DNA In Aqueous Solution Research Articles

Radiolysis of DNA in aqueous solution in the presence of a scavenger: A kinetic model based on a nonhomogeneous reaction of OH radicals with DNA molecules of spherical or cylindrical shape

Two expressions are given for the survival dose of DNA exposed to high-energy radiation in aqueous solution in the presence of a scavenger. They are derived from a model where a diffusion controlled reaction of OH radicals occurs on the surface of the DNA macromolecules in competition with scavenging in the bulk of the solution. The DNA molecules are approximated either by spheres or by cylinders. The model based on molecules of spherical shape corresponds closely to that developed by van Rijn et al. [20]. Expressions obtained from the cylindrical model are used to account for the dependence on the scavenger concentration of some experimentally measured quantities, namely the survival dose and the G value for single-strand breaks upon Co gamma-irradiation of phi X174 DNA and polyadenylic acid, respectively.

FT-IR and Raman investigation of cadmium binding by DNA

Complexes of cadmium(II) with DNA in aqueous solution under physiological conditions are investigated by means of vibrational spectroscopy. It is shown that at low concentrations, Cd 2+ binds primarily on PO 2 −, through a pure electrostatic interaction. This is followed by a cooperative coordination on both guanine and adenine 1 1 Abbreviations: Ade or A: adenine, Cyt or C: cytosine, Gua or G: guanine, Thy or T: thymine, R: ribose, NMP: nucleotide monophosphate. when large amounts of Cd(II) are present (3 eq. of Cd(II) for 1 eq. PO 2 −). Tests performed on DNA with Cd(II) concentrations met in real biological systems leave the DNA intact, indicating that the toxic effect of Cd(II) may not be directly related to its action on DNA. We demonstrate that FT-IR spectroscopy is useful in identifying the type of binding occurring on the PO 2 − sites of DNA, while Raman spectroscopy is remarkably well adapted for monitoring base complexation by Cd 2+.

Reactions of stereoisomeric non-bay-region benz[a]anthracene diol epoxides with DNA and conformations of non-covalent complexes and covalent adducts.

The reactions of the non-bay-region diol epoxides racemic trans-8,9-dihydroxy-anti-10,11-epoxy-8,9,10,11-tetrahydrobenz[a]an thracene (anti-BA-10,11-DE) and racemic trans-8,9-dihydroxy-syn-10,11-epoxy-8,9,10,11-tetrahydrobenz[a]ant hracene (syn-BA-10,11-DE) with native double-stranded DNA in aqueous solutions (5 mM sodium cacodylate buffer, pH 7.0, 23 degrees C) was investigated utilizing various spectroscopic techniques. The results of linear dichroism experiments suggest that both diastereomers form non-covalent, intercalative complexes with DNA prior to undergoing chemical reactions; the association constant for the anti stereoisomers is about twice as large (850 +/- 100 M-1) as that for the syn-diastereomers, thus qualitatively paralleling the behavior established previously for the bay-region diol epoxides of benzo[a]pyrene and benz[a]anthracene. The reaction rates of both anti- and syn-BA-10,11-DE are significantly accelerated in the presence of DNA, and the fraction of diol epoxide molecules which bind covalently to DNA is 13 +/- 2% and 3 +/- 1% respectively; these levels of covalent binding are lower by factors of about two respectively, than in the case of the bay-region diol epoxides of benz[a]anthracene. The phenanthrenyl residues in the covalent anti-BA-10,11-DE-DNA adducts are tilted with their long axes closer to the average orientations of the normals to the DNA bases; in contrast, the adducts derived from the binding of the syn diastereomers, appear to be characterized by intercalative-type conformations; however, the overall degrees of orientations are weak in the cases of these non-bay-region diol epoxide-DNA adducts. Nevertheless, these adduct conformations resemble those derived from the highly tumorigenic anti and the less active syn diasteromers of benzo[a]pyrene and benz[a]anthracene, thus providing one additional example to the previously observed correlations between adduct structure and biological activity.

