Recent Advances of DNA Origami Technology and Its Application in Nanomaterial Preparation

In recent years, the unrivalled self‐assembly properties of DNA molecules have driven the rapid development of a new class of DNA self‐assembly technology—DNA origami technology. Over the past decade, this technology has enabled the construction of DNA nanostructures with different shapes, such as 2D and 3D structures. Meanwhile, the application of DNA origami are also developed rapidly. DNA origami structures are widely used in nanomaterial preparation and drug delivery due to their characteristics of accurate addressability, excellent programmability, and good biocompatibility, especially in the field of nanomaterial preparation. Such structures provide a new platform to construct nanomaterials with high precision. Herein, the development of DNA origami technology is introduced, and the research progress of 2D and 3D DNA origami in the past two decades is systematically summarized. Then, the application of DNA origami template in nanomaterial preparation is emphasized. Finally, the main challenges and opportunities for nanomaterials fabrication based on DNA origami technology are illustrated.


Introduction
The rapid development of DNA nanotechnology has refreshed human's understanding of DNA, a biological macromolecule, which can not only be used to carry genetic information, but also participate in the construction of nanostructures to show abiotic functions. The structural diversity, flexibility, and programmable properties of DNA molecules ensure their unprecedented advantages in the fabrication of complex nanomaterials. Specific DNA molecules can be assembled to form DNA nanostructures with unique morphology by base complementary pairing. As an important part of DNA nanostructures, DNA origami is widely used in the fields of chemical biology, nanomaterial science, and synthetic biology due to the characteristics of precise addressability, good biocompatibility, and chemical stability. [1] DNA origami technology is one of DNA self-assembly technologies developed in recent years. A long single-stranded DNA (ssDNA) can pair with hundreds of shortstranded DNA or self-assemble to form DNA nanostructures with specific morphology by this technology. [2] Since reported in 2006, DNA origami technology has immediately attracted worldwide attention. [2a] With the development of the past decade, DNA nanostructures obtained by this technology have been expanded from simple 2D graphics (such as 2D nanosheet, smiling faces, and other nanopatterns) [2a] to complex 2D graphics such as dolphin structures, [3] and to even more complex 3D nanostructures. To date, various DNA origami structures were successively prepared. Meanwhile, the application about DNA origami has also been rapidly developed and was mainly reflected in two large fields. The first is the application of DNA origami in drug delivery, which is based on the good biocompatibility of DNA origami and its effective uptake by cells. For example, Ding's group used DNA origami to deliver small molecule drugs to treat tumor-related diseases. [4] The second is the application of DNA origami in the field of nanomaterials preparation. DNA origami structures are often used as templates to accurately fabricate nanomaterials with different compositions and complex geometries due to their structural programmability and location addressable characteristics. Based on the development of DNA origami technology, its advantages in the application field are currently more reflected in the field of fabricating nanomaterials.
In this review, we first introduce the development of DNA origami techniques in recent years. Subsequently, the research progress of DNA origami templates in fabricating nanomaterials is reviewed in detail. Finally, we summarize the challenges DOI: 10.1002/sstr.202200376 In recent years, the unrivalled self-assembly properties of DNA molecules have driven the rapid development of a new class of DNA self-assembly technology-DNA origami technology. Over the past decade, this technology has enabled the construction of DNA nanostructures with different shapes, such as 2D and 3D structures. Meanwhile, the application of DNA origami are also developed rapidly. DNA origami structures are widely used in nanomaterial preparation and drug delivery due to their characteristics of accurate addressability, excellent programmability, and good biocompatibility, especially in the field of nanomaterial preparation. Such structures provide a new platform to construct nanomaterials with high precision. Herein, the development of DNA origami technology is introduced, and the research progress of 2D and 3D DNA origami in the past two decades is systematically summarized. Then, the application of DNA origami template in nanomaterial preparation is emphasized. Finally, the main challenges and opportunities for nanomaterials fabrication based on DNA origami technology are illustrated. and prospects of DNA origami nanotechnology-mediated nanomaterials.

Self-Assembly of DNA Origami
The researches on DNA nanostructures started from the crossholiday structure first proposed by Professor Seeman in 1982. [5] Since then, Mao et al. expanded the cross number and successively constructed three-, four-, and six-arm DNA tiles. [6] With the development in recent years, a series of complex and diverse DNA nanostructures have been prepared successively, including DNA tile, [7] DNA origami, and DNA brick self-assemblies, [8] among which DNA origami has been the most widely studied. At present, researchers have successively obtained a variety of 2D and 3D DNA origami structures and extensively studied the potential application value of these DNA origami structures. This part will review the recent progress of 2D and 3D DNA origami structures.

M13mp18 Served as a Scaffold to Assemble 2D DNA Origami
The concept of DNA origami technology was introduced by Rothemund in 2006, which has since opened the prelude to the research of DNA origami. [2a] A series of 2D DNA origami structures such as triangle, pentagram, and smiley face ( Figure 1) were assembled by using M13mp18 ssDNA as a scaffold and specifically combining with hundreds of staple strands. [2a] Using similar technology, Qian and colleagues [9] constructed a 2D DNA origami structure similar to the map of China in the same year. Kjems and co-workers [3] designed and prepared a dolphinshaped 2D DNA origami structure using a friendly software package developed in 2008. Yan et al. designed curved 2D DNA origami by introducing curved surfaces, which provided a theoretical basis for the construction of 3D DNA origami and further promoted the development of DNA origami technology. [10] Then, Seeman and Qian et al. have successively prepared 2D DNA origami structures with different shapes such as cross, solid triangle, and hollow rectangle by using M13mp18 scaffold. [11] 2.1.2. Long Double-Stranded or SsDNA Served as a Scaffold to Assemble 2D DNA Origami Although a large number of DNA origami structures have been assembled by using M13mp18 as a scaffold, the complexity, size, and diversity of DNA origami structures are limited by the length of such scaffolds, which somewhat hampered the development of DNA origami technology. [12] To solve the problem, in recent years, researchers have used long double-stranded DNA (dsDNA) or long ssDNA instead of M13mp18 to provide a new strategy for the assembly of 2D DNA origami. [12b,13] Woolley et al. constructed a series of 2D DNA origami structures with different shapes by using polymerase chain reaction (PCR) to amplify the length of DNA scaffold and replacing M13mp18 with amplified DNA scaffolds. [13b] Yan and colleagues [12b] successfully prepared square and triangular DNA origami using dsDNA scaffolds ( Figure 2A). Fan and co-workers [13a] creatively used long ssDNA as the scaffold to obtain a layered porous 2D DNA origami structure ( Figure 2B).  The length of DNA scaffold has a great influence on the complexity and size of DNA assemblies. The commonly used scaffold M13mp18 is only 7249 nt in length. The maximum area and volume of DNA origami assembled by M13mp18 can only reach 78 Â 78 nm 2 and 24.7 Â 24.7 Â 24.7 nm 3 in 2D and 3D, respectively. [12b] How to expand the size of DNA origami has become a key challenge to promote the further development of DNA origami technology. To address this challenge, researchers have successfully expanded the size of DNA origami by trying to replace DNA scaffolds, which can be summarized in the following two ways: 1) the first way to expand the size of the assemblies was introducing a new scaffold to assemble the formed DNA origami. Yan and colleagues [12a] used phiX174 as the scaffold to assemble DNA origami constructed by M13mp18, and successfully expanded the size of DNA origami ( Figure 3A). Liu et al. divided M13mp18 into two new scaffolds and used one scaffold containing only 1146 bases to construct a sheet DNA origami with the size of 17 Â 16 nm in length and width. [14] The remaining 6103 bases were used as another scaffold to assemble the formed DNA origami sheets, thus achieving the expansion of the size of DNA origami. Sleiman et al. obtained size-expanded DNA nanoribbons by using the scaffold which was amplified by PCR to assemble rectangular DNA origami. [15] 2) The second way to expand the size of the assemblies was extending the length of DNA scaffold. Yan's group [12b] successfully constructed triangular DNA origami with enlarged size by denaturing dsDNA into ssDNA at high temperature to extend the length of the scaffold. Fan and co-workers first obtained ssDNA scaffold with a length of 26 KB by PCR amplification method and then assembled flaky DNA origami with the size of 238 Â 108 nm in length and width using this scaffold. [16] LaBean et al. [17] increased the length of M13 scaffold to 51 466 nt by introducing λ-bacteriophage and successfully assembled DNA origami with significantly increased size ( Figure 3B). Li's group [12d] used a similar method to amplify the length of DNA scaffold and successfully extended the side length of hollow triangle to 300 nm. The size of the rectangular DNA origami assembled by this method reached 235 Â 100 nm.
The size of 2D DNA origami can also be expanded by extending the sticky end of DNA origami edge chain. [18,19] Fan's group [19b] extended eight bases from the side chains at the left and right ends of rectangular DNA origami and connected DNA origami along the same direction by one-step rapid assembly method, thus producing DNA origami nanoribbons with a length of up to micron. Sugiyama et al. [18] used similar strategy to successfully expand the size of DNA origami in 2D space ( Figure 3C). Since 2011, researchers have further expanded the size of DNA origami by constructing DNA origami arrays and finally obtained 2D DNA origami with length and width of tens of microns. [11a,20] Through the construction of 2 Â 2 modules and the complementary pairing of sticky ends, Qian et al. realized the preparation of large 2D DNA origami arrays and successfully outlined nanopatterns such as Mona Lisa and bacteria on DNA origami array. [20b] In addition, DNA origami structure can be formed through self-assembly of only a long ssDNA. Yin et al. reported a strategy to construct DNA origami structures from only a long ssDNA, which was different from the conventional origami assembly method. [2b] In this work, partially complemented dsDNA and parallel crossover cohesion were used to construct a structurally complex yet knot-free structure. Meanwhile, this work demonstrated that ssDNA origami structure could be melted and used as a template for amplification by polymerases in vitro. Sugiyama and coworkers even used the RNA transcript as a template to construct RNA-templated 2D DNA origami by annealing with designed DNA staple strands. [21] 2.1.3. 2D Wire-Frame DNA Origami In addition to the aforementioned DNA origami, a class of structures known as 2D wire-frame DNA origami have also made rapid progress. Different from 2D planar DNA origami, 2D wire-frame DNA origami can realize the construction of complex 2D geometric shapes with irregular boundaries. [22] Yan's group [23] took the lead in the preparation of various 2D wire-frame DNA origami structures by using the four-arm junction connection technology ( Figure 4A). Subsequently, the same group [24] further promoted the development of wire-frame structure through n Â four-arm junction connection technology ( Figure 4B). Bathe and co-workers [25] designed and prepared a series of asymmetric 2D wire-frame DNA origami structures by using the newly developed software ( Figure 4C). At present, the research on 2D wireframe DNA origami is mainly reflected in the preparation of DNA nanostructures. With the in-depth research in the future, its application value will be continuously explored.

