Jeanette Nangreave

DNA Origami with Complex Curvatures in Three-Dimensional Space

Rationally introduced crossover positions bend networks of double-helical DNA strands into complex shapes. We present a strategy to design and construct self-assembling DNA nanostructures that define intricate curved surfaces in three-dimensional (3D) space using the DNA origami folding technique. Double-helical DNA is bent to follow the rounded contours of the target object, and potential strand crossovers are subsequently identified. Concentric rings of DNA are used to generate in-plane curvature, constrained to 2D by rationally designed geometries and crossover networks. Out-of-plane curvature is introduced by adjusting the particular position and pattern of crossovers between adjacent DNA double helices, whose conformation often deviates from the natural, B-form twist density. A series of DNA nanostructures with high curvature—such as 2D arrangements of concentric rings and 3D spherical shells, ellipsoidal shells, and a nanoflask—were assembled.

Molecular robots guided by prescriptive landscapes

Traditional robots rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment. A first step in this direction was the development of DNA walkers, which have developed from being non-autonomous to being capable of directed but brief motion on one-dimensional tracks. Here we demonstrate that previously developed random walkers—so-called molecular spiders that comprise a streptavidin molecule as an inert ‘body’ and three deoxyribozymes as catalytic ‘legs’—show elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as ‘start’, ‘follow’, ‘turn’ and ‘stop’. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour.

DNA origami: a quantum leap for self-assembly of complex structures

The spatially controlled positioning of functional materials by self-assembly is one of the fundamental visions of nanotechnology. Major steps towards this goal have been achieved using DNA as a programmable building block. This tutorial review will focus on one of the most promising methods: DNA origami. The basic design principles, organization of a variety of functional materials and recent implementation of DNA robotics are discussed together with future challenges and opportunities.

DNA origami as a carrier for circumvention of drug resistance.

Although a multitude of promising anti-cancer drugs have been developed over the past 50 years, effective delivery of the drugs to diseased cells remains a challenge. Recently, nanoparticles have been used as drug delivery vehicles due to their high delivery efficiencies and the possibility to circumvent cellular drug resistance. However, the lack of biocompatibility and inability to engineer spatially addressable surfaces for multi-functional activity remains an obstacle to their widespread use. Here we present a novel drug carrier system based on self-assembled, spatially addressable DNA origami nanostructures that confronts these limitations. Doxorubicin, a well-known anti-cancer drug, was non-covalently attached to DNA origami nanostructures through intercalation. A high level of drug loading efficiency was achieved, and the complex exhibited prominent cytotoxicity not only to regular human breast adenocarcinoma cancer cells (MCF 7), but more importantly to doxorubicin-resistant cancer cells, inducing a remarkable reversal of phenotype resistance. With the DNA origami drug delivery vehicles, the cellular internalization of doxorubicin was increased, which contributed to the significant enhancement of cell-killing activity to doxorubicin-resistant MCF 7 cells. Presumably, the activity of doxorubicin-loaded DNA origami inhibits lysosomal acidification, resulting in cellular redistribution of the drug to action sites. Our results suggest that DNA origami has immense potential as an efficient, biocompatible drug carrier and delivery vehicle in the treatment of cancer.

Structural DNA nanotechnology: state of the art and future perspective.

Over the past three decades DNA has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular Watson–Crick base-pairing interactions. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures of increasing complexity. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, DNA computing, nanorobotics, disease diagnosis, and drug delivery. This Perspective discusses the state of the art in the field of structural DNA nanotechnology and presents some of the challenges and opportunities that exist in DNA-based molecular design and programming.

