigated by differential pulse voltammetry according to the decrease in deoxyguanosine anodic oxidation signal of double-stranded calf thymus DNA. Fluorescence spectroscopy studies were performed by titrating paroxetine against double-stranded calf thymus DNA solution at four different temperatures. The fluorescent results showed that paroxetine had a great affinity to bind with double-stranded calf thymus DNA. Interaction studies demonstrate that paroxetine binds to double-stranded calf thymus DNA via intercalation binding mode, and the binding constant values ​​were calculated as 7.24 × 104 M−1 and 1.52 × 104 M−1 at 25 °C, based on voltammetric and spectroscopic results, respectively. Moreover, with the aim of elucidating the interaction mechanism between paroxetine and double-stranded calf thymus DNA, electrochemical and fluorescence spectroscopy studies along with molecular docking analysis were made.

Designing a two-stage colorimetric sensing strategy based on citrate reduced gold nanoparticles: Sequential detection of Sanguinarine (anticancer drug) and visual sensing of DNA.

Distance dependent optical properties of colloidal gold nanoparticles offer designing of colorimetric sensing modalities for detection of a variety of analytes. Herein, we report a simple and facile colorimetric detection assay for an anti-cancer drug, Sanguinarine (SNG) and Calf Thymus DNA (Ct-DNA) based on citrate reduced gold nanoparticles (CI-Au NPs). The electrostatic interaction between SNG and CI-Au NPs induce aggregation of Au NPs accompanied with visible colour change of colloidal solution. The assay conditions like salt concentration, pH and reaction time had been adjusted to achieve highly sensitive and fast colorimetric response. Furthermore, the optimized CI-Au NPs/SNG sensing system is used for the detection of Ct-DNA based on the mechanism of anti-aggregation of CI-Au NPs. The simultaneous presence of SNG and Ct-DNA prevent aggregation of Au NPs owing to preferential formation of Ct-DNA-SNG intercalation complex and colour of the Au NPs solution tends to remain red, depending on the concentration of Ct-DNA in solution. The degree of aggregation and anti-aggregation of CI-Au NPs was monitored using Transmission electron microscopic (TEM) measurements and UV-Visible spectrophotometry by analysing the ratio of absorptions for aggregated and dispersed Au NPs. The intercalation mode of binding between SNG and Ct-DNA in CI-Au NPs/SNG sensing system was determined by Fluorescence spectral studies and UV-thermal melting studies. The absorption ratio (A627/A525) of Au NPs exhibited a linear correlation with SNG concentrations in the range from 0 to 0.9μM with detection limit as 0.046μM. This optical method can determine Ct-DNA as low as 0.36μM and the calibration is linear for concentration range 0 to 5μM. The proposed sensing strategy enables detection as well as quantification of SNG & Ct-DNA in real samples with satisfactory results and finds application in drug or DNA monitoring.

Radiation Effects On Mammalian DNA - PAC Method And Theory

The flexibility of DNA and its molecular dynamics have been the subject of various studies. One of the techniques that have been employed is the PAC method, which is considered as a valuable tool for such studies. In PAC experiments we can detect the angular correlation of two γ rays emitted in succession by a radioactive probe. Using the hyperfine interaction of the probe with it's surrounding electric field gradient we can obtain information about the dynamics of the molecules to which the probe is attached. In this work, for the PAC measurements, we use m I n which is added in a buffered solution of calf thymus DNA, exposed to various doses (0- 80 Gy) of 7-rays.

DNA Complexes with Cobalt(II) Phthalocyanine Disodium Disulfonate.

The interaction of cobalt phthalocyanine disodium disulfonate (CoPc) with calf thymus DNA in solutions was investigated by UV/vis spectrophotometry, circular dichroism (CD), and hydrodynamic methods (viscosity and flow birefringence). Two types of CoPc binding to DNA were observed. Fast CoPc interactions with DNA via external binding to phosphates were accompanied by the formation of stack-type phthalocyanine structures on the periphery of the DNA helix. The optical absorption spectra of such CoPc complexes with DNA were analyzed in order to obtain a binding constant K = (4.8 ± 0.4) × 104 M–1. CD spectra show the increasing optical activity of phthalocyanines bonded to DNA. DNA plays the role of a matrix, contributing to an increase in their stacking interactions. The CD spectrum of DNA varies slightly. The second type of cobalt-to-DNA binding manifests itself over a certain time. It can be associated with the reorganization of ligands in the cobalt coordination sphere by introducing DNA atoms. In our experiments, such binding was observed after storage of solutions for approximately 20 h at a temperature of 4 °C. It was shown that the minor groove of DNA remains free in CoPc–DNA complexes. CoPc does not bind with the most important group for metal coordinating to DNA in the major groove (N7 guanine). We completely excluded the intercalation binding model. The planes of phthalocyanines in CoPc–DNA complexes are oriented predominantly normal to the axis of the DNA helix. DNA rigidity (persistent length) does not change. This follows from the data on the measurement of the optical anisotropy and intrinsic viscosity of DNA in complexes.

