Yonggang Ke

Three-Dimensional Structures Self-Assembled from DNA Bricks

Building with DNA One route for assembling three-dimensional (3D) DNA nanostructures is to start with a long natural DNA single strand and attach short strands, or “staples,” that cause the entire “origami” structure to fold into a desired shape. Ke et al. (p. 1177, see the cover; see the Perspective by Gothelf) present an alternative approach to 3D assembly that builds upon modular assembly of 2D DNA tiles. One hundred and two distinct shapes were created from four-domain, 32-nucleotide single-stranded DNAs that assembled like childrens blocks; each block could bind to four neighboring blocks through specific pairing interactions. Computer design and stepwise assembly allowed assembly of hollow shapes with a variety of internal cavities. Stepwise assembly of 32-nucleotide DNA “bricks” can create a wide variety of nanoscale objects. We describe a simple and robust method to construct complex three-dimensional (3D) structures by 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 of 2.5 by 2.5 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.

Self-Assembled Water-Soluble Nucleic Acid Probe Tiles for Label-Free RNA Hybridization Assays

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.

Self-assembled DNA nanostructures for distance dependent multivalent ligand-protein binding

An important goal of nanotechnology is to assemble multiple molecules while controlling the spacing between them. Of particular interest is the phenomenon of multivalency, which is characterized by simultaneous binding of multiple ligands on one biological entity to multiple receptors on another. Various approaches have been developed to engineer multivalency by linking multiple ligands together. However, the effects of well-controlled inter-ligand distances on multivalency are less well understood. Recent progress in self-assembling DNA nanostructures with spatial and sequence addressability has made deterministic positioning of different molecular species possible. Here we show that distance-dependent multivalent binding effects can be systematically investigated by incorporating multiple-affinity ligands into DNA nanostructures with precise nanometre spatial control. Using atomic force microscopy, we demonstrate direct visualization of high-affinity bivalent ligands being used as pincers to capture and display protein molecules on a nanoarray. These results illustrate the potential of using designer DNA nanoscaffolds to engineer more complex and interactive biomolecular networks.

Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container

We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is approximately 54 nm in dimension. The estimated total external volume and the internal cavity of the triangular pyramid are about 1.8 x 10(-23) and 1.5 x 10(-23) m(3), respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques.

Polyhedra Self-Assembled from DNA Tripods and Characterized with 3D DNA-PAINT

Engineering Larger DNA Structures Several approaches now exist for the self-assembly of DNA into nanostructures. For example, three-arm DNA tripods can be assembled into larger wireframe polyhedra, but for the most complicated shapes, assembly yields can be low, apparently because the flexibility of smaller tripods allows for misassembly. Iinuma et al. (p. 65, published online 13 March) now show that larger, stiffer tripods that have controlled arm lengths and interarm angles can be designed to form a wide variety of open wireframe polyhedra—including tetrahedra, cubes, and hexagonal prisms, with edges 100 nanometers in length. Stiff DNA tripod units enabled the assembly of wireframe polyhedra with edges 100 nanometers in length. DNA self-assembly has produced diverse synthetic three-dimensional polyhedra. These structures typically have a molecular weight no greater than 5 megadaltons. We report a simple, general strategy for one-step self-assembly of wireframe DNA polyhedra that are more massive than most previous structures. A stiff three-arm-junction DNA origami tile motif with precisely controlled angles and arm lengths was used for hierarchical assembly of polyhedra. We experimentally constructed a tetrahedron (20 megadaltons), a triangular prism (30 megadaltons), a cube (40 megadaltons), a pentagonal prism (50 megadaltons), and a hexagonal prism (60 megadaltons) with edge widths of 100 nanometers. The structures were visualized by means of transmission electron microscopy and three-dimensional DNA-PAINT super-resolution fluorescent microscopy of single molecules in solution.

DNA‐Tile‐Directed Self‐Assembly of Quantum Dots into Two‐Dimensional Nanopatterns

