Shuoxing Jiang

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.

Complex wireframe DNA origami nanostructures with multi-arm junction vertices

Structural DNA nanotechnology and the DNA origami technique, in particular, have provided a range of spatially addressable two- and three-dimensional nanostructures. These structures are, however, typically formed of tightly packed parallel helices. The development of wireframe structures should allow the creation of novel designs with unique functionalities, but engineering complex wireframe architectures with arbitrarily designed connections between selected vertices in three-dimensional space remains a challenge. Here, we report a design strategy for fabricating finite-size wireframe DNA nanostructures with high complexity and programmability. In our approach, the vertices are represented by n × 4 multi-arm junctions (n = 2-10) with controlled angles, and the lines are represented by antiparallel DNA crossover tiles of variable lengths. Scaffold strands are used to integrate the vertices and lines into fully assembled structures displaying intricate architectures. To demonstrate the versatility of the technique, a series of two-dimensional designs including quasi-crystalline patterns and curvilinear arrays or variable curvatures, and three-dimensional designs including a complex snub cube and a reconfigurable Archimedean solid were constructed.

Mapping the Thermal Behavior of DNA Origami Nanostructures

Understanding the thermodynamic properties of complex DNA nanostructures, including rationally designed two- and three-dimensional (2D and 3D, respectively) DNA origami, facilitates more accurate spatiotemporal control and effective functionalization of the structures by other elements. In this work fluorescein and tetramethylrhodamine (TAMRA), a Förster resonance energy transfer (FRET) dye pair, were incorporated into selected staples within various 2D and 3D DNA origami structures. We monitored the temperature-dependent changes in FRET efficiency that occurred as the dye-labeled structures were annealed and melted and subsequently extracted information about the associative and dissociative behavior of the origami. In particular, we examined the effects of local and long-range structural defects (omitted staple strands) on the thermal stability of common DNA origami structures. The results revealed a significant decrease in thermal stability of the structures in the vicinity of the defects, in contrast to the negligible long-range effects that were observed. Furthermore, we probed the global assembly and disassembly processes by comparing the thermal behavior of the FRET pair at several different positions. We demonstrated that the staple strands located in different areas of the structure all exhibit highly cooperative hybridization but have distinguishable melting temperatures depending on their positions. This work underscores the importance of understanding fundamental aspects of the self-assembly of DNA nanostructures and can be used to guide the design of more complicated DNA nanostructures, to optimize annealing protocol and manipulate functionalized DNA nanostructures.

Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional DNA Nanostructures

We use single-particle fluorescence resonance energy transfer (FRET) to show that organizing oligonucleotide probes into patterned two-dimensional arrays on DNA origami nanopegboards significantly alters the kinetics and thermodynamics of their hybridization with complementary targets in solution. By systematically varying the spacing of probes, we demonstrate that the rate of dissociation of a target is reduced by an order of magnitude in the densest probe arrays. The rate of target binding is reduced less dramatically, but to a greater extent than reported previously for one-dimensional probe arrays. By additionally varying target sequence and buffer composition, we provide evidence for two distinct mechanisms for the markedly slowed dissociation: direct hopping of targets between adjacent sequence-matched probes and nonsequence-specific, salt-bridged, and thus attractive electrostatic interactions with the DNA origami pegboard. This kinetic behavior varies little between individual copies of a given array design and will have significant impact on hybridization measurements and overall performance of DNA nanodevices as well as microarrays.

Steric crowding and the kinetics of DNA hybridization within a DNA nanostructure system

The ability to generate precisely designed molecular networks and modulate the surrounding environment is vital for fundamental studies of chemical reactions. DNA nanotechnology simultaneously affords versatility and modularity for the construction of tailored molecular environments. We systematically studied the effects of steric crowding on the hybridization of a 20 nucleotide (nt) single-stranded DNA (ssDNA) target to a complementary probe strand extended from a rectangular six-helix tile, where the number and character of the surrounding strands influence the molecular environment of the hybridization site. The hybridization events were monitored through an increase in the quantum yield of a single reporter fluorophore (5-carboxyfluorescein) upon hybridization of the 20-nt ssDNA, an effect previously undocumented in similar systems. We observed that as the hybridization site moved from outer to inner positions along the DNA tile, the hybridization rate constant decreased. A similar rate decrease was observed when noncomplementary single- and double-stranded DNA flanked the hybridization site. However, base-pairing interactions between the hybridization site of the probe and the surrounding DNA resulted in a reduction in the reaction kinetics. The decreases in the hybridization rate constants can be explained by the reduced probability of successful nucleation of the invading ssDNA target to the complementary probe.

Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold.

Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.

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

Controlled nucleation and growth of DNA tile arrays within prescribed DNA origami frames and their dynamics.

Controlled nucleation of nanoscale building blocks by geometrically defined seeds implanted in DNA nanoscaffolds represents a unique strategy to study and understand the dynamic processes of molecular self-assembly. Here we utilize a two-dimensional DNA origami frame with a hollow interior and selectively positioned DNA hybridization seeds to control the self-assembly of DNA tile building blocks, where the small DNA tiles are directed to fill the interior of the frame through prescribed sticky end interactions. This design facilitates the construction of DNA origami/array hybrids that adopt the overall shape and dimensions of the origami frame, forming a 2D array in the core consisting of a large number of simple repeating DNA tiles. The formation of the origami/array hybrid was characterized with atomic force microscopy, and the nucleation dynamics were monitored by serial AFM scanning and fluorescence spectroscopy, which revealed faster kinetics of growth within the frame as compared to growth without the presence of a frame. Our study provides insight into the fundamental behavior of DNA-based self-assembling systems.

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

Kinetics of DNA tile dimerization.

Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile–tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency.