DNA Nanotechnology Hot Paper DOI: 10.1002/anie.201403965 Surface-Assisted Large-Scale Ordering of DNA Origami Tiles** Ali Aghebat Rafat, Tobias Pirzer, Max B. Scheible, Anna Kostina, and Friedrich C. Simmel* Abstract: The arrangement of DNA-based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close-packing of symmetric, non-interacting DNA origami structures, or by utilizing blunt-end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins. In recent decades, self-assembly based on the specific and programmable recognition interactions between DNA mol- ecules has been shown to be a successful strategy for the generation of artificial nanoscale molecular structures. [1] Most prominently, the DNA origami technique [2] has enabled the realization of almost arbitrarily shaped molecular objects, which can further be decorated with functional molecules or nanoparticles with a spatial resolution of only a few nano- meters. This capability has already resulted in a variety of potential applications for DNA nanotechnology, ranging from nanomaterials [3] over biosensing [4] to nanomedicine. [5] For many applications, an extension of the “local order” facili- tated by DNA self-assembly also to larger length scales would be desirable, for example, when the generation of macro- scopic DNA-based materials is envisioned, or integration with top-down fabrication technologies is required. Several features have been found to be useful for the generation of large-scale assemblies based on small (i.e., non- origami) DNA tiles. Apart from the development of rigid molecular building blocks, [6] sequence-symmetric interac- tions, [7] the precise control of nucleation and growth param- eters, [8] and the utilization of weak, cooperative interactions have been shown to generate particularly well-ordered structures in two and even three dimensions. [9] Also the presence of a surface was found to promote the assembly of large ordered lattices from small T-junction units [10] or star- shaped tiles. [11] DNA origami structures can adopt a larger diversity of shapes than small DNA tiles, and also allow for more complex chemical functionalization. Previous approaches toward the generation of larger DNA origami assemblies involved litho- graphic patterning, [12] the utilization of longer scaffold strands, [13] polymerization [14] and higher order assembly using hybridization [15] or blunt-end stacking interactions. [10, 16] The only successful example of extended 2D crystallization of DNA origami tiles was demonstrated by Liu et al. [17] based on sticky-end hybridization of cross-shaped origami structures. Thermal annealing over several days resulted in crystalline 2D arrays with dimensions of up to 5  10 mm 2 . We show herein that surface-assisted assembly [10, 11] can also be achieved with DNA origami structures by simply improving their surface mobility by the addition of mono- valent salts (Scheme 1). In typical atomic force microscopy (AFM) experiments on negatively charged mica sheets, DNA origami structures are strongly adsorbed to the substrate to facilitate stable imaging. Adsorption is mediated by Mg 2+ ions (typically already contained in the origami folding buffer), which act as salt bridges between mica and DNA (Figure 1 A). This interaction can be weakened by the addition of mono- valent ions, such as Na + , which partly replace the Mg 2+ ions and form a more diffuse charge layer between the surface and the polyelectrolyte. [18] As a result, the origami structures become mobile on the surface and can then associate to form extended ordered structures on the surface. We demonstrate that non-interacting, regularly shaped origami structures, such as rectangles [2a, 19] or triangles, [2a] simply assemble into close- packed structures, dictated by the steric repulsion between the building blocks. In the case of twist-corrected, cross- shaped origami tiles [17] we also employ attractive blunt-end Scheme 1. The surface mobility of DNA origami structures on mica can be tuned by the addition of monovalent cations. Depending on the symmetry of the origami building blocks and their mutual interactions, large-scale ordered 2D assemblies can form. [*] A. Aghebat Rafat, [+] Dr. T. Pirzer, [+] M. B. Scheible, A. Kostina, Prof. Dr. F. C. Simmel Physik-Department E14 and ZNN/WSI, TU München Am Coulombwall 4a, 85748 Garching (Germany) E-mail: simmel@tum.de Prof. Dr. F. C. Simmel Nanosystems Initiative Munich Schellingstrasse 4, 80539 Munich (Germany) [ + ] These authors contributed equally to this work. [**] We gratefully acknowledge financial support by the Volkswagen Stiftung (grant no. 86 395), the EU Marie Curie Initial Training Network EscoDNA, and the Cluster of Excellence Nanosystems Initiative Munich (NIM). We thank A. Kuzyk for initial work in this project. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201403965. A ngewandte Chemi e 7665 Angew. Chem. Int. Ed. 2014, 53, 7665 –7668  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim