DNA Origami: Scaffolds for Creating Higher Order Structures

Review Cite This: Chem. Rev. 2017, 117, 12584-12640

pubs.acs.org/CR

DNA Origami: Scaffolds for Creating Higher Order Structures Fan Hong, Fei Zhang, Yan Liu,* and Hao Yan* The Biodesign Institute and School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States ABSTRACT: DNA has become one of the most extensively used molecular building blocks for engineering self-assembling materials. DNA origami is a technique that uses hundreds of short DNA oligonucleotides, called staple strands, to fold a long singlestranded DNA, which is called a scaffold strand, into various designer nanoscale architectures. DNA origami has dramatically improved the complexity and scalability of DNA nanostructures. Due to its high degree of customization and spatial addressability, DNA origami provides a versatile platform with which to engineer nanoscale structures and devices that can sense, compute, and actuate. These capabilities open up opportunities for a broad range of applications in chemistry, biology, physics, material science, and computer science that have often required programmed spatial control of molecules and atoms in three-dimensional (3D) space. This review provides a comprehensive survey of recent developments in DNA origami structure, design, assembly, and directed self-assembly, as well as its broad applications.

CONTENTS 1. Introduction 2. Fundamentals of DNA Tile Design 3. Different Designs of DNA Origami Structures 3.1. Single-Layer Designs 3.1.1. Tightly Packed Single-Layered DNA Origami Designs 3.1.2. Wireframe Single-Layered Designs 3.2. Multilayered Designs 4. Scaling Up DNA Origami Structures 4.1. Origami with Extended Scaffolds 4.2. Origami with Complex Staples 4.3. Hierarchical Assembly of DNA Origami 4.3.1. Hybridization 4.3.2. Base Stacking 4.4. Surface-Assisted Self-Assembly 4.5. DNA Origami-Templated Tile Assembly 5. Mechanisms of DNA Origami Self-Assembly 5.1. Folding Behaviors of DNA Origami 5.2. Thermodynamics and Kinetics of the SelfAssembly Process for Higher-Order DNA Origami Self-Assembly 5.3. Mechanic Properties of DNA Origami 6. Applications of DNA Origami 6.1. Molecular Robotics 6.1.1. DNA Walkers 6.1.2. Reconfigurable DNA Origami Devices 6.2. DNA Origami Templated Architectures and Nanomaterial Functionality 6.2.1. DNA Origami Templated Architectures 6.2.2. Chiral Plasmonic Structures 6.2.3. Plasmonic Hotspots 6.3. Biophysical Studies with DNA Origami 6.3.1. DNA Origami-Aided Protein Function Study

© 2017 American Chemical Society

6.3.2. DNA Origami-Based Single-Molecule Force Spectroscopy 6.3.3. DNA Origami Aided Protein Structure Determination 6.3.4. DNA Origami Nanopores 6.4. Drug Delivery with DNA Origami 6.5. Bioanalysis with DNA Origami 6.5.1. Nucleic Acid Analysis 6.5.2. Distance-Dependent Interactions 6.6. Programming the Structure and Conformation of Non-DNA Materials 6.7. Bridging Top-down and Bottom-up Fabrications 6.7.1. Molecular Lithography with DNA Origami 6.7.2. Directed Positioning of DNA Origami Structures 6.8. Self-Assembled Enzymatic Nanoreactors 6.9. Self-Assembled Light Harvesting Systems 7. Conclusions and Outlook Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments References

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1. INTRODUCTION Molecular self-assembly exists everywhere in the natural world and plays crucial roles in biology, chemistry, and material Special Issue: Bioinspired and Biomimetic Materials

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Received: December 15, 2016 Published: June 12, 2017 12584

DOI: 10.1021/acs.chemrev.6b00825 Chem. Rev. 2017, 117, 12584−12640

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Figure 1. Fundamental structural motifs used in DNA nanotechnology (A) The immobile Holliday junction. (B) A proposed self-assembled 3D DNA crystal for protein organization.36 (C) Multiarm DNA structural motifs with different numbers (2, 3, 4, and 6) of Holliday junctions tethered together. Their 2D lattice assemblies were imaged by AFM and are shown below.42−45 Images were reproduced with permission from ref 42. Copyright 1998 Macmillan Publishing Ltd. Images were reproduced with permission from ref 43. Copyright 2003 Association for the Advancement of Science (AAAS). Images were reproduced from ref 44. Copyright 2005 American Chemical Society. Images were reproduced from ref 45. Copyright 2006 American Chemical Society. (D) A DNA six-helix bundle motif with helices aligned with a hexagonal cross-section.46 The hexagonal schematic shows the end view of a 2D lattice, which was assembled with six-helix bundle units, where the vertices represent the helix axis. The AFM image of the corresponding 2D lattice is shown below. Images were reproduced from ref 46. Copyright 2005 American Chemical Society. (E) A long scaffold-directed nucleation assembly of a DNA DX tile complex.56 Five DX tiles were aligned together, sharing a long scaffold (red). Structural features (blue dots) can be added to the tiles individually to display a “barcode”. These linear barcodes were linked together, end-to-end and side-to-side, to form a 2D lattice to display repeating patterns (see the AFM image shown below). A well-defined 2D pattern (with detectable surface features) using a long scaffold-directed selfassembly was also proposed. Images were reproduced with permission from ref 56. Copyright 2003 National Academy of Sciences. 12585


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science.1,2 The interactions involved in the self-assembly process are generally weak and noncovalent interactions, such as hydrogen bonding, hydrophobic interactions, and van der Waals interactions, etc. For instance, lipid molecules form micelles in water, single chains of amino acids fold into functional proteins, chains of DNAs (DNA) form double helices with their complementary sequences, and single chains of ribonucleic acid (RNA) fold into complex tertiary structures. By learning how these interactions guide the sophisticated self-assembly processes, one may utilize the inherent rules of cellular machineries to synthesize designer molecular structures or devices to perform desired functions. A variety of molecules, such as peptides,3 proteins,4 DNA,5 lipids,6 organic molecules,7 and hybridization of metal ions and organic molecules8 have been used as self-assembly building blocks. Among these molecules, DNA is one of the most attractive candidates for nanoconstruction, due to its unique properties. First, the Watson−Crick base pairing is highly predictable and specific. The interactions between DNA strands are governed by the pairing of nucleotide (nt) bases, where adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). On the basis of this simple rule, one can easily program the interactions between DNA oligos. Second, the wellstudied molecular conformation of DNA facilitates the creation of designer DNA structures. A typical B-type DNA is a righthanded double helix with a diameter of ∼2 nm and ∼3.4 nm per helical turn. The measured twist in this molecule is ∼34.6° per base pair (bp). These parameters allow DNA nanostructures to be precisely engineered at a nanometer scale. Third, the convenient synthesis of DNA oligonucleotides (oligos) facilitates the use of DNA as building materials.9 DNA synthesis is now largely automatic, allowing thousands of different oligos to be synthesized in a short amount of time (hours to days). Additionally, a number of enzymatic manipulations further decrease the cost of DNA synthesis. Thanks to these advances, the field of structural DNA nanotechnology has developed rapidly, and a variety of DNA nanostructures have been designed and successfully constructed.10−18 The technique of DNA origami utilizes hundreds of short DNA oligos, typically 15− 60-nt long, called staple strands to fold a long single-stranded scaffold oligo into a desired target shape by one-pot annealing. The use of DNA origami has dramatically improved the complexity of DNA designs and has led to numerous uses in various fields, including electronics,19 photonics,20 artificial enzymatic reaction networks,21 and so forth.9,16,22−35 This review will discuss the structural design and self-assembly of DNA origami and its broad applications.

associations (Figure 1, panels A and B).36 After successfully constructing an immobile 4-arm junction,38 Seeman and colleagues then assembled 5-, 6-, 8-, and 12-arm junctions, demonstrating that DNA junction molecules can be highly branched.39,40 However, because these multiarm junctions are flexible and their conformations are difficult to predict, they are unsuitable to use as the basic structural units with which to assemble higher-order periodic lattice structures. To overcome this problem, they constructed a DNA double crossover (DX) molecule by joining two 4-way junctions into one structural motif. This resulted in a rigid DNA molecule with aligned, parallel, or antiparallel double helices, which were held together by two crossovers.41 In 1998, the first higher-order DNA lattice structure, a periodic two-dimensional (2D) crystalline, was successfully constructed from the DX molecule (Figure 1C).42 Yan, LaBean, and co-workers43 extended the DX tile into a rigid, branched, 4 × 4 tile with four arms pointing in four directions. Similar to the DX molecule that uses a central strand to link two Holliday junctions end to end, the 4 × 4 tile uses a long central strand to link four Holliday junctions together into a 4-fold symmetry. T4 loops were placed at each of the four corners of the central strand to reduce the probability of stacking interactions between adjacent arms. Nanoribbons or 2D gridlike lattices were successfully assembled by providing the 4 × 4 tile with proper sticky end association sites. Inspired by the DX and 4 × 4 tile, DNA units, such as the three-point star tile (Figure 1C),44 sixpoint star tile (Figure 1C),45 and the six-helix bundle (Figure 1D),46 were successfully constructed. These molecules can further self-assemble to form 2D arrays with different patterns.47 Furthermore, 3D macroscopic crystals have been successfully created via the assembly of a rationally designed tensegrity triangle motif.48 These 2D or 3D DNA crystals allow for the precise organization of periodic nanoparticles,49,50 quantum dots,51 and biomolecules.52−55 In addition to assembling tiles into periodic DNA structures, Reif, Yan, LaBean, and colleagues described their use of a long scaffold to direct the nucleation of DNA tiles into complicated aperiodic structures called barcode DNA lattices (Figure 1E).56 The long scaffold was constructed by ligating shorter synthetic oligos, in order to provide binding sites for shorter strands and to form a large individual complex with a barcode pattern that could be further assembled into latticed structures if sticky ends were provided. Shih et al. published a method whereby they constructed an octahedron with a 1.7-kilobase single-stranded DNA. The structure embeds five double-crossover struts and seven paranemic-crossover struts, which are connected by six four-way junctions at the vertexes.57 The formation of the structure proceeds via two steps: (1) five 40-nt staples bind to the 1669-nt scaffold, forming a giant branched molecule with bulges at each arm and (2) the arms associate with their partners by the paranemic-cohesions interactions, resulting in a final octahedron-shaped structure. The fundamental concepts and early explorations in structural DNA nanotechnology provided a solid foundation for the development and rapid growth of designer DNA origami structures that are highly complex, diverse, and convenient.

2. FUNDAMENTALS OF DNA TILE DESIGN Ned Seeman and colleagues pioneered structural DNA nanotechnology research, and from their achievements, there arose an interest in and rapid development of DNA origami structures. The essential foundation of structural DNA nanotechnology, including DNA origami, is that DNA can form immobile branch junctions that can be further joined together via sticky ends to form higher-order structures and lattices.36 However, the naturally occurring branched DNA structures, such as the Holliday junctions, are unstable due to the intrinsic internal sequence symmetry.37 Seeman proposed that an immobile Holliday junction could be created by minimizing the sequence symmetry. He also suggested that it was possible to use an immobile junction to generate three-dimensional (3D) networks of nucleic acids through rationally designed sticky-end

3. DIFFERENT DESIGNS OF DNA ORIGAMI STRUCTURES 3.1. Single-Layer Designs

3.1.1. Tightly Packed Single-Layered DNA Origami Designs. In 2006, Paul Rothumend22 was the first to report the 12586


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Figure 2. DNA origami design and assembly.22 (A) A target shape is first approximated with parallel cylinders that are (B) tightly aligned in parallel and that (C) utilize the DNA helices. (D) An example of a DNA origami scaffold with staple strand routing. The dark blue strand is the scaffold, and the staple strands are shown in various colors. The distance between the crossovers (along the scaffold) is 1.5 turns (∼15−16 nt) to make the staple strands transverse between the parallel helices, which are aligned approximately in the same plane. If the scaffold sequence and its routing path is given then the sequence of the short staple strands will be determined. (E) The scaffold and the staples (with a large excess of the staples) are mixed, and the designed structures self-assemble with a cooling temperature ramp in a salted buffer. (F) The AFM image of a representative assembled DNA origami structure that has a smiley face pattern. The size of the image is 165 × 165 nm. Image reproduced with permission from ref 22. Copyright 2006 Macmillan Publishing Ltd.

connect adjacent helices is usually constant along the parallel alignment of DNA helices. However, if the distance between the crossovers along the outer DNA helices is greater than that of the inner helices, tension causes the outer helices to bend. This strategy has been successfully implemented to engineer DNA into concentric rings or squares with rounded corners (Figure 3A).56 Researchers have implemented two general strategies to create 3D shapes with the DX-based single layered design. The first strategy relies on the second-step folding of the formed planar origami structure. In this case, the 2D planar structure is not completely paired with staples. Rather, the scaffold is intentionally left as a single strand in certain regions so that the relatively compact 2D planar structure could be broken into well-cut pieces, such as triangles and squares. Semirigid 2D shapes are then folded into 3D shapes when they join with the matching well-cut pieces. For example, Gothelf and co-workers constructed a boxlike structure with an external size of 42 × 36 × 36 nm3 that had a lid that could open or close in a boxlike way. They were able to create this reconfigurable DNA nanostructure by designing the M13 scaffold to route through all of the faces of the box and by introducing flexible hinges (Figure 3B).61 A small cuboidal structure with a light controlled lid was also reported.62,63 Two other groups utilized a stepwise folding mechanism that was controlled by a set of staples, to assemble a cuboidal box64 and a DNA prism.65 Also, by routing a scaffold through four triangular faces that had single-stranded hinges, a tetrahedral structure has been created (Figure 3B).66 The second strategy that was used to build 3D DNA origami was to use plane linkages and to introduce crossovers with a distance that was not equal to an integer number of half turns. In this case, the parallel DNA helices did not pack into a single plane. For example, Douglas et al. implemented this strategy to construct a DNA origami nanotube.67 By combining this approach with an in-plane curvature strategy, more complicated

DNA origami technique. In a seminal paper, he described the assembly process of several single-layered, planar structures, which ranged from simple rectangular, triangular, five-point star shapes to complex smiley faces, each with a unique size, roughly 100 nm in diameter (Figure 2 and 3A). The procedures associated with the design and self-assembly process are shown in Figure 2. (1) A target planar pattern was transformed into a number of parallel cylinders, representing the DNA double helices. (2) A number of periodic crossovers were introduced to hold all of the helices together. The distance between the neighboring crossovers along each helix was 1.5 helical turns (16 bp), which allowed one of the strands to continuously route through each adjacent helix and serve as the scaffold. Its complementary strand was cut into hundreds of shorter oligos (staple strands), usually 15−60-nt long, which traveled between the neighboring helices via the crossovers. Rather than using a chemically synthesized strand, it is much cheaper and convenient to use a strand of single-stranded DNA from a viral genome as the scaffold. This is because these genomes are already thousands of nucleotides long and originate from a bacteriophage virus with determined sequences. The genome of the M13mp18 virus is the most widely used scaffold for assembling DNA origami structures. The sequences of the staples are determined by the routing pathway of the scaffold, the locations of the crossovers used to maintain the overall shape, and the nick point positions. (3) Finally, the scaffold and the staple strands were mixed into a salted buffer and slowly annealed from 90 °C to room temperature, allowing each short staple to find its unique position on the scaffold strand and to ultimately form the desired structure. By using a similar design strategy, other groups since then have successfully constructed 2D planar patterns, such as a map of China58 and a depiction of dolphins.59 Han et al. reported on a strategy to create curvature within 2D planar structures (Figure 3A).60 The distance between the consecutive crossovers that 12587


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Figure 3. Single-layered DNA origami structures. (A and B) Compact DNA origami structures with single-layered, tightly aligned DNA helices in 2D or folded into 3D.22,60,61,64,66,68 The sizes of the rectangles, triangles, and five-point star images are all 165 × 165 nm. Images reproduced with permission from ref 22. Copyright 2006 Macmillan Publishing Ltd. Images reproduced with permission from ref 60. Copyright 2011 AAAS. Images reproduced with permission from ref 61. Copyright 2009 Macmillan Publishing Ltd. Images reproduced with permission from ref 64. Copyright 2009 Royal Society of Chemistry. Images reproduced with permission from ref 68. Copyright 2010 Macmillan Publishing Ltd. (C) Wireframe DNA origami structures that were constructed with different DNA rendering units.69−72 All of the scale bars in the AFM images are 50 nm. Images reproduced with permission from ref 69. Copyright 2015 Macmillan Publishing Ltd. Images reproduced with permission from ref 70. Copyright 2016 AAAS. Images reproduced with permission from ref 71. Copyright 2015 Macmillan Publishing Ltd. Images reproduced with permission from ref 72. Copyright 2013 AAAS.

shapes such as a 3D sphere, football, and nanosized flask were constructed (Figure 3B).60 Although the DX DNA molecule is a relatively rigid structural motif, when compared to duplex DNA on a large scale (such as ∼100 nm), the single-layered origami structure is still flexible. Furthermore, the distance between neighboring crossovers, along the same pair of DNA helices in a planar DNA origami structure, is usually 32 bp, or approximately three turns that equal 10.67 bp per turn, while natural B-type DNA is 10.50 bp per turn. This difference of 0.17 bp accumulates and causes a small structural strain, which results in a global twist in larger-sized structures. Yan and co-workers utilized these two features of single-layered origami structures and built a Möbius

strip with a DNA molecule of ∼210 nm long and ∼25 nm wide with a 180° twist, which connected back on itself (Figure 3B).68 Moreover, the DNA Möbius strip could be reconfigured through a toehold-mediated strand displacement to create catenanes and supercoiled structures. 3.1.2. Wireframe Single-Layered Designs. The DNA origami structures that were created with the design strategies mentioned above were all formed by aligning DNA helices in a parallel and compact way, as the designs are essentially based on the DX motifs. If new motifs are introduced to the origami structure, new structures can be achieved. For example, multiarm junctions allow for a mesh style of desired patterns. Woolley et al. 12588


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Figure 4. Multilayered DNA origami structures. (A−C) Multilayered DNA origami structures with hexagonal, square, and triangular packing of parallel DNA helices, where the distance between the neighboring crossovers along the scaffold strand are 7, 8, and 13 bps, respectively.79−81 (A) Images reproduced with permission from ref 79. Copyright 2009 Macmillan Publishing Ltd. (B) Images reproduced from ref 80. Copyright 2009 American Chemical Society. (C) Images reproduced from ref 81. Copyright 2012 American Chemical Society. (D) DNA origami with controlled twists and turns. In the hexagonal packing, the regular distance between the crossovers is 7 bp. If the distance of a unit is larger or smaller than 7 bp, the tension generated between the crossovers leads to a bending of the unit.82 Images reproduced with permission from ref 82. Copyright 2009 AAAS. (E) A DNA tensegrity structure with 13-helix bundles that are used as the compression members and 9 single-stranded DNA tensed cables.83 Images reproduced with permission from ref 83. Copyright 2010 Macmillan Publishing Ltd. (F) 3D framework of DNA origami structures based on the layered crossover motifs, which are shown in the colored helical model.85 Images reproduced with permission from ref 85. Copyright 2016 Wiley.

