We describe a simple and robust method to construct complex three-dimensional (3D) structures using short synthetic DNA strands that we call “DNA bricks”. In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8-base-pair interaction between bricks defines a voxel with dimensions 2.5 nanometers by 2.5 nanometers by 2.7 nanometers, and a master brick collection defines a “molecular canvas” with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features as well as intricate interior cavities and tunnels.

The DNA origami method, in which long, single-stranded DNA segments are folded into shapes by short staple segments, was used to create nucleic acid probe tiles that are molecular analogs of macroscopic DNA chips. One hundred trillion probe tiles were fabricated in one step and bear pairs of 20-nucleotide-long single-stranded DNA segments that act as probe sequences. These tiles can hybridize to their targets in solution and, after adsorption onto mica surfaces, can be examined by atomic force microscopy in order to quantify binding events, because the probe segments greatly increase in stiffness upon hybridization. The nucleic acid probe tiles have been used to study position-dependent hybridization on the nanoscale and have also been used for label-free detection of RNA.

An interesting goal of nanotechnology is to assemble biomolecules to display multivalent interactions, which are characterized by simultaneous binding of multiple ligands on one biological entity to multiple receptors on another with high avidity 1 . Various approaches have been developed to engineer multivalency by linking multiple ligands together 2-4 . However, the effects of wellcontrolled inter-ligand distances on multivalency are less understood. Recent progress in selfassembling DNA tile-based nanostructures with spatial and sequence addressability 5-12 has made deterministic positioning of different molecular species possible 8,11-13 . Here we show that distancedependent multivalent binding effects can be systematically investigated by incorporating multiple affinity ligands into DNA nanostructures with precise nanometer spatial control. Using atomic force microscopy (AFM), we demonstrate direct visualization of high avidity bivalent ligands being used as pincers to capture and display protein molecules on a nanoarray. Our results set forth a path for constructing spatial combinatorics at the nanometer scale.The model system ( Fig. 1) we chose to demonstrate the distancedependent multivalent ligandprotein binding consists of two different aptamers positioned into a multi-helix DNA tile to bind a single protein target, such that the distance between them can be precisely controlled by varying the spatial arrangement of the aptamers on the DNA tile. Aptamers are oligonucleotidebased recognition regions that are selected to bind small molecules or proteins 14 . The two aptamers used here both are thrombin (a coagulation protein involved as a key promotor in blood clotting) binding aptamers which were previously selected and well characterized 15,16 . Each has a unique sequence and binds to a nearly opposite site on the thrombin molecule 15,17, 18 . Aptamer A (apt-A: 29 mer, 5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′) binds to the heparin binding exosite, 15 and aptamer B (apt-B: 15 mer, 5′-GGT TGG TGT GGT TGG-3′) binds primarily to the fibrinogen-recognition exosite. 16 It is proposed that, when these two aptamers are linked together by a rigid spacer, by varying the length of the space, an optimal inter-aptamer distance will be achieved, so that the two aptamers will act as a bivalent single molecular species that displays a stronger binding affinity to the protein than any one of the individual aptamers alone does.The multi-helix DNA tile was designed and constructed from either a fourhelix bundle (4HB) structure 19 or a five-helix bundle (5HB) structure (generated by narrowing an eight-helix bundle tile 19 ) that are modified with the closed-loop aptamer sequences extending out from (Fig. 1b). The spacing between the two aptamers can be controlled at a subnanometer precision. For example, the 5HB DNA tile can provide 2, 3.5, 5.3 and 6.9 nm inter-aptamer distances. This was accomplished by integrating apt-A into helix 1 (the left-most helix) and moving apt-B from helix 2 to 5 (to the right). Th...

Molecular self-assembly using DNA as a structural building block has proven to be an efficient route to the construction of nanoscale objects and arrays of increasing complexity. Using the remarkable "scaffolded DNA origami" strategy, Rothemund demonstrated that a long single-stranded DNA from a viral genome (M13) can be folded into a variety of custom two-dimensional (2D) shapes using hundreds of short synthetic DNA molecules as staple strands. More recently, we generalized a strategy to build custom-shaped, three-dimensional (3D) objects formed as pleated layers of helices constrained to a honeycomb lattice, with precisely controlled dimensions ranging from 10 to 100 nm. Here we describe a more compact design for 3D origami, with layers of helices packed on a square lattice, that can be folded successfully into structures of designed dimensions in a one-step annealing process, despite the increased density of DNA helices. A square lattice provides a more natural framework for designing rectangular structures, the option for a more densely packed architecture, and the ability to create surfaces that are more flat than is possible with the honeycomb lattice. Thus enabling the design and construction of custom 3D shapes from helices packed on a square lattice provides a general foundational advance for increasing the versatility and scope of DNA nanotechnology.