DNA-Directed Artificial Light-Harvesting Antenna

Designing and constructing multichromophoric, artificial light-harvesting antennas with controlled interchromophore distances, orientations, and defined donor-acceptor ratios to facilitate efficient unidirectional energy transfer is extremely challenging. Here, we demonstrate the assembly of a series of structurally well-defined artificial light-harvesting triads based on the principles of structural DNA nanotechnology. DNA nanotechnology offers addressable scaffolds for the organization of various functional molecules with nanometer scale spatial resolution. The triads are organized by a self-assembled seven-helix DNA bundle (7HB) into cyclic arrays of three distinct chromophores, reminiscent of natural photosynthetic systems. The scaffold accommodates a primary donor array (Py), secondary donor array (Cy3) and an acceptor (AF) with defined interchromophore distances. Steady-state fluorescence analyses of the triads revealed an efficient, stepwise funneling of the excitation energy from the primary donor array to the acceptor core through the intermediate donor. The efficiency of excitation energy transfer and the light-harvesting ability (antenna effect) of the triads was greatly affected by the relative ratio of the primary to the intermediate donors, as well as on the interchromophore distance. Time-resolved fluorescence analyses by time-correlated single-photon counting (TCSPC) and streak camera techniques further confirmed the cascading energy transfer processes on the picosecond time scale. Our results clearly show that DNA nanoscaffolds are promising templates for the design of artificial photonic antennas with structural characteristics that are ideal for the efficient harvesting and transport of energy.

DNA Gridiron Nanostructures Based on Four-Arm Junctions

Rewiring DNA Origami Complex DNA nanostructures can be formed from a long scaffold strand of DNA by binding many shorter “staple” strands. In these DNA origami structures, the path of the scaffold has been restricted by a double-crossover motif to form parallel helices. Han et al. (p. 1412) now describe a more flexible approach based on a “gridiron unit” in which four four-arm junctions link together to form a two-layer square frame. A variety of two- and three-dimensional structures were created, including highly curved structures, such as a sphere and a screw. Flexible DNA wireframe nanostructures have double-helical domains as edges and modified Holliday junctions as vertices. Engineering wireframe architectures and scaffolds of increasing complexity is one of the important challenges in nanotechnology. We present a design strategy to create gridiron-like DNA structures. A series of four-arm junctions are used as vertices within a network of double-helical DNA fragments. Deliberate distortion of the junctions from their most relaxed conformations ensures that a scaffold strand can traverse through individual vertices in multiple directions. DNA gridirons were assembled, ranging from two-dimensional arrays with reconfigurability to multilayer and three-dimensional structures and curved objects.

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.

Molecular Behavior of DNA Origami in Higher-Order Self-Assembly

DNA-based self-assembly is a unique method for achieving higher-order molecular architectures made possible by the fact that DNA is a programmable information-coding polymer. In the past decade, two main types of DNA nanostructures have been developed: branch-shaped DNA tiles with small dimensions (commonly up to ∼20 nm) and DNA origami tiles with larger dimensions (up to ∼100 nm). Here we aimed to determine the important factors involved in the assembly of DNA origami superstructures. We constructed a new series of rectangular-shaped DNA origami tiles in which parallel DNA helices are arranged in a zigzag pattern when viewed along the DNA helical axis, a design conceived in order to relax an intrinsic global twist found in the original planar, rectangular origami tiles. Self-associating zigzag tiles were found to form linear arrays in both diagonal directions, while planar tiles showed significant growth in only one direction. Although the series of zigzag tiles were designed to promote two-dimensional array formation, one-dimensional linear arrays and tubular structures were observed instead. We discovered that the dimensional aspect ratio of the origami unit tiles and intertile connection design play important roles in determining the final products, as revealed by atomic force microscopy imaging. This study provides insight into the formation of higher-order structures from self-assembling DNA origami tiles, revealing their unique behavior in comparison with conventional DNA tiles having smaller dimensions.

A replicable tetrahedral nanostructure self-assembled from a single DNA strand.

We report the design and construction of a nanometer-sized tetrahedron from a single strand of DNA that is 286 nucleotides long. The formation of the tetrahedron was verified by restriction enzyme digestion, Ferguson analysis, and atomic force microscopy (AFM) imaging. We further demonstrate that synthesis of the tetrahedron can be easily scaled up through in vivo replication using standard molecular cloning techniques. We found that the in vivo replication efficiency of the tetrahedron is significantly higher in comparison to in vitro replication using rolling-circle amplification (RCA). Our results suggest that it is now possible to design and replicate increasingly complex, single-stranded DNA nanostructures in vivo.