A. Rupprecht

Specific formation of electron gain and loss centres in X-irradiated oriented fibres of DNA at low temperatures

Electron spin resonance (e.s.r.) spectra have been measured at X- and Q-band frequencies (ca. 9.5 and 34.1 GHz) from six different DNA specimens prepared as oriented A-DNA fibres after irradiation between 4.2 and 77 K. The DNA either varied in its guanine–cytosine base-pair content or contained 5-chloro- or 5-bromo-uracil instead of the natural base thymine. Amended by single-crystal studies at 4.2 K of the 5-halogenouracils and by spin-density calculations and associated spectral simulation, the DNA data have been analysed in terms of two primary species stabilized at low temperatures in thymine-containing DNA which are assigned to the cation of guanine and the anion of thymine. The latter is very probably replaced by the respective 5-halogenouracil anions in the substituted specimen. The average total radical yield is ca. 2.3 per 100 eV. The relative contribution of the guanine cation depends on the base composition.

Low-Frequency Raman Spectra of Lysozyme Crystals and Oriented DNA Films: Dynamics of Crystal Water

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.

DNA: a molecular wire?

Abstract Radiation-induced conductivity measurements on hydrated DNA provide evidence for highly mobile charge carriers within the B-DNA superstructure. The lack of anisotropy in the conductivity for aligned fibre samples and the second-order nature of the decay argue against one-dimensional conduction via a ‘π-way’ type mechanism involving electron transport confined to the base-pair core.

Competitive binding of Mg2+, Ca2+, Na+, and K+ ions to DNA in oriented DNA fibers: experimental and Monte Carlo simulation results.

Competitive binding of the most common cations of the cytoplasm (K(+), Na(+), Ca(2+), and Mg(2+)) with DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH) containing different combinations and concentrations of the counterions. The affinity of DNA for the cations decreases in the order Ca > Mg >> Na approximately K. The degree of Ca(2+) and/or Mg(2+) binding to DNA displays maximum changes just at physiological concentrations of salts (60-200 mM) and does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na(+) or K(+)). Ca(2+) is more tightly bound to DNA and is replaced by the monovalent cations to a lesser extent than is Mg(2+). Similarly, Ca(2+) is a better competitor for binding to DNA than Mg(2+): the ion exchange equilibrium constant for a 1:1 mixture of Ca(2+) and Mg(2+) ions, K(c)(Ca)(Mg), changes from K(c)(Ca)(Mg) approximately 2 in 65% EtOH (in 3-30 mM NaCl and/or KCl) to K(c)(Ca)(Mg) approximately 1.2-1.4 in 52% EtOH (in 300 mM NaCl and/or KCl). DNA does not exhibit selectivity for Na(+) or K(+) in ethanol/water solutions either in the absence or in the presence of Ca(2+) and/or Mg(2+). The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, uniformly and discretely charged polyions with the density and spatial distribution of the charged groups modeling B DNA. A quantitative agreement with experimental data on divalent-monovalent competition has been obtained for discretely charged models of the DNA polyion (for the uniformly charged cylinder model, coincidence with experiment is qualitative). The GCMC method gives also a qualitative description of experimental results for DNA binding competitions of counterions of the same charge (Ca(2+) with Mg(2+) or K(+) with Na(+)).

Aggregated DNA in ethanol solution

A recently developed mechanochemical method has provided a new, efficient tool for studies on the thermal stability and structure of aggregated DNA in ethanol‐water solutions. At low ethanol concentrations DNA is fully soluble and is in the B form. However, with increasing ethanol concentration the melting temperature of DNA, T m, decreases. At a critical ethanol concentration dependent on the nature and concentration of the counterion, aggregation of the DNA molecules sets in. This is reflected in a marked increase in T m indicating that the aggregated DNA molecules are thermally more stable than the dissolved ones. However, they are still in the B form. In general, T m of aggregated DNA also decreases with further increasing ethanol concentration and is dependent on the nature of the counterion, but T m is not affected by the concentration of the counterion (excess salt) in the ethanol‐water solution. When the ethanol concentration reaches the range of 70–80% (v/v), the B‐to‐A conformational transition occurs in the case of Na‐, K‐ and CsDNA. Above this transition point the A form is more stable than the B form due to the reduced water activity and to increased interhelical interactions. At very high ethanol concentrations, above 85% and dependent on the nature of the counterion, a drastic change in the thermal behaviour is observed. Apparently such a strong interhelical interaction is induced in the aggregated DNA that the DNA is stabilized and cannot adopt a random coil state even at very high temperatures. This stability of DNA in the P form is fully reversed if the ethanol concentration is lowered and the activity of water, thereby, is restored.