3D DNA Origami
Seeman et al. constructed the first 3D DNA nanostructure in 1991. [26] The plane formed by complementary pairing of DNA strands was further assembled to form a cubic DNA nanostructure through steps such as cyclization and T4 enzyme ligation. Thereafter, Joyce and co-workers constructed DNA octahedron by using a long ssDNA paired with several short-stranded DNA and confirmed the structural framework of DNA octahedron through freeze transmission electron microscopy (TEM) for the first time. [27] With the rapid development of DNA nanotechnology, new breakthroughs have been made in 3D DNA origami structures in the same period. [28] To date, 3D DNA origami structures can be divided into the following categories according to the morphological characteristics and properties: single-layer 3D DNA origami, single-layer 3D wire-frame DNA origami and multilayer DNA origami.

Single-Layer 3D DNA Origami
The assembly method of single-layer 3D DNA origami mainly includes two strategies. The first strategy is to fold the formed planar 2D DNA origami to assemble the single-layer 3D DNA origami structure. Using this strategy, Sugiyama et al. first constructed three-arm, four-arm, and six-arm DNA origami, followed by a second step of folding through complementary base-pairing to achieve the preparation of 3D hollow prism structures. [29] Yan's group successfully constructed the hollow tetrahedron structure by using similar strategy ( Figure 5A). [30] Komiyama and co-workers also used the similar strategy to realize the preparation of DNA origami cube structure for the first time. [31] The six planes required to form the cube were first obtained by assembling M13mp18 and short strands and then folded with each other by the addition of the connecting strands to finally assemble into a 44 Â 42 Â 35 nm 3 cube structure. In the same year, Kjems and co-workers [32] constructed a similar 3D DNA origami cube structure with the size of 42 Â 36 Â 36 nm 3 ( Figure 5B). Since then, the team used a similar method to prepare a cubic DNA origami structure containing a switchable lid, which was only 18 Â 18 Â 24 nm 3 in size ( Figure 5C). This reversible switching process opened up the possibility for the controlled delivery of subsequent drugs and the application of molecular computing. [33] Church et al. developed a 3D aptamergated DNA nanorobot ( Figure 5D). The structure sensed incoming signals from the cell surface and then was reconfigured for efficient drug delivery by triggering DNA complementary strands to activate logic gates that control aptamer coding. [34] The second strategy is to construct 3D DNA origami structures with curved surfaces by introducing crossover structures and guiding surface formation. In 2011, Yan et al. took the lead in the construction of 3D curved DNA origami. [10] The 3D DNA origami structures with different shapes such as hemispherical, spherical, and vase ( Figure 5E) were successfully constructed. They further explored the fabrication of circular DNA nanoribbons ( Figure 5F), providing technical support for the creation of unprecedented programmable topologies. [35] According to the second strategy, Nasr and co-workers [36] prepared the circular DNA origami structure ( Figure 5G). Dekker's group also achieved the preparation of 3D DNA nanoring [37] to simulate the bionic nuclear pore complex, allowing accurate control over the position of nuclear pore complex components through the addressable characteristics of DNA origami. In addition, Firrao and Shih also used similar strategies to fabricate 3D DNA nanotubes and barrel-shaped DNA nanorobots. [38] 2.2.2. Single-Layer 3D Wire-Frame DNA Origami With the development of DNA nanotechnology, single-layer 3D wire-frame DNA origami has achieved rapid development in recent years, presenting diverse morphology and functions. Liedl and co-workers creatively constructed hollow rigid tetrahedral frame structure with a length of 75 nm by bottom-up assembly method. [39] Yan et al. prepared 2D and 3D spherical wire-frame DNA origami structures by using four-arm knot connection technology. [23] Through the construction of latitudinal and longitudinal frames, the diameter of the assembled spherical wire-frame structure reached 56 nm ( Figure 6A). The molecular weight of 3D DNA origami prepared by traditional methods was usually less than 5 MDa. Through a simple and universal one-step self-assembly strategy, Yin's group [40] successfully broke through the molecular weight limit of 3D DNA origami and realized the preparation of diversified 3D wire-frame DNA origami ( Figure 6B). Hogberg and co-workers developed a method for folding arbitrary polygonal digital grid structures, which mainly constructed target structures through the routing algorithm process based on graph theory and tracking scaffolding ropes. [41] Different from the traditional design of closed spiral origami, the structure formed by this method has more open conformations, making it easier to assemble structures that were difficult to achieve by the conventional method ( Figure 6C).
By connecting arbitrarily selected vertices in 3D space, Yan et al. assembled a wire-frame DNA nanostructure with highly complex and programmable properties. [24] In this method, the vertices were composed of n Â 4 multi-arm knots (n = 2-10) and the lines were composed of antiparallel DNA crossover blocks with variable length. The vertices and lines were integrated by scaffolds to form the complex 3D wire-frame semiregular solid structure. Bathe et al. reported a strategy for the autonomous design of 3D DNA origami based on specific  [23] Copyright 2013, American Association for the Advancement of Science. B) Complex DNA wire-frame structure constructed by four-arm junction DNA. Reproduced with permission. [24] Copyright 2015, published by Springer Nature. C) Sequence design path and AFM image of asymmetric wire-frame structure. Reproduced with permission. [25] Copyright 2019, American Association for the Advancement of Science.  [30] Copyright 2009, American Chemical Society. Reproduced with permission. [32] Copyright 2009, Springer Nature. Fabrication of C) small DNA box origami, Reproduced with permission. [33a] Copyright 2012, American Chemical Society. D) aptamer-gated DNA nanorobot, Reproduced with permission. [34] Copyright 2012, American Association for the Advancement of Science. and E) 3D DNA origami with different shapes containing complex 3D curvatures Reproduced with permission. [10] Copyright 2011, American Association for the Advancement of Science. F) Construction of 3D Mobius DNA strip. Reproduced with permission. [35] Copyright 2010, Springer Nature. G) Schematic and TEM image of DNA-corralled nanodisc. Reproduced with permission. [36] Copyright 2018, American Chemical Society. Figure 6. A) Schematic and TEM image of spherical grid wire-frame DNA origami structure. Reproduced with permission. [23] Copyright 2013, American Association for the Advancement of Science. B) Construction of polyhedral DNA origami. Reproduced with permission. [40] Copyright 2014, American Association for the Advancement of Science. C) Schematic diagrams and TEM images of 3D mesh DNA origami structures with different shapes. Reproduced with permission. [41] Copyright 2015, Springer Nature. shapes, through which various grid-based polyhedral DNA origami could be achieved. [42] This work mainly demonstrated the utility of such structures in both biotic and abiotic directions by exploring the stability of DNA origami in serum and low-salt buffers. Hogberg and coworkers not only achieved the construction of hexagonal wire-frame DNA origami rod structures, but also explored the uptake and distribution of the structures in the cell spheroid tissue models. [43] In this work, two rodlike DNA origami structures with similar geometry were constructed by using either a compact lattice-based-or a wire-frame-design scheme. Subsequently, the structural properties and their cell uptake and distribution in cell spheroid tissue models of these two structures were compared. The experiment demonstrated that wire-frame DNA origami remained outside, or on the cellular membrane while compact DNA origamis were internalized into cells to a larger extent. In contrast, wire-frame DNA origami displayed a higher penetration ability in cell spheroid tissue models than compact DNA origami.