resulted in a series of 2D lattice DNA origami structures. The strategy of bending DX structures with tension can also be applied to the gridiron units to create curved structures in the shape of an S or a sphere. Zhang et al. subsequently created another method to make much more complex wireframe structures through a number of multiarm junctions (Figure 3C).71 By controlling the length of the Tn loops that were inserted into the staple strands that surround each of the vertices

used a square junction to create branched structures, which connected the stem of the branch region to only one of the base helices of the desired feature, and allowed the connecting helix to remain at a right angle.73 Han et al. was the first to create the wireframe DNA origami with a gridiron pattern (Figure 3C).72 The essential gridiron unit consists of four 4-arm junctions that are joined together to produce a square frame. Connecting a number of these units together with a consecutive scaffold strand 12589


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honeycomb lattice alignment. A more densely packed 3D origami was developed by Ke et al. as well, in which each helix was surrounded by six helices. The basic unit could be regarded as a three-helix bundle in a triangular lattice (Figure 4C).81 To create a relative orientation angle of 60° between adjacent helices, the crossovers created by the staple strands were separated by either 13 bp (7/6 of 10.5 bases) or 9 bp (5/6 of 10.5 bases). They further demonstrated that the three different packaging styles could be incorporated into a single DNA origami structure. Dietz et al. expanded the multilayered design to include more complex structures that have programmable twists and curvatures (Figure 4D).82 They systematically adjusted the number of base pairs in selected units to create global twists or bends due to mechanical forces between adjacent helices at junctions where base pair insertions or deletions occurred. By varying the curvature radii between the ranges of 64 to 6 nm, the bending of the structure can be rationally tuned between the angles of 30° and 180°. A different approach used the combination of multilayered structure rigidity and single-strand flexibility to achieve tensegrity architectures (Figure 4E).83 Tensegrity is a structural principle based on the use of isolated components that are under compression, inside a net of continued tension, which gives the structures extremely high strength-to-weight ratios and great resilience. Liedl et al. successfully created a tensegrity prism that consisted of three multilayered DNA bundle structures that acted as compression components and nine single-stranded DNA (ssDNA) regions that acted as tension cables.83 The multilayered DNA origami structures exhibit much higher rigidity than the single-layered structures. However, a longer folding procedure is usually required to avoid kinetic traps, and a high magnesium (Mg2+) concentration is necessary in the buffer (about 15−30 mM) to suppress the electrostatic repulsion of negatively charged phosphate backbones between adjacent helices that are closely packed. The yield of compact multilayered structures is generally lower than that of the single-layered DNA origami, which is due to the lower accessibility and shorter nucleation region of the scaffold strands. The yield could be improved through the careful design of the scaffold pathway and staple breaks.84 A 3D DNA origami structure does not necessarily require parallel alignments of DNA helices in a layer-by-layer fashion. Hong et al. developed a method to engineer a layered 3D framework of DNA structures.85 They introduced a layered crossover motif that allowed the scaffold or helper strands to travel through different layers and control the relative orientations of the DNA helices in the neighboring layers (Figure 4D). Using this strategy, Hong et al. successfully constructed a nine-layered framework structure and demonstrated that the angles between the neighboring helices could be controlled from 20° to 90° (Figure 4F). By using 3D point-star junctions, 3D latticed structures can also be achieved; however, it would be challenging to reliably control its relative orientation in a 3D space. Origami design methodologies rely on the use of DNA molecules to render objects. Scientists can use DNA origami or a 3D modeling software to generate an object. With both DNA origami and 3D modeling software, objects can be visualized as outlined meshes, folded from a 2D plane, or raster filled with layered lines. The preparation pipeline for DNA origami structures is well-established now: (1) conceive a target shape, (2) design the scaffold-staple layout and determine the sequence with a software program,86,87 (3) prepare the scaffold and staple

and the number of unpaired nucleotides in the scaffold, the angles between the arms can be tuned. Intricate wireframe patterns, such as quasi-crystalline and even arbitrary patterns without any symmetry, such as a flower-and-bird image, were created by joining many different angled multiarm junctions together. In this design, each edge of the lattice consists of two DNA helices. The scaffold had to travel between each edge twice to connect all arms to form the final desired patterns. Tuning the junctions manually is tedious for some complicated objects. A design algorithm called DAEDALUS, based on the multiarm junction, was developed to fully automate the scaffold routing and staple assignment for specific target structures (Figure 3C).70 The software can easily design and construct Platonic, Archimedean, Johnson, and Catalan solids, asymmetric structures, and polyhedrons with nonspherical topologies. In addition, Benson et al. rendered arbitrary target objects into triangulated meshes as an alternative way to engineer DNA nanoobjects in a wireframe style (Figure 3C).69,74 The majority of the edges in their design consisted of a single DNA helix rather than two DNA helices, which required the scaffold to travel through all of the edges just once, except for a few edges that require the scaffold to travel twice. The method started with a scaffold routing that was based on A-trails by an computational algorithm,75 and then a physical model was implemented to relax the tension at the junctions by using the DNA helices as rigid cylinders that were joined by stressed springs. The validity of the method was demonstrated through the successful construction of a set of meshed 3D objects, which included a ball, a bottle, and a Stanford bunny. A similar design concept was also applied to build DNA trusses.76 Depending on the design method that a research scientist uses, single-layered DNA origami structure can be created in many different ways. Using the structures for practical uses may have several limitations, despite their successful formation and high yield. For example, molecular dynamics simulations have shown that single-layered origami structures have conformational flexibility and structural heterogeneity, which may limit its usage in precise addressiblities.77 Flexibility is difficult to avoid in the single-layered wireframe design, due to the discontinuity of the DNA helices at the vertices. Molecular threading behavior has also been observed in tightly compacted designs, where DNA scaffolds open and close on their own.78 3.2. Multilayered Designs

To overcome these problems and expand design methodologies, single-layered DNA origami can be stacked to create multilayered 3D structures with increased rigidity. Douglas et al. published the first article on engineering 3D solid DNA origami structures, where they used a six-helix bundle as the basic unit (Figure 4A).79 A honeycomb-shaped lattice is the cross-section of the 3D structures, which were positioned perpendicularly to the DNA helices. Every DNA helix was connected to three adjacent helices by crossovers which were orientated at an angle of approximately 120°, separated by 7 bps [1/3 of 21 bases (= two helical turns), or 240°]. Douglas et al. demonstrated the success of this design strategy by assembling a monolith, square nut, and slotted cross based on a honeycomb alignment. Ke et al. reported another approach, based on a four-helix bundle, to achieve a square alignment of the helices (Figure 4B).80 In this design, for every 8 bp (∼3/4 of 10.5 bases, 270°), the staple strand would rotate along the helices and travel to a neighboring helix with a relative orientation angle of 90°. The packing density of the square lattice alignment was higher than that of the 12590


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Figure 5. Scaffold engineering for constructing large-sized DNA origami structures. (A) A 26-kb-long scaffold is produced based on PCR and enzymeassisted digestions.109 Images reproduced with permission from ref 109. Copyright 2012 Royal Chemical Society. (B) A super long DNA scaffold of ∼51 kb is produced using a λ/M13 hybrid virus, and DNA staples are synthesized by an inkjet printing process on a chip.110 Images reproduced from ref 110. Copyright 2014 American Chemical Society. (C and D) A double-stranded DNA scaffold is used to produce two distinct origami structures and one integrated origami structure.114,115 The scale bars are 100 nm. (C) Images reproduced from ref 114. Copyright 2009 American Chemical Society. (D) Images reproduced from ref 115. Copyright 2012 American Chemical Society.

4. SCALING UP DNA ORIGAMI STRUCTURES

oligoes, (4) allow DNA to self-assemble in a salted buffer through a temperature ramp, (5) functionalize and purify the structure if needed, and then (6) visualize and analyze the structure.88−90 A variety of methods have been developed to optimize the structure preparation processes, such as increasing the scaffold production efficiency,91−96 assembling the structure in a Mg2+free environment97 under an isothermal condition,98,99 and purifying the DNA origami structure to remove excess staples as well as any misfolded structures.100−104 A few strategies have been developed to help scientists use DNA origami under a wider range of experimental conditions, such as the use of a photoinduced cross-linking that improves the thermostability,105 the use of external triggers such as light and chemicals to help control the formation of DNA origami structures,106 and the assembly of the structure in anhydrous and hydrated deepeutectic solvents.107

Large DNA origami structures, in both size and molecular weight, are highly desired because they can be used to create more complex structures and to perform more sophisticated tasks.32 However, the size of a DNA origami structure is limited by the length of the scaffold. The most commonly used scaffold is a single-stranded, 7249-nt circular DNA, M13mp18, which was extracted from bacteriophage M13. To scale up the DNA origami structure assembly, extending the scaffold length has been attempted with a variety of biotechnological methods, such as polymerase chain reaction (PCR) and rolling circle amplification (RCA). Alternative methods, such as hierarchical assembly, can also be implemented by using individual DNA origami structures as the tile units.108 In comparison with the small DNA tiles, the large dimensions of the DNA origami allows for the application 12591


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Figure 6. Super DNA origami with complex tiles. (A) A set of eight helix tiles were employed as staples to fold a scaffold into large structures.117 The scale bar is 20 nm. Images reproduced with permission from ref 117. Copyright 2010 Wiley. (B) DNA origami were used as staples to interact with the prefolded scaffold frames to generate the desired structures.118 The scale bars are 100 nm. Images reproduced from ref 118. Copyright 2011 American Chemical Society.

printing process on a chip, which was embossed with functionalized micropillars. Schmidt et al. developed another oligo synthesis method, called a circle-to-circle amplification, which could produce a nanomolar amount of ssDNA in a highthroughput manner.111 Ducani et al. described a method whereby they used a rolling-circle amplification method to generate single-stranded DNA with precisely controlled stoichiometry.112 Reducing the amount of staples needed to fold an origami structure is another way to efficiently reduce the cost of DNA origami. Niekamp et al. recently demonstrated that they needed only 10 staples in order to fold a 6k-nt synthetic scaffold, with a repetitive sequence, into a complex origami nanostructure.113 In their design, each one of the staples in the structure was able to bind to a single scaffold about 10 times, which efficiently reduced the material cost of the DNA origami. Instead of extending the scaffold, multiple scaffolds could also be incorporated into one design to achieve a larger size. Naturally occurring double-stranded DNA (dsDNA) is a good source for researchers to use. However, dsDNA cannot be used directly because the enthalpy change required for breaking the two

of more diverse self-assembly strategies, as opposed to solely relying on sticky end associations. 4.1. Origami with Extended Scaffolds

Making longer scaffolds is a straightforward and efficient way to increase the size of DNA origami, since the scaffold length limits the size of a DNA origami structure. Zhang et al. used PCR to amplify 26-kb fragments from a lambda DNA and to selectively digest one of the two strands to obtain ssDNA (Figure 5A).109 The ssDNA was then used as the scaffold to form a rectangular DNA origami with the dimensions of 238 × 108 nm. Marchi et al. developed a method to produce a 51k-nt long, circular, ssDNA scaffold by using a λ/M13 hybrid virus, which is the largest biologically derived single-stranded scaffold to date (Figure 5B).110 However, a scaffold with a larger size leads to an increase in the number of staple strands needed to fold the scaffold. Although the price of synthesized oligos has decreased dramatically over the past decade, the cost is still relatively high when it is necessary to obtain approximately 200 strands of custom synthesized oligoes in order to fold a regular-sized DNA origami. To reduce the cost associated with the synthesis of the staple strand, Marchi et al. generated oligos with an inkjet12592


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Figure 7. Scaling up DNA origami structures via sticky end hybridization. (A) Polyhedron with a molecular weight of 60 MD that was assembled from 12 DNA origami tripods.119 Images reproduced with permission from ref 119. Copyright 2014 AAAS. (B and C) 2D DNA origami arrays that were assembled from cross-shaped and hexagonal-shaped DNA origami tiles, respectively.131,135 (B) Images reproduced with permission from ref 131. Copyright 2011 Wiley. (C) Images reproduced from ref 135. Copyright 2016 American Chemical Society.

individual tiles to fill in the cavities of the crudely prefolded main scaffold and forced it to form the desired super DNA origami structure. An 85% yield was achieved through an optimization of the design. Four different structures were successfully constructed by using DNA origami tiles with different geometries. The largest structure was composed of 10 origami tiles and had a molecular weight of 45.5 MD (137962 nt).

complementary scaffolds is much higher than the enthalpy change that is gained for binding the staples to the scaffolds. Högberg et al. solved this issue by using the denaturing agent formamide to separate a dsDNA into two separate strands.114 Högberg et al. then used two unique sets of staple strands to fold the two scaffolds into two distinctly shaped origami structures (Figure 5C). The folding procedure was unlike the stepwise cooling-down protocol that researchers use when regular DNA origami is assembled with ssDNA scaffolds. To assemble origami from dsDNA scaffolds, researchers quickly change the reaction temperature from 90° to 25° to reduce the probability of interactions between the two ssDNA scaffolds. To construct integrated structures that used both ssDNA scaffolds, Yang et al. successfully used dsDNA scaffolds without a denaturing agent (Figure 5D).115 Two important strategies are used to maximize the yield of a folded structure: symmetric routing pathways and a periodic convergence of the two ssDNA scaffolds. Marchi et al. reported a method where he used a one-pot reaction to fold a double-stranded M13-bacteriophage DNA into a heterodimeric structure, while the staples that were used were produced by a nicking strand-displacement amplification.116

4.3. Hierarchical Assembly of DNA Origami

Large-scale structures can be achieved by combining DNA origami molecules. Each DNA origami molecule can act as a DNA tile unit to form infinite crystal-like structures, or finite structures, through hierarchical self-assembly. Entropy decreases dramatically from a disordered mixture of individual particles to a well-ordered and structured system. Organized systems have lower enthalpy. Base-pairing and base-stacking during DNA structure self-assembly decreases the enthalpy in a system and stabilizes DNA duplexes. There are two basic rules for the hierarchical assembly of DNA origami structures. The first rule relies on the sequence specific Watson−Crick base pairing that occur via the sticky ends that extend from the DNA units. The second rule relies on the nonsequence specific base stacking that is located at the blunt ends of the DNA helices. These blunt ends reside along the edges of the DNA origami and are strong enough to hold the origami units together. The large dimension of DNA origami makes a large number of DNA helical ends available for binding. To make the interactions more specific, geometric coding can be applied to the edges of the DNA origami. Both of the two driving forces (sticky end associations and blunt end base stacking) have been implemented in the higher-order selfassembly methodology that utilizes DNA origami units. 4.3.1. Hybridization. A straightforward method to create specific association sites is to extend sticky ends from the DNA origami structure unit. Iinuma et al. developed a general strategy for the hierarchical self-assembly of 3D polyhedrons from DNA origami “tripod” monomers, whose inter arm angles can be tuned by supporting struts and strengthened by vertical helices (Figure 7A).119 With the use of tripod monomers with various angles, a series of polyhedral structures were constructed from tetrahedrons of 4 monomers to hexagonal prisms of 12 monomers. The maximum molecular weight of the assembled structure was 60 MD. This strategy is similar to the one used to assemble DNA

4.2. Origami with Complex Staples

Rather than extending the scaffold length to increase the size of a DNA origami, conventional single-stranded staples can be replaced with complex nanostructures, such as DNA tiles that consist of multiple strands, or even the origami structure itself. This method, proposed by Zhao et al., is called “super DNA origami” and drastically increases the size of an individual origami structure. They used an 8-helix, rectangular, DNA tile that was assembled from 18 staple strands. The two strands extended out as single-stranded overhangs to base pair with different M13 scaffold locations (Figure 6A).117 The largest structure achieved through this method used 56 tile staples, yielding a superstructure with a molecular weight of about 20 MD and ∼30000 base pairs. The overall number of staple strands was 248, which is comparable to the number in a regular DNA origami structure. They later expanded the method by using individual DNA origami as staples to fold another scaffold strand assisted with short staple strands (Figure 6B).118 First, a small set of regular, short DNA staples folded the main scaffold into a structure with large cavities. Then, a set of preformed DNA origami acted as 12593


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Figure 8. Scaling up DNA origami structures via base stacking. (A) A linear DNA origami array is assembled by using combinatorial codes, which are created by active (1) or inactive (0) patches that allow blunt-end stacking and shape complementarities.136 The scale bars are 60 nm. Images reproduced with permission from ref 136. Copyright 2011 Macmillan Publishing Ltd. (B) Shape complementarity relies on base stacking and is used to create higher-order DNA origami structures.137 Images reproduced with permission from ref 137. Copyright 2015 AAAS. (C) DNA origami tiles are assembled by using a combination of sticky-end association and blunt-end stacking for pattern construction with programmable disorders.142 The size of images are 880 × 880 nm2. Images reproduced with permission from ref 142. Copyright 2016 Macmillan Publishing Ltd.

polyhedra from multiarmed tiles.120 When the DNA connectors were functionalized with responsive moieties, external signals such as light,121 pH,122 metal ions,123 and small chemical molecules124 could be utilized to trigger the higher-order selfassembly of the DNA origami.