Radiation-induced free radicals in oriented bromouracil-substituted DNA.

Electron paramagnetic resonance has been used to study the free radicals induced by γ irradiation in 5-bromouracil-substituted DNA from Bacillus subtilis. The DNA samples were oriented by wet spinning and contained 30% humidity. As in normal DNA, irradiation at 77 K produced two major radical components. One is assigned to guanine cation radicals, similar to those in normal DNA but produced in concentrations 20-50% higher than in normal DNA. The other component is assigned to 5-bromouracil anion radicals, apparently replacing the thymine anion radicals in normal DNA. The G value for total radical formation is about 2.0, not significantly higher than the G value of 1.5 determined for normal DNA. Warming the sample above 77 K results in a radical transformation, which may be the 5-bromouracil anion radical transforming into a hydrated uracil-5-yl radical. Above 200 K all radicals decay. As in normal DNA no secondary radicals could be observed from the guanine cation radicals. Replacement of thymine by 5-bro...

Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.

Competitive substitution of hexammine cobalt(III) for Na+ and K+ ions in oriented DNA fibers

Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) >> Ca2+ > Mg2+ >> Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+.

A mechanochemical study of MgDNA fibers in ethanol-water solutions

Highly oriented calf-thymus MgDNA fibers, prepared by a wet spinning method, were studied with a simple mechanochemical set-up. The relative fiber length, L/Lo, was measured with the fibers submerged in ethanol-water solutions. In one type of experiment L/Lo was measured as a function of ethanol concentration at room temperature. No substantial decrease in L/Lo with increasing ethanol concentration was observed, indicating that MgDNA fibers stay in the B form even when the water activity is very low. For low ethanol concentrations the fiber structure is stable and does not dissolve even at very high water activities. In a second type of experiment, the heat-induced helix-coil transition was manifested by a marked contraction of the fibers. The transition temperature decreases linearly with increasing ethanol concentration between 52 and 68% ethanol. At higher ethanol concentrations the helix-coil transition temperature increases due to strong aggregation within the DNA fibers, and above 77% ethanol the fibers do not contract at all, not even at the upper temperature limit of the experiments, approximately 80 degrees C. This behavior is discussed with reference to dried DNA and the P form of DNA. The helix-coil transition temperature of the MgDNA fibers in 70% ethanol does not show any dependence on the MgCl2 concentration. It is shown that the Poisson-Boltzmann cylindrical cell model can account qualitatively for this lack of salt dependence.

Relationships in Raman intensity theories, in particular the Mayants-Averbukh theory

Abstract Brief surveys are given of the Mayants-Averbukh Raman intensity theory, and of the polar tensor Raman intensity theory recently presented by Bogaard and Haines. It was found that these intensity theories in essence are equivalent. In addition, the appearances of the symmetry invariant parameter matrices F n 0 of the Mayants-Averbukh theory were derived and tabulated for various symmetries of bond n . These matrices, and a single bond coordinate system, can be used as a convenient alternative to the Mayants-Averbukh treatment of bonds which have some kind of symmetry with respect to the midpoint of the bond. A modification of the Mayants-Averbukh treatment is also suggested. The rotational mode equations of the Mayants-Averbukh theory have been investigated to elecudate the constraints which they impose on Raman intensity theories based on the bond polarizability model. It was found that the valenceoptical theory is in conformity with the rotational modes only if all electrooptical parameters ∂α ii (n) ∂γ p are neglected, where α ii ( n ) ( i = 1, 2, 3) are the diagonal components of the polarizability α ( n ) of bond n , and γ p is the p th internal angular coordinate. Furthermore, the valence-optical theory was found to be strictly applicable only for cylindrical bond symmetry, C mv ( m ≥ 4). A generalized valence-optical Raman intensity theory, allowing also for non-zero off-diagonal components α ij ( n ) , was found to be incompatible with the rotational mode equations of the Mayants-Averbukh theory. However, its basic polarizability equation was useful for suggesting a unique interpretation of a set of f parameters (elements of F n 0 ) in terms of components of the anisotropic part of a symmetric bond polarizability.