Multilayer DNA Origami
Despite the high generation efficiency of single-layer DNA origami, these structures have some limitations in practical applications. Molecular dynamics simulation experiments show that the conformational flexibility and structural heterogeneity of single-layer DNA origami limit their addressable accuracy. Meanwhile, the inevitable structural flexibility due to the discontinuities of the single-layer wire-frame structure greatly limits their applications. In view of the previous problems, a new class of 3D DNA origami structures-multilayer DNA origami have been developed in recent years. These structures are composed by a honeycomb mesh lattice or tetragonal lattice helical arrangement, which can be roughly summarized into multilayer 3D DNA origami and higher-order multilayer 3D DNA origami. [44] Shih's group designed a series of 3D multilayer DNA origami structures through an open-source software package with a graphical user interface and confirmed the feasibility of the software design through experiments. [45] Since then, the software has been widely used in the design of DNA origami, realizing the preparation of various 3D multilayer DNA origami. The group then prepared different 3D multilayer DNA origami structures ( Figure 7A) by using the mixed packaging of honeycomb grid, square lattice, and hexagonal lattice structures. [46] A barrelshaped 3D multilayer DNA origami structure was designed by Kostiainen and co-workers ( Figure 7B). Glucose oxidase and horseradish peroxidase were assembled inside the barrel structure which formed the nanoreactors. [47] Gothelf 's team constructed a hollow cube DNA origami structure ( Figure 7C). [48] Shih et al. have extended the shape of 3D multilayer DNA origami, forming different shapes of DNA origami, such as square nuts, railing bridges, stacked crosses, and slotted crosses by layered assembling the helical folds of a honeycomb lattice. [49] All the structures reported earlier were directly obtained by complementary pairing of scaffold with short-stranded DNA. In addition, another higher-order 3D multilayer DNA origami can be obtained by further assembly of 3D DNA origami. [44a,c] Yin et al. assembled higher-order barrel structure by using this strategy. [50] Based on the ability of DNA to distort complex shapes at the nanoscale, Shih et al. guided DNA sequences to form double helices that were tightly cross-linked and arranged parallel to the helix axis, creating a variety of complex DNA origami structures by directional insertion or deletion of base-pairs. [51] Dietz Figure 7. A) Simulated diagrams and TEM images of multilayer DNA origami with different amounts of honeycomb mesh. Reproduced with permission. [46] Copyright 2012, American Chemical Society. B) Schematic diagram and TEM images of bucket DNA origami. Reproduced with permission. [47] Copyright 2015, Royal Society of Chemistry. C) Construction of hollow cube DNA origami. Reproduced with permission. [48] Copyright 2017, Wiley-VCH GmbH. D) A schematic diagram of the assembly of multilayer DNA origami through the complementary orthogonal DNA bricks with four arms. Reproduced with permission. [52] Copyright 2015, American Association for the Advancement of Science. E) Schematic diagram and cryo-electron microscopy of tubular DNA origami assembled by self-limiting cyclic oligomers. Reproduced with permission. [55] Copyright 2017, Springer Nature.
www.advancedsciencenews.com www.small-structures.com and co-workers used a similar method to synthesize a series of complex high-order 3D multilayer DNA origami structures ( Figure 7D) and further assembled them to form a higher-order structure. This work endowed high-order 3D DNA origami good dynamics by regulating the concentration of Mg 2þ , which could be used for the fabrication of nanorobots. [52] Simmel and colleagues constructed a dynamic high-order DNA origami rotaxane structure by using a similar strategy. [53] Mao et al. first fabricated DNA nanobuckets with different diameters and then further assembled them to produce more complex higher-order DNA nanostructures. [54] Dietz's group [55] formed serrated self-limiting ring oligomer DNA origami by assembling "V"-shaped multilayer DNA origami and then further assembled them to obtain tubular DNA origami ( Figure 7E). The size of the high-order 3D DNA origami finally formed by this assembly method could be up to micron level.

Application of DNA Origami for the Fabrication of Nanomaterials
Due to the unique optical, electrical, and chemical properties, nanomaterials are widely used in biomedical, chemical, electronic, and aerospace fields. How to controllably fabricate nanomaterials with special morphology and size has become one of the major challenges in the field of materials science. Using the traditional top-down precision machining technology to fabricate nanomaterials not only wastes the raw materials seriously, but also restricts the micromorphology of nanomaterials greatly. The bottom-up self-assembly strategy developed in recent years provides the possibility for the fine fabrication of nanoscale materials and has become an important research field in materials science. DNA origami, with its programmable and addressable properties, can be used to regulate the growth of nanomaterials at the molecular scale, customize various nanomaterials with predesigned patterns, and achieve the precise control of material morphology. This technology has been widely used in the controllable fabrication of nanomaterials by bottom-up strategy, thus bringing a potential revolution to the field of nanomaterials science. In recent years, new achievements have been made by using DNA origami as a template to control the fine fabrication of organic and inorganic nanomaterials. In this section, we systematically discuss the application of DNA origami for the fabrication of polymer, inorganic metal, and inorganic nonmetallic materials.

Fabrication of Polymer Materials on DNA Origami Template
The template method has become an important method to prepare polymer materials. The commonly used templates currently can be divided into several categories, such as protein molecules, mineral skeletons, various inorganic materials, and biological macromolecular DNA. Among them, DNA molecule is one of the most widely used template for the fabrication of nanomaterials. Through the design of DNA-based sequence and the precise control of DNA molecule length, DNA molecule can be selectively functionalized and used to arrange heterogeneous nanoscale materials on DNA origami, which provides a powerful manufacturing technology for high-precision nanomaterials. In recent years, new breakthroughs have been made in the fabrication of polymer materials by DNA origami template. For example, Seeman et al. used DNA nanotubes to sheathe rodlike species and arranged them into organized patterns by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin on DNA origami. [56] Zhong's group anchored curli-specific gene B onto triangular DNA origami templates to direct curli-specific gene A monomers to form oligomers and even fibril. [57] In this section, we will discuss three commonly used preparation methods: 1) through electrostatic adsorption, positively charged polymers can be controllably distributed on DNA origami to form polymer nanocomposites. 2) By DNA hybridization, the polymer can be arranged on DNA origami to form specific nanopatterns. 3) Through in situ polymerization, polymer materials with specific morphology can be fabricated on DNA origami.