2D Crystalline DNA origami structures can also be achieved with sticky end hybridizations. Latticed DNA structures have been successfully constructed from various small DNA tiles,47 and their associations have been systematically studied.125−127 DNA origami, in comparison to small DNA tiles, possess larger 12594


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may also need to consider ways to allow self-correction during the assembly process, such as minimizing the global twist accumulation and avoiding strong association bonds. 4.3.2. Base Stacking. Base stacking, which is a major force that stabilizes DNA duplex structures, is often overlooked during the assembly of higher order DNA structures. Helical blunt-end base stacking was reported as an important factor that affects the higher-order self-assembly of DNA nanostructures by Yan et al. in 200356 and was systematically studied as an assembly rule by Woo et al. in 2011.136 The latter group found that base stacking is a directional bond force that favors antiparallel stacking polarity and can be used as an interaction force to guide DNA origami structure self-assembly. When base stacking is implemented into the higher-order self-assembly of DNA origami, three main factors need to be considered: (1) the sequences of the bases involved in the stacking, which dictates the strength of the interaction; (2) global twist, which influences the accessibility of the stacking; and (3) crossovers around the blunt-end region, which may deform the blunt-end stacking conformation and reduce its efficiency. Base stacking along a continuous edge is unspecific, which is one of the main reasons underlying DNA origami structure aggregation. A simple method of extending the origami edge with a poly-T sequence, or leaving unpaired scaffold loops, can prevent the unwanted base stacking and yield wellseparated origami monomers. To utilize the stacking force, a set of base stacking interactions can be programmed into binary or programmed into complementary codes to achieve specific hybridization associations (Figure 8A). For example, the edge of a rectangular DNA origami is divided into a number of patches. The patches that have protruding blunt-ended DNA helices that allow stacking are designated as “1”, while the nonprotruding patches that avoid stacking due to the presence of a loop are regarded as “0”. Thus, a binary sequence can be created. In terms of shape complementarity, the DNA origami’s interfacing edges can be divided into three different patches with different depths (0, 1, or 2). Only when the two edges perfectly match each other will the DNA base stack (Figure 8A). Dietz et al. recently expanded the rule of shape complementarity with 3D multilayered DNA origami bricks (Figure 8B).137 They showed that shape complementarity can be used to assemble 2D latticed DNA structures. They also showed that experimental parameters, such as temperature and salt concentration, could be used to switch on or off interactions between origami units. The mechanism underlying the switch control is that the equilibrium of association taking place through the shape complementarity is sensitive to ion concentrations and temperature. Both factors affect attraction/repulsions between the negatively charged surfaces of DNA. By varying the temperature and Mg2+ concentrations, the balance between the forces of base stacking and surface charge repulsion can be adjusted and controlled. Thus, the stacking and nonstacking conformations of the DNA origami molecules can be switched. A set of higher-order assembly DNA structures can be reconfigured (e.g., from a compacted DNA lattice structure to a grid lattice structure). In a follow up study, small-angle X-ray scattering was applied by the same group to study the conformational changes quantitatively.138 In addition, base stacking based on shape complementarity and hybridization can be combined and simultaneously utilized by using the concept of “jigsaw pieces.”139−141 Sugiyama et al. designed a set of origami structures with sticky ends that could be attached to regions of concavity or convexity in a DNA origami molecule. Thus, the sticky end hybridization can be enforced by

dimensions and more complex dynamics. Thus, more factors such as the global twist of the structure and the position and strength of the connectors need to be considered. Jungmann et al. investigated three kinds of rectangular DNA origami polymerization and found that the most efficient approach was to use the staple strands that were bridging the structure edges to directly link the origami units.128 B-form DNA has a helical twist of 10.5 bp per turn, while the regular origami structures are designed using 10.67 bp per turn. The increase in number of base pairs per turn in the design results in a significant global twist, which may play an important role in the higher-order selfassembly of DNA origami structures because small distortions will accumulate when hundreds or thousands of units are assembled. In the work of Jungmann et al., a helical twist with a periodicity of ∼200 nm was found in the final long ribbon products. Li et al. constructed a set of rectangular-shaped DNA origami tiles with a zigzag design to avoid global twist and investigated their self-assembly into a higher-order structure.129 They found that the dimensional ratio of the origami tiles and intertile connections also greatly influenced the final products. Walther et al. found that the number of connectors between DNA origami units can cause the higher-order self-assembly of the DNA to form a linear fiber,130 due to the connector’s influence on the melting temperature of the hierarchical assembly. Seeman and co-workers constructed a DNA origami array by using a cross-shaped origami tile with the DNA helices propagated in two independent directions (Figure 7B).131 Large 2D arrays were created by using sticky end associations that were located at the ends of the cross-shaped origami tiles. The corrugated design was required to avoid curvature accumulations in either of the two dimensions during crystalline growth. If the curvature was not minimized or eliminated then the DNA origami was much more likely to form a tube-shaped structure.132 Sugiyama et al. found that a loop arrangement of a scaffold on the single-layered DNA origami affected the 2D crystallization process.133 Since the distance between crossovers in a DNA origami is usually 10.67 bp per turn, rather than 10.5 bp, the origami structure was curved. The attachment of structural features (such as loops) on the surface of DNA origami helps to eliminate unwanted curvature and leads to a more efficient assembly between origami tiles.134 Recently, Wang et al. designed a set of hexagonal DNA origami tiles that successfully formed lattices (Figure 7C).135 N-point-star DNA origami tiles have also been tried, but only aggregates were obtained, which indicated that the relative spatial relationships between the arms of the origami tiles are flexible and are difficult to keep in the same plane during assembly. They also varied the designs of the connection (e.g., bp length and the gap between the connected domains) between origami tiles to investigate their effect on lattice formation. They found that eight 2-bp connectors resulted in mostly small aggregations because the thermodynamic driving force of the formation process (of the optimal bound state) was counteracted by the slow escaping process (from the ill-formed state), which resulted in strong kinetic traps. Aggregation was repressed when the connector length was reduced to 1 bp. However, tubes rather than 2D arrays were formed, which indicated that curvature was present in the origami unit. A gap of one base was introduced to create a 1-bp quasi-gap connector, which resulted in a large 2D lattice structure. The rigidity and formation of the origami monomers are very important for their further higher-order assembly. Moreover, one 12595


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Figure 9. Surface-assisted DNA origami self-assembly. (A and B) The DNA origami self-assembly on a mica surface is mediated by the balance between electronic interactions of the DNA nanostructures with the mica surface and the enhanced diffusion mobility of DNA origami on mica in the presence of the proper concentration of Na+.146,148 Both blunt end stacking and close packing could be the driving forces that help guide the self-assembly. The scale bar in B is 200 nm. (A) Images reproduced with permission from ref 146. Copyright 2014 Wiley. (B) Images reproduced with permission from ref 148. Copyright 2014 Macmillan Publishing Ltd. (C and D) Supported lipid bilayers on mica surfaces have been used to provide the surface mobility (lateral diffusions) to guide the assembly of the DNA origami structures.154,155 (C) Images reproduced with permission from ref 154. Copyright 2015 Macmillan Publishing Ltd. (D) Images reproduced with permission from ref 155. Copyright 2015 American Chemical Society.

of the final structure due to very short sticky ends with specific sequences that were located on the DNA origami structures. The origami tiles formed an infinite array with dimensions up to 16 × 16 μm. Truchet tiles were created when the four-fold rotational connection symmetry was broken and arc or T-shaped surface patterns were decorated with double-stranded biomarkers. Truchet tiles are square tiles that are decorated with surface patterns that have no rotational symmetry. When placed within a square tiling plane, they can display varied patterns.144 The DNA

shape complementarity. Recently, Qian and co-workers designed a square-shaped DNA origami with a 4-fold symmetry by using short single-stranded domains to bridge 4 pieces of triangles (Figure 8C).142,143 The connection between the origami units was created through the combination of both blunt-end stacking and associations between very short sticky ends. The weak interactions from the blunt-end stacking, allowed the system’s self-correction when binding errors occurred. Units were able to find their correct positions and to form the desired connections 12596


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Figure 10. DNA origami nucleated/templated tile self-assembly. (A) DNA origami is used as a seed to control the nucleation and growth pathway of DNA tiles to realize algorithmic self-assembly.159 Images reproduced with permission from ref 159. Copyright 2009 National Academy of Sciences. (B) The DNA tiles are assembled within hollow DNA origami structures.162 Images reproduced from ref 162. Copyright 2014 American Chemical Society. (C) A DNA origami bundle is used to control DNA nanotube nucleation and growth.163 Images reproduced from ref 163. Copyright 2013 American Chemical Society.

origami Truchet tiles have been used to program the assemblies that display various aperiodic patterns, such as loops, trees, and mazes. Moreover, by controlling the connection geometry, the origami tiles can be assembled into a finite grid with a determined size in a one-pot reaction. The key design principle is to encourage the sequential stages of origami tile binding to occur during the annealing step and by allowing the assembly process to occur in a step-by-step process in order to reduce spurious interactions. Using this method, mazes with predetermined sizes and designed entrances and exits have been successfully formed. The combination of different association strategies have extensively expanded the complexity of self-assembled patterns that can be created.

DNA origami self-assembly can be assisted by mica or lipid surfaces, where the diffusion is controlled by the electrostatic force between the surfaces and structures, as well as the surface’s intrinsic mobilities.145 Simmel et al. reported that monovalent cations, such as sodium (Na+), could guide DNA origami array formation by electrostatically controlling the adhesion and mobility of the origami on the mica surface (Figure 9A).146 Keller et al. implemented the method to create ordered protein patterns by depositing negatively charged proteins onto the cavities of a DNA origami array on a mica surface.147 Woo et al. comprehensively studied how DNA-mica binding guides the higher-order assembly of DNA origami structures (Figure 9B).148 They explored the competition between Mg2+ and Na+ in the DNA-mica interactions and utilized the knowledge to control the stepwise diffusion of origami onto a surface to achieve higher-order latticed DNA origami structures. They discovered that when the fractional surface density of divalent cations was between 0.04 and 0.1, more than 90% of the rectangular DNA origami tiles would assemble into lattices.

4.4. Surface-Assisted Self-Assembly

The self-assembly process can be mediated by external templates. For example, the provision of a surface that constrains the unit’s Brownian diffusion into a small space could lead to an increased local concentration and a higher probability for the units to meet. 12597


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pathway. By controlling the nucleation barrier with rationally designed tiles, Schulman and Winfree constructed a DNA tile self-assembly system where tiles grow into ribbon-like structures with a predetermined width.158 In the system, there are two types of tiles: regular DX tiles with four full association sites which are used as the growth tiles and double-DX tiles with association sites on a single edge which are used as the barrier to control tiles. Under a slightly supersaturated condition, attachment by two or more sticky ends is favorable, while attachment by a single sticky end is unfavorable. This allows researchers to rationally design the energy barrier of the homogeneous nucleation by tuning the number of required unfavorable bindings during the nucleation steps to control the width of the final crystal products. In comparison with DNA tiles, DNA origami is able to carry much more information in terms of the algorithm input that is necessary to guide more complicated tile assembly behaviors. The DNA origami seeds help the tiles to overcome nucleation barriers and predetermine its assembly pathway to execute computations such as copying,159 counting,160,161 and forming fractal patterns, such as a Sierpinski triangle (Figure 10A).156 Li et al. utilized a 2D DNA origami seed with a hollow interior and selectively positioned association sites to guide the tile assembly inside of an L-shaped DNA origami structures (Figure 10B). They found that the tile assembly rate was increased in the presence of DNA origami seeds.162 Using a designer seed from a DNA origami tube, Schulman and co-workers observed that DNA nanotube nucleation and growth were greatly accelerated, while undesired nucleation was avoided (Figure 10C).163 They also showed that the DNA origami tubes that were located at two different landmarks on a surface allowed nucleated DNA tiles to grow into tubes, which joined together and created a single tube that connected both of the landmarks. The growth of the tube could be controlled with a defined geometry as well by using a DNA origami seed with predetermined orrientations.164 The distances between the connected landmarks spanned over 1−10 μm.165 Zhang et al. showed that the DNA tile’s self-assembly process could be logically controlled to fill into the hollow regions of the DNA origami.166,167

Instead of mica, a lipid bilayer is another ideal surface medium to control DNA origami self-assembly. DNA origami can be attached to a lipid surface by electrostatic adhesions or hybridization with an anchoring oligo strand, which is labeled with lipid molecules (such as cholesterol) that is embedded in the lipid membrane. Then, the intrinsic lipid membrane flexibility and fluidity can facilitate DNA origami assembly.149 By using single molecule fluorescence microscopy, Johnson-Buck et al. discovered that the cholesterol labeled DNA origami can become anchored in a supported lipid bilayer within a few minutes. While the cholesterol labeled DNA origami is bound to the lipid bilayer, it undergoes a two-dimensional Brownian motion with diffusion coefficients similar to that of typical lipid-linked membrane proteins.150 A variety of factors, such as the number and positions of the cholesterol labels, composition of the membrane, buffer conditions, and the interaction surface of the DNA origami affects the association and diffusion of DNA origami within a lipid bilayer. Czogalla et al. studied the rotation and translation of a needle-shaped origami on a lipid membrane and found that both the rotational and translational diffusions were strongly suppressed upon an increase in the density of the origami on the surface.151,152 With high speed atomic force microscopy (AFM), Suzuki et al. directly visualized the DNA origami’s assembly/ disassembly process on a lipid bilayer.153 Later, they visualized the growth of a DNA origami 2D lattice on a zwitterionic bilayer (Figure 9C).154 DNA origami structures are electrostatically adsorbed onto the zwitterionic bilayer if divalent cations are present. Both blunt-end stacking and close packing can be utilized to guide the self-assembly process. Kocabey et al. studied the use of cholesterol functionalized DNA origami structures to grow 2D arrays on a lipid bilayer and found that the diffusion of a DNA origami on a lipid bilayer was associated with the size of the DNA origami and the number of cholesterol anchors on the origami structure (Figure 9D).155 The orientation of the DNA origami with respect to the lipid bilayer is predefined by the DNA anchors that extend from the origami structure. The confined motions in 2D and the well-aligned orientation of the units help direct the growth of DNA origami into 2D arrays, which may be at least 1 order of magnitude larger than those assembled in solution.

5. MECHANISMS OF DNA ORIGAMI SELF-ASSEMBLY Since the first article describing DNA origami was published in 2006, it has become an extremely feasible and powerful technique to construct tens of nanometer scale molecular objects of customized shapes. However, the mechanism of origami assembly is still under investigation by different groups in the field. For example, the DNA origami structures rarely have a 100% formation yield. The percent yield depends on the complexity of the design and the experimental conditions. Various incorrectly folded structures often appear among the final products. Although a longer annealing time or a strict adherence to empirical rules can improve the yield, more information about the self-assembly process will provide more guidance on designing a way to improve DNA origami structure yield and quality.168

4.5. DNA Origami-Templated Tile Assembly

Periodic DNA crystalline structures assembled from small DNA tiles require less programmed information, while DNA origami structures with full addressability need a large amount of programmed information. The information is provided by hundreds of distinct short oligoes and a long scaffold. An important question is whether information-rich DNA origami structures can be used to guide the self-assembly of DNA tiles into complex patterns by controlling the growth pathway. Generally, the assembly of a crystalline structure proceeds through two phases: nucleation and growth. Nucleation is the rate-limiting step for crystal formation, when the system lacks binding sites and several units start to encounter and associate. The intensive entropic cost in this phase leads to a high kinetic barrier to overcome. A subtle variance in the nucleation step will result in a huge difference in the morphology of the final crystal products. For example, short DNA oligoes can be used as seed precursors to guide nanoparticle growth. Varying the sequence of DNA oligos will lead to different shapes of the final nanoparticles.156,157 A preformed information-rich seed can be used to assist a DNA tile assembly system to overcome the energy barrier of nucleation and precisely predetermine the growth

5.1. Folding Behaviors of DNA Origami

DNA origami self-assembly involves hundreds of short DNA staple strands that are incorporated onto a long scaffold. To dissect the assembly process of DNA origami, the key is to understand how the short staples dominate and control the long scaffold folding. Dietz and colleagues studied the folding and unfolding processes as a function of annealing time and temperature by using real-time fluorescence monitoring and 12598


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Figure 11. Mechanistic study of DNA origami folding. (A) Fluorometrically obtained folding rates and agarose gel analysis of folding products during the folding and unfolding process of a gear-like DNA origami structure.169 Images reproduced with permission from ref 169. Copyright 2012 AAAS. (B) One-pot annealing of combinatorial origami structures and the corresponding AFM images of the products.170 Images reproduced from ref 170. Copyright 2013 American Chemical Society. (C) A melting temperature map for rectangular DNA origami.171 Images reproduced from ref 171. Copyright 2012 American Chemical Society. (D) Melting and cooling curves of DNA origami folding as revealed by FRET analysis.172 Images reproduced from ref 172. Copyright 2013 American Chemical Society. (E) Temperature-dependent morphological evolutions of DNA origami during the cooling and heating processes.173 Images reproduced from ref 173. Copyright 2016 American Chemical Society. (F) Folding pathway study of an origami structure with a dimer scaffold.176 The scale bars are 50 nm. Images reproduced with permission from ref 176. Copyright 2015 Macmillan Publishing Ltd.

shifted toward higher temperatures (60 °C), as compared with the peaks of the folding process. The narrow peaks indicated a strong cooperativity during the folding and unfolding process, since the hybridization of two independent DNA strands usually happens over a wider temperature range. Because of the strong cooperativity and narrow transition temperature, it was

cryogenic reaction quenching followed by gel electrophoresis and TEM imaging (Figure 11A).169 They found that both the folding and disassembling rates showed a sharp and narrow peak at specific temperatures (∼55 °C for their design method) and otherwise fluctuated around zero. During the unfolding process, the disassembling rate featured distinctly narrow peaks that were 12599


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it could bind to the scaffold to form an internal linkage or (2) a pair of cross-links between the two copies in the scaffold. These two conformations allowed the origami system to form a set of distinguishable shapes, representing various minimal free energy states that were created from the two different folding pathways. By analyzing the final products, the folding pathway could be revealed. They found that DNA origami folding is similar to that of proteins, in that the folding is highly cooperative and requires a reversible step to avoid incorrectly folded structures. The way in which staples bind in order to connect scaffolds is a key step in controlling the folding pathway and final structures. Controlling the initial staple binding could be used to guide the folding pathway. A quantitative model based on a heterotypic DNA junction178 and a direct simulation using a coarse-grain DNA model179 has proved that cooperativity is a key factor in how DNA origami folds. Usually, the formation of DNA origami requires a temperature ramp to remove the secondary structures of the scaffold and to facilitate the correct binding of the staple strands. Bae et al. introduced a mechanical force to stretch the scaffold and to remove kinetic traps during the folding process.180 The self-assembly process can be dissected into three steps. The first step is a mechanical force, which includes the mechanic stretching of the scaffold, the second step is the base pairing of the staples with the scaffold, and the third step is the displacement among the staples. DNA origami folding is as complicated as protein folding,181 and different origami structures may have completely distinct folding behaviors. Even for similarly shaped origami structures, a change in the routing pathway of the scaffold and the staple strands will dramatically change the thermodynamics and kinetics of the folding behaviors. For example, Dietz and coworkers found that the distribution of a backbone nick positions of the staples strongly influenced the success rate of DNA origami.97 Rather than imaging the folded structure by microscopy or gel electrophoresis, Myhrvold et al. recently reported a method of using next-generation sequencing to quantitatively measure the incorporation of the staples into a origami structure.182 In this method, the staples and products are separated by gel electrophoresis after self-assembly and then ligated with barcodes for sequencing analysis. The sequencing results provide information related to the staple sequence and the abundancy through a read count that has been incorporated into the origami structure or remained in the staple pool. Thus, allowing the reconstruction of the structure with a read count can enable researchers with the ability to tell which strand is part of the assembled structure and which ones are not. The main advantage of this method is that it is able to provide detailed information on each of the staples on an assembled origami structure. However, it cannot tell whether the structure formed correctly or not. If the origami formed an undesired structure with all of the staples in it, the sequencing method would give misleading information in regard to whether the structure was well-formed or not. This method could be a good supplement to imaging with microscopy, which is the current study methodology. Overall, the reported results indicate that DNA origami folding is highly cooperative. Unlike the complexities and difficulties associated with studying protein folding, investigating DNA origami folding could be much easier because of DNA’s programmability and the well-established methods that are used to characterize DNA origami. As the DNA origami structure is usually constructed by repeating a small unit, examining their folding behaviors may shed light on the overall folding process of

hypothesized that DNA origami may be able to fold at a constant temperature. The quick folding of three-dimensional DNA objects within 5 min was successfully achieved at an optimized constant temperature (at the lower end of the transition temperature) with a yield that approached almost 100%. Fan and colleagues also described the rapid folding of a DNA origami structure in 10−20 min (Figure 11B).170 In an in situ study, through temperature-controlled AFM real-time imaging, Song and co-workers showed that DNA origami started to disassemble at ∼55 °C.171 A melting temperature map for a rectangular DNA origami structure was also created, and they found that the region with the higher melting temperature was GC rich. Yan and colleagues incorporated a Forster resonance energy transfer (FRET) dye pair into the two selected staples within various positions, located in 2D and 3D DNA origami structures.166 They monitored the FRET efficiency under different temperatures to capture the association and dissociation behaviors of the two staples under different scenarios (Figure 11D).172 They found that the melting of the various staple strands, placed at different locations in the DNA origami structure, showed highly cooperative behaviors. They also revealed that local defects significantly decreased the melting temperature, while the influence of defects further away from the staple strands were negligible. The existence of incorrectly folded DNA origami structures in the final annealing products suggested that there were various folding pathways available during the experiment. Some incorrectly folded structures could not be removed by extending the annealing time. These structures may be due to kinetic or topological traps of some individual staples. Information about the dynamic folding behaviors of an origami structure still remains to be explored. Wah and co-workers published a study on DNA origami folding that employed AFM imaging during a temperature ramp.173 They were able to see six different intermediate states during the folding and unfolding process (Figure 11E). They found that the nucleation was likely to happen at the upper and lower edges of the rectangular DNA origami, where the staple binding was more thermodynamically favorable. The connecting distance along the scaffold is usually much smaller as compared with that in the middle. They also showed that the folding pathway could be tuned by changing the concentrations of the staples during the assembly process. By increasing the concentration of staples in the middle of the experiment, the folding pathway dramatically changed and the DNA origami began to assemble from the center portion instead of from the edges. Majikes et al. also found a similar phenomenon by exploring a competitive annealing system that consisted of two sets of staples with the intent to form two different structures with the use of only one scaffold.174 The two sets of staples competed with each other to guide the folding of the scaffold. When the concentration of one of the sets of staples increased, the amount of the corresponding structure in the final products was increased as well. With the use of AFM in combination with nanomechanical spectroscopy under various temperatures, Song et al. studied the kinetics of how a DNA strand was able to fill the central region of a DNA origami.175 As the temperature increased, the factors that effected the filling-in process and the local hybridization rate increased exponentially. Dunn and colleagues investigated the folding behavior of DNA origami via a special circular, ssDNA scaffold that contained two identical copies of the monomer that was joined head-to-tail and that could bind with two copies of each of the staple strands (Figure 11F).176,177 In this case, each staple had two conformations: (1) 12600


