Dongran Han

Challenges and opportunities for structural DNA nanotechnology

DNA molecules have been used to build a variety of nanoscale structures and devices over the past 30 years, and potential applications have begun to emerge. But the development of more advanced structures and applications will require a number of issues to be addressed, the most significant of which are the high cost of DNA and the high error rate of self-assembly. Here we examine the technical challenges in the field of structural DNA nanotechnology and outline some of the promising applications that could be developed if these hurdles can be overcome. In particular, we highlight the potential use of DNA nanostructures in molecular and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics for human health.

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.

Folding and cutting DNA into reconfigurable topological nanostructures

Topology is the mathematical study of the spatial properties that are preserved through the deformation, twisting and stretching of objects. Topological architectures are common in nature and can be seen, for example, in DNA molecules that condense and relax during cellular events. Synthetic topological nanostructures, such as catenanes and rotaxanes, have been engineered using supramolecular chemistry, but the fabrication of complex and reconfigurable structures remains challenging. Here, we show that DNA origami can be used to assemble a Möbius strip, a topological ribbon-like structure that has only one side. In addition, we show that the DNA Möbius strip can be reconfigured through strand displacement to create topological objects such as supercoiled ring and catenane structures. This DNA fold-and-cut strategy, analogous to Japanese kirigami, may be used to create and reconfigure programmable topological structures that are unprecedented in molecular engineering.

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.

Colloidal synthesis of metastable zinc-blende IV–VI SnS nanocrystals with tunable sizes

Here we report the colloidal synthesis of size-tunable SnS nanocrystals that have an unusual meta-stable cubic zinc-blende phase instead of the more stable layered orthorhombic phase. The single-crystalline zinc-blende SnS nanocrystals with sizes of 8 nm, 60 nm, and 700 nm were achieved by injecting the sulfur-oleylamine precursor into tin-oleylamine solution in the presence of hexamethyldisilazane (HMDS) at different temperatures. The morphology and structure of the SnS nanocrystals were studied by high-resolution electron microscopy techniques. The small SnS nanoparticles (∼8 nm and ∼60 nm) are nearly spherical and have the polyhedral shape. The large (∼700 nm) crystals display a unique crystal morphology that have T(d) symmetry with a truncated tetrahedron configuration, and the four truncated surfaces each outgrow to form a convex triangular pyramid corner. Careful structural analysis revealed that each of the crystal is enclosed by 4 low-index {111} hexangular facets and 12 high-index {220} triangular facets using a lift-out technique with a focused ion beam (FIB) and followed by high resolution electron microscope imaging. The direct band gaps of the different sized SnS nanocrystals range from 1.63 eV to 1.68 eV. These heavy-metal-free and low cost nanocrystals are highly efficient absorptive materials in the whole UV-visible range, suitable for applications in photovoltaic cells.

Single-stranded DNA and RNA origami

Large origami from a single strand Nanostructures created by origami-like folding of nucleic acids are usually formed by base-pairing interactions between multiple strands. Han et al. show that large origami (up to 10,000 nucleotides for DNA and 6000 nucleotides for RNA) can be created in simple shapes, such as a rhombus or a heart. A single strand can be folded smoothly into structurally complex but knot-free structures by using partially complemented double-stranded DNA and the cohesion of parallel crossovers. The use of single strands also enables in vitro synthesis of these structures. Science, this issue p. eaao2648 Large nanostructures of up to 10,000 nucleotides can be formed by folding a single nucleic acid strand. INTRODUCTION Self-folding of an information-carrying polymer into a compact particle with defined structure and function (for example, folding of a polypeptide into a protein) is foundational to biology and offers attractive potential as a synthetic strategy. Over the past three decades, nucleic acids have been used to create a variety of complex nanoscale shapes and devices. In particular, multiple DNA strands have been designed to self-assemble into user-specified structures, with or without the help of a long scaffold strand. In recent years, RNA has also emerged as a unique, programmable material, offering distinct advantages for molecular self-assembly. On the other hand, biological macromolecules, such as proteins (or protein domains), typically fold from a single polymer into a well-defined compact structure. The ability to fold de novo designed nucleic acid nanostructures in a similar manner would enable unimolecular folding instead of multistrand assembly and even replication of such structures. However, a general strategy to construct large [>1000 nucleotides (nt)] single-stranded origami (ssOrigami) remains to be demonstrated where a single-stranded nucleic acid folds into a user-specified shape. RATIONALE The key challenge for constructing a compact single-stranded structure is to achieve structural complexity, programmability, and generality while maintaining the topological simplicity of strand routing (to avoid putative kinetic traps imposed by knots) and hence ensuring smooth folding. The key innovation of our study is to use partially complemented double-stranded DNA or RNA and parallel crossover cohesion to construct such a structurally complex yet knot-free structure that can be folded smoothly from a single strand. RESULTS Here, we demonstrate a framework to design and synthesize a single DNA or RNA strand to efficiently self-fold into an unknotted compact ssOrigami structure that approximates an arbitrary user-prescribed target shape. The generality of the method was validated by the construction of 18 multikilobase DNA and 5 RNA ssOrigami, including a ~10,000-nt DNA structure (37 times larger than the previous largest discrete single-stranded DNA nanostructure) and a ~6000-nt RNA structure (10 times larger than the previous largest RNA structure). The raster-filling nature of ssOrigami permitted the experimental construction of programmable patterns of markers (for example, a “smiley” face) and cargoes on its surface, its single-strandedness enabled the demonstration of facile replication of the strand in vitro and in living cells, and its programmability allowed us to codify the design process and develop a web-based automated design tool. CONCLUSION The work here establishes that unimolecular DNA or RNA folding, similar to multicomponent self-assembly, is a fundamental, general, and practically tractable strategy for constructing user-specified and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology. Folding of DNA or RNA ssOrigami structures. (A) Multiple DNA strands have been designed to self-assemble without (left) or with (middle) a long scaffold strand. Here, we fold single long DNA or RNA strands into target shapes (right). (B) Schematics and atomic force microscopy images of single-stranded DNA (top three rows) and RNA (bottom row) nanostructures