Supramolecular Organization in Calf-Thymus DNA Solutions under Flow in Dependence with DNA Concentration

DNA dynamics and flow properties are of great importance for understanding its functions. DNA is a semiflexible polymer chain characterized by having a large persistence length of around 50 nm and high charge density; DNA chains are interacting efficiently at high concentrations, in dependence of the ionic concentration. In relation with DNA molecular characteristics, it is also known that DNA solutions are able to form liquid crystalline phases over a critical polymer concentration. In this work, the supramolecular organization in calf-thymus DNA solution, with low degree of entanglement, appearing under flow was studied in a wide DNA concentration range from 2 to 10 mg/mL, at a pH of 7.3 and 20 °C. The rheological behavior of the system was studied using steady state flow and oscillatory measurements. Transient regimes were also tested by imposing controlled shear rates on a short time up to steady state. Furthermore, a combination of visual observations and flow birefringence measurements was proposed to reach a better understanding of the obtained rheological behavior. The presence of a shear-induced texture is revealed under flow for the calf-thymus DNA solutions at CDNA> 5 mg/mL and attributed to organized domains of DNA molecules, named in the text as crystalline parts, which are progressively oriented under shear. Finally, at high shear rates (over 100 s–1), it is shown that for the DNA solutions the orientation of these organized DNA domains and connecting chains under flow goes to an anisotropic monodomain.

Formation of pyrimidine-pyrimidine type DNA intrastrand cross-links: a theoretical verification.

Pyrimidine-type radicals have been demonstrated to be able to attack their 3' or 5' neighboring purine nucleotides forming diverse DNA intrastrand cross-links, but whether or not these radicals can attack their surrounding pyrimidine nucleotides forming pyrimidine-pyrimidine type DNA intrastrand cross-links remains unclear. To resolve this question, probable additions of the uracil-5-methyl (˙UCH2) radical to the C5[double bond, length as m-dash]C6 double bond of its 3'/5' neighboring pyrimidine nucleotides in the four models, 5'-T(˙UCH2)-3', 5'-C(˙UCH2)-3', 5'-(˙UCH2)T-3', and 5'-(˙UCH2)C-3', are explored in the present work employing density functional theory (DFT) methods. The C6 site of its 5' neighboring thymidine is the preferred target for ˙UCH2 radical addition, while additions of the ˙UCH2 radical to the C6 and C5 sites of its 5' neighboring deoxycytidine are found to be competitive reactions. The ˙UCH2 radical can react with both the C6 and C5 sites of its 3' neighboring pyrimidine nucleotides, but the efficiencies of these reactions are predicted to be much lower than those of the corresponding addition reactions to its 5' neighboring pyrimidine nucleotides, indicating the existence of an obvious sequence effect. All the addition products could be finally transformed into closed-shell intrastrand cross-links, the molecular masses of which are found to be exactly the same as certain MS values determined in a recent study of an X-irradiated deoxygenated aqueous solution of calf thymus DNA. The present study thus not only definitely corroborates the fact that the reactive ˙UCH2 radical can attack its 3'/5' neighboring pyrimidine nucleotides forming several pyrimidine-pyrimidine type DNA intrastrand cross-links, but also provides a plausible explanation for the identities of these structurally unknown intrastrand cross-links.