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(honeycomb lattices, single-layer rectangles, two-layer lattices, and three-layer lattices) on the nanopores and studied their ionic conductivity under various voltages.190 They revealed that DNA origami plates are very permeable to small ions and that the DNA origami plates could be strongly deformed and pulled through the nanopore under a high voltage. Among the four different types of designs, the honeycomb lattice showed the least ion current leakage through the nanopore and the two-layer lattice exhibited the best mechanical stability. The voltage-dependent properties of the DNA origami nanopores due to structural deformation has also been studied by Keyser and co-workers.191 Li et al. conducted a comprehensive study on the permeability and structural deformation by means of an all-atom molecular simulation and nanocapillary electric current recording.192 They found that the bending of the DNA origami plate reduced the current while the separation of the DNA origami layers increased the current. The mechanical properties rely heavily on the design of the DNA origami structures. For example, Shrestha et al. found that the Holliday junctions that are spread throughout a structure play a very important role in the mechanical properties.193 Using optical tweezers, they discovered that the mechanical stability of the DNA origami depended on the density of the Holliday junctions that were located along the stretching direction. Meanwhile, the cooperative transition between the short and long DNA tube isomers was due to the collective mechanic isomerization of many individual Holliday junctions. The change in the mechanical behavior of the DNA origami is responsive to external inputs and could be utilized for single molecule detection.194 In addition to experimental methods, computational modeling may provide a convenient way to evaluate the mechanical properties of a given origami. Kim et al. constructed a computational model that included the origami’s features, such as canonical bends, twists, stretch stiffness, backbone nicks, and entropic elasticities, to predict the shape and flexibility of origami in solution. Their computational results agreed with the experimental results.195 Pan et al. proposed another model that was based on the multiway junction topologies that were constrained by dsDNA to predict the DNA nanostructures.196 With consideration to the steric, electrostatic, and solventmediated forces, an all-atom molecular dynamics model that simulated DNA origami’s structure in aqueous solutions revealed that DNA origami undergo considerable temporal fluctuations on both a local and global scale.197 It also showed that the lattice type of DNA helical arrangements have a considerable influence on the structure’s mechanical properties.

biomacromolecules. More experimental and computational studies are needed to obtain a deeper understanding of the process by which DNA origami forms. 5.2. Thermodynamics and Kinetics of the Self-Assembly Process for Higher-Order DNA Origami Self-Assembly

DNA origami can be used as a unit tile to create higher-order structures, but its assembly behavior is different from that of the small tiles. This difference arises from the relatively large dimensions and structural complexities of the DNA origami tiles. A fundamental understanding of the thermodynamics and kinetics of how origami tiles associate with one another can help improve assembly efficiency and quality. Schulman and coworkers systematically studied the thermodynamics and kinetics of DNA origami dimerization under various interface architectures, such as different lengths and different levels of floppiness associated with the sticky ends.183 Thermodynamic data from various interface designs feature a nonlinear van’t Hoff behavior for dimerization. They assume that the nonlinear effect should be attributed to the origami dimer’s lack of a well-formed interface. The dimerization rate constants were on the order of 105−106 M−1 s−1, which is similar to the rate constants of DNA strand hybridization and small tile association. They also studied the thermodynamics of four origami assemblies during the time it took them to form a tetrameric ring. This research revealed that the interface’s position and strength controlled the dimerization free energies and weak cooperativity during assembly.184 The tetrameric origami assembly does not favor a one-by-one association process; rather, it preferentially forms pairs of dimers, which then bind to each other to form the tetramer. 5.3. Mechanic Properties of DNA Origami

Since a DNA origami structure consists of a large number of DNA helices, the mechanical properties of DNA origami is significantly different from the mechanical properties of dsDNA. The persistence length of dsDNA has been determined at different salt concentrations and temperatures, resulting in values ranging from 30 to 80 nm,185 generally estimated as 50 nm. The wormlike chain model describes that dsDNA is able to sustain a mechanic force up to 10 pN.186 Magnetic torque tweezers were implemented to measure the torsional stiffness of dsDNA and revealed that the effective torsional stiffness C of dsDNA exhibits a force-dependent behavior, with C = ∼40 nm at low forces and up to C = ∼100 nm at high forces.187 The current most widely used DNA origami structure is a parallel alignment of dsDNA in 2D or 3D. Measuring the mechanical properties of these types of structures will provide us with useful information for both design rules and future applications. Kauert et al. applied magnetic tweezers to directly measure the mechanical properties of four- and six-helix bundle DNA origami structures.188 When compared to dsDNA, the bending rigidities greatly increased (15 and 38-fold for the fourand six-helix bundle, respectively), while the torsional rigidities only moderately increased (4.0 and 5.5-fold for four- and six-helix bundles, respectively). They used a model of DNA cylinders, which were connected by 1 nm connectors to simulate the mechanical properties and found that the results aligned with the experimental results. The rectangular 2D DNA origami is relatively more flexible along the direction perpendicular to the DNA helices, and it can be folded edge-by-edge to create tubular structures, if the global twisting and entropy loss were overcome by the decrease in enthalpy.189 Ionic transportation through DNA origami had been used to study mechanical properties as well. Plesa et al. deposited four types of DNA origami structures

6. APPLICATIONS OF DNA ORIGAMI In nanoscience, the precise manipulation of objects in the nanometer scale is a critical challenge. DNA origami has provided a way to engineer molecular structures with unprecedented complexity and nanometer scale resolution. DNA origami has found a variety of applications, from engineering functional devices to studying fundamental mechanisms in nanoscience. 6.1. Molecular Robotics

The concept of molecular robotics extends beyond traditional robotics by using molecules as components and fuels to drive the molecular robot to execute actions following external cues. The 2016 Nobel Prize in Chemistry was awarded to Jean-Pierre Sauvage, Sir J. Fraser Stoddart, and Bernard L. Feringa for the design and synthesis of molecular machines that used chemical synthesis to bridge functional chemical groups together. For 12601


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(Figure 12B).217 The spider walker had a streptavidin body, three DNAzyme legs, and another DNA strand leg that was complementary to a docking station on the DNA origami. When researchers extended a series of staple strands with a sequence complementary to the DNAzyme legs, the researchers were able to form a track for the spider to walk along on the DNA origami surface. A DNA trigger strand was added to release the spider from the docking station when it was given the “start” order. The spider would then walk along the track and simultaneously degrade it through catalytic DNA cleavage. Wickham et al. designed a DNA walker that was able to move 16 steps along a track on the DNA origami.218 After paving the route with strand displacement reactions, enzymatic cleavages and intramolecular strand displacement reactions would drive the movement of the DNA walker on the track. Moreover, the track could be branched into a network that could provide multiple routes for the walker to move along.219 External DNA molecules could navigate the movements of the DNA walker to achieve a programmable motion in response to different inputs. If the DNA walker was equipped with light responsive moieties, its movement could be triggered with light. 220 Instead of programming the motion on a single DNA origami unit, Liber et al. demonstrated that the movement could be engineered in both back and forth directions between two origami units.221 The DNA walkers achieved so far have allowed us to engineer dynamic DNA systems that have the capability to perform more sophisticated motions in the future. Besides walking, DNA structure’s reconfiguration can be performed on a DNA origami substrate as well. Gu et al. engineered a dynamic patterning device that was able to capture specific DNA tiles that fit by conformation requirements.222 The operation of the device started with a DNA origami containing two slots for the cassettes in the PX (paranemic crossover) state or the JX2 (paranemic crossover with two juxtaposed sites) state to fill in, enabling recognition of orientation. Afterward, the states of the two cassettes that were bound to the DNA origami determined the particular type of molecules that could be captured onto the substrate. Olejko et al. immobilized a Gquadruplex on a DNA origami structure that was capable of stretching as a single strand or shrinking as a compacted quadruplex in response to the presence of potassium ions.223 6.1.2. Reconfigurable DNA Origami Devices. In 2009, Anderson et al. created a 3D DNA box with a controllable lid, which was the first dynamic device based on DNA origami.61 They designed a “lock-and-key” system to control the closing and opening of the box. Before the addition of the “key” DNA strands, the lid was closed because the two extended strands on its edge were bound to the strands on the edge of the neighboring face. Once the key strands were presented, it separated the hybridized strands and opened the lid through strand displacement. The DNA’s hybridization and disassociation through strand displacement has been used to build reconfigurable DNA devices. Marini et al. constructed a DNA origami actuator capable of autonomous internal motion (Figure 13A).224 It was made of two subunits: an external ring and an internal disk that were constrained in two opposing locations. The disk in the center was designed to have flexibility via an unpaired region in the middle, which allowed the disk to bend like a wing. The edge of the disk was bridged by a long ssDNA probe that could bind a hairpin molecule, which provided the tension necessary to bend the disk. In AFM images, a hole could be observed upon the addition of the hairpin molecules. Yan and co-workers implemented a “fold-release-fold” strategy based on rationally

example, the pH- or light-sensitive chemical groups enabled the machine to be driven by changes in pH198 or light.199 These types of molecular machines can be designed at an angstrom level and perform motions such as rotating and shuttling.200,201 However, their functions and complexities are constrained by the difficulties of chemical synthesis. DNA is an easily programmable material that is useful for building molecular machines.202−204 Prior to DNA origami development, DNA had already been utilized to build dynamic robots such as molecular walkers202,205−209 and tweezers.210 These molecular robots could be used for autonomous organic synthesis211,212 or to regulate enzymatic reactions.213,214 DNA origami makes the creation of more complicated and functional nanorobots possible. Three kinds of driving forces, toeholdmediated strand displacement reactions, enzyme catalyzed reactions, and base stacking, can power DNA machines. 6.1.1. DNA Walkers. In 2010, Seeman and co-workers reported a system that consisted of a tensegrity triangle walker and a DNA origami platform.215 They provided the system with a track and stations with cargoes (gold nanoparticles) to load (Figure 12A). Upon the addition of fuel (i.e., DNA strands), the

Figure 12. DNA walkers that walk along tracks on DNA origami. (A) The Seeman walker is able to pick up cargo at each station along an assembly line with DNA strand guidance.215 (B) A streptavidin spider consisting of three deoxyribozyme legs can walk along a designed track by hydrolyzing the nucleic acid track.217

tensegrity triangle walker would move along the track and load the intended cargoes at the stations. Additional DNA strands controlled whether or not the walker was loaded with the cargo at each station. The additional DNA strands controlled the station conformation and therefore the cargo availability. All movements were processed through strand displacement reactions. This system revealed that the control of molecular movements can be completed at a nanometer scale, through DNA’s programmability in both structure and dynamics. Catalytic reactions by enzymes can drive the DNA walker to move on the DNA origami platform as well. Polycatalytic assemblies were reported to exhibit diffusion behaviors on a substrate-displaying matrix that are driven by continuous catalytic cleaving of substrates.216 With the programmability of DNA origami, the substrate pattern could be designed and programmed, leading to a programmable control of movement. Lund et al. designed a DNA spider walker that could execute “start,” “flow,” “turn,” and “stop” commands 12602


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Figure 13. Reconfigurable DNA structural robotics. (A) A DNA origami hatch.224 Images reproduced from ref 224. Copyright 2011 American Chemical Society. (B) A DNA origami quasi-fractal structure.225 Images reproduced from ref 225. Copyright 2012 American Chemical Society. (C) A four-bar DNA machine that has Bennett linkages and that can traverse a complex 3D motion path between closed and opened states.228 Images reproduced with permission from ref 228. Copyright 2015 National Academy of Sciences. (D) A heterotrimeric reconfigurable nanorobot that can change between three different conformational states based on the surrounding magnesium concentration.137 Images reproduced with permission from ref 137. Copyright 2015 AAAS. (E) Detection of small molecule with DNA origami tweezers.233 Images reproduced with permission from ref 233. Copyright 2011 Macmillan Publishing Ltd. (F) A DNA origami robot consisting of four arms in a rhombus shape that can control distances between interacting molecules.234 Images reproduced with permission from ref 234. Copyright 2016 Macmillan Publishing Ltd.

successfully achieved a set of DNA origami robots that were capable of performing complex 3D motions. These origami robots included a hinge, a slider, and a crank-slider (Figure 13C).228 They fabricated two versions of the slider with different ranges of motion and stiffness, which were controlled by the lengths of the ssDNAs that were used as connection cables. They

designed strand displacement reactions to reconfigure a simple square frame origami structure into a complex, quasi-fractal pattern (Figure 13B).225 To achieve more complicated reversible motions and mechanics, Castro and co-workers built DNA nanomachineries by implementing the design principles of macroscopic mechanical machines.226,227 For example, they 12603


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Figure 14. DNA origami-templated nanomaterial architectures. (A) Plasmonic nanostructures that are rationally organized with DNA origami templates in a variety of patterns, including lines, 3D clusters, and 2D lattices.251−253,258−262 Images reproduced from ref 251. Copyright 2010 American Chemical Society. Images reproduced with permission from ref 252. Copyright 2010 Wiley. Images reproduced from ref 253. Copyright 2016 American Chemical Society. Images reproduced with permission from ref 258. Copyright 2011 Wiley. Images reproduced from ref 259. Copyright 2016 American Chemical Society. Images reproduced from ref 260. Copyright 2013 Macmillan Publishing Ltd. Images reproduced with permission from ref 261. Copyright 2015 Macmillan Publishing Ltd. Images reproduced with permission from ref 262. Copyright 2016 Macmillan Publishing Ltd. (B) Carbon nanotubes perpendicularly aligned to a rectangular DNA origami.19 Images reproduced with permission from ref 19. Copyright 2010 Macmillan Publishing Ltd. (C) Quantum dots are positioned with a programmable periodicity on a DNA origami nanotube.276 Images reproduced from ref 276. Copyright 2010 American Chemical Society.

continuously rotating without any energy input.230 The rotary apparatus was assembled from three DNA origami parts, a rotor unit, a clamp unit and a socket unit, through the interaction of base-stacking or sticky end hybridization. Lin et al.231 and Simmel et al.232 constructed DNA origami structures with topological connections like rotaxanes. The devices are capable of performing sliding and rotational movements along a defined track. Other than DNA hybridization and strand displacement, the geometrical arrangement of base stacking can also be implemented to drive the motion of DNA origami structures. Dietz and co-workers discovered that the geometrical arrangement of base stacking tends to be broken under conditions that involve higher temperatures or lower divalent ion concentrations.137 On the basis of this knowledge, they built a set of

found that the design with shorter connections resembled a linear spring with a stiffness of 0.42 pN/nm, while the design with the longer connection cable exhibited nonlinear force−extension behavior with a stiffness of 0.07 pN/nm at a shorter extension and 0.21 pN/nm at a longer extension. Next, a DNA device consisting of two rigid arms that were joined along the edge through a scaffold was designed to perform purely rotational motions. Theoretically, rotational and linear motions can be integrated to perform any complex motion. On the basis of this, the slider and the hinge were combined to create a crank-slider to perform complex 3D motions. Later, they designed a DNA origami compliant joint with tunable mechanical properties.229 The tunable torsional stiffness was tuned from 107 pN nm/rad to 367 pN nm/rad by just adding a few strands. Dietz et al. built a nanometer scale rotary apparatus that was capable of 12604


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strand that protrudes out from a structure.250 Yan and coworkers reported the novel use of DNA origami to arrange gold nanoparticles in a linear fashion on a triangular DNA origami (Figure 14A).251,252 They were the first to demonstrate that they could precisely control the number and position of nanoparticles on a discrete architecture. The origami-nanoparticle architectures have since then been expanded and have exhibited novel properties.237,253−256 For example, DNA origami was used to study distance-dependent single-fluorophore quenching by gold nanoparticles.257 Zhao et al. reported a method of encapsulating gold nanoparticles in a DNA origami cage and attaching nanoparticles onto the outsides of the cage at specific sites (Figure 14A).258 Recently, Shen et al. designed a DNA origami clamp to envelope and specifically functionalized a gold nanorod with a controlled number of nanoparticles at specific positions (Figure 14A).259 Gold nanoparticle clusters in planet-satellite fashion were assembled using DNA origami nanotubes to connect the nanoparticles (Figure 14A).260 To better control the geometry between the nanoparticles, Gang et al. designed a DNA origami structure that was shaped like an octahedron with attachment sites on the vertices.261 These attachment sites captured gold nanoparticles and achieved an octahedral arrangement of gold nanoparticles (Figure 14A). The assembled complex was visualized with cryoEM and small-angle X-ray scattering. They further demonstrated that the octahedral DNAnanoparticle complex could be further assembled into a higherorder lattice structure by using the nanoparticles as the connectors. They also used a planar DNA origami with gold nanoparticles that were held centrally and assembled into predetermined patterns by base pairing between origami units (Figure 14A).262 By programming the interactions between the origami-particle units, a nanoparticle complex analog of Leonardo da Vinci’s Vitruvian Man was successfully formed.262 Gold nanoparticles, on the other hand, are capable of organizing the arrangement of DNA origami as well. For example, Turberfield et al. reported a strategy to embed a nanoparticle with a number of DNA origami bundles to form a flower-shaped core−shell structure.263 This kind of structure is capable of growing a higher-order lattice, optical trapping,264 and delivering drug.265 Fan et al. developed a gold-nanoparticle-mediated Jigsaw-puzzle-like assembly strategy to control the formation of DNA origami-nanoparticle complexes.266 Other than gold nanoparticles, silver nanoparticles have also been positioned on DNA origami substrates that have well-defined geometries and distances.267 Ding et al. directed the assembly of silver nanoparticles onto a triangular origami that was embedded in a chain structure.252 Eskelinen et al. constructed a bow-tie silver structure with a rectangular DNA origami.268 Besides nanoparticles, Maune et al. showed that DNA origami can be used to precisely arrange single-wall carbon nanotubes (Figure 14B).19 In their experiment, a nanotube was modified with short oligos that had three domains: a dispersal domain to bind the carbon nanotube, a protection domain to prevent the DNA strand from being adsorbed by the carbon nanotube, and a toehold domain to initiate the strand displacement reaction via the capture strands that extended from the DNA origami template. Through strand displacement reaction, carbon nanotubes could be immobilized onto the DNA origami surface. A similar strategy was reported by Zhao et al.269 as well. Mangalum et al. reported that a carbon nanotube that bound to the ssDNA and extended out from the edge of a DNA origami could organize the DNA origami into linear 1D higher-order structures.270 Alternatively, a method employing streptavidin−biotin inter-

dynamic devices, such as dynamic configurable frames and a heterotrimeric nanorobot that could be driven by the changes in the Mg2+ concentrations. For example, a reconfigurable lattice that could move between compacted and wireframe states was created by the 2D polymerization of a dynamic switch.137 This dynamic switch was capable of switching between crossed and linear states. A heterotrimeric nanorobot was constructed that could switch between three different states: disassembled, assembled with open arms, and assembled with closed arms (Figure 13D). Reconfigurable DNA devices may have a variety of applications. Kuzuya et al. designed a DNA tweezer-like origami with target recognition sites that could be used as a singlemolecule beacon. The single-molecule targets for this beacon that could actuate the device and could be directly visualized under AFM ranged from materials such as metal ions to proteins (Figure 13E).233,235,236 Recently, Ke et al. designed a nanoactuator that consisted of four stiff arms that were connected in a rhombus shape.234 The angle between the opposing joints had to be mirrored for this rhombus shape to be created (Figure 13F).237 Two different mechanisms were used to actuate the device. One mechanism was the addition of extra structurelocking strands to form two rigid DNA double helices that would determine the distance between the connecting sites and the angle of the rhombus. A probe was extended from the staples on the interior sides of the arms, which bound to the target molecules and induced a long-range allosteric conformational change to the device. Ke et al. applied the allosteric conformational change to regulate the formation and separation of a split green fluorescence protein complex. The second mechanism was a compressed spring that maintained its closed state via a locking mechanism on the top and bottom hinges of the DNA rhombus. The locking action could have been caused by a variety of molecular interactions such as DNA hybridization, RNA-DNA hybridization, and G-quadruplex formation. A similar strategy was used to study molecular interactions.238 Instead of regulating molecular function by controlling the interaction distance, dynamically blocking an enzyme from and exposing it to its substrate could be another control. For example, Torelli et al. constructed a capsule-like DNA origami structure with a controllable window and a DNAzyme that was attached onto the inner surface of the window.239 In the absence of a target DNA strand, the window was closed and the DNAzyme was protected from accessing its substrate in the solution. When the target DNA strand was present, it would open the window and expose the DNAzyme, which resulted in a colorimetric reaction. The actuation of DNA devices could also be actuated by light if light-responsive moieties were integrated into the device.240 6.2. DNA Origami Templated Architectures and Nanomaterial Functionality