Electrostatic Adsorption
Electrostatic adsorption was used to regulate the distribution of polymer on DNA origami, aiming to improve the physical and chemical properties of DNA origami through polymer modification. Kostiainen et al. used the capsid proteins of cowpea spotted virus to interact with DNA origami to obtain protein-coated nanocomposites through electrostatic interactions ( Figure 8A). The nanocomposite not only improved the stability of DNA origami, but also significantly enhanced the cell uptake efficiency of DNA origami. [58] By selecting other proteins to interact with DNA origami, they continued to explore the effect of different protein packages on the physicochemical properties of DNA origami. [59] Bovine serum albumin (BSA) was modified with dendrimers (G2) to generate BSA-G2 conjugated polymer (CP), which resulted in the change of its electrical properties. As a cationbinding domain, the dendritic part of copolymer was uniformly wrapped on DNA origami by electrostatic interaction, forming a complex of DNA origami-BSA-G2 ( Figure 8B). Similarly, our group coated DNA origami with cationic human serum albumin (cHSA) via electrostatic interaction, which significantly improved the stability of DNA origami. [60] Keller's group investigated the effects of two different DNA origami nanostructures on human islet amyloid polypeptide aggregation by strong electrostatic interactions between the negatively charged DNA origami nanostructures and the positively charged peptide. [61] In addition to natural proteins and polypeptide, synthetic polymer materials can also be wrapped on DNA origami through electrostatic adsorption to form polymer coatings. Shih et al. used positively charged oligolysine (K 10 ) and polyethylene glycol (PEG)ylated oligolysine co-polymer (k 10 -PEG 5k ) to interact with the negatively charged barrel DNA origami, resulting in a tubular distribution of polymer adsorbed on DNA origami ( Figure 8C), which significantly improved the stability of DNA origami in low salt state. [62] Schmidt et al. used cationic PEG-b-poly(l-lysine) block copolymers (PEG-PLys) to enwrap DNA origami ( Figure 8D). The block copolymer covered DNA origami uniformly, forming polyplex micelles to protect DNA origami. [63] Barišić's group used chitosan and linear polyethyleneimine to obtain polycationic shells with specific morphology by wrapping different 3D DNA origami structures. [64] This work explored the effect of charge density on the stability of packaged DNA origami by varying the degree of polymerization and the N/P charge ratio (ratio of the amines in polycations to the phosphates in DNA skeleton).

DNA Hybridization
DNA hybridization technology has become an important method for the fabrication of polymer nanomaterials. Due to the complementary pairing of DNA fragments of the polymer material with DNA origami, the polymers can be arranged on DNA origami to form specific nanopatterns. How to obtain amphipathic assemblies with controllable size and shape has become a major challenge for the development of amphiphilic molecular selfassembly field. Liu et al. provided a possible opportunity for the assemble of amphiphilic molecules through DNA hybridization and successfully constructed a layer of hydrophobic polymer nanosheets with controllable shape on flake and triangle DNA origami structures. [65] Amphiphilic molecules modified by ssDNA binded specifically to DNA origami, forming a thin hydrophobic layer on DNA origami and resulting in a local concentration of hydrophobic groups on DNA origami that was much higher than the concentration of hydrophobic groups in solution. The excess amphiphilic molecules in the solution then entered the hydrophobic thin layer through hydrophobic interaction and finally formed the hydrophobic nanosheet structure on DNA origami ( Figure 9A). The same group subsequently constructed cuboid and dumbbell hetero-vesicles by the similar frame-guided assembly strategy ( Figure 9B), demonstrating the versatility of the frame-guided assembly strategy for guiding complex amphiphilic assemblies. [66] Shih and co-workers used a similar strategy to construct a phospholipid micelle layer on DNA origami. [67] Oligonucleotide-modified liposomes were precisely arranged on DNA origami by hybridizing with ssDNA extended from 3D wire-frame DNA origami. The hydrophobic interaction of liposomes promoted the adsorption of various surfactants, ultimately simulating the morphology of encapsulated virus particles, resulting in the formation of a compact phospholipid micellar layer with controllable thickness on DNA origami. The formed micellar layer provided a good protection to DNA origami by preventing the contact between nuclease and DNA origami. Compared with the control group, the immune activity of DNA origami wrapped by micelle layer was reduced by two orders of magnitude, and the pharmacokinetic bioavailability was increased by nearly 17 times. Lin et al. reported a strategy to guide liposome formation using DNA origami nanocages as templates. [68] This work provides a solution to the longstanding challenge of controlling liposome shape, arrangement, and dynamics by regulating the size of DNA origami ring, the pillar length, and rigidity, as well as the number and position of the handles and teeth on DNA origami nanocages.
In addition, the introduction of amphiphilic molecules such as DNA-lipid conjugates and DNA block-copolymers can not only form hydrophobic polymer layers on DNA origami, but also guide the assembly of DNA origami to form higher-order structures through hydrophobic interaction. [69] Simmel's group reported an approach to assemble single-layered DNA origami structures into sandwich-like bilayer structures by hydrophobic interactions.
[69b] DNA-cholesterol conjugates anchored on DNA origami by DNA hybridization presented a strong tendency to form aggregates in aqueous solutions, which induced singlelayered DNA origami sheets to form sandwich-like structures. Liu et al. developed a novel strategy to fold the same 2D DNA sheet into multiple complex structures (Termed as origami þ ) driven by introducing hydrophobic interaction. [70] As shown in Figure 8. A) Formation of the DNA origami-protein complex through electrostatic adsorption between virus capsid proteins and rectangular DNA origami. Reproduced with permission. [58] Copyright 2014, American Chemical Society. B) Formation of DNA origami-BSA-G2 protein complex through electrostatic interaction. Reproduced with permission. [59] Copyright 2017, Wiley-VCH GmbH. C) Formation of DNA origami-peptide complex by coating DNA origami with peptides K 10 -and K 10 -polyethylene glycol (PEG) 5 K . Reproduced with permission. [62] Copyright 2017, Springer Nature. D) Formation of DNA origami polyplex micelles by coating DNA origami with the block polymer PEG-b-poly(l-lysine) block copolymers (PEG-PLys). Reproduced with permission. [63] Copyright 2017, Wiley-VCH GmbH.  Figure 9C, three trapezoidal domains in the triangle DNA origami were modified with cholesterol molecules by DNA hybridization in a stepwise manner and self-folding in each domain were realized to form origami þ nanostructures. Although synthetic polymers are ubiquitous in modern society, it is still a great challenge to accurately control the molecular conformation of single polymer at the 1D linear level. In recent years, the use of DNA origami to accurately regulate the molecular conformation of polymers has made a breakthrough, which provides us with the possibility to study the single-molecule properties of synthetic polymers. Gothelf et al. achieved precise regulation of 1D linear polymers by DNA hybridization for the first time. [1c] Oligonucleotide-modified synthetic polymers were arranged on DNA origami along arbitrary paths to form specific nanopatterns ( Figure 9D). DNA origami technology not only provides the possibility to accurate regulate the morphology of materials, but also can be used to develop nanomechanical devices. Through DNA hybridization, Gothelf et al. developed a nanomechanical device that could freely switch the position of singlemolecule-CPs. [71] The ssDNA extended on DNA origami was hybridized with DNA fragments of the polymer to guide the copolymer to be arranged on DNA origami in a well-defined geometric shape. Based on the end-mediated chain replacement strategy, the polymer exhibited a switch between left-handed and right-handed conformations on DNA origami. This nanotechnology for controlled conformational conversion of conjugated organic polymers demonstrated unique control over the shape of linear polymers, providing new methods for reconstructing nanophotonics and electronic devices.
CPs have been widely studied due to their unique optical and electrical properties. However, the ability to regulate these polymers at the nanoscale is still very lagging behind. Gothelf et al. expanded the means of regulating CPs by synthesizing polyfluorene-DNA graft-type polymer (poly[F-DNA]). [72] The DNA fragment of the copolymer provided special addressing capability for the material. The copolymer could be distributed on DNA origami in a controlled manner by DNA hybridization ( Figure 9E). This work confirmed the existence of energy transfer between two different copolymers through the regulation of copolymer arrangement and the fluorescence directed quenching of poly(F-DNA), which provided a way to study polymer-polymer interaction and intramolecular energy transfer. The work thus provided an important step for us to realize the control of nanoscale circuits based on single-conjugated-polymer molecules. Mertig et al. connected π-CPs to DNA origami by DNA hybridization, achieving the controllable arrangement of CPs at the nanoscale. [73] Thiophene-based block copolymers were directionally linked to DNA origami by terminal functionalized oligonucleotides. The optical properties of polymers densely fixed on DNA origami could be fine-tuned by π-π-stacking interactions between the CPs. The destruction of π-π stacking by surfactants significantly enhanced the fluorescence of the polymer, which Figure 9. A) The assembly of amphiphilic polymer layer via 2D DNA origami frame-guided process. Reproduced with permission. [65] Copyright 2016, Wiley-VCH GmbH. B) Construction of hydrophobic cubic vesicles via 3D DNA origami frame-guided process. Reproduced with permission. [66] Copyright 2017, Wiley-VCH GmbH. C) Schematic illustration of triangle 2D origami folding process through the hydrophobic interactions. [70] Reproduced with permission. Copyright 2019, Wiley-VCH GmbH. D) Schematic diagram of controlled arrangement of copolymer on DNA origami by DNA hybridization. Reproduced with permission. [1c] Copyright 2015, Springer Nature. E) Controlled arrangement of two different polymers on DNA origami. Reproduced with permission. [72] Copyright 2017, Wiley-VCH GmbH. confirmed that the CP had the characteristics of manipulating optical properties at the molecular level. This characteristic broadened the application range of DNA-polymer hybrid materials and laid a solid foundation for the design of complex optoelectronic nanodevices in the future.

