ABSTRACT
Auxetic structures are unique with a negative Poisson’s ratio. Unlike regular materials, they response to external loading with simultaneous expansion or compression in all directions, rendering powerful properties advantageous in diverse applications from manufacturing to space engineering. The auxetic behaviors are determined by structural design and architecture. Such structures have been discovered in natural crystals and demonstrated synthetically with bulk materials. Recent development of DNA-based structures has pushed the unit cell size to nanometer scale. DNA nanotechnology utilizes sequence complementarity between nucleotides. By combining sequence designs with programmable self-assembly, it is possible to construct complex structures with nanoscale accuracy and to perform dynamic reconfigurations. Herein, we report a novel design of auxetic nanostars with sliding behaviors using DNA origami. Our proposed structure, inspired by an Islamic pattern, demonstrates a unit cell with two distinct reconfigurations by programming directed sliding mechanisms. Compared to previous metamaterials, the DNA nanostars show an architecture with tunable auxetic properties for the first time. We envision that this strategy may form the basis of novel metastructures with adaptability and open new possibilities in bioengineering.
INTRODUCTION
Auxetic metamaterials are synthetic architectures that respond to external loadings in a unique manner. Regular materials under compressive (or extensive) forces will expand (or contract) in orthogonal directions. In contrast, auxetic structures deform simultaneously in all directions. Poisson’s ratio measures such a property which describes deformation behaviors quantitatively: where εx and εy are strains, and x is the loading direction.1 Ordinary materials thus have positive Poisson’s ratios, while auxetic structures show negative values. Architected metamaterials have several distinct advantages including light-weight high-strengths2-4 and the ability to absorb impact forces5,6. They are widely used in design and manufacturing ranging from commodities (e.g., shoes and clothes) to aerospace engineering.7-9
Most metamaterials have periodic cellular structures and their auxetic behaviors arise from unit cell designs, which will deform in unison upon external forces. The unit cells range over various length scales. For example, naturally occurring crystals such as α-cristobalite and cubic metals demonstrate auxetic properties with unit cells of sub-nanometer sizes.10-12 Conventional auxetics have been manufactured with metals,13 polymers,14 and other materials15 in larger scales from microns to centimeters. There is a lack of studies in auxetic metamaterials at the nanoscale.16 Recently, Li et al. bridged this gap in lengthscale.17,18 In their studies, nanoscale auxetic units were constructed using DNA origami exploiting sequence complementarity of DNA molecules. DNA self-assembly has been developed as a powerful bottom-up strategy with excellent programmability and precision19 and demonstrated for complex architectures,20-24 reconfigurable designs,25-28 and dynamics processes.29-32 Their origami architectures were designed with ‘jack’ edges, whose lengths were adjusted by two-step DNA reactions for global structural transformations. The ‘chemical deformation’ resulted in negative Poisson’s ratio (NPR) behaviors.
While the work opened new opportunities for nanoscale metamaterials, the structures were limited in that their auxetic deformations were pre-determined and could not change. In fact, this invariable mechanics is also similar for most metastructures at other lengthscales. Herein we ask if it is possible to program distinct pathways of NPR reconfigurations. To achieve such tunable auxetic properties, we propose a novel design of origami-based DNA nanostars that can reconfigure in different directions upon external loading via chemical deformations. The DNA architecture was inspired by one of the Islamic pattern designs that have been used in arts and buildings. Our DNA origami design consists of 3 nanostars that can slide against each other in two distinct directions, thus resulting in two NPR values. We have investigated their structures and behaviors with coarse-grained molecular dynamics (MD) simulations and atomic force microscopy (AFM). This work opens a new horizon towards smart materials with adaptive mechanical properties for applications involving complex and ever-changing environments.
EXPERIMENTAL SECTION
Computer-Aided Origami Design
The design of the wireframe DNA origami was conducted using caDANano2.33,34 The edges were designed as four double-stranded (ds) bundles with staples routed around to increase rigidity. The vertices were designed with single-stranded (ss) DNA to allow flexibility in turning angles. In our design, regular right-handed B-form DNA was assumed to have a rise on the axis of 0.332 nm/bp. Positional staples on edges used for performing sliding actions contained binding domains of 10 nt. For strand displacement reactions, 8-nt ss-toeholds were added at one end of the strands. Detailed sequence information is presented in Table S1 and S2.