6.2.1. DNA Origami Templated Architectures. The organization of nanomaterials such as metal nanoparticles, quantum dots, and carbon nanotubes with nanometer-scaled precision is one of the central challenges in nanotechnology. DNA origami-guided self-assembly provides a feasible method to address this problem.241 Simple DNA structures such as DNA duplexes or tiles can help organize metal nanoparticles.242−249 DNA origami with custom designer structures and unique addressability may enable more complicated nanomaterial architectures to be built. To decorate the DNA origami structure with nanoparticles, the nanoparticles are required to be modified with nucleic acids that enable them to hybridize with a docking 12605


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Figure 15. 3D Nanoparticle superlattices that were assembled with a DNA origami frame. (A) A diamond nanoparticle lattice can be assembled with a tetrahedron DNA origami frame by using nanoparticles as connectors at the vertex.280 Images reproduced with permission from ref 280. Copyright 2016 AAAS. (B) Various nanoparticle superlattices are assembled from DNA origami frames.281 Images reproduced with permission from ref 281. Copyright 2016 Macmillan Publishing Ltd.

reported that the lifetime of the quantum dot could be tuned by the numbers, relative positions, and sizes of the gold nanoparticles that were placed close by.279 Engineering 3D nanoparticle superlattices with a prescribed geometry is a daunting challenge. DNA origami provides support with a well-defined geometry for the 3D lattice of nanoparticles growth. Gang and co-workers designed a tetrahedron-shaped DNA origami with gold nanoparticles attached to the four vertices (Figure 15A).280 These nanoparticles then acted as connectors to link the tetrahedron origami-nanoparticle units into a well-ordered face-centered cubic lattice structure. If another gold nanoparticle was positioned inside the tetrahedronshaped origami structure, the final lattice structure achieved would be in the shape of a diamond. By varying the size and number of particles inside, a zinc blend and a “wandering” zinc blend lattice style was achieved. Recently, they successfully

actions successfully demonstrated that carbon nanotubes could be arranged onto a DNA origami substrate with predetermined directions and positions.271 DNA origami structures have also been used to organize quantum dots, which are semiconductor nanocrystals that exhibit unique optical and electronic properties.272−274 Deng and colleagues developed a method to synthesize DNA-functionalized core/shell quantum dots for the DNA origami aided organization.275 Bui et al. used a tubelike origami structure decorated with biotin to organize quantum dots that were coated with streptavidin (Figure 14C).276 Liddle et al. explored a variety of factors that affected the yield of quantum dots that were able to bind onto a target DNA origami structure, such as the valency of the binding site, biotin linker length, and spacing between the binding locations.277 They then used a single particle-tracking system to monitor the binding in real time and 3D.278 They also 12606


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Figure 16. Anisotropic plasmonic structures were assembled with DNA origami. (A) Gold nanorods and nanoparticles were positioned to form anisotropic nanoarchitectures with light-tailoring capabilities.288−290 Images reproduced from ref 288. Copyright 2011 American Chemical Society. Images reproduced from ref 289. Copyright 2013 American Chemical Society. Images reproduced from ref 290. Copyright 2013 American Chemical Society. (B) Gold nanoparticle helices, nanorod helices, and toroidal superchiral plasmonic structures were assembled with DNA origami as the template.291−293 Images reproduced with permission from ref 291. Copyright 2012 Macmillan Publishing Ltd. Images reproduced from ref 292. Copyright 2015 American Chemical Society. Images reproduced from ref 293. Copyright 2016 American Chemical Society.

strained into the small region between the particles and would exponentially decay outside that region. To experimentally achieve plasmonic coupling, a method that allows the precise positioning of metallic nanoparticles at a nanometer resolution is required. The DNA origami structure is an ideal platform to address the challenge of assembling plasmonic nanostructures with new optical properties.284−286 For example, Klein et al. used a DNA origami scaffold to align a series of gold nanoparticles (10 nm in diameter, 14 nm away from each other) for visible spectrum subdiffraction plasmonic wave-guiding.287 When the metal-nanostructure assembly is asymmetric, its plasmonic coupling can interact with the incident light according to the chirality of the assembled plasmonic structure. For example, Pal et al.,288 Wang et al.,289,294,295 and Ding et al.296,297 constructed a series of anisotropic gold nanorod nanostructures using DNA origami as scaffold and studied their properties of tailoring light chirality. Gold nanorod dimer structures with various predetermined inter rod angles and distances led to tunable optical responses to opposite circular polarized lights

designed a variety of DNA origami structures with polyhedron shapes (Figure 15B), including an octahedron, a cube, an elongated square bipyramid, a prism, and a triangular bipyramid, to assist in the construction of a 3D nanoparticle lattice.281 The geometries of the origami frames and the DNA-assisted binding of the gold nanoparticles helped to define the connections between the gold nanoparticles and to determine the different crystallographic lattices that could be achieved. 6.2.2. Chiral Plasmonic Structures. Metallic nanoparticles have surface plasmonic resonance that affects a very small surrounding area. Mie282 and Prodan et al. predicted that metal nanoparticles can absorb light and a plasmonic coupling between nanoparticles can occur if the distance between the nanoparticles is short enough.283 The coupling of plasmonic resonance is analogous to the hybridization of electronic wave functions from atomic orbitals in a molecule that form molecular bonds. In general, the plasmonic coupling requires the edge-to-edge distance between particles to be less than 2.5 times the particle diameter.283 In this way, near-field coupling would be con12607


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(Figure 16A). Chirality was also achieved through creating asymmetric nanoparticle tetramer clusters by using a DNA tetrahedron. The symmetry was broken either by controlling the distances between the particles or by varying the sizes of the particles (Figure 16A).290,298,299 Except for the structure of the dimeric nanorods and tetrameric particles, chiral plasmonic structures can also be achieved by assembling helical superstructures of gold nanorods or particles. Liedl and co-workers folded a DNA origami of 24helical bundles with 9 helically arranged binding sites that were available for attachment by the gold nanoparticle (Figure 16B).291 The optical responses of the helical arranged nanoparticle assemblies can be tuned in handedness, color, and intensity. The gold nanoparticle helices could also be constructed by folding a rectangular DNA origami with diagonally arranged nanoparticles into a tube.300 Other than the helical arrangement of nanoparticles along a cylinder, Urban et al. constructed a plasmonic toroidal structure through the hierarchical selfassembly of four identical origami units that were bent in a 90 deg angle (Figure 16B).293 Lan et al. achieved superhelical structures by assembling two gold nanorods into an X-shape on the bottom and top surfaces of a rectangular DNA origami structure (Figure 16B).292 By taking advantages of the strand displacement reaction, plasmonic structures can be reconfigurable and dynamic. Liu and co-workers used a switchable DNA origami template consisting of two connected DNA bundles to hold two gold nanorods.301 DNA locks that extended from the origami bundles controlled the relative angle between the gold nanorods through a toeholdmediated displacement reaction (Figure 17A). The plasmonic structure could reconfigure to either a left- or right-handed state by using different fuel strands.301 Later on, they successfully constructed a walker system where gold nanorods could execute directional, progressive, and reversible walking on a DNA origami platform with real-time switchable optical properties (Figure 16C).302,303 The dynamical chirality change could be engineered to be responsive to external signals, such as light if the linkage strand was modified with light sensitive moieties.304 Liedl and co-workers built switchable gold nanoparticle helices by immobilizing one end of the origami bundle onto a surface (Figure 16B).305 Their optical responses were switched by controlling the orientation of the nanoparticle helix to either stand up or lay flat on the surface. 6.2.3. Plasmonic Hotspots. Plasmonic structures can create a strong local field that enhances the brightness of the dye’s fluorescent emission when the dye is sitting at a specific local spot, called a hot spot. This concept has been implemented to create nanoantenna to enhance the excitation field in a very small local area,306 to direct single-molecule emissions,307 and to increase quantum yields for the detection of low-quantum yield dyes.308,309 DNA origami’s addressability with high spatial resolution provides a convenient platform to engineer enhanced antenna structure based on metallic nanoparticles. Tinnefeld and co-workers reported a self-assembled nanoantenna that was constructed by positioning a dye between two gold nanoparticles with a distance of several nanometers on a DNA origami to enhance the dye fluorescence intensity in a plasmonic hotspot (Figure 18A).310 They first immobilized a pillar-shaped DNA origami with a length of 220 nm and a diameter of 15 nm on a surface through a biotin−streptavidin interaction. Two gold nanoparticles and an ATTO747N dye were bound to the pillar origami structure by hybridizing it with the capturing strands at prescribed positions. A maximum fluorescence enhancement of

Figure 17. Reconfigurable chiral plasmonic devices. (A) 3D plasmonic metamolecules can switch between left- and right-handed states, and CD spectra are monitored to verify the structural switching in real time.301 Images reproduced with permission from ref 301. Copyright 2014 Macmillan Publishing Ltd. (B) Nanoparticle helices that were sitting on a surface with switchable CD by controlling the orientations.305 Images reproduced with permission from ref 305. Copyright 2013 Macmillan Publishing Ltd. (C) A plasmonic nanorod walks stepwise on a DNA origami staircase with six different states. TEM images and CD spectra are used to monitor the movements.302 The frame size of each TEM image is 80 nm. Images reproduced with permission from ref 302. Copyright 2015 Macmillan Publishing Ltd.

117-fold was achieved when the dye was positioned in a gap of 23 nm between a dimer of 100 nm gold nanoparticles. They showed that the technique could be used for single-molecule analysis by 12608


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Figure 18. Plasmonic hotspots on the DNA origami surface. (A) A DNA origami pillar with two gold nanoparticles forming a dimer that surrounded a dye molecule. On the right, a simulated electric field intensity is shown for a monomer and dimers, which can extensively enhance dye fluorescence.310 Images reproduced with permission from ref 310. Copyright 2012 AAAS. (B−E) Plasmonic hotspots for the surface-enhanced Raman scattering by using DNA origami to position nanoparticles with nanometer resolution.315,318,321,322 (B) Images reproduced from ref 315. Copyright 2013 American Chemical Society. (C) Images reproduced from ref 318. Copyright 2014 American Chemical Society. (D) Images reproduced with permission from ref 321. Copyright 2014 Macmillan Publishing Ltd. Images reproduced from ref 322. Copyright 2014 American Chemical Society.

al. first used a triangular-shaped DNA origami to position a pair of gold nanoparticle dimers with a distance of about 25 nm.315 This method makes the observation of several molecules possible (Figure 18B) by SERS. They further expanded the system to Au−Ag-core−shell nanoparticles and hybrid structures with graphene to study the surfaced-enhanced SERS.316,317 Minimizing the gap between the nanoparticles was the key to improve and enhance the SERS signal. Liedl et al. designed a three-layered rectangular DNA origami structure with two gold nanoparticles attached to the top and bottom, resulting in a gap of ∼6 nm (Figure 18C).318 In their following study, they reported that the optothermal-induced shrinkage of a DNA origami template was capable of tuning the distance between two nanoparticles in a range from 1 to 2 nm and the design was employed to control the plasmon-exciton coupling.319,320 Thacker et al. constructed a multilayered DNA origami structure with two grooves that were separated by a ridge of DNA double helices (Figure 18D).321 The

visualizing the binding and unbinding events of the short DNA strands, as well as the dynamic conformational changes of the DNA Holliday junction with FRET. They further improved the design and parameter settings for the DNA origami based nanoantennas and reached more than a 5000-fold fluorescence enhancement.311 Using this method, single-molecule detection at 25 μM can be achieved. They also demonstrated that the photobleaching of a dye can be tuned by controlling the distance between the dye and the gold nanoparticles on a DNA origami platform.312 Wang et al. investigated the fluorescence enhancement effect of gold nanoparticles on an organized ensemble of fluorophores with various distances.313 With a DNA origami platform, Aissaoui et al. revealed that gold nanoparticle enhanced the FRET efficiency through improving the energy transfer rate.314 A surface-enhanced Raman spectroscopy (SERS) signal can also be amplified by using the same strategy. For example, Bald et 12609


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Figure 19. Applications of DNA origami structures in biophysical studies. (A) A molecular tug-of-war study using DNA origami to control the number and position of two motor proteins.323 Images reproduced with permission from ref 323. Copyright 2012 AAAS. (B) A DNA origami device that is able to position molecules down to a Bohr radius level329 and (C) its employment to study nucleosome interactions.331 (B) Images reproduced with permission from ref 329. Copyright 2015 Macmillan Publishing Ltd. (C) Images reproduced with permission from ref 331. Copyright 2016 AAAS. (D) A DNA origami bundle serving as a rigid linker to repress the noise of optical tweezer experiments.335 Images reproduced with permission from ref 335. Copyright 2013 Wiley. (E) Base-stacking study at a single-molecule level on a tethered-beam platform.336 Images reproduced with permission from ref 336. Copyright 2016 AAAS. (F) A DNA origami nanoscopic force clamp for single molecule force spectroscopy.338 Images reproduced with permission from ref 338. Copyright 2016 AAAS. (G) A DNA origami nanotube that is employed as a detergent-resistant liquid crystalline medium to induce a weak alignment of a membrane protein to help NMR structure determination.67 Images reproduced with permission from ref 67. Copyright 2007 National Academy of Sciences. (H) A synthetic DNA origami ion channel with modified cholesterol can be inserted into lipid membranes.357 Images reproduced with permission from ref 357. Copyright 2012 AAAS. (I) A synthetic DNA nanopore inserted into a solid-state nanopore by an electric field.359 Images reproduced from ref 359. Copyright 2012 American Chemical Society. (J) A DNA origami structure is used as a support to host proteins and control their orientation for CryoEM structure determination.346 Images reproduced with permission from ref 346. Copyright 2016 National Academy of Sciences.

6.3. Biophysical Studies with DNA Origami

gold nanoparticles were arranged to sit in the two grooves, and the ridge maintained a distance of ∼5 nm between them. PiloPais et al. created a cluster of four gold nanoparticles that were attached on the four corners of a rectangular DNA origami (Figure 18E).322 Upon irradiation with light at the resonance wavelength, the small gap between the gold nanoparticles formed a strong electromagnetic field in the center spot that could be harnessed to enhance the SERS intensity from the molecules located at the hot spot.

6.3.1. DNA Origami-Aided Protein Function Study. The programmability of DNA origami enables us to create a variety of nanodevices that can help answer biophysical questions and solve challenging problems. Motor protein is a type of protein that is able to move along the surface of a suitable substrate and transport cargo in cells. DNA origami with predetermined geometric and numerical arrangements of motor proteins enables us to study their movements in detail. Derr et al. designed a DNA nanotube that was used as a scaffold to attach to cytoplasmic dynein and kinesin-1, microtubule-based motors 12610