In Situ Polymerization Reaction
In situ chemical reaction on DNA origami provides a novel way for the fabrication of polymer nanomaterials. DNA sequence with catalytic function anchored on DNA origami can catalyze monomers to form nanopatterned polymer materials. Ding et al. prepared polyaniline nanomaterials by extending the terminal G-quadruplex (G4) structure on DNA origami. [74] G4 sequence bound hemin to form a DNA enzyme with a horseradish peroxidase-mimicking H 2 O 2 -mediated oxidation ability, which catalyzed the oxidation of aniline monomer by hydrogen peroxide in acidic environment and generated conductive polyaniline polymer on DNA origami in situ. This research realized the controllable preparation of polymer materials on DNA origami for the first time and provided a new way for the customized development of nano organic circuits. Similar strategies can also be used for the fabrication of other polymer materials. Our group used this strategy to achieve the accurate fabrication of polydopamine (PDA) nanomaterial. [75] G4 sequences were precisely arranged on DNA origami to construct specific patterns. Under the action of hydrogen peroxide, G4/hemin catalyzed the oxidation of dopamine monomers to form PDA nanostructures ( Figure 10A). This strategy provided a controllable method for manufacturing functional DNA nanodevices and PDA nanostructures, which created a new research hotspot in the fields of DNA nanotechnology, materials science, and nanomedicine. By anchoring the photocontrolled polymerization system on DNA origami, our group proposed a new method for the fabrication of PDA nanostructures, which could accurately control the formation of PDA nanostructures from the dimensions of time and space. [76] G4 sequence acted as a binding site to anchor the photosensitizer protoporphyrin IX, which induced the oxidation of dopamine monomers in a specified region to form PDA nanostructures under visible-light irradiation ( Figure 10B). This strategy mainly regulated the polymerization process through an optical switch. Subsequent studies showed that the generated polymer nanomaterials significantly improved the stability of DNA origami templates, providing a good solution for biomedical and chemical applications that were usually hindered by DNA instability. Based on this photo-controlled polymerization system on DNA origami, our group added eosin Y and methylene blue to G4 sequence to establish broad wavelength flexibility and subsequently achieved catecholamines polymerization ( Figure 1C). [77] Our team further constructed a larger DNA template by concatenating several DNA tiles together and achieved the synthesis of complex digital nanopatterned PDA structures on DNA origami ( Figure 10D). [78] In addition to using G4/hemin to mimic horseradish peroxidase to fabricate PDA nanostructures, our team developed another method based on atom-transfer radical polymerization (ATRP) by functionalizing DNA ends to achieve controllable preparation of nanopatterned polymer materials on DNA tiles. The initiator required for polymerization was linked to ssDNA by chemical modification and then combined with DNA origami by DNA hybridization. The initiator reacted with monomer through ATRP and finally nanopatterned polymer material was successfully formed on DNA origami ( Figure 11A). [79] The same group further developed a 3D nanoreactor by using tubular DNA origami template. [80] The outside of DNA tubes was connected to the initiator by extending multiple ssDNA sequences, which induced ATRP polymerization reaction on the surface of the nanotube to generate a densely cross-linked polymer shell, while the inner space was functionalized by G4/hemin to trigger dopamine polymerization ( Figure 11B). By integrating these two different polymers, this strategy provided an elegant approach for the fine fabrication of nanoscale 3D polymer materials.
Lithographic method offers another opportunity to fabricate polymer materials by using DNA origami templates. Liu et al. reported a general method to fabricate polymer stamps by using DNA nanostructure master templates with high fidelity. [81] Polymer stamps fabricated by the lithographic method present diverse nanoscale features with dimensions ranging from several tens of nanometers to microns. At present, there are few reports about the fabrication of polymer materials by lithographic method based on DNA origami technology, which needs further exploration. However, the technology has been widely used in the fabrication of inorganic metallic and nonmetallic materials. Therefore, the research progress of this technology in the field of material preparation will be discussed in detail in Section 3.2 and 3.3.

Fabrication of Inorganic Metal Materials on DNA Origami Template
In addition to polymer nanomaterials, a variety of metal materials such as Au, Ag, Cu, Pd, and Pt can also be fabricated by DNA origami templates in a controllable manner. [82] At present, the fabrication methods of inorganic metal materials using DNA origami templates can be mainly divided into the following four ways: DNA hybridization, seed growth, in situ chemical reaction, and lithographic method.