DNA Origami Synthesis
All DNA origami structures were prepared at a concentration of 5 nM scaffold DNA with 4× staple strands in 1× TAE buffer solution (containing 40 mM trisaminomethane, 20 mM acetic acid, and 1 mM ethylenediaminetetraacetic acid (EDTA) disodium salt with pH ∼ 8). A final concentration of 6 mM Mg2+ was provided. The mixture was put in a BIO-RAD S1000 Thermal Cycler under a custom-designed thermal cycle. The sample was heated to 75 °C for 18 minutes and then cooled down at a ramping rate of -0.1 °C/15 mins until it reached 25 °C. The solution was then stored at 4 °C before further experiments.
Sliding Experiment
Toehold-mediated strand displacement was used for origami sliding motions. The sliding was performed in two steps: (1) removal of previous positional staples and (2) insertion of new positional staples. The first step was carried out by adding 50× releaser strands and incubating the mixture in the thermal cycler at 40 °C for 12 hours before cooling down to 25 °C at a rate of - 0.1°C/6 seconds. Then the mixture was purified by using centrifugal filters (Amicon) to remove excess strands. For centrifugation, approximately 55 μL of DNA origami mixture was added to the filter and additional TAE buffer with 6 mM Mg2+ was added to reach a final volume of 500 μL. Then the mixture was centrifuged at 5,000 rpm for 3 minutes and the solution remaining in the filter was retrieved. After the first step, the origami structures were released to an undefined position. In the second step, new staples defining the relative positions of the three-stars were added at a 10-fold higher concentration. The mixture was subsequently reannealed in the thermal cycler at 40 °C for 12 hours and then cooled down to 25 °C at a rate of -0.1°C/6 seconds. The sample was then stored at 4 °C for further measurements or experiments.
Coarse-grained MD Simulations
We performed MD simulations to evaluate the DNA design and verify structural integrity. In the coarse-grained MD model, pseudo-atoms are used to represent a group of atoms in order to reduce the complexity of calculations. For a predetermined simulation time, particles representing the structures follow designated interactions to demonstrate the development of the system dynamically. In our study, oxDNA platform35,36 was used for computing the equilibrium conformations of the origami structures. In the oxDNA model, DNA strands are represented by a string of rigid nucleotides, where multiple interactions are taken into consideration including sugar-phosphate backbone connectivity, excluded volume, hydrogen bonding, etc. To perform the computation, we used the designed static structures from caDNAno and converted them into topology and configuration files using real sequence information as initial conformations in oxDNA. The environmental parameters were set based on the experimental conditions. Temperature was the same as in experiments, while magnesium concentration of [Mg2+] = 6 mM was replaced by [Na+] = 0.5 M due to limited options in the computational platform. The initial configurations of DNA origami were first relaxed by setting the phosphate backbone connection 10 times stronger to pull the edges to their places. Then, a threshold of 3% relative fluctuations was set to start the second stage of simulations, where the structures were computed for more than 106 steps to reach an equilibrium state for observation. For each conformation, the simulation took about two days. The resulting structures were visualized with oxView37 and the quantitative measurements were performed on the platform. The simulated edges have a length of approximately 41 nm, a thickness of around 4 nm, and the angle at the vertices of ∼ π/4, which agree well with our design parameters.
AFM Imaging
The planar origami structures were characterized by AFM in air. The samples prepared were deposited on mica surfaces for measurements. The target sample was diluted to ∼0.5 nM in 1× TAE buffer at 6 mM Mg2+. A 10 μL aliquot of the diluted sample was added onto a freshly peeled mica surface and deposited for 5 minutes. Then the liquid was blown away by compressed air. Approximately 80 μL of DI water was added to the mica surface afterward and immediately blown away to avoid salt accumulation. AFM imaging was carried out with a Bruker Dimension Icon AFM using ScanAsyst-Air probes in the Peak-Force tapping mode. We followed the same procedure for sample preparation and measurement as previously described.17 The lengths of edges and the angles at vertices were measured and matched with our designs.