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interest in a single-molecule mechanical study is usually linked to two micron-sized polymer beads through molecular linkers such as dsDNA.334 The stiffness of the linkers plays an important role in the signal-to-noise ratio of the measurements, which may bury the signal of weak interactions. To make the linker more rigid, Pfitzner et al. used DNA origami structures as rigid beams for measurements of high-resolution single-molecular mechanics (Figure 19D).335 The rigid DNA origami bundle linkers led to a higher resolution at low forces and enabled the mechanical analysis of a weak DNA hairpin with 6-bp stem. Dietz and coworkers applied this technique to study the forces of base stacking at a single-molecule level (Figure 19E).336 They designed DNA origami structures as optical tweezers to position the blunt-ends of the DNA helices and to measure the weak base stacking force. Briefly, their experimental design consisted of three components: (1) parallel arrays of base pair stacks that allowed them to study the stacking effect and signal transportation from weak stacking interactions into force regimes as well as the time scales accessible for optical trapping; (2) stiff DNA origami bundles to help them to reduce the noise occurring at low forces that have fast kinetics; and (3) a flexible tether linking two beams to help them to increase the frequency of the binding and rebinding events, making repeatable observation and classification of the events possible. All 16 sequence combinations of the stacking forces had been studied in the presence of 20 mM Mg2+ or 500 mM Na+, mimicking the in vitro DNA environment for the DNA nanotechnology and cellular environments, respectively. Their study showed that the strength of the base stacking shared the same sequence-dependent rule with hybridization: among all of the base stacking pairs, the strongest stacking force (−3.42 kcal/mol) occurred between a CG:CG pair, and the weakest stacking was between TA:CG (−0.81 kcal/ mol).337 Usually the single-molecule force spectroscopy is required to have the target molecule of interest attached to a microsized object. The throughput of the measurements is relatively low. To overcome these limitations, Nickels et al. constructed a nanoscopic force clamp via DNA origami to study molecular interactions under mechanical forces (Figure 19F).338 The entropic spring behavior of ssDNA was used to exert defined and tunable forces on the target molecule. The tuning occurred by controlling the length of the ssDNA that was connected to the rigid DNA origami substrate and the target molecule through anchor points. The fixed distance was imparted by the DNA origami and the given length of the ssDNA provided an approximately constant force to the target molecule over time. The conformation change to the target molecule under mechanical forces was monitored with single-molecule FRET. They applied the DNA origami nanoscopic force clamp to study the conformational exchange of a Holliday junction and the binding of a protein to a dsDNA under various mechanical forces. The results revealed that the DNA Holliday junctions had a preferential conformation when it is under a relatively strong force. The protein’s binding to the dsDNA was inhibited if a strong force was applied to stretch the dsDNA substrate, which indicated that the protein/DNA binding caused a mechanical force to bend the DNA. Recently, Shrestha et al. reported the usage of DNA origami to study the folding of molecules in confined space based on single molecule force spectroscopy.339 The stability of folded molecules is predicted to increase in a confined space because of entropic effects. Shrestha et al. studied the stability of folded G-quadruplex inside DNA origami cages with difference sizes and found that

with opposite polarity that transport cargo in cells, aiming to study their motion mechanisms and cooperative behaviors (Figure 19A).323 By varying the number of proteins from one to seven and the types of motors attached to the DNA nanotube cargo, they found that the number had a minimal influence on the directional velocity if motors of the same polarity were attached. When a set of motors with opposing polarities were attached, they became engaged in a tug-of-war that was resolvable by replacing one of the motor species. Hariadi et al. studied the collective behavior of myosin motors by constructing artificial myosin filaments on DNA origami scaffolds. They found that neither the myosin density nor their spacing influenced the gliding speed of the actin filaments, supporting the idea that myosin ensembles act as energy reservoirs that buffer individual stochastic events to achieve a smooth and continuous motion.324−326 Iwaki et al. studied the influence of local heat on the myosin movement by using a DNA origami bundle to precisely control the distance between the myosin and the nanoparticle heat source.327 They further applied a DNA origami nanospring to study the movement of myosin under a mechanical tension and found that mechanical force could induce the transition of myosin VI heads from the nonadjacent binding state to the adjacent binding state.328 The study of nucleosomes that consist of DNA that is wrapped around a disc-shaped protein octamer is another example that DNA origami can be used to study protein function in an unprecedented way. Dietz and co-workers first developed an exquisite DNA origami hinge that was able to position molecules at a resolution down to a Bohr radius scale (Figure 19B).329 The angle between the two rigid arms of the hinged DNA origami device could be controlled by an adjuster helix. Molecular distance was adjusted by varying the position of the DNA origami device relative to the adjuster helix location. Using this DNA origami device, they were able to adjust the average distance between fluorescent molecules or reactive chemical groups from 1.5 to 9 nm in 123 discrete steps. The smallest step size possible was an unprecedented 0.04 nm that is comparable to the Bohr’s radius. Afterward, they used the hinge origami device to explore the forces between the nucleosome and the salt-induced nucleosome unwrapping (Figure 19C).330,331 By placing two nucleosomes on the two arms of a DNA origami hinge separately and observing their interaction through cryo-electron microscopy (cryoEM) and FRET, they derived a Boltzman-weighted distance-dependent energy landscape for a nucleosome pair interaction. Similarly, through the observation of conformational changes to the origami hinge from bend to more extended states, caused by the unwrapping of the nucleosome leads, the binding equilibrium constant and the energetic penalties were extracted. Their investigation revealed that upon an increase in the salt concentration, nucleosomes are more likely to dissociate. Le et al. used a similar DNA origami design to study the stability of the nucleosome’s wrapping as well.332 They investigated the wrapping efficiency of the nucleosome with different lengths of DNA through the angle distribution of the DNA hinges. They found that a 75 bp linker DNA was able to fully wrap the nucleosome, while the DNA linkers with a shorter length, such as 6, 26, and 51 bp, only partially wrapped the nucleosome. These studies demonstrated that DNA origami devices could be good tools to study protein interactions and dynamics. 6.3.2. DNA Origami-Based Single-Molecule Force Spectroscopy. Single-molecule mechanical studies play a crucial role in learning dynamic conformational changes of molecules and deriving their energy states.333 The molecule of 12611


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tight orientation control of the protein relative to the DNA origami. 6.3.4. DNA Origami Nanopores. Membrane nanopores are of great importance to control molecule transport across cell membranes. Natural nanopores such as α-hemolysin, MspA, and solid-state synthetic nanopores made from Si3N4 and polyethylene terephthalate (PET) have shown great promise in single-molecule analysis and next-generation sequencing technology.347−349 De novo designed molecular nanopores offer great opportunities to study cellular activities and provide useful tools for bioanalysis. DNA is one of the ideal molecules to achieve this goal because of its high programmability that allows custom shape design and easy modification.350−356 In 2012, Langecker et al.357 designed a synthetic origami nanopore that consisted of a stem that was capable of penetrating and spanning into a lipid membrane. It also had a barrel-shaped cap that could adhere to the cis side of the membrane (Figure 19H). The nanopores constructed out of DNA conducted electricity when a voltage was applied, and it displayed similar gating properties as natural protein nanopores due to the structure’s thermal fluctuations. Langecker et al. also demonstrated that the origami nanopore was useful for single-molecule analysis to investigate DNA hairpin unzipping and G-quadruplex unfolding. Krishnan et al. constructed a DNA origami nanopore that was 4 nm and had stable electrical properties.358 DNA origami nanopores could be incorporated into a lipid membrane either by using a large number of hydrophobic functionalization on their sides or by using streptavidin linkages between biotinylated nanopores and lipids. Bell et al. constructed a funnel-like origami nanopore and interfaced it with solid-state nanopores (Figure 19I).359 In their experiment, the DNA origami structure was assembled and added to the solution, and then a voltage was applied across the solid-state nanopores. This voltage drove the origami nanopores into the solid-state nanopores. The origami nanopores were ejected when an opposite voltage was applied. The DNA origami nanopores could offer either physical control or chemical control to slow down the translocation of the DNA through the nanopores360 and might function as gatekeepers.190,191,361 Recently Barati Farimani et al. constructed a DNA origamigraphene hybrid nanopore that was capable of distinguishing four types of DNA bases.362

the stability of G-quadruplex increases when the size of DNA origami cage decreases. 6.3.3. DNA Origami Aided Protein Structure Determination. DNA structures are also useful for organizing proteins in order to determine the protein structures through nuclear magnetic resonance (NMR), X-ray diffraction, or single particle CryoEM. Douglas et al. showed that DNA origami nanotubes can be used to induce weak alignments of membrane proteins for NMR structure determination (Figure 19G).67,340 In the method described, a 0.8-μm-long DNA origami nanotube was assembled to form a detergent-resistant liquid crystal for the weak alignment of a membrane protein and a ξ−ξ transmembrane domain of the T cell receptor. The measured residual dipolar couplings (RDCs) could serve as the NMR structural information source. The results agreed with a previously determined structure, demonstrating the feasibility of the DNA origami-based method for resolving membrane protein structures. The method was then successfully applied to help determine the local and secondary structures of mitochondrial uncoupling protein 2, by piecing together molecular fragments from the Protein Data Bank that best fit the experimental RDCs from samples that were weakly aligned in the DNA nanotube liquid crystal.341 The method of using 3D DNA lattices as a scaffold to organize proteins for X-ray crystallography was first proposed by Seeman in 1982.36 The successful construction of macroscopic DNA crystals48,342 indicates that researchers have become closer to reaching the goal of determining protein structures with the aid of DNA lattices. Recently, thanks to efficient direct electron detectors and image processing algorithms, cryo-EM has emerged as a powerful method for determining protein structures to near-atomic resolution.343,344 The cryo-EM structure of DNA origami has already been reported by Dietz et al. with an estimated resolution of 9.7 Å at the core, to 14 Å at the periphery.88 Despite being able to obtain the atomic structures of proteins without crystallization, it is still difficult to determine the structure of proteins that are smaller than 200 kDa because it is difficult to identify the individual protein particles from the background noise in the electron transmission images.345 The problem may be overcome if the proteins are tethered to a DNA origami structure because the DNA origami particle has a relatively large size and known structure and can thus serve as an index to help locate the much smaller protein particles. In addition, it is possible for the DNA origami structures to control the orientation of the protein structures to facilitate the image processing because it would make the classifications of the 2D projections much easier. Martin et al. designed a hollow cylinder DNA origami structure with a doublestranded DNA helix across its center, which allowed the binding of a transcription factor p53 (Figure 19J).346 The electron images of a protein were collected through the hollow center of a cylinder. The hollow DNA origami support also prevented the protein from unfolding, by keeping it inside of the cylinder’s hole, during the sample preparation from adsorption to the air−water interface. Meanwhile, the DNA helix across the cylinder could control the orientation of the transcription factors by shifting the binding domain along the helix. With this design, a cryoEM structure of the protein with ∼15 Å resolution was achieved. There is still room for improvement, and the possible improvement strategies may include the following: increasing the rigidity of the DNA origami by cross-linking the DNA helices and sealing the nicks of the staples, shortening and increasing the rigidity of the linkers for the protein binding domain, or using multivalent binding for the protein of interest so that there is a

6.4. Drug Delivery with DNA Origami

Disease therapeutics require targeted deliveries of drugs to kill abnormal cells and reduce side effects. Various materials such as self-assembled polymers363,364 and nanoparticles365−368 have been used as drug delivery carriers. DNA origami is one of the most promising materials because of its biocompatibility, minimal cytotoxicity, and higher programmability for targeted and controlled release.369−374 As DNA origami structures are assembled from biomolecules, they can be digested by cellular machineries without causing an accumulation of debris within the cell. It is desirable if the DNA origami structures remain intact in intercellular and cellular environments before completing their delivery missions. Yan and co-workers demonstrated that DNA origami possessed an innate resistance to nucleases in cell lysates for at least 12 h at room temperature.375 The stability of DNA origami have also been studied in other media, such as in tissue culturing,376 serum,377 in the presence of chaotropic agents,378 protein crystallization buffer,379and organic solvent.380 DNA origami is capable of delivering anticancer drugs. Ding and co-workers published an article on the use of variously 12612


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Figure 20. DNA origami as a drug delivery vehicle. (A) A DNA origami triangle and nanotube can be used to load drugs, photosensitizers, and gold nanorods for chemotherapy, photodynamic, and photothermal therapies.381,390,391 (B) DNA origami structures for CpG delivery that enhances immunoresponse.392 Images reproduced from ref 392. Copyright 2011 American Chemical Society. (C) Logic computation-controlled antibody delivery carried out by a DNA origami box that opens upon binding with an aptamer target.398 Images reproduced with permission from ref 398. Copyright 2012 AAAS. (D and E) Lipid bilayer- and virus-coated DNA origami structures with enhanced cellular uptake efficiency.405,406 (D) Images reproduced from ref 405. Copyright 2014 American Chemical Society. (E) Images reproduced from ref 406. Copyright 2014 American Chemical Society.

could also circumvent the efflux pump-mediated drug resistance in leukemia cells with clinically relevant drug concentrations.387 Other therapeutic methods, such as photothermal and photodynamic therapy, are also possible with using DNA origami.388,389 For example, Ding et al. delivered a DNA origami-gold nanorod complex into a MCF7 breast cancer cell, and the heat generated from the gold nanorod under nearinfrared red irradiation was able to kill the cancer cells (Figure 20A).390 Afterward, they developed an optoacoustic imaging agent by assembling DNA origami with gold nanorods.389 The hybrid complex not only improved the imaging quality of the tumor tissues but also responded to near-infrared irradiation (NIR) for effective photothermal therapy to inhibit tumor growth and to prolong the survival of the mice that had tumors. To demonstrate the use of DNA origami for photodynamic therapy, Zhuang et al. loaded the photosensitive agent BMEPC, by intercalating it into the DNA origami structure, and delivered the complex into the cell.391 BMEPC was able to produce free radicals and singlet oxygen upon light irradiation to kill cancer cells and simultaneously destroy the DNA origami structure through photocleavage (Figure 20A). Immunostimulatory drugs can also be transported with DNA origami carriers. Liedl and co-workers studied immune responses in spleen cells that were induced by an origami tube, which was coated with up to 62 cytosine-phosphate-guanine (CpG)

shaped DNA origami structures that could kill cancer cells via the transportation of doxorubicin (Dox) inside of those cells (Figure 20A).381 Dox is a chemotherapy drug that intercalates DNA to inhibit cell biosynthesis.382−384 DNA origami has a high load capacity of Dox because of its abundant duplex structures. The Origami-Dox complex exerts a high cytotoxicity on both regular breast adenocarcinoma cancer cells and Dox-resistant cancer cells. However, the cellular internalization of the drug could be greatly improved with an origami carrier. The Origami-Dox complex inhibits lysosomal acidification and causes a cellular redistribution of the drug to the nucleus. Ding et al. further studied the performance of the origami-Dox complex by injecting it into a tumor mouse model and found that it exhibited remarkable antitumor efficacy without any observable systemic toxicity.385 Since DNA origami structures have such a high degree of customization and the mechanism of loading Dox into DNA origami structure is by simple intercalating to DNA duplexes, Högberg et al. hypothesized that the drug load and release efficiency could be tuned by changing the origami design.386 They verified this idea by using two origami structures, twisted and untwisted tubes, as Dox carriers to target three different breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF-7). They found that the twisted DNA nanotube had a superior drug delivery rate for its loading efficiency and releasing rate. Halley et al. found that the Dox-DNA origami complex 12613


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Figure 21. DNA origami as a platform for nucleic acid analysis by AFM. (A) A DNA origami chip can detect RNA targets with probes and target hybridization. The barcode encoded on the origami surface allows for the simultaneous analysis of multiple targets.407 The dimension of each origami is about 60 × 90 nm. Images reproduced with permission from ref 407. Copyright 2008 AAAS. (B) A DNA origami chip can differentiate diverse SNP targets.410 All of the scale bars are 50 nm. Images reproduced from ref 410. Copyright 2011 American Chemical Society. (C and D) DNA origami is used as a frame to study nucleic acid conformational changes by using fast AFM scanning.416,419 The size of the AFM images are 130 × 130 nm. (C) Images reproduced from ref 416. Copyright 2013 American Chemical Society. (D) Images reproduced with permission from ref 419. Copyright 2014 Wiley. 12614


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biomolecules on a molecular level. AFM and fluorescence microscopy are usually implemented to read the interaction signals. 6.5.1. Nucleic Acid Analysis. Nucleic acid detection is usually carried out by using enzymatic reactions, such as polymerase chain reaction (PCR) and reverse transcriptase (RT-) PCR to amplify the target, as the level of expression is usually low in the cellular environment. DNA origami that allows the direct observation of nucleic acids under microscopy makes the detection of nucleic acids at a single molecular level possible. In 2008, Yan and co-workers reported the use of DNA origami as a label-free bioanalysis platform in solution (Figure 21A).407 They engineered a DNA origami chip with hybridization probes that extended out for RNA hybridization. When the RNA targets were present in the environment, they would bind to the probe through Watson−Crick base pairing and form a V-shaped structure that could be readily visualized as a bright spot under AFM. They found that the exact position of the probe made a remarkable difference on the hybridization efficiency. To overcome this problem, they created a barcode system with a group of dumbbell-shaped DNA loops that protruded out of the surface of the origami chip as topographic registration markers. These origami chips were implemented to allow optimal probe placement and to make multiplexing possible. Using this technique, they were able to simultaneously detect three gene targets: C-myc, Rag-1, and β-actin. Other methods, such as the use of an asymmetric DNA origami as chips408 and the logical computation on an origami surface409 have been reported to detect nucleic acids as well. To reach the single nucleotide polymorphism (SNP) resolution of nucleic analysis, Seeman and co-workers combined two techniques. They used an effective kinetic method to differentiate SNPs based on a strand displacement reaction and the AFM of origami patterns to give direct visual readouts (Figure 21B).410 Strand displacement happens when one single strand hybridizes with a single-stranded overhang of a double strand that has partial or full complementarity. The process initiates from the hybridization of part of the single strand with the complementary overhang (also called a toehold) and proceeds through a branch migration to displace the shorter one of the double strand.411,412 The rate of strand displacement is affected by the toehold length and fidelity of the Watson−Crick base pairing in the branch migration region. Any mismatch in the branch migration region will cause a strong kinetic trap for the branch migration, leading to the pausing or failure of the strand displacement. The significant change in the strand displacement reaction was utilized to detect SNP. The rectangular DNA origami chips were encoded with DNA duplexes that displayed four unique alphabetical patterns. The difference between each of the DNA duplexes was a mutation by only one nucleotide at the same position. Consequently, DNA duplexes at each alphabetic character could only react with the invading strand of interest that had the perfectly matched sequence, leading to the disappearance of the alphabetic pattern. Therefore, by observing the disappearing pattern under AFM, one could recognize the SNP of the invading strand. Zhang et al. published a similar strategy to detect SNP on an origami chip as well.413 DNA origami has also enabled the study of the conformational changes of nucleic acids during their interaction with DNA binding proteins, enzymes, and ions.414,415 Sugiyama and colleagues employed DNA origami frames with cavities in the middle, where the target of interest could be captured to study biological interactions under AFM. By adding different stimuli,

sequences (Figure 20B).392 CpG sequences are present in bacterial genomes at a high frequency but are present in mammalian cells at a much lower frequency. The mammalian immune system has the capability to sense foreign DNA from bacteria or viruses through various receptors and can trigger an immune response. In this way, CpG can be used as an immunostimulatory drug that is recognized by a TL9 receptor to trigger a mammalian immune system and to improve its resistance against bacterial or viral infection.393 Natural CpG is not durable due to the endonuclease in the cellular environment. Therefore, CpG is usually chemically modified to increase its endonuclease resistance.394 DNA origami structures are able to load a substantial number of CpG fragments via tethering the Cpg fragments to their surfaces and may also protect the Cpg fragments from nuclease digestion. From the study of Liedl and co-workers, CpG-coated DNA origami structures were able to traverse the cell membrane and induce a strong immune response without a toxic response. Fan and colleagues proposed another approach based on a rolling circle amplification to fold well-defined DNA structures that had CpG sequences and only several staple strands for immunostimulatory drug delivery.395 To improve targeted drug delivery specificity, dynamic origami can be integrated with DNA aptamers to build a lock-and-key system to create an intelligently controlled release system. DNA aptamers are DNA species that are selected by the systematic evolution of ligands by exponential enrichment (SELEX) to bind various molecular targets, such as small molecules, proteins, virus, and cells.396 DNA aptamers have already been used for some cancer cell-targeted drug deliveries.396,397 Douglas and coworkers built an aptamer-gated DNA origami nanorobot with five antibody fragments that worked against human leukocyte antigens (HLA)-A/B/C (Figure 20C).398 In the absence of the correct key, the robot remained inactive, and the antibody could not access the cell surface. However, if the robot encountered the correct keys, the DNA origami box would open and the antibodies would be exposed. The antibodies could then bind to the cell’s surface to repress its growth. Amir et al. further explored the device to perform logical computations in a living animal.399 The system consists of multiple origami robots that used two growth factors as input signals (PDGF-BB and VEGF165) and processed the signals with a set of DNA strand displacement circuits. The system was capable of emulating various logic gates, such as AND, OR, XOR, NAND, NOT, CNOT, and a half adder. Surface modification of DNA origami is necessary to facilitate translocation across the cell membrane. The efficiency of the cellular uptake of a bare DNA origami structure is relatively low. Various methods have been developed to improve the efficiency of cellular uptake.400−404 For example, Mikkila et al. reported a method using capsids from viruses to encapsulate DNA origami structures, which resulted in a more than 10 fold increase in cellular uptake efficiency as compared to the unenhanced origami structures (Figure 20E).405 Shih and co-workers enveloped the DNA origami with a PEGylated lipid bilayer that decreased immune activation (by 2 orders of magnitude) and improved pharmacokinetic bioavailability (by a factor of 17) (Figure 20D).406 6.5. Bioanalysis with DNA Origami