Molecular hydrogen attenuates radiation-induced nucleobase damage to DNA in aerated aqueous solutions

Purpose: The main aim of the present study is to gain mechanistic insights into the modulating effect of molecular hydrogen on the γ-radiation-induced alteration pathways of DNA nucleobases.Materials and methods: Aerated aqueous solutions of calf thymus DNA were exposed to a 60Co source at doses ranging from 0 to 55 Gy under normoxic conditions, in the presence or not of 0.7 MPa hydrogen or helium. The measurement of several modified bases was performed using HPLC associated with electrospray ionization tandem pass spectrometry (HPLC-ESI-MS/MS). Bleaching of aqueous solutions of p-nitrosodimethylaniline (p-NDA) solutions was also used to allow the quantification of hydroxyl radical (•OH) formation.Results: pNDA bleaching was significantly reduced in the presence of hyperbaric hydrogen. This is undoubtedly due to •OH scavenging by H2 since, under the same conditions, He had no effect. Similarly, base alterations were significantly reduced in the presence of hydrogen, as compared to controls under normal atmosphere or in the presence of helium. The relative proportions of modified nucleobases were not changed, showing that the only effect of H2 is to scavenge •OH without exhibiting reducing properties.Conclusions: Our findings demonstrate that H2 exerts a significant protection against radiation-induced DNA base damage in aqueous solutions, •OH scavenging being the only mechanism involved.

Conformation and Rheological Properties of Calf-Thymus DNA in Solution.

Studies of DNA molecule behavior in aqueous solutions performed through different approaches allow assessment of the solute-solvent interactions and examination of the strong influence of conformation on its physicochemical properties, in the presence of different ionic species and ionic concentrations. Firstly, the conformational behavior of calf-thymus DNA molecules in TE buffer solution is presented as a function of temperature. Secondly, their rheological behavior is discussed, as well as the evidence of the critical concentrations, i.e., the overlap and the entanglement concentrations (C* and Ce, respectively) from steady state flow and oscillatory dynamic shear experiments. The determination of the viscosity in the Newtonian plateau obtained from flow curves η () allows estimation of the intrinsic viscosity and the specific viscosities at zero shear when C[η] < 40. At end, a generalized master curve is obtained from the variation of the specific viscosity as a function of the overlap parameter C[η]. The variation of the exponent s obtained from the power law η~−s for both flow and dynamic results is discussed in terms of Graessley’s analysis. In the semi-dilute regime with entanglements, a dynamic master curve is obtained as a function of DNA concentration (CDNA > 2.0 mg/mL) and temperature (10 °C < T < 40 °C).

Study on the Interaction between Isatin-β-Thiosemicarbazone and Calf Thymus DNA by Spectroscopic Techniques.

The interaction between isatin-β-thiosemicarbazone (IBT) and calf thymus DNA (CT-DNA) was investigated in physiological buffer (pH 7.4) using Neutral Red (NR) dye as a spectral probe by UV-Vis absorption and fluorescence spectroscopy, as well as viscosity measurements. The IBT is stabilized by intercalation in the DNA (K [IBT -DNA] = 1.03×10(5) M(-1)), and displaces the NR dye from the NR-DNA complex. The binding constants Kf and number of binding sites (n≈1) of IBT with DNA were obtained by fluorescence quenching method at different temperatures. Furthermore, the enthalpy and entropy of the reaction between IBT and CT-DNA showed that the reaction is enthalpy-favored and entropy-disfavored. The changes in the base stacking of CT-DNA upon the binding of IBT are reflected in the circular dichroic (CD) spectral studies. The viscosity increase of CT-DNA solution is another evidence to indicate that, IBT is able to be intercalated in the DNA base pairs.

Photodynamic inactivation mechanism of Streptococcus mitis sensitized by zinc(II) 2,9,16,23-tetrakis[2-(N,N,N-trimethylamino)ethoxy]phthalocyanine

Photoinactivation of Streptococcus mitis induced by zinc(II) 2,9,16,23-tetrakis[2-(N,N,N-trimethylamino)ethoxy]phthalocyanine (ZnEPc4+) was studied under different experimental condition in order to obtain information about the photodynamic processes and the cellular damage. A 3 log decrease in S. mitis survival was found in cell suspensions (∼2×108 cells/mL) incubated with 2μM ZnEPc4+ and irradiated for 30min with visible light (54J/cm2). Also, S. mitis cells growth was not detected in broth treated with 5μM ZnEPc4+ under continuous irradiation. Studies of photodynamic action mechanism showed that the cells were protected in the presence of azide ion, while the addition of mannitol did not produce a significant effect on the survival. Moreover, the photocytotoxicity was increased in D2O indicating the interference of singlet molecular oxygen. On the other hand, it was found that ZnEPc4+ interacts strongly with calf thymus DNA in solution but photocleavage of DNA was only detected after long irradiation periods. After S. mitis photoinactivation, modifications of genomic DNA were not observed by electrophoresis. In contrast, the transmission electron microscopy showed structural changes in the S. mitis cells, exhibiting mesosome-like structures. After 2h irradiation, the cytoplasm showed segregation patterns and PDI appeared to have effects on the cell wall, including variability in wall thickness. Also, the presence of bubbles was detected on the cell surface by scanning electron microscopy. However, the photodamage to the cell envelope was insufficient to cause the release of intracellular biopolymers. Therefore, modifications in the cytoplasmic biomolecules and alteration in the cell barriers could be mainly involved in S. mitis photoinactivation. It can be concluded that photosensitization by ZnEPc4+ mainly involved a type II photoprocess, while alteration in the cytoplasmatic components and modifications in the cell envelope were the major cause for the photoinactivation of S. mitis.