Organizing nanoparticles (NPs) into rationally designed ensemble structures is of great scientific interest because architecturally defined collective properties from multiple NPs could lead to applications such as photonic antennas and controlled plasmonic interactions.[1] Recently, structural DNA nanotechnology has opened a new avenue for directed self-assembly of NPs [2] and other molecular species [3] into patterned nanostructures, taking advantage of the exciting progress in design and construction of artificial nanostructures with complex geometry or patterns via DNA self-assembly.[4] Among these, success of using DNA tile based nanostructure to organize NPs has only been limited to metallic gold NPs. To our knowledge, there has been no report demonstrating DNA tile directed self-assembly of semiconducting NPs (QDs) into rationally designed architectures, partly may be due to the significantly different surface properties of QDs and gold NPs. The difficulty of making QDs compatible to DNA tile based nanostructure has prohibited many interesting studies of multi-component NP photonic systems, e.g. distance dependent plasmonic quenching or enhancement between metallic NPs and QDs. Herein, we worked out a strategy to use two-dimensional DNA tile arrays to direct the assembly of streptavidin conjugated CdSe/ZnS core/shell QDs into well-defined periodic patterns. We anticipate that this first example of DNA tile based QD assembly would pave the way for controlling more sophisticated nanopatterns of QDs and beyond. The strategy and schematic process of the DNA tile directed QD assembly is illustrated in Figure 1. We used a set of four double crossover (DX) molecules, named the ABCD tile system,[5] as scaffolds for QD assembly. Each different DX tile (DX-A, DX-B, DX-C and DX-D) is shown by a different color in Fig. 1. The ‘A’ tile contains a short DNA stem protruding out of the tile plane that carries a biotin group (illustrated as yellow star) at the end (see supporting information for sequence information). Upon self-assembly, the four-tile system gives 2D arrays displaying parallel lines of biotin groups, with a periodic distance between two neighboring biotin lines ~ 64 nm, and a distance between two biotins within the line about 4–5 nm. After adding streptavidin coated CdSe/ZnS QDs (Invitrogen, QdotR 545 ITK streptavidin conjugate, or STV-QD) to the DNA array, streptavidin molecules (illustrated as yellow blocks) specifically bind to the biotin groups so that the QDs (illustrated as black ball) will be organized onto the DX tile arrays (see supporting information for detailed methods). Figure 1 Schematic showing the process of DNA tile directed self-assembly of QD arrays. AFM images and cross-section profiles shown in Figure 2 clearly demonstrate that STV-QDs bind specifically to the DX array and get organized into periodical stripes of QD arrays with the designed distances between the parallel lines. Due to the short stem on A tile carrying the biotin group, the patterned 2D array of the ABCD tile system alone (left image and blue trace) show a small height change across the line of the biotin groups, ~ 0.5 nm. When STV-QDs bind to the DX array (middle image and green trace), the average height across the biotin sites increases to ~ 9 nm. A control experiment by adding streptavidin to the same type of biotin modified DX arrays shows a height change of only ~ 2 nm (right image and red trace). The height change resulting from the STV-QD binding is significantly higher than that of streptavidin binding to the array, the line widths are also much wider, owning to much larger sizes of the STV-QDs (diameter estimated ~ 10 nm including the surface polymer and protein coating), compared to that of the protein molecules alone ~ 2–3 nm. Figure 2 a) From left to right: AFM images of DX-ABCD array alone with each A tile bearing a biotin; the biotinylated DX-ABCD array incubated with STV-QD conjugate; and same array incubated with streptavidin only; b) Cross section analysis of the AFM images. Each ... TEM imaging further reveals the patterning of QD arrays templated by the DX tiles. TEM images shown in Figure 3 represent the periodic alignment of QDs into parallel stripes with measured periodicity of ~ 64 nm between the lines, matched well with the designed parameters. The diameter of each individual QD particle is measured ~ 4 nm, corresponds to the size of CdSe/ZnS QDs with green emission. The protein and polymer coating on the surface of the QDs are not visible due to low electron density thus low contrast in the TEM image. It is notable the QDs within each stripe sometimes slightly shift out of the line, which may be due to the orientational flexibility of the short protruding DNA stems bearing the biotin group and the tendency of neighboring QDs to avoid steric crowdedness. The center to center distance between QDs within the same line measures from 7 to 15 nm, which is larger than the distance between neighboring biotin groups, 4–5 nm in the DX array. This can be explained by the large size of the protein coated QDs causing steric hindrance within the line. In addition, the multiple streptavidin molecules conjugated on each QD and the multivalency of streptavidin-biotin binding may also contribute to this effect. It is possible that multiple streptavidin molecules on a single QD may obtain orientations that allow for binding of two or three neighboring biotin groups in the same line. Taking the size of the QD with the protein and polymer coating on surface into account, this 7–15 nm distance is close to the highest possible packing density of the protein coated QDs in the line. More TEM and AFM images of larger sample areas are also included in the supplemental materials, demonstrating the fidelity of the successful assembly of the STV-QDs using the DNA tile scaffolds. Figure 3 a) TEM images of the periodic patterns of the organized QD arrays. b) High resolution TEM images with an insert in the right corner reveal the crystalline structure of the QDs. c) EDX spectrum verifies the composition of the CdSe/ZnS QDs. High resolution TEM images (insert in Fig. 3b) clearly reveals the crystalline structure of the QDs and the energy dispersive X-ray (EDX) data supports that the NPs in the image are composed of CdSe/ZnS, as shown in Figure 3c. In addition to AFM and TEM imaging, we further used laser fluorescence imaging and photo-bleaching experiments to demonstrate that the QDs were assembled onto the DX array. In this experiment, a DNA strand in the B tile of the DX array was modified with an organic fluorophore with red emission, Cy5 (λem = 648 nm). The DX arrays carrying both Cy5 and STV-QD of green emission (λem = 545 nm) was imaged by fluorescence imaging (Figure 4a), revealing the co-localization of red Cy5 and green QDs on the DNA array (see superimposed fluorescent image in Fig. 4a, rightmost panel). It is well-known that QDs have higher photostability than organic fluorophores. A rectangular shaped region of 11×15 μm2 was selected from the imaged area to be photobleached (Fig. 4b). This region was constantly irradiated by a focused 405 nm laser beam at the power of 0.9 mW for 81 s. Images were taken using the same sequential scanning set-up with 9 s intervals during the photobleaching process. The change of the relative intensity of the red and green fluorescence in the bleached region was also plotted in Figure 4b. It is clear that the organic fluorophore was photobleached with a 90% drop of intensity within 30 s, while the emission intensity of the green QDs still persists after 80 s. This experiment further indicates that QDs are successfully organized onto the DNA tile arrays. Figure 4 a) Confocal fluorescent microscope images and b) photobleaching on the DNA arrays. Scale bar: 20 μM. In summary, we have constructed well-aligned two-dimensional arrays of QDs with controlled periodicity by coupling DNA self-assembly with streptavidin coated QDs. As an elegant bottom-up method, DNA self-assembly has the inherent advantage in generating programmable nanostructures with rationally designed functionality and nanometer precision in addressability. Our work demonstrates the capability to direct QDs into designer nanoarchitectures, this opens up opportunities to construct discrete nanostructures of multi-component NP systems for energy and biosensing applications. It is worthy to point out that both the surface and bioconjugation chemistry of QDs are much more complicated than gold nanoparticles, this led us to optimize many important experimental parameters to achieve successful QD assembly on DNA tile arrays (see supporting information for further comments). We acknowledge that more robust bioconjugation chemistry on QDs to obtain DNA sequence coded QDs (desirable to have discrete number of DNA oligos displayed on the QD surface) is needed for sequence addressable organization of QDs into more sophisticated architectures. Combining QD assembly strategies with previous success of metallic NP organization, we expect that many new properties of the well-controlled multi-component nanophotonic structures will be revealed. Indeed, the organizational power of structural DNA nanotechnology demonstrated so far is only at its horizon.