RESULTS AND DISCUSSION
Design of Architected Metastructures from DNA
Figure 1a shows the proposed auxetic design inspired by an Islamic geometric pattern. The Islamic pattern consists of periodic 2D arrays of large and small stars shown in blue and orange. The unit cell includes one small and two large stars which can slide along each other’s edges as depicted in Figure 1b. Upon loadings on horizontal directions, the sliding mechanism results in auxetic reconfigurations globally.38 This Islamic pattern design allows only one reconfiguration due to the limitations on sliding directions. By modifying the structure, we propose a new design which enables multiple reconfigurations with a single structure. To model the system, we simplified the geometric pattern to a design of sliding 4-point stars with identical sizes as illustrated in Figure 1c and 1d. The angles at four vertices of each star are designed to be π/4. Sliding of neighboring blue stars along the edges of the orange stars simultaneously in horizontal directions will result in a contraction of the structure both horizontally and vertically (Figure 1c). Note that the shrinkage in x and y directions will not be equal. By calculating the displacements in both directions, we can find the Poisson’s ratio:
In contrast, a completely different structure will emerge if one of the two adjacent blue stars slides vertically, while the other moves horizontally. In this case, the entire structure will rotate for an angle of π/8, as shown in Figure 1d. This centrosymmetric movement will result in an auxetic behavior as well as a global rotation. Calculating the displacement in both x and y directions yields: The rotation with ν = -1 is consistent with that of other centrosymmetric designs such as rotating squares.17 Overall, the auxetic properties of the sliding stars can be modulated by directing the sliding behaviors into a desired combination.
We further simplified the design into a unit cell of three (2 blue and 1 orange) stars which can be constructed by DNA origami (Figure 1e and 1f). Each edge of the stars is designed to have a length of approximately 40 nm long and the vertices have angles of π/4. These design parameters were later confirmed by measurements from oxDNA simulations and AFM imaging. Three sliding routes are made available for each unit cell. The horizontal sliding of the left blue star is named Route 1, while its vertical movement is termed Route 2. Route 3 represents the horizontal sliding of the right blue star. Note that the vertical sliding of the right blue star is omitted due to symmetry. By programming the sliding behaviors, we can generate two combinations (Route 1, 3 or Route 2, 3) between the routes, which will give rise to two distinct auxetic properties. In a periodic cellular structure, the distinct Poisson’s ratios indicate the difference in mechanical properties.
In this work, a unit cell of the structure capable of the sliding behaviors is demonstrated with a wireframe DNA origami method. This strategy uses edges and multi-arm joints to represent geometric patterns.39,40 In wireframe origami, edges are composed of dsDNA bundles for structural integrity and are connceted by ssDNA at joints for flexibility. This method is efficient for material usage and allows for designing larger structures with a limited number of nucleotides (nt).41 To enhance the stiffness of the edges of the stars, we followed the principles previously described by Li et al.17 They demonstrated that edge thickness t must be sufficiently large for a given length L and ss-joints at vertices must experience a certain level of tension (termed joint stretch η) in order to avoid significant flexure or distortion during reconfiguration. Our edges and joints are designed with four duplex bundles (t/L ≈ 0.1) and a stretch level of η ≈ 55% to meet the design requirements. In addition, the edges are designed in a honeycomb arrangement to avoid internal strains in the structure as shown in Figure 2a. Here, the long blue arrows represent the routing of scaffold sequences, and the short gray lines denote staple strands. A three-dimensional molecular model from oxDNA is also shown in Figure S1. Given the design parameters of our proposed structure, single origami may not provide enough nucleotides. Therefore, we adopted a double scaffold strategy developed by Dietz and coworkers42 who used multiple orthogonal scaffolds with minimal interferences for larger origami structures. Figure 2(c)-(e) illustrates the routing of our origami design, where two kinds of scaffolds are differentiated by colors: 9072-nt scaffold (named 9k scaffold for simplicity) provided by the Dietz group is shown in blue color and commercially purchased 8064-nt scaffold (8k scaffold) is shown in orange color. The three-star structure is composed in a manner that each scaffold winds into one and a half stars, and the two scaffolds are connected by connecting staples to form the complete three stars. It is worth noting that the left one and a half stars in blue have a one-way connection given the linearity of the 9k scaffold, while a two-way connection is used for the right one and a half stars in orange as the 8k scaffold is circular. To differentiate between the left and right stars, a ss-loop of scaffold is designed on the top of the right star.