Viscosity of aqueous DNA solutions determined using dynamic light scattering

Viscosity is a key parameter for characterising the behaviour of liquids and their flow. It is, however, difficult to measure precisely, reproducibly and accurately for aqueous solutions on a micro-litre volume scale, which is what is usually needed for biological samples. We report the development of a new method for measuring dynamic viscosity by measuring dynamic light scattering (DLS) data for a range of particles of well-defined size. Most applications of DLS involve determining particle size for samples of known viscosity. We inverted the usual protocol and endeavoured to determine viscosity for samples of known particle size. Viscosity measurements for water and aqueous solutions of calf thymus DNA made using DLS were compared with those from a U-tube viscometer. The styrene particles, frequently used as particle size standards, gave unsatisfactory results for our DNA samples as did C-6 derivatized silica and positively charged amino polystyrene microspheres. However, negatively charged carboxylate polystyrene microspheres particles readily gave accurate viscosity measurements over a range of temperatures (0-100 °C). The sample volume required depends on the cuvette used to measure DLS, but can be performed with samples sizes ranging from 40 to 3000 μL. The sample can then be recovered for subsequent experiments. The DLS method is simple to perform at different temperatures and provides data of accuracy significantly above that of a U-tube viscometer. Our results also indicate a way forward to account accurately for solution viscosity in the normal applications of DLS to particle size determination by including the appropriate non-interacting particles as an internal standard.

Kinetics of Porphyrin Adsorption and DNA-Assisted Desorption at the Silica−Water Interface

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to study in situ the kinetics of the adsorption of 5,10,15,20-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) from pH 7.4 phosphate buffer solution (PBS) to the silica-water interface and the interaction of calf thymus DNA (CT-DNA) with the resulting TMPyP-functionalized surface. TMPyP was delivered to the silica surface using an impinging jet technique to allow relatively fast surface kinetics to be accessed. Adsorption was first-order in TMPyP, and the initial adsorption rate constant at the bare surface was found to be k = (4.1 +/- 0.6) x 10(-2) cm s(-1). A deceleration in the adsorption kinetics was observed at longer times that could be described semiquantitatively using an Elovich-type kinetic expression. The limiting value of the absorbance corresponded approximately to monolayer coverage (6.2 x 10(13) molecules cm(-2)). Exposure of the TMPyP-modified silica surface to CT-DNA, achieved by flowing CT-DNA solution over the functionalized surface, resulted in efficient desorption of the TMPyP. The desorption process was driven by the interaction of TMPyP with CT-DNA, which UV-vis spectroscopy indicated involved intercalative binding. The desorption kinetics were also simulated using complementary finite element modeling of convection-diffusion coupled to a surface process.

Synthesis of Diquinolineno[1,3,7,9]Tetraazacyclododecine-7, 15 (14H, 16H)-Dibenzene, and DNA Binding Studies of Macrocyclic Co(II), Cu(II) Complexes: As New Class of Antimicrobial Agent

The synthesis of macrocyclic ligand, diquinolineno[1,3,7,9]tetraazacyclododecine-7, 15 (14H, 16H)-dibenzene(L), is described. The metal complexes of the type [MLX2], where (M = Co(II) (1), Cu(II) (2) and X = (Cl), have been synthesized by the reaction of ligand(L) with the corresponding metal salts, and characterized by elemental analysis, FT-IR, 1H-NMR and electronic spectra. The binding property of the complexes with CT-DNA was studied by absorption spectra, viscosity measurements, as well as thermal denaturation studies. The absorption spectral results indicate that the complexes (1) and (2) are binds with base pairs of DNA. The intrinsic binding constant Kb had the value 3.8 × 104M −1 for (1) and 3.3 × 104 M−1 for (2), respectively, in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2. The viscosity measurement results show the viscosity of sonicated rod-like DNA fragments increased when the complex were added to the solution of calf thymus-DNA. The synthesized ligand and its metal complexes have been screened for antibacterial and antifungal activities.

