LETTERS Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra Yu He 1 , Tao Ye 1 , Min Su 2 , Chuan Zhang 1 , Alexander E. Ribbe 1 , Wen Jiang 2 & Chengde Mao 1 DNA is renowned for its double helix structure and the base pair- ing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute molecular computations 1–4 . Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional 5–9 . Examples of three-dimensional DNA struc- tures include cubes 10 , truncated octahedra 11 , octohedra 12 and tetrahedra 13,14 , which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large numbers (hundreds) of unique DNA strands poses a challenging design problem 9,15 . Here, we demon- strate a simple solution to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger three-dimensional structures. We test this hierarchical self-assembly concept with DNA molecules that form three-point-star motifs, or tiles. By controlling the flexibility and concentration of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometres in size and comprised of four, twenty or sixty individual tiles, respectively. We expect that our assembly strategy can be adapted to allow the fabrication of a range of relatively complex three-dimensional structures. Our approach to forming DNA polyhedra is a one-pot self- assembly process illustrated in Fig. 1: individual single strands of DNA first assemble into sticky-ended, three-point-star motifs (tiles), which then further assemble into polyhedra through sticky-end asso- ciation between the tiles. The three-point-star motif contains a three- fold rotational symmetry and consists of seven strands: a long repetitive central strand (blue-red; strand L or L9), three identical medium strands (green; strand M), and three identical short peri- pheral strands (black; strand S). At the centre of the motif are three single-stranded loops (coloured red). The flexibility of the motif can be easily adjusted by varying the loop length, with increased loop length increasing tile flexibility. The termini of each branch of the tile carry two complementary, four-base-long, single-stranded over- hangs, or sticky ends. Association between the sticky-ends allows the tiles to further assemble into larger structures such as the poly- hedra described here. The three-point-star motif has been used for the assembly of flat two-dimensional (2D) crystals 16,17 , where neighbouring units face in opposite directions of the crystal plane to cancel the intrinsic curvature of the DNA tiles. Because polyhedra are closed three- dimensional (3D) objects containing a finite number of component tiles, we reasoned that three factors would promote polyhedron formation. (1) If all component DNA tiles face in the same direction, their curvatures would add up and promote the formation of closed structures. For example, some closed DNA tubular structures have been observed when all DNA tiles face the same side of the crystal plane 7 . This requirement can be easily satisfied by choosing the length of each pseudo-continuous DNA duplex in the final structures to be four turns (42 bases). (2) Self-assembly is an inter-unit process. This means that higher (micromolar) DNA concentrations favour large assemblies such as flat 2D crystals, whereas lower DNA concen- trations favour small assemblies such as polyhedra. This concentra- tion-dependent kinetic effect should also provide some control over polyhedral size. (3) 2D crystal formation was found to require loops that are two to three bases long 17 . Elongating the loops increases tile flexibility; this should prevent the assembly of DNA stars into large 2D crystals and instead promote the formation of smaller structures. We first tested this hypothesis by assembling a DNA tetrahedron from four three-point-star tiles. Each tile sits at a vertex, and its branches each associate with a branch from another tile to form the edges of the tetrahedron. The assembled tiles at the four vertices retain the threefold rotational symmetry of the free, individual star tiles, but are no longer planar. In fact, they are significantly bent and thus need to be quite flexible. To provide this flexibility, the loop length is designed to be five bases long. This ensures that the DNA stars will associate with each other under hybridization conditions to form highly flexible assemblies, which allows the free sticky-ends in the assemblies to meet and associate with each other to yield closed 1 Department of Chemistry, 2 Markey Center for Structural Biology and Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA. S M LL′ Tetrahedron Loop: 5 bases DNA: 75 nM Dodecahedron Loop: 3 bases DNA: 50 nM Buckyball Loop: 3 bases DNA: 500 nM Figure 1 | Self-assembly of DNA polyhedra. Three different types of DNA single strands stepwise assemble into symmetric three-point-star motifs (tiles) and then into polyhedra in a one-pot process. There are three single- stranded loops (coloured red) in the centre of the complex. The final structures (polyhedra) are determined by the loop length (3 or 5 bases long) and the DNA concentration. Vol 452 | 13 March 2008 | doi:10.1038/nature06597 198 Nature Publishing Group ©2008