The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

ABST4RACTThe structure of the tetramer d(CpGpCpG) has been solved by x-ray analysis in two different crystal forms with and without spermine cations. The molecules crystallize in hexagonal unit cells and they form a left-handed double helix of Z-DNA similar to that previously reported for the hexamer d(Cp~pCpGpCpG). In the crystal lattice the molecules stack together to form a virtually continuous left-handed double helix in which every fourth phosphate group is missing. The stacking of bases upon each other is similar to that seen in the hexamer. However, the base pairs have a slightly different orientation in that the cytosine residues are slightly removed from the axis of the molecule compared to the position they occupy in the hexamer. The structures are similar in two crystal forms with and without spermine cations. Most of our earlier knowledge of the organization of the DNA double helix came from fiber x-ray diffraction studies. These studies had the advantage that diffraction data are relatively easy to obtain but they had the substantial disadvantage that the data are limited in resolution and consequently the finer structural details are lost. It has been apparent for at least 7 years that fragments of nucleic acid double helices can be seen in a single crystal diffraction analysis which, in principle, can lead to atomic resolution data (1, 2). Our (Table 1). In addition, a fiber diffraction pattern of poly(dG-dC) has yielded a pattern consistent with a polymeric version of Z-DNA (7). In all of the crystals we find some slight modifications in the basic geometry of the left-handed helix. These variations can be seen because of the relatively high resolution of the crystal diffraction data. This emphasizes the fact that there is configurational variability in the left-handed double helix, a variability which no doubt will eventually be found when right-handed double-helical DNA is studied by these same methods. A major biological feature of double-helical DNA involves its interactions with small molecules and ions as well as with proteins, and all of these are likely to induce variations in conformation. Many of these configurational variations are sequence-dependent and therefore reflect the informational content of these long molecules. EXPERIMENTAL METHODSThe ammonium salt of the deoxy tetramer d(CpGpCpG), designated d(CG)2, was prepared by modification of the recently developed phosphotriester method (8,9). Two different crystal forms were obtained by using the vapor diffusion method with 5% isopropanol as the precipitating agent. Both crystal forms were obtained from solutions containing 30 mM sodium cacodylate buffer (pH 7.0), 15 mM MgCl2, and 2 mM d(CG)2. One solution also contained 10 mM spermine tetrachloride. The crystals grew in the form of hexagonal rods measuring up to 0.5 X 0.5 X 2.0 mm. They were mounted in sealed glass capillaries with a droplet of mother liquor, and three-dimensional data were collected to a resolution of 1.3 A by using a Picker diffractometer, although the data beyo...