By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first-order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the lessdense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This (10,11). The range of osmotic pressures accessible through this method is substantially larger (especially at high stress) than by the equilibrium sedimentation approach (7).Because DNA is equilibrated against a vast excess of a polymer and water solution of known chemical potential, it is always in a single phase at thermodynamic equilibrium. This behavior should be contrasted with multiple-phase equilibria that usually emerge from stoichiometric mixtures.In this work, we combine both the structural and thermodynamic approaches to the condensed DNA phases so that structural and dynamical parameters of DNA packing and ordering (interhelical separation, bond orientational order parameter, 31P-NMR spectra) are all measured concurrently with the free energy and/or its derivatives. We report here the structural and dynamic changes that occur in the DNA concentration region from 120 to 600 mg/ml corresponding to interaxial separations of 25-55 A. We show that at lower densities (or higher spacings) DNA packing is characterized by short-range positional order, measured by x-ray diffraction, long-range cholesteric order, revealed by optical birefringence, and high mobility of the DNA backbone, inferred from 31P-NMR spectroscopy. At high densities (or small spacings) DNA packing is characterized by short-range positional order and long-range bond orientational order in the plane perpendicular to the average nematic director, revealed by the tOn leave from: J. Stefan Institute, Ljubljana, Slovenia. IITo whom reprint requests should be addressed.

We have used Raman scattering to study the water O-H stretching modes at approximately 3450 and approximately 3220 cm-1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220-cm-1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen-bond network of water is disrupted in the primary hydration shell (which therefore cannot have an "ice-like" structure). The number of water molecules in the primary hydration shell was determined from the intensity of the approximately 3200-cm-1 band as about 30 water molecules per nucleotide pair. The approximately 3400-cm-1 O-H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5-6 tightly bound water molecules per nucleotide pair remain. The frequency of the approximately 3400-cm-1 O-H stretch mode is lower by 30 to 45 cm-1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the approximately 3200-cm-1 band and the shift of the approximately 3400-cm-1 O-H stretch band provide a reliable way of measuring the hydration number of DNA.

We report conclusive high resolution small angle x-ray scattering evidence that long DNA fragments form an untwisted line hexatic phase between the cholesteric and the crystalline phases. The line hexatic phase is a liquid-crystalline phase with long-range hexagonal bond-orientational order, long-range nematic order, but liquidlike, i.e., short-range, positional order. So far, it has not been seen in any other three dimensional system. By line-shape analysis of x-ray scattering data we found that positional order decreases when the line hexatic phase is compressed. We suggest that such anomalous behavior is a result of the chiral nature of DNA molecules.

Competitive binding of K+, Na+, and Li+ to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions in the range of ethanol concentration from 65 to 90% (by volume) and for salt concentrations, C s, from 3 to 300 mM. The affinity of DNA for the cations decreases in the order Na ≈ K > Li, and this is opposite to the sequence determined for DNA in aqueous solution. The ion exchange equilibrium constant, K c K Li, determined in the system DNA fibers−ethanol/water solutions of KCl and LiCl, varies between K c K Li ≈ 1.4 in 70% EtOH (K/Li = 1/1) and K c K Li ≈ 2.5−2.7 in 84−90% EtOH (K/Li = 1/1) or K c K Li ≈ 3.7−4.0 in 84% EtOH (K/Li = 1/9). Between 76 and 84% EtOH, the value of K c K Li increases steeply, which is due to the B−A transition of KDNA occurring in this concentration range of EtOH. Neither the A nor the B form of DNA exhibits selectivity for Na+ or K+ in mixtures of KCl and NaCl in ethanol/water solutions. Computer simulations based on the grand canonical Monte Carlo (GCMC) method were applied for modeling the experimental conditions. These calculations were performed within the approximations of describing the solvent as a dielectric continuum and the DNA polyion as a uniformly charged cylinder or a cylinder with arrays of spherical charges representing phosphate groups of the B or A form of DNA. It is found that the GCMC method explains qualitatively the ion selectivity of DNA in K/Li mixtures with respect to the dependence on the ethanol concentration, K/Li ratio, and A or B structural form of DNA.