DNA modifications as a means of detecting the irradiation of wheat

Irradiation produces a variety of modifications in DNA which, if detectable, would provide a means of determining whether food containing DNA had been irradiated. This paper presents initial attempts to detect thymidine glycol, a known DNA irradiation product. Two methods have been employed, a fluorescence assay and an enzyme immunoassay. The reliability of the fluorescence assay was first tested by using it to determine the radiolytic yields of thymidine glycol in the much simpler system of aqueous thymidine solutions saturated with either N 2O or N 2O/O 2(4:1). HPLC was employed to independently assess the radiolytic yields of thymidine glycol in these systems. Results from the fluorescence (values from HPLC experiments given in brackets) gave G-values for glycol of 0.11(0.07) μmol J -1 and 0.16(0.07) μmol J -1 in N 2O and N 2O/O 2 respectively. The fluorescence assay has also been applied to determine G(thymidine glycol) produced on irradiating aqueous solutions of calf thymus DNA, values of 0.03 μmol J -1 and 0.07 μmol J -1 being found in N 2O and N 2O/O 2 saturated solutions respectively. Techniques have been developed to enable DNA to be extracted from small samples of wheat. The preparation of antibodies specific for thymidine glycol-DNA required for the immunoassay is described. DNA from wheat has been tested both by the fluorimetric test and immunoassay. In both cases DNA from unirradiated wheat appeared to contain significant amounts of thymidine glycol. The fluorimetric assay showed increasing levels of glycol with increasing doses of radiation (0–1 kGy). No such correlation was apparent in the immunoassay, this is most probably due to the unsuitability of the initially selected antibody, which although recognising DNA treated with OsO 4 to produce intramolecular thymidine glycol, failed to interact with irradiated calf thymus DNA. Antibodies from other clones are now being screened in an endeavour to find one more suitable.

Dielectric behavior of aqueous solutions of plasmid DNA at microwave frequencies

The relative permittivity and dielectric loss of aqueous solutions of plasmid (pUC8.c1 and pUC8.c2) DNA have been measured at 20 degrees C over the frequency range 100 MHz-10 GHz. The solutions had a concentration of 0.1% DNA, and were studied both in the relaxed and the supercoiled form. The dielectric measurements were made using a variety of techniques including frequency domain and time domain methods of operation. No evidence of any resonance absorption, nor of any other kind of enhanced absorption, was observed.

Energetic electron tracks and DNA strand breaks

A theoretical model with no adjustable parameters is presented to evaluate the strand break yields for incident electrons between 100 eV and 1 MeV. Indirect mechanisms as well as direct mechanisms are included for the production of strand breaks. The model includes the following features: (i) multiple scattering of low energy electrons; (ii) decay of hydroxyl radicals in an aqueous solution containing Tris buffer; (iii) Monte Carlo simulation of the motion of hydroxyl radicals for interaction with the DNA sites; and (iv) stochastic aspects of the direct ionization on the DNA sites and the use of oscillator strength of a DNA molecule. The model is presented using numerical values characteristic of a dilute aqueous solution of SV40 DNA (10 μg/ml) containing 10 mM of Tris. The results have been expressed in terms of yields (indirect and direct) and D 37 (indirect only) values as a function of electron energy. The yields have been normalized to breaks/rad/dalton. In the absence of experimental data with different energy electrons, the results of the present calculations have been folded into the estimation of strand breaks induced by heavy charged particles. When these results are compared with experimental data for mammalian cells under conditions such that enzymatic strand break repair is negligible, there is good qualitative agreement with the model. With the expectation that experimenal data will soon be available with photons, the present model has been used to predict the strand break yields with electromagnetic radiation for thick as well as thin targets.

ChemInform Abstract: Effects of Ionizing Radiation on Deoxyribonucleic Acid. Part 5. The Role of Thiols in Chemical Repair.

AbstractExposure of frozen, deoxygenated, aqueous solutions of DNA to 60Co γ‐rays at 77 K results in the formation of guanine‐centered radical‐cations and thymine‐centered radical‐anions.