AFM imaging characterized the assembled DNA units. The structures from a single scaffold (e.g., 9k or 8k strands) are tested initially with a scaffold concentration of 5 nM in 1× TAE buffer with 6 mM Mg2+. Both scaffolds formed half structures as designed; the 9k scaffold shapes the left star and half of the middle star (Figure 2(f)), while the 8k scaffold forms the right star and the other half of the middle star with a small loop at the top of the right star (Figure 2(g)). Finally, a whole structure assembled from both scaffolds is shown in Figure 2(f). Note that the right and left stars are connected with the middle star by unpaired segments of the scaffolds as indicated by 3 lines (one in blue and two in orange) in Figure 2(e). However, the relative positions between the stars are not determined (i.e., undefined position). Their locations are close to each other but random due to the deposition on mica surfaces – positional linker strands will determine the exact positions between the stars (vide infra). The structures are also examined with agarose gel electrophoresis. As shown in Figure S2, the half structures with the 8k scaffold or the 9k scaffold as well as the full three-star structures all have clear bands in the gel where the 8k-structure moves the fastest and the complete three stars were the slowest corresponding to their molecular weights.
Demonstration of Auxetic Sliding Behaviors
With well-built structures, we designed sliding behaviors of the auxetic stars via toehold-mediated strand displacement as illustrated in Figure 3. For each route, the three stars are arranged to start with undefined position, where no binding is provided to determine relative positions between stars (state (i)). On one of two edges sliding against each other, three locations are modified with a 10-nt extension as binding entities (as shown in edge 1). Initially, the binding strands on the opposite edge (edge 2) are not provided, thus the stars remain in an undefined position as shown as state (i) in Figure 3(b). Then, each route can be programmed to take three positions to guide the sliding behaviors. To initiate the sliding, staples on edge 2 with toeholds are replaced by new staples (P1 staples in green color) via strand displacement. This allows the stars to initially bind to each other at the opposite vertices (position 1, state (ii)). Next, the binding sequences (shown in green) can be replaced with new sets of linker strands via toehold mediated strand displacement to direct the edges to overlap halfway where the stars slide more into each other (position 2, state (iii)). The binding staples for position 2 are shown in red color. Finally, with the same strand displacement mechanism, the edges can be programmed to slide to fully overlap with each other resulting in a configuration of position 3 (state (iv)). The staple designs for position 3 are shown in purple color. With the strand displacement mechanism used, this process may also be reversed to direct the stars to move back from position 3 to position 2 and then to position 1.
The sliding mechanism is firstly tested on individual routes to ensure independent sliding beahviors. All routes start from the undefined positon as presented in Figure 2(e) and (h). On the edges, staples used for determining the relative postions are designed with a 10-nt toehold. The reconfigurations take place in two steps: (1) previous staples are displaced by adding complementary releaser strands. These strands bind to the staples defining positions on the edges and remove them. (2) The undefined structures are reannealed with new positional staples. These new staples relocate the edges to a new relative position as designed.
For each route and each position, we performed MD simulations using oxDNA to verify the formation of the structure. Figure 4 presents the computed structures and corresponding, reconfigured origami units from AFM imaging. Figure 4(a)-(c) shows the sliding movement on route 1, where the left star starts from the tip of the vertices (position 1) and moves to the midpoint (position 2) and finally to the center of the middle star (position 3) as the staples are replaced sequentially. Route 2 sliding is demonstrated similarly as shown in Figure 4(d)-(f). The left star on route 2 slides vertically along the edge of the middle star. Starting from the corner (position 1), the star slides to a lower position (position 2) and reaches the destination at the junction in the center (position 3). Figure 4(g)-(i) shows the sliding on route 3, where the right star begins at the correct position (position 1) and then moves horizontally from right to left, reaching the middle point and the center point in the end (positions 2 and 3, respectively). More experimental results are included in Figure S3. For all the positions, both the experimental and simulation results show correct formation of the structures, and the relative positions of the stars are placed as designated.