Prescribed nanoparticle cluster architectures and low-dimensional arrays built using octahedral DNA origami frames

Three-dimensional mesoscale clusters that are formed from nanoparticles spatially arranged in pre-determined positions can be thought of as mesoscale analogues of molecules. These nanoparticle architectures could offer tailored properties due to collective effects, but developing a general platform for fabricating such clusters is a significant challenge. Here, we report a strategy for assembling three-dimensional nanoparticle clusters that uses a molecular frame designed with encoded vertices for particle placement. The frame is a DNA origami octahedron and can be used to fabricate clusters with various symmetries and particle compositions. Cryo-electron microscopy is used to uncover the structure of the DNA frame and to reveal that the nanoparticles are spatially coordinated in the prescribed manner. We show that the DNA frame and one set of nanoparticles can be used to create nanoclusters with different chiroptical activities. We also show that the octahedra can serve as programmable interparticle linkers, allowing one- and two-dimensional arrays to be assembled with designed particle arrangements.

DNA brick crystals with prescribed depths.

We describe a general framework for constructing two-dimensional crystals with prescribed depth and sophisticated three-dimensional features. These crystals may serve as scaffolds for the precise spatial arrangements of functional materials for diverse applications. The crystals are self-assembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micron-size in the lateral dimensions with precisely controlled depth up to 80 nanometers. They can be designed to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels, and to pack DNA helices at parallel and perpendicular angles relative to the plane of the crystals.

Metallized DNA nanolithography for encoding and transferring spatial information for graphene patterning

The vision for graphene and other two-dimensional electronics is the direct production of nanoelectronic circuits and barrier materials from a single precursor sheet. DNA origami and single-stranded tiles are powerful methods to encode complex shapes within a DNA sequence, but their translation to patterning other nanomaterials has been limited. Here we develop a metallized DNA nanolithography that allows transfer of spatial information to pattern two-dimensional nanomaterials capable of plasma etching. Width, orientation and curvature can be programmed by specific sequence design and transferred, as we demonstrate for graphene. Spatial resolution is limited by distortion of the DNA template upon Au metallization and subsequent etching. The metallized DNA mask allows for plasmonic enhanced Raman spectroscopy of the underlying graphene, providing information on defects, doping and lattice symmetry. This DNA nanolithography enables wafer-scale patterning of two-dimensional electronic materials to create diverse circuit elements, including nanorings, three- and four-membered nanojunctions, and extended nanoribbons.

Controlled delivery of DNA origami on patterned surfaces

Due to its capacity for programmable self-assembly, wellestablished modes of chemical synthesis, and exceptional stability, DNA serves as a powerful nanoscale structural material. In particular, the recent invention of DNA origami technology has established a paradigm in which DNA’s capacity for deterministic self-assembly into essentially any discrete two-dimensional (2D) shape can be exploited for the construction of molecular ‘‘bread boards’’. For example, previous groups have demonstrated the delivery of nanoparticles to specific positions within an origami scaffold with nanometer-scale precision and the weaving of DNA aptamers into origami for the assembly of protein arrays. However, in order to fully harness the potential of DNA as a universal nanoscale template, it is important not only to control the position of cargo material within the origami scaffold but also to accurately control the position and orientation of the