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...

DNA tile based self-assembly provides an attractive route to create nanoarchitectures of programmable patterns. It also offers excellent scaffolds for directed self-assembly of nanometer-scale materials, ranging from nanoparticles to proteins, with potential applications in constructing nanoelectronic/nanophotonic devices and protein/ligand nanoarrays. This Review first summarizes the currently available DNA tile toolboxes and further emphasizes recent developments toward self-assembling DNA nanostructures with increasing complexity. Exciting progress using DNA tiles for directed self-assembly of other nanometer scale components is also discussed.

Experimental MethodsMaterials. The DNA sequences were designed using the SEQUIN program (Seeman, N. C. J. Biomol. Struct. Dyn. 1990, 8, 573-581). DNA strands were commercially synthesized by Integrated DNA Technologies, Inc. (IDT). All unmodified helper strands were purchased from IDT in the format of 96-well plates at 6 nmole synthesis scale and used without further purification. Helper strands modified with aptamer sequences, control sequences and index sequences were purchased from IDT in the format of individual vials and purified by denaturing polyacrylamide gel electrophoresis (PAGE) before use. Strands used in the DX arrays were also purchased from IDT in the format of individual vials and purified by denaturing PAGE. Concentration of purified DNA strands was calculated by measuring optical density at 260 nm wavelengh. Microcon Centrifugal Filter Devices (100,000 MWCO) was purchased from Millipore Corporation. Thrombin was from Haematologic Technologies, Inc., recombinant human PDGF-BB was from BioVision, Inc., and stains all was purchased from Sigma-Aldrich. Both the proteins were reconstituted into aqueous solutions by adding ultrapure water (18 MΩ). M13 viral DNA was purchased from New England Biolabs, Inc. (NEB). (Catalog number: #N4040S. The sequence of the product was confirmed by NEB before they ship to the customers).Assembly of 2-D DNA scaffold. The ABCD arrays were formed by mixing equimolar quantities of all the constituting strands (see Figure S1) at a concentration of 1 µM (as estimated by OD 260 ) in 1 x TAE-Mg 2+ buffer (Tris, 40 mM; Acetic acid, 20 mM; EDTA, 2 mM; and Magnesium acetate, 12.5 mM; pH 8.0). The mixture was cooled slowly from 90 °C to room temperature over 48 hours. Individual DX-B and DX-D tiles were also assembled following the same procedure.

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.DNA nanotechnology ͉ immobile DNA junction ͉ self-replication ͉ synthetic biology T he notion that DNA is merely the gene encoder of living systems has been eclipsed by the successful development of DNA nanotechnology (1-2). Using branched DNA as the main building block (also known as a ''tile'') and cohesive singlestranded DNA (ssDNA) ends to designate the pairing strategy for tile-tile recognition, one can rationally design and assemble complicated nanoarchitectures from specifically designed DNA oligonucleotides. Objects in both two and three dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield (3-9); this development enables the construction of DNA-based nanodevices (10-11) and DNA template directed organization of other molecular species (12)(13)(14)(15)(16)(17)(18)(19). The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. However, the synthetic scale of oligonucleotides limits the potential applications of DNA nanotechnology, especially when the nanostructure is designed to be made from long ssDNA (Ͼ100 bases). Replicable DNA nanostructures are thus a highly desirable goal to help overcome this barrier.To this end, several possible solutions have been explored. For example, a three-point star motif has been replicated by chemical methods in which the parental nanomotif serves as the template to direct the chemical ligation of nonidentical DNA strands to form the next generation of the nanomotif (20). Another instance involves a 1.7-kb ssDNA that was used to assemble a DNA octahedron (together with five short strands); this molecule was cloned in a plasmid in bacteria (9). A nicking end...

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