Two-dimensional DNA origami assemblies using a four-way connectorw

Directionally controlled self-assembly of multiple molecules is an important technique for constructing multidimensional structures. DNA molecules are widely used for controlled self-assembly because of their programmability, defined structure, and acceptance of various functionalizations. The scaffolded DNA origami method developed for the preparation of twodimensional (2D) structures has been utilized for the selective positioning of the functional molecules and nanoparticles, and for designing various 3D architectures. We have recently developed a programmed assembly method in 1D space using multiple DNA origami components with selective connections. For further extension of the self-assembly of the origami structures in 2D directions, novel designs and approaches should be explored. In this communication, we intended to expand the direction of the DNA origami assembly into both horizontal and vertical directions. The designed dual-directional DNA origami connector, which can allow connection at all four edges, has sequence-programmed connection sites to both horizontal and vertical edges (Fig. 1a). As reported, the edges of the helical axis promote connection with the neighboring DNA origami via the p-stacking interactions. The important point we need to emphasize is that we utilize the p-stacking interactions at the end of the helical axis to facilitate association of the DNA origami. Therefore, we designed a four-way connector that oriented the helical-axis element of all four edges outside (Fig. 1a). In addition, we introduced tenons and mortises to allow the selective connection of different DNA origami components via these connection sites. We planned to create the 2D assemblies such as cruciate and closed hollow square forms as given in Fig. 1c and e. Three principle rules for the programmed assembly of DNA origami components are: (1) p-stacking interactions of the edges, which can enhance the stability of the complex formation and lead to a precise connection of the edges; (2) sequence complementarity of the DNA strands in the connection sites for the selective base-pairing; and (3) shape complementarity of the edges that was introduced for the selective connection and exclusion of undesired pairing of DNA components. The selective connection between a tenon and a mortise is shown in Fig. 1f. The tenon and the mortise were introduced to all four edges of a four-way connector. In this four-way connector, two domains at the top and bottom, whose helical orientations were vertical to the central domain, fitted to the central domain via tenon–mortise-like connections introduced inside the structure as described in Fig. 1a. This arrangement is important for the stabilization and keeping the plane of the involved four domains. The four-way connectors A and B are nearly the same except that in the connector B the sequence complementarity was removed at the tenon and the mortise that were not involved in the connection. We also prepared five linear connectors to connect at the desired position of the four-way connector (Fig. 1b and d). Each linear connector had a tenon and a mortise that could be used for the selective base-pairing as employed previously. For the identification of the individual linear DNA connectors, hairpin DNAs were introduced as markers adjacent to the tenon and the mortise (Fig. 1 and ESIw). The additional 2D domain was introduced to the bottom edge for identification of the orientation of the linear connectors. DNA origami formation was carried out using M13mp18 single-stranded DNA with custom-designed complementary DNA strands (staple strands) in a solution containing tris buffer (pH 7.6), EDTA, and Mg. The detailed design and sequences of the staple strands of the DNA origamis are illustrated in ESI.w The M13mp18 strand and staple strands were annealed from 85 1C to 25 1C by decreasing the temperature at a rate of 1.0 1C min 1 for monomer formation. After annealing, the formed structures were imaged using an atomic force microscope (AFM) in the same buffer solution (Fig. 2a and b). The four-way connector formed a predesigned shape with two tenons and two mortises (Fig. 2a and b). The size of the four-way connector was ca. 130 70 nm, a Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: [email protected], [email protected]; Fax: +81 75-753-3670 CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan w Electronic supplementary information (ESI) available: DNA sequences of the DNA origami nanostructures. See DOI: 10.1039/ c0cc05306f ChemComm Dynamic Article Links