Size-Selective Incorporation of DNA Nanocages into Nanoporous Antimony-Doped Tin Oxide Materials

A conductive nanoporous antimony-doped tin oxide (ATO) powder has been prepared using the sol-gel method that contains three-dimensionally interconnected pores within the metal oxide and highly tunable pore sizes on the nanoscale. It is demonstrated that these porous materials possess the capability of hosting a tetrahedral-shaped DNA nanostructure of defined dimensions with high affinity. The tunability of pore size enables the porous substrate to selectively absorb the DNA nanostructures into the metal oxide cavities or exclude them from entering the surface layer. Both confocal fluorescence microscopy and solution FRET experiments revealed that the DNA nanostructures maintained their integrity upon the size-selective incorporation into the cavities of the porous materials. As DNA nanostructures can serve as a stable three-dimensional nanoscaffold for the coordination of electron transfer mediators, this work opens up new possibilities of incorporating functionalized DNA architectures as guest molecules to nanoporous conductive metal oxides for applications such as photovoltaics, sensors, and solar fuel cells.

Construction and Structure Determination of a Three-Dimensional DNA Crystal

Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.

Unidirectional Scaffold-Strand Arrangement in DNA Origami†

Since the origin of DNA nanotechnology over 30 years ago, branched DNA tiles have been developed to construct a variety of DNA nanostructures. Among the unique building blocks that have been demonstrated, rigid, antiparallel double-crossover (DX) tiles have had great significance as both the unit motif in the first two-dimensional DNA crystal reported and as the basic repeating unit in most DNA origami structures. In contrast, parallel DX tiles have yet to be developed, despite that they were initially reported more than 20 years ago, most likely because their assembly yields are not comparable to anti-parallel DX tiles. Herein we demonstrate construction of DNA origami architectures based on modified parallel DX tiles, in which a single-crossover linkage, rather than reciprocal crossovers, is present at each junction point between neighboring helices. The yields of these novel origami structures are similar to their counterparts that are constructed based on antiparallel DX tile units containing double-reciprocal crossovers at each junction point. We demonstrate that a unidirectional arrangement of the scaffold strand can be used in the assembly of a variety of 2D and 3D DNA origami. This new design will greatly expand the diversity of DNA origami achieved and enable their further assembly into larger structures. The DX tiles, defined by Fu and Seeman in 1993, each contain two independent crossover points that join two adjacent DNA helices to form a rigid tile with the axes of the helices between the crossovers arranged roughly coplanar. Five types of DX tiles were initially reported: two antiparallel and three parallel molecules that differed in the relative orientation of the helical domains, the arrangement of the constituent strands, and the distance between the crossover points. DAE and DAO molecules (double-crossover antiparallel molecules with an even number or odd number of half turns between crossover points along the same two helices) were later applied to DNA origami designs with great success, largely because these tiles contain two unperturbed DNA strands of opposing polarity that are easily linked to form a scaffold strand. In that case, the antiparallel scaffold is wound back and forth in a raster fill pattern to form a particular shape, through the use of hundreds of staple strands. In contrast, DPE (double-crossover parallel molecules with an even number of half turns between crossover points) molecules were not thoroughly explored for the construction of higher-order structures. Compared to antiparallel DX molecules, parallel DX molecules are generally regarded as less stable presumably because of stronger electrostatic repulsion between the opposing DNA backbones. Later, Sherman and co-workers reported that strand end-pinning and misfolding caused by the structural bias of nominally flexible junctions might affect the proper structural formation of parallel DX molecules. Breaking the continuity of one of the two strands that comprise each junction in a DPE tile at the junction point will produce parallel, double-helical units with single-crossover linkages between the helices. Such modified DPE tiles provide a direct solution to both the possible steric hindrance at the crossover points and the kinetic trap noted by Sherman and co-workers. Herein, we demonstrate that the unperturbed strands in these modified DPE tiles can be linked to form a long scaffold strand that is directed by a collection of staple strands to form DNA origami structures. The scaffold strand in adjacent helices possesses the same 5’–3’ polarity and we therefore refer to these structures as parallel helix (PH) origami. Assembling PH origami required that we develop unique, coiled-scaffold folding paths rather than the typical raster fill patterns adopted by the scaffold in DNA origami structures with antiparallel helices (AH). We also designed and assembled hybrid DNA origami structures that contain both parallel and antiparallel scaffold regions. The DAE, DPE, and modified DPE tiles are shown in Figure 1A–C for comparison. The single-stranded DNA (depicted in gray) remains unperturbed at all junction points and the relative polarities of these two strands determine whether the molecules are classified as antiparallel or parallel. The remaining DNA strands are shown in various colors and correspond to the staple strands that direct/hold the linear strands in a coplanar arrangement. The staple strands in the antiparallel tiles (Figure 1A) reverse direction at each crossover point while those in the parallel tiles (Figure 1B) do not. Note that the backbones of the strands at the crossover points in the parallel tiles are directly opposite one another, which imparts a certain degree of instability to the parallel tiles. Thus, in the modified DPE tiles (Figure 1C), a single-crossover linkage replaces the typical reciprocal [*] Dr. D. Han, S. Jiang, A. Samanta, Prof. Y. Liu, Prof. H. Yan The Biodesign Institute, Arizona State University Tempe, AZ 85287 (USA) E-mail: hao.yan@asu.edu