The vital staining of nuclear chromatin and the chromosomes of HeLa cells with the fluorochrome AMHA binding to DNA

The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF less than or equal to 10(-4) M and short incubation periods tI less than or equal to 2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die. In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm-1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm-1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission. Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nucleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1 = 1.1.10(5) M-1, binding parameter n1 = 0.15; the pre-intercalative or external binding 2, KF2 = 6.9.10(5) M-1, n2 = 0.21; the external binding 3, KF3 = 2.8.10(5) M-1, n3 = 0.55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1 = 1.1.10(5) M-1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1 = (1-4).10(4) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydroxyl Radical-induced Strand Break Formation in Single-stranded Polynucleotides and Single-stranded DNA in Aqueous Solution as Measured by Light Scattering and by Conductivity

Combining conductivity measurements and molecular weight determination by means of low-angle laser light scattering, we have found for the polyribonucleotides (polyuridylic acid (poly(U], polyadenylic acid (poly(A], polycytidylic acid (poly(C] and polyguanylic acid (poly(G] and for single-stranded DNA (ssDNA) that, on average, 8.5 counterions per single-strand break (ssb) are liberated under salt-free conditions. This relationship allows us to estimate, from conductivity measurements alone, G-values of single-strand break formation (G(ssb] for the polydeoxyribonucleotides (polydeoxyriboadenylic acid (poly(dA], polydeoxyribocytidylic acid (poly(dC], polydeoxyribothymidylic acid (poly(dT], polydeoxyribouridylic acid (poly(dU] and polydeoxyriboguanylic acid (poly(dG]. The following G(ssb) values (units of mumol J-1) have been obtained for anoxic conditions: poly(dA), 0.23; poly(dC), 0.14; poly(dT), 0.06; poly(dU), 0.046 and poly(dG), 0.009. Time-resolved conductivity measurements in pulse radiolysis enable us to measure the rate of strand break formation. The rate has been found to be similar for poly(dA) and ssDNA over a range of pH values. Poly(dC) and poly(dU) exhibit conductivity increase components with half-lives similar to those of poly(dA) and ssDNA at corresponding pH values. The implications of these results are discussed.

Inorganic phosphate release from gamma-irradiated DNA solutions

Dilute aqueous solutions of DNA were γ-irradiated. The effect of added chemical modifiers (viz. SH compounds and sensitizers) on inorganic phosphate (P i) release as the end product was studied in O 2 and N 2. The dependence of P i release on DNA concentration and dose was explained in terms of competition between two sites of damage on the DNA molecule. The observed enhanced protection by SH compounds in N 2 over O 2 was attributed to H donation. While radiobiological sensitizers alone did not sensitize either in O 2 or in N 2, they showed sensitization in presence of SH compounds. A possible mechanism for the same has been proposed.

Formation and reaction of peroxyl radicals of polynucleotides and DNA in aqueous solution

Radiation-induced strand breaks of polyuridylic acid [poly(U)] in aqueous solution in the presence of oxygen are known to proceed via peroxyl radicals of the uracil residues. The peroxyl radicals abstract H atoms from sugar moieties, and reaction of the sugar radicals leads to chain breaks. The lifetimes of the peroxyl radicals of poly(U), polyadenylic acid [poly(A)] and of single- and double-stranded DNA were determined by time-resolved e.s.r. spectroscopy and found to be in the range of seconds whereas the peroxyl radicals of low-molecular-weight model compounds have lifetimes of several milliseconds. In poly(U), the rate of decay of the peroxyl radicals was shown to coincide with the rate of strand-break formation, determined from pulse conductivity measurements. This leads to the conclusion that the reaction of peroxyl radicals with the sugar moieties is the rate-limiting step in strand-break formation. The peroxyl radicals of poly(U), poly(A) and DNA react with various thiols with rate constants of ⋍10 4–10 5 M −1 s −1. Strand-break formation in poly(U) was found to be prevented by thiols. The consequences of these results for the radioprotection of living cells are discussed.