The two main advantages of DNA origami are its full addressability at a resolution of several nanometers and its high degree of shape customization. Furthermore, the relatively large dimensions and easy modification of oligoes make DNA origami an ideal substrate with which to study interactions between 12615


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Figure 22. Fluorescence-based study of nucleic acid binding event studies on DNA origami. (A) Single-molecule kinetics study of DNA oligo binding, where bright red fluorescence is only observed upon the binding of a red fluorophore-labeled strand. The observed fluorescence intensity versus time is obtained to solve the binding kinetics of DNA strand.445 Images reproduced from ref 445. Copyright 2010 American Chemical Society. (B) The study of DNA strand binding onto DNA origami with different densities of hybridization probes.459 Images reproduced from ref 459. Copyright 2013 American Chemical Society. (C) Super-resolution imaging with DNA-PAINT based the transient docking of fluorophore labeled DNA strand.447 The size of DNA origami is 70 × 100 nm. Images reproduced with permission from ref 447. Copyright 2014 Macmillan Publishing Ltd. (D) Discrete molecule imaging technique allows the discrimination of molecules with distance of 5 nm.449 The scale bar is 10 nm. Images reproduced with permission from ref 449. Copyright 2016 Macmillan Publishing Ltd.

the conformational changes of the target DNA in the cavity was examined with high-speed AFM in real time. For example, to observe the motion of a DNA motor based on a B-Z conformational transition, two dsDNAs containing a (5meCG)6 sequence (for BZ transition control) and a random sequence were introduced to the top and bottom of the cavity in the DNA origami frame (Figure 21C).416 A flag marker in the dsDNA was used to observe the switching of the rotation during a B-Z transition. Recently, they used the system to study how torsional constrains of DNA impacted the cleaving function of Cas9.417

The platform enabled them to study a series of dynamic conformational changes of the DNA under various scenarios and the dynamics of the proteins that interact with DNA.416,418−433 Through AFM imaging, single-molecule reactions can be detected if reactants are immobilized on a DNA origami.434−442 For example, Gothelf and co-workers studied cleavage (DDT and singlet oxygen induced cleavage) and chemical coupling reaction (click reaction) on a DNA origami substrate.443 In their experiments, the reactants were modified with streptavidin to attach onto a DNA origami substrate for observation. The 12616


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Figure 23. Distance-dependent biological interaction study with DNA origami. (A) Bivalent binding of thrombin with its two aptamers with different distances.460 The size of each AFM frame is 150 × 150 nm. Images reproduced with permission from ref 460. Copyright 2008 Macmillan Publishing Ltd. (B) DNA origami with ephrin ligands displayed to study the distance-dependent influence of EphA2 receptor activation in human breast cancer cells.461 Images reproduced with permission from ref 461. Copyright 2014 Macmillan Publishing Ltd.

center of a regular rectangular DNA origami, the binding kinetics at this location showed a 30% increase of koff as compared to the rate at the edge positions. They also found that the unbinding rate koff depends exponentially on the length of a duplex formed, while the binding rate kon only weakly depends on this length. When the binding length is about 7-nt long, the DNA strand exhibits a transient binding behavior on the DNA origami. Inspired by super-resolution imaging methods based on PAINT (point accumulation for imaging in nanoscale topography),446 they developed the DNA-PAINT assay that uses the transient binding of fluorescently labeled DNA imager strands. The technique of DNA-PAINT has been applied to the character-

movement or disappearance of the bright spots in the AFM images indicated whether the reactions had happened or not. AFM is a powerful tool that is used to observe the morphologies of nucleic targets on origami chips. However, the kinetics of nucleic acid binding events are difficult to obtain because of their subsecond duration. The binding of fluorophore labeled DNA strands onto a DNA origami substrate can be engineered to display nanobarcodes.444 Jungmann et al. presented a single-molecular fluorescence assay to study the binding and unbinding kinetics of a DNA strand onto a DNA origami structure (Figure 22A).445 The positional dependence of the binding kinetics on the DNA origami can be observed. At the 12617


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Figure 24. Programming non-DNA materials with DNA origami. (A) A DNA origami-templated gold cluster growth.479 The scale bar is 200 nm. Images reproduced with permission from ref 479. Copyright 2011 Wiley. (B) A DNA origami is used as a mold to control the shape of gold nanoparticles.483 Images reproduced with permission from ref 483. Copyright 2014 AAAS. (C) The precise control of lipid size by using DNA as a template.491 Images reproduced with permission from ref 491. Copyright 2016 Macmillan Publishing Ltd. (D) Flexible polymer routing by using a DNA origami hybridization.493 Images reproduced with permission from ref 493. Copyright 2015 Macmillan Publishing Ltd.

ization of 3D nanostructures,119 super-resolution cellular imaging by conjugation of DNA to an antibody,447 quantitative imaging,448 and individual molecule imaging in high density449,450 (Figure 22, panels C and D). Due to DNA origami’s precise addressability and ability to position molecules, it has been widely used as a benchmark to verify the reliability of fluorescence based super-resolution imaging.451−458 This kind of ruler is currently commercially available for researchers to verify

their own custom super-resolution imaging methods or to evaluate the microscopy working statues. The kinetic behavior of the DNA binding onto a DNA origami inside of a crowded neighbor environment is different from the scenarios without neighbors. Through single-molecule FRET analysis, Walter and co-workers investigated how a singlestranded oligo array on a DNA origami affected the kinetics and thermodynamics of their hybridization with complementary targets in solution (Figure 22B).459 They discovered that the rate 12618


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resulted from a very close distance (direct physical contact) between the gates. When the DNA gates were well-separated, the circuit did not gain any benefit in reaction speed in comparison with the reaction speeds in bulk solutions. The reaction speed was boosted with a proper distance,and the leakage was suppressed with an optimized distance. This fundamental study provided useful information for designing localized circuits464−466 and devices.467

of dissociation of a target strand was reduced by an order of magnitude in the densest oligo arrays. Two distinct mechanisms are proposed to explain the slow dissociation: direct hopping of targets between adjacent sequence-matched oligos on an origami surface and nonsequence-specific, salt-bridged attractive electrostatic interactions of the oligoes with the DNA origami pegboard. 6.5.2. Distance-Dependent Interactions. Distance plays a crucial role in the chemical and biological interactions. For example, multivalent binding is a ubiquitous phenomenon that cells use to regulate intracellular activities or intercellular recognitions. Cooperative intercellular activities are dominated by interactions between membrane-bound receptors that specifically bind to ligands on the surfaces of neighboring cells. The distribution of ligands, on a nanometer scale, is hypothesized to regulate membrane receptor signals. Subtle differences in ligand nanoarchitectures may cause different cellular responses. Besides cellular signaling, artificial multivalent interactions (such as the binding between an aptamer and its target) are also highly dependent on spatial relationships. These underlying distancedependent mechanisms are not well-understood because it is difficult to control the spatial orientation and interactions of molecules at nanometer scale distances. A well-controlled interligand distance with multivalent binding can provide a comprehensive understanding about how the distance influences binding events and biological responses. Yan and co-workers systematically studied the distancedependent bivalent binding effects between two aptamers and a thrombin (a coagulation protein involved as a key promoter in blood clotting) at the single-molecule level by using AFM (Figure 23A).460 By varying the distance between the two aptamers on the edge of the DNA structures, they found that the optimal distance to bind a 4 nm thrombin between two aptamers was 5.3 nm. A smaller or larger distance would decrease the binding affinity. This was the first example of taking advantage of the full addressability of DNA origami to study spatially controlled interactions. The DNA aptamers that extend from the structure can be easily replaced with other biomolecules such as peptides and proteins in order to study its corresponding distance-dependent behaviors. Shaw and colleagues presented a method for investigating the effect of the spatial distribution of ephrin ligands on the Eph’s function of cell membrane-bound receptors (Figure 23B).461 Ephrin receptors bind ephrins in neighboring cells, causing ephrin phosphorylation and activating intracellular signaling pathways that primarily regulate cell migration and proliferation. They developed a tubelike 18-helix DNA origami with multiple docking sites and prepared the origami structure with no ligand, one ligand, and two ligands (which were about 100 or 40 nm apart) or a saturated amount of ligands. They found more efficient EphA2 phosphorylation in human breast cancer cells when the ligand distance was 40 nm. In addition, in a chemical reaction, the traveling distance of the reactions species, or intermediate between two reaction centers, generally dominate the reaction efficiency and yield. Helmig et al. revealed a distance-dependent oxidation of organic moieties by positioning a photosensitizer that produces singlet oxygen in the middle of an origami and a DNA strand with1 an O2 cleavable linker around it.462 When the light was on, the cleavage of the DNA strands around the photosensitizer strand was observed. Similar strategies have been applied to study the distancedependent quenching of a single fluorophore by gold nanoparticles.257 Teichmann et al. studied the impact of distance on the localized strand displacement cascade that was assembled on a DNA origami surface.463 They found that a strong leakage

6.6. Programming the Structure and Conformation of Non-DNA Materials

Controlling the shape of inorganic materials is of great importance because they can be used for diverse applications such as biosensing,468 light harvesting,469 and nanophotonics.470 As DNA is a highly programmable material that can be folded into arbitrary 2D or 3D shapes, researchers have been trying to program the synthesis of inorganic materials with DNA. It is hard to synthesize custom shapes of inorganic materials with conventional methods.471−478 For example, in 2011, Liedl and co-workers designed a set of DNA origami structures for shapecontrolled metallization (Figure 24A).479 They used positively charged 1.4 nm gold clusters that were bound to the negatively charged DNA origami as seeds for the growth of the gold clusters, which would then continuously metallize to form DNA origami nano-objects of predetermined shapes. Bundles, circles, planar structures, and X-shaped structures have been synthesized. PiloPais et al.480 and Woolley et al.481,482 reported a similar strategy that they used to position gold nanoparticle seeds with designed patterns and to grow them into continuous shapes. Although these strategies provide a way to roughly control the shapes of the target materials, the obtained products still consist of a numerous amount of clusters that do not have clear boundaries because growth occurs in an open environment. To improve the quality and resolution of inorganic material synthesis, Yin et al.483 and Seidel et al.484 created a method of using DNA origami molds (like a barrel) to hold a single gold nanoparticle seed and to grow the nanomaterial within a cavity that is embedded in a DNA origami (Figure 24B). With this strategy, DNA origami was designed to have a cavity in the shape of the target inorganic material. Then, a gold nanoparticle was anchored to the interior surface of the cavity, and the origami barrel was fully enclosed with two lids, resulting in a boxlike structure with a single gold nanoparticle seed trapped inside. The seed in the box would grow to fill in the box under suitable chemical conditions. Using this method, a variety of inorganic materials of different shapes were programmed and synthesized at a resolution of ∼3 nm, such as a cube, triangle, and Y-shaped structure. The self-assembly and the conformation of organic molecules can be programmed as well. For example, Udomprasert et al. reported a method of using DNA origami to organize amyloid fibrils.485 Amyloid fibrils are a type of protein aggregate that is related to neurodegenerative conditions such as Alzheimer’s and are of great interest for the development of self-assembly and biotechnology applications.486 To program the assembly of amyloid fibrils, an amyloid peptide is positioned in the interior side of a DNA origami tube in order to nucleate the assembly of the amyloid and to shape the amyloid into a tubular form. The strategy of controlling the nucleation and the growth by an origami frame was applied to liposome assembly as well. Liposomes are an important material that are used to mimic cellular membrane dynamics and to provide a good platform to study membrane cellular activities.487 Engineering custom12619


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Figure 25. DNA origami as a mask for molecular lithography. (A) Pattern transferred from DNA origami to a SiO2 surface. Triangular DNA origami on a SiO2 surface (left), concave pattern transferred (middle), convex pattern transferred (right).502 Images reproduced from ref 502. Copyright 2011 American Chemical Society. (B) DNA origami patterned on a chemically modified graphene surface.507 Images reproduced with permission from ref 507. Copyright 2012 Wiley. (C) Graphene nanopatterns that were transferred from DNA nanostructures.508 The scale bars are 100 nm. Images reproduced with permission from ref 508. Copyright 2013 Macmillan Publishing Ltd.

shaped and custom-sized liposomes can provide more opportunities for drug delivery and biological sensing applications.488−490 Lin and co-workers designed a DNA origami structure that was shaped like a ring and that had a customized size.491 They decorated the interior surface with lipid molecules to guide liposome formation and to limit their sizes (Figure 24C). With the use of this method, the size distribution of the final liposome products was narrow, indicating that the process was reliable and that the sizes of the liposome products could be rationally controlled. Recently, Dong et al. used a cuboid DNA

origami structure that was conjugated with vesicles that were seeded on the external surface to construct cuboid-shaped vesicles.492 Polymers are usually random chains without any preferable conformations. Gothelf and co-workers used DNA origami to route individual polymers into designed shapes (Figure 24D).493 They synthesized a conjugated (2,5-dialkoxy) paraphenylenevinylene (APPV) brush polymer containing 9 nt of ssDNA that extended out from the majority of the phenylene groups. SsDNA probes that were complementary to those that extended out from 12620


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Figure 26. Directed DNA origami self-assembly for nanofabrication. (A) Directing the positions of the DNA origami and their orientations (left) with gold nanoparticles attached (right) on a lithographically patterned surface.513 Images reproduced with permission from ref 513. Copyright 2010 Macmillan Publishing Ltd. (B) Engineering and mapping nanocavity emissions via the precise placement of DNA origami. Top: the x−y location of a Cy5-origami within a photo cavity center within a 40 × 15 PCC array. The size of the array is 176 × 77 μm. Bottom: Van Gogh’s The Starry Night with approximately 65536 cavities, each having zero-to-seven binding sites.515 Images reproduced with permission from ref 515. Copyright 2016 Macmillan Publishing Ltd. (C) DNA nanotubes that were aligned into different shapes by gold islands.509 (C) Images reproduced from ref 509. Copyright 2010 American Chemical Society.

6.7. Bridging Top-down and Bottom-up Fabrications

the backbone of the polymer were immobilized at specific locations on the origami. The binding of the DNA-polymer hybrid molecules on the DNA origami surface created predetermined patterns. The conformational reconfiguration of the polymer has been demonstrated by the use of strand displacement reactions.494 Recently, Tokura et al. performed an atom-transfer radical polymerization reaction on a DNA origami surface to create different nanopatterned polymeric nanostructures.495

Bottom-up fabrication is based on molecular self-assembly, such as DNA origami, and is a very powerful tool for engineer nanoscale objects with a nanometer resolution. However, scaling up the structure to the size of a millimeter or larger is very challenging. The top-down fabrication method is based on lithography and is robust when engineering structures at the micrometer scale, but it is difficult to achieve a resolution smaller than 20 nm with this method. Therefore, a goal of nano12621


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treated with glutaraldehyde to enable seeding with silver and then subsequently coated with gold. The gold surface on the DNA origami structure protected the DNA from being destroyed during the subsequent etching process. An argon/oxygen plasma-reactive ion etching process removed the uncovered grapheme, while the spatial information from the metalized DNA origami structure was transferred to the final etched graphene products after metal removal. The disadvantage of etching with a DNA origami mask is that the position and orientation cannot be controlled. This results in an unordered pattern on a surface. 6.7.2. Directed Positioning of DNA Origami Structures. Yan and co-workers explored a new method in order to direct DNA origami self-assembly into a predetermined pattern. They used electron beam lithography to pattern gold nanoarrays into well-defined shapes, such as squares and hexagonal patterns, on a silicon surface (Figure 26C).509 The gold islands of the array each had a diameter of ∼20 nm and had a length that was close to the length of DNA origami nanotubes (∼350 nm). These DNA origami tubes were modified with thiols at the two ends and were then bound to the gold islands. After that, the DNA origami tubes spanned between the gold islands to form the predetermined pattern. Pearson et al. reported a similar strategy to align DNA origami on a gold island, which was patterned with block copolymer.510 Later on, Morales et al. further demonstrated that rectangular DNA origami structures could be suspended on four gold dots.511 To control the position and orientation of the DNA origami structures on the substrate surface, Kershner et al. developed an electron-beam lithography and dry oxidative etching process. This new process helped to create DNA origami-shaped binding sites on SiO2 to direct the DNA origami positions and orientations with a high selectivity and a proper orientation (Figure 26A).512 The method was also used to organize ordered gold nanoparticles on a SiO2 surface by putting the gold nanoparticles onto a triangle-shaped DNA origami structure.513 Rothemund and co-workers further optimized the assembly by modifying the concentrations of the DNA origami structures and Mg2+.514 Other optimizations included changing the buffer pH, incubation time, and process by which the DNA origami was bound onto a predetermined position. This binding could result in yields of 94% and 90% of the sited origami’s orientation within ±10° of the target orientation. Recently, they successfully used the method to couple molecular emitters to photonic crystal cavities (PCCs) (Figure 26B).515 This coupling enabled them to amplify the optical properties of molecules or nanoparticles through strongly localized optical fields. This amplification helped create new properties such as lasing516 and nonlinear phenomena.517 It is very challenging to place a well-defined number of molecular or nanoparticle emitters into a single cavity and properly position them relative to the antinodes of the cavity. To address this challenge, the authors synthesized a triangle DNA origami structure with a defined number of modified dyes (3 or 15) and positioned the origami structure on the PCC center. Using this method, they were able to reproduce Van Gogh’s The Starry Night with 65536 cavities, each having from zero to seven binding sites to tune the light intensity of each cavity. Tinnefeld et al. harnessed the DNA origami structures as nanoadapters to match the small hole size, called zeromode waveguides.518 DNA origami could be selectively modified with a single dye, in order to optimize the use of each hole and to improve photophysical properties. The combination of the top-down lithography fabrication with bottom-up DNA origami assembly enables researchers to study

technology is to combine the molecular self-assembly and conventional microfabrication methods. DNA origami is considered to be an ideal intermediate that bridges the two methods because it is programmable and can self-assemble into almost any arbitrary shape. Additionally, its size is large enough to match the smallest features generated by lithography. 6.7.1. Molecular Lithography with DNA Origami. Lithography is a technique that is used to transfer a pattern from one object to another. DNA structures with custom patterns can dramatically improve the fabrication resolution of lithography technique. First, DNA origami with high addressability can transfer its encoded address information to other molecules. Edwardson et al.496 and Zhang et al.497 demonstrated the concept of “molecular printing” by transferring pattern information, which was encoded on a DNA nanostructure, to a nanoparticle. A protein pattern, which was encoded on a DNA origami, could be transferred to a functionalized substrate as well.498 Eritja et al. showed that the encoded information on a DNA origami can be transferred to a gold surface with sub-10 nm resolution.499 Furthermore, the pattern of the DNA origami structure itself could transfer diverse and complicated patterns from custom-designed DNA structures to other materials, such as silicon oxide (SiO2) and graphene.500,501 The dry and wet etching processes used to etch SiO2 occurred through plasma treatment and buffered HF, respectively. However, DNA nanostructures have a limited chemical stability and a poor adhesion to common inorganic substrates, both of which make it difficult to transfer shapes to the substrates. Liu and co-workers published a one-step pattern transfer process to enable shapes to be moved from a DNA origami to an inorganic substrate. This process involved the use of DNA to modulate the vapor-phase of SiO2 at the single-molecule level (Figure 25A).502 Water is the catalyst used for the HF to etch the SiO2. The reaction does not occur without water. A DNA backbone is made up of phosphates that can bind water molecules, and a DNA origami can absorb a massive amount of water. If the humidity is relatively high, DNA can absorb more than 100% (w/w) of water.503 Furthermore, when present on the substrate surface, DNA nanostructures can inhibit the diffusion of the HF to the SiO2. Therefore, DNA nanostructures can manipulate the etching kinetics. At higher humidity levels, DNA structures enhance etching because a higher amount of water is absorbed, while at lower relative humidity DNA will slow down etching. By carefully controlling the time and speed of etching, subten-nanometer patterns in depth can be transferred from the DNA origami to the SiO2 layer.504 Surwade et al. reported a room-temperature chemical vapor deposition (CVD) method that they used to create custom-shaped inorganic oxides by using a DNA origami template.505 The method was capable of generating both positive- and negative-tone patterns on a wide range of substrates, such as mica, gold, and TiO2. Another method has been reported that can program the growth of carbon nanostructure with DNA origami substrates.506 Yun et al. showed that bottom-up self-assembled DNA origami structures can be deposited on a graphene film that has been fabricated by a top-down photolithography method (Figure 25B).507 Yin and co-workers developed a metallized DNA nanolithography technique that allowed spatial information of DNA nanostructures to transfer to the graphene in order to prepare a customized graphene sheet (Figure 25C).508 In their experiment, the DNA origami template was self-assembled in solution and then deposited on the graphene surface with a 1pyrenemethylamine adhesion layer. DNA structures were then 12622