Sensitive determination of a glyoxal–DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.

Targeting the inverted CCAAT Box-2 of the topoisomerase IIα gene: DNA sequence selective recognition by a polyamide–intercalator as a staggered dimer

The synthesis and DNA binding characteristics of a polyamide–intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIα promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5′-AAGCTA-3′). Formation of ligand-induced CD bands at ∼330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6 × 10 6 M −1), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 μM. DNaseI footprinting, using the topoIIα promoter sequence, highlighted the specificity of the conjugate for the cognate site (5′-AAGCTA-3′). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIα. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIα by blocking the binding of NF-Y.

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties

Novel –COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding –COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time ( t 1/2) of the coated beads increases to 537 s—seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 μg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

Oxidative DNA cleavage mediated by a new copper (II) terpyridine complex: Crystal structure and DNA binding studies

The copper (II) complex [Cu(Itpy) 2](ClO 4) 2 ( 1), (Itpy = imidazole terpyridine) has been synthesized and structurally characterized. Crystal structure of the complex shows the complex to be a monomeric copper (II) species with two Itpy ligands coordinated to the metal ion to give a six coordinate complex. The complex has a distorted octahedral geometry with axial elongation. Variable temperature crystal structure data shows dynamic nature of the Jahn-Teller distortion. The complex is an avid DNA binder with a binding constant of 4.26 ± 0.20 × 10 3 M −1. Observed changes in the viscosity and circular dichroic spectrum of calf thymus DNA solution in the presence of complex 1 suggests intercalative binding of complex 1 to DNA. The complex cleaves supercoiled pBR322 DNA oxidatively in the presence of hydrogen peroxide.

A new chemically amplified electrochemical system for DNA detection in solution

A newly developed chemically amplified electrochemical detection system was applied to the quantitation of DNA in solution. The system employed Ru(bpy) 2dppz (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2- a :2′,3′- c]phenazine), a high-affinity DNA intercalator, as the electrochemical indicator, oxalate as the sacrificial electron donor to chemically amplify the electrochemical signal, and tin-doped indium oxide as the working electrode to suppress background current. Intercalation of Ru(bpy) 2dppz into calf thymus DNA in solution led to a reduction in the oxalate-amplified electrochemical current as compared to a DNA-free solution. The degree of reduction was a function of the concentration of DNA, thus forming the basis for DNA detection. To illustrate the advantages of the new system, a direct comparison was made between amplified (with oxalate) and non-amplified (without oxalate) DNA detection. In the presence of 100 mM oxalate, anodic current of Ru(bpy) 2dppz was amplified by more than 60 folds, resulting in substantial improvement in signal-to-background ratio. Furthermore, as the DNA concentration was increased, the amplified current decayed much faster than the non-amplified signal, giving rise to higher detection sensitivity. The steeper decay was attributed to slower redox reaction between DNA-intercalated Ru(bpy) 2dppz and oxalate, as the negatively charged phosphate groups on DNA repelled the anions. Effect of ionic strength was investigated to provide support for the interpretation. As expected, the decay of the amplified response with increasing concentration of DNA became less steep when more NaCl was added into the solution. The opposite effect was observed when tri-propylamine, a cationic electron donor, was used instead of oxalate. With an optimized concentration of 30 mM oxalate and 5 μM Ru(bpy) 2dppz, calf thymus DNA of as low as 1 pM was detected in solution, which was close to the detection limit of some fluorescence measurements.

Changes in DNA flexibility after irradiation with gamma rays and neutrons studied with the perturbed angular correlation method.

Neutron and gamma irradiation of buffered solutions of calf thymus DNA resulted in changes in the dynamics of the macromolecule. In the low-dose region (0.8-10 cGy of 239Pu-Be neutrons and 0.34-3 Gy of 60Co gamma rays), the flexibility of DNA decreased as indicated by slower rotation of the molecules. Neutrons appeared to be approximately 35 times more effective than 60Co gamma rays. The rotational correlation time, tau C, was measured using the perturbed angular correlation (PAC) method. Its variation appears to follow a linear-exponential behavior. An attempt is made to formulate this behavior as a function of the energy deposited on the macromolecule (radiation dose), the average threshold energy (dose) required to form new lesions, and the available population of intact DNA sites.