DNA Hybridization
DNA hybridization has become one of the most commonly used methods for fabricating metal-DNA hybrid materials using DNA origami templates. ssDNA-modified nanoparticles can be controllably distributed on DNA origami by base complementary pairing. Liedl and Ding et al. reported how to construct unique 3D spiral gold nanoparticles on DNA origami by DNA hybridization. [1a,83] Wang et al. assembled gold nanoparticles with a particle size of 30 nm using a hollow 2D DNA origami template and obtained high-yield dimer and tetramer plasmonic nanostructures. The Raman signal of plasmonic nanostructures was significantly enhanced by molecular covalent attachment, which provided a new way for the development of high-sensitivity Raman sensors. [84] Ke and co-workers [85] achieved self-assembly of AuNPs (10 nm in diameter) by using DNA origami template ( Figure 12A). This work successfully obtained optomagnetic circuits and high-fidelity plasmonic metamaterials through the regulation of DNA origami, which promoted the development of controllable DNA-based optomagnetic circuits. Gang et al. presented a simple and general assembly strategy for planar structures through the assembly of gold nanoarrays. [86] DNAfunctionalized gold nanoparticles bound to 2D rigid DNA origami through DNA hybridization to form the nanoscale modules. Subsequently, the nanoscale modules linked between themselves in a planar manner via fourfold DNA-encoded interactions to form 2D gold nanoarrays with different shapes ( Figure 12B). In addition, other DNA nanostructures can also be used to assemble gold nanoparticles. For instance, Sleiman and coworkers reported how to selectively encapsulate citratecoated gold nanoparticles using DNA nanotubes with longitudinal variation and DNA nanotubes with alternating large and small capsules along their length. [87] Figure 10. A) Fabrication of polydopamine (PDA) nanomaterials with controllable shapes by DNA origami-templated polymerization. Reproduced with permission. [75] Copyright 2018, Wiley-VCH GmbH. B) Fabrication of PDA nanomaterials by photo-controlled polymerization system. Reproduced with permission. [76] Copyright 2020, Wiley-VCH GmbH. C) Multiple wavelength photopolymerization on DNA origami tubes. Reproduced with permission. [77] Copyright 2022, Wiley-VCH GmbH. D) Construction of digital nanopatterned PDA nanomaterials on DNA origami template. Reproduced with permission. [78] Copyright 2021, Wiley-VCH GmbH. DNA origami can be used to regulate not only the distribution of gold nanoparticles, but also the distribution of gold nanorods. Wang et al. successfully arranged DNA-modified gold nanorods on both sides of DNA origami by DNA hybridization ( Figure 12C). The gold nanorods on DNA origami still had the characteristic of base complementary pairing and can continue to be connected Figure 11. A) Fabrication of polymers on DNA origami templates using in situ atom-transfer radical polymerization (ATRP) method. Reproduced with permission. [79] Copyright 2016, Wiley-VCH GmbH. B) Construction of polymer nanoreactor by DNA origami template. Reproduced with permission. [80] Copyright 2018, Royal Society of Chemistry. Figure 12. A) Construction of plasmonic nanoparticle networks by using 2D DNA origami to assemble gold nanoparticles. Reproduced with permission. [85] Copyright 2019, Wiley-VCH GmbH. B) Schematic diagram of assembling 2D gold nanoarrays. Reproduced with permission. [86] Copyright 2016, Springer Nature. C) Assembly of gold nanorods by using DNA origami templates. Reproduced with permission. [88] Copyright 2013, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com with other DNA origami, which provided the possibility for the assembly of layered 3D superstructures and opened a new door for the manufacturing of complex functional 3D nanostructures. [88]

Seed Growth
The synthesis of metal nanoparticles by seed growth has become an important method in the field of metal material preparation. [89] Due to the lack of effective control in the fabrication of metal materials by traditional crystal seed method, the morphology of the prepared metal materials is often uncontrollable. The strategy of introducing seed into the cavity of 3D DNA origami template provides an ideal path for the controllable fabrication of metal materials with specific shapes. [50,90,91] Yin et al. reported a strategy to synthesize metallic nanomaterials with arbitrary 3D shapes. [50] As shown in Figure 13A, the DNA strands were self-assembled to form 3D cavity nanostructure and then the gold seed was loaded into the 3D cavity by DNA hybridization. Under mild conditions, the gold seed continued to grow and fill in the 3D cavity, finally forming the gold nanorods. Through the regulation of DNA origami templates, this strategy has realized the controllable fabrication of a series of gold and silver nanomaterials with different shapes. Seidel et al. used a similar strategy to controllably fabricate conductive gold nanowires on DNA origami templates at the nanoscale ( Figure 13B). The morphology and size of conductive gold nanowires could be regulated by adjusting the shape of DNA origami templates. The method of introducing seed into DNA origami template has been extended to the preparation of more complex inorganic materials, which shows broad application prospects in biosensing, photonics, and nanoelectronics. [90,91] The previous paragraph describes the preparation of metal materials inside the cavity of 3D DNA origami. In fact, even earlier, the controllable fabrication of metal materials on the surface of DNA origami has been achieved by chemical deposition, which depended on the accumulation of seed grains in the early stage. [92] Woolley's group has made outstanding contributions to the rapid development of this field. DNA origami containing gold or silver seeds could achieve controllable fabrication of metal nanomaterials with specific morphology by continuous deposition of metal through chemical-coating technology. [92a,93] Woolley et al. reported a method using site-specific attachment of gold nanoparticles to modified staple strands and subsequent metallization to fabricate conductive wires from DNA origami templates. [94] As shown in Figure 14A, DNA-modified gold nanoparticles were attached to "T" DNA origami structure via complementary base-pairing. Then, the attached gold nanoparticles grew by plating Au with the commercial plating solution until gaps between particles were filled and uniform continuous nanowires were formed. The team improved over previous results by using multiple Pd seeding steps to increase seed uniformity and density, further enabling the fabrication of conductive copper nanostructures on DNA origami. [93a] These metal nanostructures will theoretically show great potential in the field of electrically connected small structures. Following earlier work, Woolley et al. explored how to prepare two different metals simultaneously on a single-DNA origami template. [95] Through the Figure 13. A) Programmable 3D DNA origami structures were used to cast gold and silver metal particles with controllable shapes. Reproduced with permission. [50] Copyright 2014, American Association for the Advancement of Science. B) Fabrication of conductive gold nanowires by DNA origami template. Reproduced with permission. [90] Copyright 2019, American Chemical Society. use of an organic layer or chemical mask to prevent unwanted deposition during the metallization, a Cu-Au metal junction was successfully fabricated on a single-DNA origami template ( Figure 14B). In addition, the team has made some progress in recent years by exploring how to use DNA origami templates for anisotropic metallization to obtain metal lines with different morphologies. [96] As shown in Figure 14C, Woolley's group attached DNA-functionalized gold nanorods to DNA origami templates via base-pairing to obtain nanorod-seeded DNA templates. Subsequently, nanorod-seeded DNA templates were introduced to an electroless gold-plating solution to fabricate continuous metal structures of rectangle, square, and T shapes. Although the method is currently limited to the fabrication of simple metal materials such as gold nanorods, it opens the possibility of building complex nanocircuits.

In Situ Chemical Reaction
The introduction of catalytic DNA sequences or functional groups on DNA origami provides a new way for the controllable fabrication of metal materials through in situ chemical reaction. Yan et al. introduced glycosylated DNA sequences onto DNA origami and subsequently synthesized water-soluble fluorescent silver nanoparticles by Tollens reaction. [97] This research realized the controllable fabrication of inorganic metal materials on DNA origami by in situ chemical reaction for the first time. Different from the work of Yan's group, Ding et al. successfully fabricated fluorescent silver nanoparticles on DNA origami by using DNA sequences with specific adsorption capacity for Ag þ . [98] Ag þ was adsorbed by the short DNA strands extended from triangular DNA origami and then reduced by NaBH 4 to generate Ag nanoparticles with fluorescent properties, which were deposited on DNA origami to form specific patterns ( Figure 15A). Three different DNA sequences were used for the fabrication of Ag nanoparticles. The different nucleic acid chains resulted in different sizes of the generated nanoparticles, which showed differences in fluorescence wavelength. The research provided a way for us to understand the spatial effect mechanism of surface chemical reactions by manipulating the spatial position of the backbone to create metal nanomaterials with controllable shape. Fan et al. used a similar strategy to achieve controllable fabrication of copper nanoparticles. [82d] Due to the strong coordination effect between Cu 2þ and protruding clustered DNA, Cu 2þ can be enriched on DNA origami and then reduced to copper nanoparticles under the action of reducing agent to realize the metallization of DNA nanostructures ( Figure 15B). Referring to the previous preparation method of Ag nanoparticles, Ding et al. creatively realized the controllable fabrication of various metals and metal oxides, such as Fe, Co, and Ni, on DNA origami by introducing sulfacyl-modified DNA chains ( Figure 15C). [82c]

