Structure Elucidation Very Important Paper DOI: 10.1002/ange.201309066 Crystal Structure of Metallo DNA Duplex Containing Consecutive Watson–Crick-like T–Hg II –T Base Pairs** Jiro Kondo,* Tom Yamada, Chika Hirose, Itaru Okamoto, Yoshiyuki Tanaka, and Akira Ono Abstract: The metallo DNA duplex containing mercury- mediated T–T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B- form DNA duplex containing two consecutive T–Hg II –T base pairs. The Hg II ion occupies the center between two T residues. The N3-Hg II bond distance is 2.0 . The relatively short Hg II - Hg II distance (3.3 ) observed in consecutive T–Hg II –T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B-form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg II . The structure of the metallo DNA duplex itself and the Hg II - induced structural switching from the nonhelical form to the B- form provide the basis for structure-based design of metal- conjugated nucleic acid nanomaterials. The DNA molecule is an attractive material in pharmaceut- ical technology and nanotechnology because of its chemical stability, ease of chemical synthesis and sequence design, and relatively simple structure resulting from base complemen- tarity. For example, the antigene and antisense DNAs, deoxyribozymes, and DNA aptamers have been extensively developed as nucleic acid medicines and biotechnological tools. [1] Recent discoveries and extensive studies of metal- mediated pairs of natural and artificial bases expand consid- erably the design possibility of functional DNA molecules. [2] The metal-mediated base pairs have been applied to DNA molecular devices, such as ion sensors, [3] electric transport nanowires, [4] and DNA magnets. [5] However, only some structural information of nucleic acid duplexes containing metal-mediated base pairs are available. [6] The mercury-mediated T–T base pair (T–Hg II –T) has been investigated for more than fifty years. [7] In our recent study, we observed that Hg II significantly stabilizes a DNA duplex by binding selectively to a T–T mispair. [8] Based on the phenomenon observed, we developed a DNA-based sensing system which selectively and sensitively detects Hg II in aqueous solution. [3a] The Hg II binding mode in the T–Hg II –T base pair was determined in solution both with 15 N NMR and Raman spectroscopy. [9] The binding of Hg II was also observed in the pioneering crystallographic study of a 2 :1 complex of 1- methylthymine/Hg II . [10] However, structural information of a metallo DNA duplex containing the T–Hg II –T base pair is missing, even though structures of some artificial DNA duplexes containing metal-mediated base pairs have been studied. [5a,c, 6d–f] Very recently, we have successfully solved the solution structure of a DNA duplex containing two consec- utive T–Hg II –T base pairs by using NMR spectroscopy. [11] In the present study, we have performed the X-ray analysis of a DNA duplex both in the presence and absence of Hg II to obtain more detailed structural information and to unveil the effect of Hg II binding on the whole structure of the DNA duplex. The data acquired in crystalline and solution states provide a reliable structural basis for advanced design and further development of metallo DNAs. The DNA dodecamer d(CGCGATTTCGCG), 12TT hereafter, was designed to fold as a self-complementary duplex containing tandem T–T mispairs at the center. Such DNA fragments have been extensively used as successful models in the crystallographic studies of B-form DNAs containing natural and modified bases. [12] For phase determi- nation by the multiple anomalous diffraction (MAD) method, the DNA dodecamer containing 5-bromocytosine at the third position, 12TT-Br hereafter, was also synthesized. Crystals were obtained both in the presence and absence of Hg II (Table 1). Crystal structures of DNA duplexes with and without Hg II , 12TT/Hg II , 12TT, and 12TT-Br hereafter, have been deposited in the Protein Data Bank (PDB) with the ID codes 4L24, 4L25, and 4L26, respectively. A detailed descrip- tion of materials and methods of this study is included in Supporting Information. In the presence of Hg II (12TT/Hg II crystal in Table 1), two DNA strands form an antiparallel right-handed double helix (Figure 1). At both ends of the double helix, canonical Watson–Crick G-C and A-T base pairs are formed (base pairs are named according to the Leontis/Westhof classification). [13] In the central part of the double helix, two Hg II ions bridge the [*] Dr. J. Kondo, T. Yamada, C. Hirose Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University 7-1 Kioi-cho, Chiyoda-ku, 102-8554 Tokyo (Japan) E-mail: j.kondo@sophia.ac.jp Dr. I. Okamoto, Prof. A. Ono Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, Yokohama 221-8686 (Japan) Dr. Y. Tanaka Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University Sendai 980-8578 (Japan) [**] This work was supported by a Grant-in-Aid for Scientific Research (A) (No. 24245037), and in part by a Strategic Research Foundation Grant-aided Project for Private Universities (No. S1201015) from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (MEXT). We thank the Photon Factory for provision of synchrotron radiation facilities (No. 2011G630) and acknowledge the staff of the BL-1A and NW12A beamlines. We are grateful to Dr. V. Sychrovsky ´ and Prof. E. Westhof for helpful discussions and critical reading of the manuscript. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201309066. A ngewandte Chemi e 2417 Angew. Chem. 2014, 126, 2417 –2420  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim