DNA Structures DOI: 10.1002/ange.201003647 A Geometric Approach to the Crystallographic Solution of Nonconventional DNA Structures: Helical Superstructures of d(CGATAT)** Iæaki Martínez de Ilarduya, Daniela De Luchi, Juan A. Subirana, J. Lourdes Campos,* and Isabel Usón* Dedicated to Professor Herbert W. Roesky on the occasion of his 75th birthday Oligonucleotides have been used extensively to build nano- structures [1] and nanodevices. [2] Mostly, rather long oligonu- cleotides (10–100 bases) are used to form either flat tiles or closed objects based on standard Watson–Crick base pairs. [3] Only recently, self-assembled three-dimensional DNA latti- ces have been described. [4] Such structures feature large cavities, allowing incorporation of globular shaped molecules. Also, short oligonucleotides (2–12 bases) may assemble into intricate lattices, such as cubes [5] and other complex struc- tures, [6] containing large voids. X-ray crystallography provides the indispensable three-dimensional view into the atomic structure of such nanomaterials, but their crystals usually diffract far from atomic resolution, and thus their structures cannot be solved by direct methods. [7] Herein, we present a new geometrical approach to solve nonconventional DNA structures and its application to the solution of the superstructure of new topology formed by d(CGATAT). Such unprecedented structures could result in materials with new properties, and their characterization should not be hindered for lack of a suitable phasing method. As it is well known from the pioneering work on the structure of the DNA double helix using fiber diffraction, [8] in contrast to protein crystallography, meaningful information can be derived already from the diffraction pattern. Previous knowledge leads to the expectation that DNA forms base- paired, double-stranded helices in A, B, or Z geometry. Such helical moieties tend to stack on piles or to lean their ends on the grooves of other helices. Other motifs may play a role: quadruplexes, [9] three-way and Holliday-junctions, [10] or loop- ing out unpaired bases [11] have been described in the structures of oligonucleotides and their complexes. Major base-stacking directions can be identified from the diffraction images by the strong Bragg reflections at 3.3  spacing and fiber streaks. Thus, analysis of the diffraction data fixes the preferred orientation of piled base pairs (Figure S1a in the Supporting Information). The unit-cell geometry and the symmetry, along with the estimation of the solvent content from the atomic volumes, allow one to predict whether they fit a simple packing of regular helices or a distortion is required to build a three-dimensional structure. Figure S1b illustrates the relationship between the dimensions of a hexagonal projection and the requirements on the helical radii. Thus, examination of the geometrical parameters can be exploited to set up structural hypotheses as to the building blocks present and their packing, to be confirmed or discarded through molecular replacement or refinement of the models. To identify such models, we automated the analysis of the packing of all DNA structures deposited with the Protein or Nucleic Acid Databases (PDB/NDB). Our program SUBIX (Figure 1) allows one to establish the geometrical require- ments of different projections and to classify DNA materials according to their building blocks, thus identifying or assem- bling the best candidates to be used alone or in combination Figure 1. Flow diagram illustrating the elements of the geometric approach to structure solution. A SUBIX test version is available from the authors upon request. The program will be distributed freely to academic users once testing and debugging is complete. [*] I. Martínez de Ilarduya, [+] Prof. Dr. I. Usón Institucio Catalana de Recerca i Estudis Avançats (ICREA) at Instituto de Biología Molecular de Barcelona IBMB-CSIC, Barcelona Science Park, Baldiri Reixach, 13, Barcelona 08028 (Spain) E-mail: uson@ibmb.csic.es Dr. D. De Luchi, [+] Prof. Dr. J. A. Subirana, Dr. J. L. Campos Departament d’Enginyeria Química, Universitat Politcnica de Catalunya, Diagonal 647, Barcelona 08028 (Spain) E-mail: lourdes.campos@upc.edu [ + ] These authors contributed equally to this work. [**] This work was supported by grants BFU 2009-10380 and BIO 2009- 10576, MICINN, Spain. Data collection was supported by the ESRF (BM16) and the EU. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201003647. Zuschriften 8092  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2010, 122, 8092 –8094