Radiolysis of Spin-Labeled DNA: An Electron Spin Resonance Investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.

"Resonances" in the dielectric absorption of DNA?

An attempt was made to confirm previous reports of resonant-like dielectric absorption of plasmid DNA in aqueous solutions at 1-10 GHz. The dielectric properties of the sample were measured using an automatic network analyzer with two different techniques. One technique used an open-ended coaxial probe immersed in the sample; the other employed a coaxial transmission line. No resonances were observed that could be attributed to the sample; however, resonance-type artifacts were prominent in the probe measurements. The coaxial line technique appears to be less susceptible to such artifacts. We note two important sources of error in the calibration of the automatic network analyzer using the probe technique.

Evaluation of complex permittivity of aqueous solution by time domain reflectometry

Abstract A time domain reflectometry (TDR) method has been developed in order to measure dielectric relaxation process with a weak relaxation strength of the order of 0.1 in aqueous solution. Application of the TDR measurement have been made for poly(L-glutamic acid) in aqueous solution, which exhibits a helix-coil transition with changing the pH value. Dielectric relaxation process observed around 100MHz shows a definite transition in its strength in the vicinity of pH=6. The TDR method has been also applied to a DNA in aqueous solution with 0.1SSC buffer. A double helix structure of DNA melts at about 75°C to a coiled structure. Relaxation process around 100MHz shows a transition in the strength and also in the relaxation time around this temperature. In both cases, relaxation process caused by water molecules could be observed separately from the process observed around 100MHz. The relaxation strength and the relaxation time are nearly the same as those of the free water. A bilinear analysis developed by Cole has been used to measure methanol-water mixtures. A relaxation process could be observed continuously with the composition. It has been concluded that the bilinear analysis is quite powerful if the present TDR method is used together for the dielectric measurement covering a wide frequency region from 1MHz to 15GHz.

Microwave absorption in aqueous solutions of DNA.

In a recent Nature leading article the possibility of the occurrence of resonance absorption at microwave frequencies in DNA was discussed in terms of the latest available theoretical and experimental evidence, and its implications were assessed. Because of the far-reaching nature of these implications it is important that the existence or otherwise of such absorption be firmly established. Here we report on a concerted effort in two independent laboratories which has involved the measurement of the dielectric properties of aqueous solutions of circular DNA molecules in the frequency range 1-10 GHz. No resonance absorption or any form of enhanced absorption was demonstrated.

Laser-induced photoionization and single-strand break formation for polynucleotides and single-stranded DNA in aqueous solution: model studies for the direct effect of high energy radiation on DNA

Quantum yields for the formation of hydrated electrons (Φ e−) and single-strand breaks (Φ ssb) were measured for polyadenylic acid (poly A), polycytidylic acid (poly C), polyuridylic acid (poly U) and single-stranded (ss) DNA in deoxygenated aqueous solution at room temperature upon nanosecond laser excitation at 248 nm. The values Φ e − = (1.7 − 3) × 10 −2 and Φ ssb = (0.5 − 5) × 10 −4 at intensities of (0.5 − 1.5) × 10 7 W cm −2 were obtained. The results show that base radical cations of these polynucleotides and of ssDNA lead to ssb formation. The large difference between Φ e − and Φ ssb (a factor of 60 – 380) is explained as being the result of back reactions of geminate pairs of radicals and/or radical ions. Conductivity changes after the laser pulse, representing ssb formation, reveal a fast increase and a consecutive show increase for all nucleic acids studied. The activation energies and pre-exponential factors, calculated from the rate constant k obs of the slow conductivity increase at pH 7.3, are reported. Dithiothreitol (DTT) inhibits ssb formation of poly C (rate constant k DTT = 1.7 × 10 6 M −1 s −1) and poly U whereas for poly A and ssDNA a smaller effect was found ( k DTT < 0.7 × 10 6 M −1 s −1. The pH dependence of k obs is very similar for poly A and ssDNA, but different from that of poly C and poly U. This indicates that the slow strand break formation in ssDNA upon laser excitation is related to the purine moiety.