To generate tunable auxetic motions, we have explored the route combinations. As demonstrated above, the right star can slide in horizontal directions while the left star moves to two different routes upon the addition of replacement sequences (designed for intended routes). If routes 1 and 3 are selected, for example, the unit cell will translate into a Poisson’s ratio of -0.414. The combination between routes 2 and 3 will result in ν = -1. Here, the formation of the two combinations is confirmed both with oxDNA simulation and AFM imaging.Figure 5(a)-(c) present the combination between routes 1 and 3. Both left and right stars move horizontally along the edges to the middle star and meet in the center point (routes 1 and 3) with specific displacement sequences added. The unit cell shrinks in both horizontal and vertical directions. The reverse movement is also possible as shown in Figure 5(d)-(f). Two outer stars are directed to slide horizontally from the center (position 3) to sequentially move to position 2 and then to position 1.
This results in expansion not only horizontally but also vertically, restoring the initial conformation. Figure 5(g)-(i) shows programmed sliding on route 2 and 3, where the stars start from position 1 and slide toward the center via position 2 and 3 sequentially. Similarly, Figure 5(j)-(l) shows sliding on route 2 and 3 from the center to the vertices going in a reverse direction from postion 3 to position 1. Additional experimental results for these combinations are shown in Figure S4. Since the sliding mechanism is based on the two step DNA reactions, it is possible to move directly to desired locations rather than sliding at sequential positions. Some examples of those positionings are demonstrated in Figure S5. The results demonstrate that our designed DNA origami can perform auxetic behaviors with tunable properties as well as reversibility as a one single unit cell structure.
CONCLUSIONS
In this work, we proposed and demonstrated a novel design of metastructures with tunable auxetic properties by using DNA origami. In our design, a unit cell of three stars is arranged such that the left and right stars can slide along the edges of the middle star. Given the symmetry, three independent routes are used. The left star is designed to have two sliding orientations, horizontally and vertically, respectively as routes 1 and 2. The right star slides along the horizontal direction (route 3). A strand displacement mechanism is used to program the sliding behaviors. By using replacement strands, relative motions between the left and right stars can be directed in two combinations. Routes 1 and 3 will result in a Poisson’s ratio of ν = -0.414, while the combination of routes 2 and 3 will demonstrate a centrosymmetric rotation with ν = -1. In periodic structures, this can give rise to auxetic materials with tunable mechanical behaviors. Numerical simulations and experimental demonstrations show that our proposed origami structure can perform the designed sliding movement on each route individually. Combinations between the routes can be programmed with tunable auxetic behaviors with reversibility.
Given the biocompatibility and versatility, tunable DNA metastructures may be designed to interact with biological or chemical environments in a programmed manner; for example, as force-responsive sensors for biophysical studies and targeted drug delivery carriers.26,30,43,44 We envision that DNA-based structural design can be used to build metamaterials with multimode reconfigurability that can detect and respond to complex surroundings and show adaptive mechanical properties. For example, combined with aptamers, i-motifs or enzymatic reactions,45-50 DNA-based metamaterials will have strong potential to respond to environmental changes (e.g., pH change or the presence/absence of target molecules) with mechanical responses (e.g., changing stiffness, young’s modulus, etc). The strategy may be extended to various applications such as wound healing and vascular scaffolds. In addition, novel DNA materials may be developed as biological or chemical sensors, which respond to cues with reconfigurations or mechanical changes, thus opening new opportunities.
CONFLICTS OF INTEREST
There are no conflicts to declare.
ACKNOWLEDGEMENTS
The authors thank Dr. Hendrik Dietz, Max Honemann, and Michael Pinner at the Technical University of Munich in Germany for providing the 9072-nt scaffold strands. This work was supported by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES) under award no. DE-SC0020673 (concept, design, and experiment) and the U.S. National Science Foundation under award no. 2025187 and 2134603 (computation).