Lithographic Method
Lithographic method using DNA origami templates can also be used to fabricate metal materials due to the superior spatial addressability of DNA origami. Single-DNA origami structure can be directed and anchored to the selected areas of lithographically fabricated substrates to form desired patterns. Toppari et al. reported a high-throughput technique for fabricating uniform and tailored metallic nanostructures such as gold, copper, and silver with special patterns on a silicon chip by exploiting the high spatial addressability of the tailored DNA nanostructures. [99] As shown in Figure 16A, a thin silicon dioxide layer was selectively grown on top of the silicon substrate to support the deposited DNA origami shapes. The aforementioned layer was used as a mask for plasma etching of the silicon beneath the opening to form smooth and rounded wells in the silicon, which had the SiO 2 window with the origami-shaped opening on the top. Subsequently, the origami silhouette was used as a mask for depositing metal by evaporation onto the chip. The SiO 2 layer was finally removed by hydrofluoric acid and hydrochloridebased wet etching to form the origami-shaped metallic nanostructures on the silicon chip. The same group further proposed a high-throughput DNA-assisted lithography method that combined the high resolution and structural versatility of DNA origami with the robustness of traditional lithography to fabricate of arbitrary-shaped plasmonic nanostructures with well-defined shapes. [100] As seen from Figure 16B, DNA origami with different shapes was employed as masks to transfer the pattern to metallic nanostructures with designed plasmonic properties. This method facilitated the production of large plasmonic metasurfaces with small feature sizes and opened up completely new avenues for the fabrication of metal nanomaterials. Linko's team developed a highly parallel fabrication method dubbed biotemplated lithography of inorganic nanostructures that enabled large-scale versatile substrate patterning of metallic and semiconducting nanoshapes with various aspect ratios. [101] This work presented high-throughput fabrication of plasmonic (Au and Ag), semiconducting (Ge), and metallic (Al and Ti) nanoparticles on substrates such as indium tin oxide-coated glass and silicon wafers by employing custom DNA origami structures and tobacco mosaic virus as biotemplates for pattern mask formation. In addition to the four methods reported earlier, electrostatic adsorption can also be used for the fabrication of metal materials with specific morphology. Liedl et al. developed a universal  strategy for the generation of arbitrary-shaped metal nanoparticles by a two-step procedure. [102] The 1.4 nm gold clusters were coated with positively charged amines and then bound to negatively charged DNA origami structures to provide seeding sites for the gold cluster growth. Subsequently, the seed coating the DNA origami structures acted as nucleation sites for further metallization by deposition of gold ions (Au þ ) from solution to the Au clusters as metallic gold (Au 0 ). Similarly, Davis and co-workers fabricated DNA origami-templated gold-plated nanowires through electrostatic interactions between hexadecyltrimethyl ammonium bromide (CTAB)-functionalized gold nanorods and DNA origami. [103] Although the strategy of electrostatic adsorption can be used to fabricate metal materials with specific morphology on DNA origami, few studies have been reported due to the lack of relevant research. We believe there will be further research in this area in the future.

Fabrication of Inorganic Nonmetallic Materials on DNA Origami Template
Inorganic nonmetallic materials, as one of the three major materials in parallel with organic polymer materials and metal materials, have the advantages of complex crystal structure, various varieties, and different properties. These materials not only have various preparation methods, but also have a wide range of applications. DNA origami, with its reasonably designed shape and spatial resolution at the nanoscale, can be used as an excellent nanofabrication template. Along this direction, a number of methods have been developed to transfer patterns of DNA origami to a wide range of inorganic nonmetallic materials. In this part, we will focus on four methods for the fabrication of inorganic nonmetallic materials using DNA origami templates, which are mainly divided into the following categories: Figure 16. A) Fabrication of gold nanostructures by lithographic method. Reproduced with permission. [99] Copyright 2015, Royal Society of Chemistry. B) A step-by-step fabrication procedure of metal nanomaterials by lithographic method. Reproduced with permission. [100] Copyright 2018, American Association for the Advancement of Science.

Electrostatic Adsorption
Electrostatic adsorption can be used not only for the fabrication of polymer materials and inorganic metal materials, but also for the fabrication of inorganic nonmetallic materials. In recent years, the technology of biomimetic preparation of inorganic nonmetallic materials by simulating the principle of forming inorganic materials through biological processes has developed rapidly. Under the action of specific templates, new mineral materials with similar morphology and functions have been prepared successively. [104] DNA origami has diverse morphology, controllable size, accurate molecular structure, and programmable characteristics, which can be used for the fine fabrication of inorganic nonmetallic materials. At present, the principle of using DNA origami templates to prepare inorganic nonmetallic materials is mainly to combine inorganic materials with negatively charged phosphate skeleton specifically through electrostatic adsorption, and then form uniform and dense inorganic mineralized layer on DNA origami, so as to realize the controllable preparation of inorganic materials. [105] ssDNA or dsDNA templates have been widely used to regulate the growth of inorganic nonmetallic materials. [104c,106] However, it is still a great challenge to regulate the growth of inorganic nonmetallic materials by using DNA nanostructures. Based on the well-known Stöber method, Fan and co-workers achieved the first preparation of DNA origami silicification (DOS) nanostructures in 2018. [13a,105a] The silicon precursor molecules first formed nanoclusters and then the nanoclusters were specifically combined with the phosphate skeleton of DNA origami through electrostatic adsorption to form DNA-silica hybrid materials ( Figure 17A). The morphology of the hybrid material was regulated by DNA origami template and the mineralization of silica nanomaterial significantly enhanced the mechanical strength of DNA origami template. Jungemann et al. simultaneously realized the silicification of rod-shaped DNA origami by sol-gel reaction. [107] Different from the work of Fan's group, the group stored the rod-shaped DNA origami in buffer solution with low Mg 2þ concentration. By adding the positively charged silicon precursor N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride and another silicon precursor tetraethoxy orthosilicate, finally, a silicon dioxide-mineralized layer was formed on DNA origami. The mineralization of silica significantly enhanced the mechanical properties and thermal stability of DNA origami in solution. Kuzyk et al. continuously explored new ways to fabricate silica by optimizing silicon precursor concentration and finally achieved controllable fabrication of ultrathin silica coating by using 3D DNA origami. [108] In the earlier provided series of work, DNA origami was used to fabricate silica nanomaterials in a controllable manner and the obtained silica materials were evenly covered on DNA origami. However, the nanomaterials prepared by the earlier provided series of methods have lost the addressability of DNA origami, which hindered the further functionalization of DNA origami. To address this problem, Ding et al. developed a new strategy to fabricate silica materials ( Figure 17B), which achieved the controllable preparation of nanopatterned silica materials on DNA origami through the controlled Stöber reaction in an aqueous environment. [109] This strategy not only enabled the precise fabrication of silica, but also preserved the addressability of DNA origami. Since the silicon precursor had a stronger electrostatic affinity for the prominent dsDNA, the precursor would  [109] Copyright 2020, Wiley-VCH GmbH.
www.advancedsciencenews.com www.small-structures.com preferentially interact with the prominent dsDNA and finally form silica nanomaterials with specific patterns on DNA origami. DNA origami templates can be used not only for the fabrication of silica materials, but also for the biomimetic preparation of inorganic mineral calcium phosphate (Ca-P) crystals. [105b] As shown in Figure 18A, Fan et al. successfully achieved controllable preparation of Ca-P crystals with specific morphology on DNA origami by using a method similar to DOS. [105b] DNA origami stored in Ca 2þ buffer solution was deposited on the mica surface and then a certain proportion of phosphate solution was slowly added to the surface. The Ca-P mineralization were formed on DNA origami by electrostatic interaction after maintaining the solution at 37°C for 24 h. By regulating the morphology of DNA origami, this research realized the control of the crystal structure of Ca-P nanocrystals and promoted our further understanding for the fabrication of inorganic nonmetallic materials by DNA origami template through the systematic study of the complex crystallization process of Ca-P nanocrystals. Our group successfully fabricated ultrathin Ca-P nanomaterials on DNA origami by particle attachment strategy. [110] This research provided a bottom-up strategy for fabricating nanomaterials by which arbitrary shapes of Ca-P nanomaterials could be prepared ( Figure 18B) while preserving the structural details of DNA templates to a large extent.
In addition, electrostatic adsorption can also be used to fabricate semiconductor nanomaterials. Woolley et al. fabricated multiple electrically connected metal-semiconductor junctions on individual DNA origami by location-specific binding of gold and tellurium nanorods. [111] Au nanorods were attached to DNA origami via DNA hybridization and Te semiconductor nanomaterials were bound to DNA origami by electrostatic interaction. CTAB directed Te nanorod growth and stabilized the nanorods by providing a cationic surfactant with a net positive surface charge on the nanorods. Subsequently, the gaps between Au and Te nanorods were filled with electroless gold-plating to create nanoscale metal-semiconductor interfaces. This work demonstrated that Au-Te-Au junctions were electrically connected through the characterization of two-point electrical measurement, which opened up potential opportunities in nanoelectronics. The same group further developed a new approach to create DNA origami-templated Au/Te/Au heterostructures with multiple junctions by spin coating a heat-resistive polymer onto the Au/Te/Au structures. [112] In this work, Te nanorods were still deposited onto bar-shaped DNA origami by electrostatic Figure 18. A) Preparation of calcium phosphate inorganic minerals by using DNA origami templates. Reproduced with permission. [105b] Copyright 2020, Cell Press. B) DNA origami-templated biomimetic mineralization via crystallization by a particle attachment strategy. Reproduced with permission. [110] Copyright 2020, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com adsorption. The Au/Te/Au structures were characterized by twopoint probe electrical measurement of current-voltage (IÀV) curves, which proved that the annealing approach successfully connected Au and Te nanorods. Such metallic-semiconductor junctions offered the possibility for the manufacture of complex electronic components such as transistors, logic gates, or integrated circuits.