ChemInform Abstract: Application of ESR Spectroscopy to the Study of the Effects of Ionizing Radiation on DNA and DNA Complexes.

AbstractExposure of aqueous solutions of DNA to 60Co γ‐rays at 77 K gives radicals whose ESR spectra suggest the presence of guanine cations and thymine anions in about equal yield.

Induction of DNA crosslinks in vitro upon reduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131

The interaction of the nitroimidazole-aziridines RSU-1069 and RSU-1131, as parent or radiation-reduced species, with plasmid DNA in aqueous solution at pH7 results in strand breakage. The yields of DNA single strand breaks (ssb), “alkali-labile” damage and DNA crosslinks induced by these alkylating agents have been assessed. It is shown that DNA crosslinks are induced only by the reduced nitro-compounds. RSU-1069, as parent or reduced compound, is more efficient at producing these effects than the equivalent form of RSU-1131. Further, RSU-1069 is about 2× more susceptible to nucleophilic attack by inorganic phosphate and deoxynucleotides than RSU-1131. RSU-1069 also shows greater selectivity for reaction with the nucleotide base moiety than does the less-reactive monomethyl analogue, RSU-1131. The yields of ssb and “alkali-labile” damaged sites induced by the two agents reflect their respective chemical reactivities and appear largely to determine their aerobic cytotoxicities. In contrast, the yield of DNA crosslinks induced by the reduced compounds appears to correspond rather better with the observed hypoxic cytotoxicities. From these findings it is suggested that the induction of DNA crosslinks by these agents may play a major role in their effectiveness as hypoxia-selective cytotoxins.

Spin trapping of precursors of thymine damage in X-irradiated DNA.

A spin-trapping method combined with ESR spectroscopy was utilized to obtain evidence for the presence of precursor radicals leading to damage in X-irradiated DNA. Two technical improvements were introduced to the conventional spin-trapping method to make possible its application to large molecules such as DNA: prior to X irradiation, sonolysis of aqueous DNA solution by 19.5-kHz ultrasound was made to get a highly concentrated DNA solution and to lower the viscosity of the solution; after precursor radicals in X-irradiated DNA were trapped by a spin-trapping reagent, the DNA was digested to oligonucleotides by DNase I to get an ESR spectrum with a well-resolved hyperfine structure. Thus, it was recognized that the ESR spectrum obtained after X irradiation of the aqueous solution containing DNA and the nitroso spin-trapping reagent 2-methyl-2-nitrosopropane consisted of at least three sets of signals in the DNA. Identification of free radicals was made by comparing the spectrum with that of thymidine, which was precisely examined by a spin-trapping method combining two kinds of spin traps (nitroso and nitrone compounds) with liquid chromatography. As a result, all the signals were identified as the spin adducts of radicals produced at the thymine base moiety of DNA. The 5-hydroxy-5,6-dihydrothymin-6-yl radical was identified as a precursor of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), the 6-hydroxy-5,6-dihydrothymin-5-yl radical as a precursor of 6-hydroxy-5,6-dihydrothymine, and the 5-methyleneuracil radical as a precursor of 5-(hydroxymethyl) uracil.

Sodium ion and solvent nuclear relaxation results in aqueous solutions of DNA.

AbstractThe field‐dependent 23Na nuclear relaxation in aqueous DNA solutions has been obtained for a range of temperatures, including the DNA melting region. At least two correlation times are needed to characterize the spectral density function for the 23Na relaxation. For the slow process (with the largest correlation time), the temperature dependence of the coupling constant and the correlation time were determined, and important premelting effects were observed. Possible origins of the slow process are discussed. The last process is shown to be correlated with the properties of the hydration water of DNA as reflected by the 17O relaxation rates in these solutions. The influence of the polyelectrolyte and NaCl concentrations on the 23Na relaxation rate is compared with previous results from solutions of linear flexible polyelectrolytes.