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Figure 27. Enzymatic nanoreactors. (A) Channeling the diffusion of intermediates with a protein bridge on a rectangular DNA origami between enzymes.536 Images reproduced from ref 536. Copyright 2012 American Chemical Society. (B) An enzymatic cascade of xylose and xylulose on a DNA origami surface.537 Images reproduced from ref 537. Copyright 2016 American Chemical Society. (C) Directional regulation of enzyme pathways by controlling substrate channeling on a DNA origami scaffold.540 Images reproduced with permission from ref 540. Copyright 2016 Wiley. (D) A modular and tubular DNA origami-based enzyme HRP-GOx cascade.541 Images reproduced with permission from ref 541. Copyright 2015 Royal Chemical Society. (E) Enhanced catalytic activity and increased stability of DNA origami-caged enzymes.543 Images reproduced with permission from ref 543. Copyright 2016 Macmillan Publishing Ltd.

ligands.521 They also developed another strategy to arrange DNA origami on biomimetic lipid patches that were prepared by dip-pen nanolithography with phospholipids.

biological interactions in a high-throughput fashion. Simmel et al. used electron beam lithography to construct a DNA origami array on a glass substrate. By combining the DNA origami arrays with the single molecule fluorescence technique, they studied DNA binding and strand displacement on the array.519 They found that there was considerable variability of DNA binding events within the array, due to structural variations and stochastic reaction dynamics. Niemeyer et al. constructed a DNA origami array as an interface for cells in order to study the activation of epidermal growth factor (EGF) receptors in living MCF7 cells.520 The DNA origami structures were arranged with a topdown micropatterning method and were decorated with welldefined ligands. Niemeyer et al. had full control over the number, stoichiometry, and precise orientation of the well-defined

6.8. Self-Assembled Enzymatic Nanoreactors

Enzymatic reaction cascades play an important role in maintaining the complex metabolic networks in living organisms. To improve enzymatic coupling efficiency and metabolic network robustness, the living system evolves a strategy to control the spatial relationships of many related enzymes with a variety of nanostructure scaffolds. This spatial relationship leads to a compartmentalized environment that can hasten the transportation of the intermediates between different enzyme stations. Mimicking such an efficient way to improve enzymatic 12623


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reactions outside of the cell can help improve the biosynthetic product yield. Self-assembled designer DNA structures with high addressability offer an ideal platform to allow the precise positioning of enzymes with prescribed distances and orientations.52,522−534 To achieve this goal, researchers have developed methods to attach proteins on DNA origami that have well-defined patterns. Saccà et al. demonstrated that proteins can be encoded on a DNA origami structure to achieve a pattern that mimics the shape of a human face.525 Kohman et al. reported a method of encapsulating a protein in a DNA origami cage and releasing it with light.535 In 2012, Yan and co-workers used a rectangular DNA origami platform to investigate distance-dependence for the activity of a glucose oxidase (GOx)/horseradish peroxidase (HRP) cascade (Figure 27A).536 GOx simultaneously oxidizes the glucose to gluconic acid and produces hydrogen peroxide (H2O2), which is the substrate for HRP to transform ABTS2− to ABTS−. Therefore, H2O2 is the intermediate that couples the two enzymatic reactions. They changed the distance between the two enzymes from 10 to 65 nm and observed a strong and enhanced activity when the enzymes were closely packed at a distance of 10 nm. The activity decreased dramatically when the distance was increased to 20 nm. However, further increases in the distance resulted in weak distance dependence. To explain this observation, they proposed that short distances between the enzymes resulted in a 2D-restricted diffusion of intermediates, while more distant enzyme pairs led to a 3D Brownian diffusion of intermediates. The distance-dependent effect was also reported by Morii and co-workers when they assembled xylose reductase and xylitol dehydrogenase on a DNA origami molecule with a controllable distance via DNA-binding adaptors to reconstruct the xylose metabolic pathway (Figure 27B).537 More recently, a geometric effect on enzymatic cascade activity was investigated on a three-enzyme system by Liu et al.538 They found that the activity of triangularly arranged enzymes outperformed that of linear geometric arrangements. Instead of using distance or geometry to control the diffusion of intermediates, Fu et al. used an artificial swinging arm with an NADH moiety that directly transferred substrates between a two-enzyme system that consisted of glucose-6-phosphate dehydrogenase (G6pDH) and malic acid dehydrogenase (MDH) on a DX DNA tile.539 With a DNA origami platform, Ke et al. was able to demonstrate more complicated behavior regulation when they reconfigured the swinging arm between different enzyme pathways (G6pDH-MDH and G6pDH-LDH) to control their ON−OFF state (Figure 27C).540 Besides controlling the relative distance and orientations of the enzymes, DNA origami is able to provide a confined environment to improve the coupling efficiency of enzyme cascades. Several groups tried to envelope multienzyme systems into spatially limited nanostructures in order to restrain the diffusion of the intermediates and to increase enzyme activity. Linko and colleagues designed an enzyme nanoreactor by assembling two tube-like DNA origami structures with GOx and HRP separately attached to the inside of a single tube (Figure 27D).541 Fu et al. encapsulated a GOx-HRP pair into a DNA nanotube by wrapping the enzymes over a rectangular DNA origami structure.170 After wrapping, a higher coupling efficiency of the enzyme pair was observed, as compared with an unwrapped enzyme pair on a rectangular origami. Wang et al. designed a DNA nanochannel with a dynamic cap to help with enzyme cascades.542 Zhao et al. used a similar assembly strategy to encapsulate the enzyme pairs into a fully closed cubic cage.543

They assembled two half-cage DNA origami structures to create the full cage (Figure 27E). A negative relationship was found between the activity enhancement and the size of the proteins that were encapsulated. To explain this phenomenon, they proposed that the distal polyanionic surfaces that were provided by a DNA origami environment could enhance the stability of the active enzyme conformations through a strongly bound hydration layer. Protein folding has been reported to be more stable in a highly ordered, hydrogen-bound water environment.544−546 The success of artificial enzymatic regulation has laid a solid foundation to help researchers achieve more complicated networks of enzymatic reactions due to the powerful addressability of DNA origami. Recently, Sprengel et al. developed a method of employing noncovalent weak interactions to host a single protein in a defined DNA origami envelope without any protein modification.547 In their method, a number of ligands were attached to a DNA origami that was used to provide a position for a target protein’s binding through supramolecular interactions. In this case, protein conjugation with a linker was not needed. 6.9. Self-Assembled Light Harvesting Systems

Natural light harvesting systems are highly efficient in transferring solar energy to the reaction center, where the light energy is converted into chemical energy. These light harvesting systems contain intermediate light transporters that are rationally organized with an optimized geometry and stoichiometry. Learning from the natural photosynthetic systems and mimicking them is important. The high addressability and custom design of DNA nanostructures make it possible to engineer complex electron networks to transfer and harvest energy in a way that is similar to that of the natural systems.548−551 Stein et al. successfully constructed an energytransfer cascade on a DNA origami pegboard and demonstrated that he was able to control the direction of the energy transfer (Figure 28A).552 They used a blue fluorophore (ATTO 488) as the light input dye, and a red fluorophore (ATTO647N) and an infrared (IR) fluorophore (Alexa 750) as two alternative light output dyes. The distance between the input and output dyes was set far enough apart to inhibit direct energy transfer. The direction of the energy transfer to the red or IR dye was determined by the position of the green jumper dye (ATTO565). Recently, Hemmig et al. presented a more complex light harvesting system. They used DNA origami to systematically study the influences of geometry and stoichiometry on antenna efficiency (Figure 28B).553 In their system, a central acceptor dye (Cy5) was surrounded by six donor dyes (Cy3) that formed a donor ring. Another Cy5 acceptor was attached to the outside of the ring but was equidistant from the nearest donor that could act as a central acceptor. Using this well-defined system, they found that when the acceptor dye was located in the middle of six donor dyes, the antenna effect was much higher than when the acceptor dye was positioned outside of the ring. Furthermore, a linear relationship was observed between the antenna effect and the number of the adjacent donors.

7. CONCLUSIONS AND OUTLOOK Researchers in the field of DNA nanotechnology have successfully demonstrated that DNA is not simply a genetic material in cells but that it is also a powerful building material in the nanometer scale world. Individuals who use the technique of DNA origami are capable of engineering custom structures with a high addressability that helps with their nanoscience research. 12624


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origami may be a better way to shed light on the behavior of DNA origami assembly. Current DNA origami structures are self-assembled from hundreds of DNA strands. Designing a single-stranded origami structure with a size comparable to that of regular DNA origami structures is an interesting and challenging goal. To address this challenge, ssDNA molecules can be prepared by hybridizing multiple strands and ligating them with enzymes into a continuous strand. An alternate way to create a large ssDNA origami structure is to program the sequence of an ssDNA and thus control its folding pathway. ssDNA architecture is replicable with enzymes or with cloning, which can be used to prepare a large amount of structures at a low cost. For example, Yan and coworkers first demonstrated the in vivo replication of simple, artificial, ssDNA structures.559 Geary et al. demonstrated that an artificially designed ssRNA is capable of cotranscriptional folding and can be folded into desired patterns.560 The ssDNA architectures also make it possible to engineer complicated topological molecules. For example, Liu et al. published a method of using 4-way junctions as nodes to construct DNA structures with complex topologies.561 By programming a DNA folding pathway, Jerala and co-workers successfully engineered a knotted DNA nanostructure with a single strand.562 Designing and constructing complex topological DNA nanostructures is a challenging and exciting direction to explore in the future. The key point of constructing a single-stranded complex nucleic acid structure is to ensure that the correct folding pathway is used in order to avoid topological traps. Further, the complexity and functionality of DNA origami designs may benefit from integrating nucleic acid programmability with sequence specific protein binding. In this way, the concept of origami is not solely limited to nucleic acid selfassembly. In nature, complicated molecular structures rely on the self-assembly between various types of molecules and through sophisticated spatial interactions at an angstrom level. An example of this self-assembly is demonstrated by the ribosome, a complex molecular machine that translates genetic information from RNA to functional proteins. The ribosome is assembled through the sophisticated intra- and intermolecular interactions between RNAs and proteins. Considering the hybrid structure of a ribosome, it would be very interesting to mimic the process where DNA and protein mutually assist one another’s folding to achieve a predetermined pattern. A variety of peptides and proteins that are produced by nature or artificially engineered can specifically bind to DNA according to the sequence. For example, a zinc finger protein,563 TALEN,564 and Cas9,565 can be engineered to bind to arbitrary DNA sequences. A predetermined hybrid structure may be achieved by rationally engineering the DNA sequences and the DNA binding proteins. For example, a fused chain of a hundred zinc finger proteins may act as the scaffold, while hundreds of DNA strands may act as the staples to guide the folding of the protein scaffold. To achieve this, new modular interaction motifs and their corresponding design rules need to be explored and established. Furthermore, the concept of DNA origami does not necessarily have to be restricted by the DNA. Recently, Dietz et al. reported a method of constructing DNA−protein hybrid structures based on the interactions between TAL (transcription activator-like) effector proteins and duplex DNA.566 They employed a duplex DNA as scaffold and double-TAL proteins as stables. The stable proteins are able to specifically bind to two regions of the scaffold to mimic the strategy of DNA origami. The strategy is demonstrated to be successful by constructing a series of

Figure 28. Light harvesting systems that use DNA origami. (A) The energy transfer pathway can be controlled by positioning fluorophores on the DNA origami.552 Images reproduced from ref 552. Copyright 2011 American Chemical Society. (B) A systematic study on the numerical and geometric influences on energy transfer by using a DNA origami platform.553 Images reproduced from ref 553. Copyright 2016 American Chemical Society.

Besides DNA origami, custom DNA structures can be achieved with DNA bricks as well.554−557 DNA bricks consist of hundreds of short single-stranded DNA (usually less than 60nt long) without a long scaffold. Since a short DNA strand has much less secondary structures than a long scaffold, a singlestranded DNA brick is able to assemble isothermally because of less kinetic traps.558 Furthermore, the sequences of the bricks are not limited by the scaffold. However, without the aid of a scaffold, the yield of the assembly of DNA bricks decreases significantly as compared with that of the yield associated with a DNA origami structure of a similar size. The high yield (generally >90%) of the scaffolded assembly is the main reason that DNA origami is more widely used for applications. Despite how successful researchers have been in creating custom molecular structural designs with DNA origami, there are still many challenges and opportunities. For example, although a couple of computer software systems are available to help in designing and verifying the final structures, it is still difficult to obtain a satisfying yield for some of the complicated designs. The current simulations of DNA origami are generally based on the final well-formed structures. We still have limited information about the folding process of DNA origami. More knowledge about the folding process would help us to improve the yield of desired structures and to avoid the formation of undesired structures. Additionally, current experimental studies are generally quite specific for some particular designs and are limited by the cost of the materials needed to create the structures or the time needed to create the structures. Being able to computationally simulate the dynamic folding of the DNA 12625


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transfer systems that use enzymatic cascades have also been successfully built. The next objective is to scale these transfer systems up so that they can perform more sophisticated tasks. For example, enhanced energy transport in protein-engineered excitonic networks have been successfully demonstrated.576 DNA origami may be useful to further improve the efficiency and complexity of the excitonic networks. The technically challenging part is to make different functional modules compatible with each other and to achieve a collective goal. DNA origami nanostructures have been designed, studied, and used by researchers with backgrounds in chemistry, physics, biology, material science, and computer science to tackle important questions. The highly interdisciplinary research among different fields will continuously generate new approaches and ideas to broaden molecular engineering, assembly, and applications. It will be exciting to see how DNA origami may contribute to the study of nanoscale-level molecular interactions and to find real life applications in human healthcare, materials fabrications, and biomimetic systems.

single-layered and multilayered hybrid structures. By programming the interactions of the RNA or the peptides according the rules developed in the DNA origami design, it is also possible to make custom RNA-origami560,567,568 or protein-origami nanostructures.569 A genetically encoded DNA origami structure is another daunting challenge. However, with a genetically encoded DNA origami structure, we could build artificial machineries in a cell and program cellular activities. RNA self-assemblies have already been achieved in vivo by encoding RNA sequences into the genome and allow cells to continuously express RNA strands that self-assemble cotranscriptionally.570 Since DNA is the stable information storage material in a cell, it is usually difficult to produce ssDNA with a desired length and sequence in vivo. However, with the recent development of synthetic biology, ssDNA with an arbitrary sequence and length can be produced by using a retron.571 Elbaz et al. successfully implemented a method to produce four ssDNAs that could then self-assemble into a crossover motif in Escherichia coli.572 Other than creating ssDNA in vivo, how to control the successful formation of complex origami structures isothermally in vivo is another problem that researchers need to address. Typically, the annealing process, during the preparation of DNA origami structures, is used to remove the secondary structures of the scaffold and to avoid the kinetic traps that occur during the folding process. Consequently, it is optimal to design a scaffold with the minimum amount of kinetic traps that can occur due to the folding process. Furthermore, another issue that needs to be considered is that the folding of the nucleic acids in cellular environments may be different from the folding that occurs in our buffer. If a scaffold is designed with minimal secondary structures, this may help us to avoid kinetic traps during the folding process and the formation of complex DNA origami in vivo would be highly possible. DNA origami is a promising material to use for human healthcare diagnostics and therapeutics. DNA nanostructures such as tetrahedrons573 have been able to cross the cell membrane and can be readily modified with transport siRNA, antibodies, or small molecular drugs. The most striking capability of DNA origami-based therapeutics and diagnostics is that it can sense the environment and perform computations to decide whether or not to release a drug. The conditional release of the drug could decrease potential side effects in patients and increase the specificity of drug binding and release. The aptamer selection technique is mature enough to select a DNA sequence that can recognize disease biomarkers. Furthermore, a nucleic acid is now able to build a reliable and sophisticated circuit.574,575 By combining aptamers as the sensor, dynamic DNA circuits as the signal processing system, and medicines as the weapon, in combination with the DNA origami scaffold structures, it is possible to build a smart DNA nanotheranostic device that can simultaneously perform complex diagnostics and therapeutic tasks based on the analysis of tens of millions of molecular markers in a living cell.18 To achieve this goal, the efficiencies of the actuation in response to an external signal and the drug load and unload should be high enough to achieve a sensitive drug release and less spontaneous leakage. Further, the stability, immune response, and amount of time that DNA origami structures are circulating in a biologically relevant environment needs to be improved and verified. DNA origami is also a useful tool to organize photonic components to construct complicated photonic networks. In the past few years, a variety of energy transfer systems have been constructed based on inorganic or organic materials. Mass

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hao Yan: 0000-0001-7397-9852 Notes

The authors declare no competing financial interest. Biographies Fan Hong received his B.S. degree in Chemistry from Huazhong University of Science and Technology (HUST) in 2014. He is currently a graduate student in the Yan Lab. He is interested in the design of functional and structural nucleic acids. Fei Zhang received her B.S. degree in Chemistry from Peking University and Ph.D. degree in Chemistry and Biochemistry from Arizona State University. She is currently an Assistant Research Professor in the School of Molecular Sciences and the Biodesign Institute at Arizona State University (ASU). Yan Liu received her B.S. degree in Chemistry from Shandong University and a Ph.D. degree in Chemistry from Columbia University. She is currently an Associate Professor in the School of Molecular Sciences and the Biodesign Institute at Arizona State University (ASU). Hao Yan received his B.S. degree in Chemistry from Shandong University and a Ph.D. degree in Chemistry from New York University. He is currently the Milton D. Glick Distinguished Professor in the School of Molecular Sciences and the Biodesign Institute at Arizona State University (ASU). He directs the Biodesign Center for Molecular Design and Biomimetics at ASU.

ACKNOWLEDGMENTS This work was supported by grants from the Army Research Office, National Institute of Health, Office of Naval Research and National Science Foundation to H.Y. and Y.L. REFERENCES (1) Whitesides, G. M.; Grzybowski, B. Self-Assembly at All Scales. Science 2002, 295, 2418−2421. (2) Zhang, S. Fabrication of Novel Biomaterials through Molecular Self-Assembly. Nat. Biotechnol. 2003, 21, 1171−1178. 12626


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DNA Origami: Scaffolds for Creating Higher Order Structures

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