DNA Hybridization
DNA hybridization provides a possible path for the fabrication of inorganic nonmetallic materials on DNA origami templates. Winfree et al. reported a method for arranging single-walled carbon nanotubes (SWNTs) on 2D DNA origami template by DNA hybridization. [113] As shown in Figure 19A, SWNTs were modified by ssDNA and then aligned along lines of complementary ssDNA on DNA origami to form SWNTs-DNA origami composites. This work demonstrated the possibility of the simultaneous nanoscale positioning and alignment of multiple populations of SWNTs based on the sequence of their DNA linkers by organizing two populations of SWNTs in a single step. Meanwhile, other materials labeled with DNA, such as gold nanocrystals, could theoretically continue to be attached to SWNTs-DNA origami composites, which providing opportunities for the preparation of composite structures with new electronic, optical, or electrochemical properties. Ke and colleagues demonstrated the precise control of DNA origami on the number and location of magnetic iron oxide nanoparticles (IONPs) through DNA hybridization. [114] This research modulated the generation efficiency of MRI contrast agent by varying the number and spacing of IONPs. This work also showed how to modularize the function of IONPs clusters by using this technology and demonstrated the value of this technology in the field of biosensing. Richard et al. achieved controllable self-assembly of semiconductor CdS nanorods on DNA origami by DNA hybridization. [115] The high colloidal stability of CdS nanomaterials and their application in DNA origamimediated self-assembly methods were ensured by functionalizing CdS nanomaterials with oligonucleotide to transfer them toward the aqueous phase ( Figure 19B). This approach also can be used to bind other materials, which potentially allows for self-assembly of nanoelectronic device structures.

Streptavidin-Biotin Interaction
Streptavidin-biotin interaction provided a feasible way for the accurate distribution of inorganic nonmetallic materials on DNA origami. Törmä et al. proposed a method for the assembly of carbon nanotubes (CNTs) on DNA origami templates by taking advantage of simple streptavidin-biotin interaction. [116] Rectangular DNA origami with biotin-modified staple strands was constructed and then streptavidin was anchored to DNA origami templates through streptavidin-biotin interaction. CNTs wrapped with biotin-modified ssDNA were finally immobilized on DNA origami templates in a well-defined geometric shape ( Figure 20A). The simplicity and cost efficiency of the method reported in this work made it a promising tool for accurate Figure 19. A) Schematic diagram of anchoring carbon nanotubes (CNTs) on DNA origami templates by DNA hybridization. Reproduced with permission. [113] Copyright 2010, Springer Nature. B) Assembly of 1D semiconductor rod-shaped cadmium sulfide regulated by DNA origami templates. Reproduced with permission. [115] Copyright 2019, Wiley-VCH GmbH.
www.advancedsciencenews.com www.small-structures.com assembly of devices and circuits at the nanoscale. Belcher's group introduced an approach for cutting SWNTs with predetermined lengths by using DNA origami and G4 hybrid complexes. [117] In this work, biotin-modified DNA-wrapped SWNTs were bound to DNA origami templates by streptavidin-biotin interaction ( Figure 20B). Subsequently, G4-hemin complex activated hydrogen peroxide to produce radical species for CNT cutting. Similarly, Liddle and coworkers successfully anchored streptavidinfunctionalized quantum dots (Qdots) to DNA origami templates by streptavidin-biotin interaction. [118] A rectangular-shaped origami template was used to investigate the influence of binding location separation and steric hindrance on the yield of the Qdot-DNA complex ( Figure 2C). This work not only quantitatively assessed the kinetics of streptavidin-functionalized Qdots binding to biotinylated DNA origami, but also explored to what extent the reaction rate and binding efficiency were controlled by the valence state of binding location, biotin linker length, and the organization, and spacing of the binding location on DNA origami. Wind's group realized, for the first time, the successful organization of Qdots and gold nanoparticles with precisely controlled positions on both sides of DNA origami template by streptavidin-biotin interaction and DNA hybridization. [119] Keller et al. selectively bound streptavidin-coated Qdots to biotin-modified DNA origami nanostructures to evaluate the homogeneity of DNA origami lattice assembly over the entire surface area by imaging more than 450 locations per sample with fluorescence microscopy. [120] 3.

Lithographic Method
Although the lithographic method based on DNA origami template is rarely used in the preparation of polymer and metal nanomaterials, it has made rapid progress in the fabrication of inorganic nonmetallic materials. Due to the easily controlled shapes and nanometer-scale spatial resolution, DNA origami shows promises as a master template for advanced lithography. Through continuous exploration in recent years, Liu's group has made outstanding contributions to the fabrication of inorganic nonmetallic materials by the lithographic method. [121] Liu et al. achieved pattern transfer to SiO 2 by modulating the rate Figure 20. A) Schematic diagram of anchoring CNTs on DNA origami templates by streptavidin-biotin interaction. Reproduced with permission. [116] Copyright 2011, Wiley-VCH GmbH. B) Schematic diagram of regulating the length of single-walled carbon nanotubes (SWNTs) by DNA origami template. Reproduced with permission. [117] Copyright 2018, American Chemical Society. C) Schematic illustration of the fabrication process of quantum dot (Qdot) nanopatterns on DNA origami templates by streptavidin-biotin interaction. Reproduced with permission. [118] Copyright 2012, Wiley-VCH GmbH.
www.advancedsciencenews.com www.small-structures.com of hydrogen fluoride (HF) vapor phase etching and exploiting the difference in adsorption of water between DNA origami and SiO 2 . [121a] In this work, DNA origami template was used to control the etching rate of SiO 2 , resulting in negative or positive tone pattern transfers to the substrate, respectively. Based on the same principle, the same group developed a novel strategy to pattern custom-shaped inorganic oxide nanostructures by using DNA origami templates. [121b] DNA origami was used to modulate the rate of chemical vapor deposition (CVD) of SiO 2 and TiO 2 with nanometer-scale spatial resolution ( Figure 21A). The area-selective CVD strategy provided a hard mask for conventional semiconductor nanofabrication and opened up the possibility to integrate DNA origami technology with conventional nanolithography to create high-resolution patterns. By areaselective atomic layer deposition strategy, they further fabricated Al 2 O 3 , TiO 2 , and HfO 2 metal oxides on both 2D and 3D DNA origami structures deposited on a polystyrene substrate. [121d] Subsequently, the formed DNA-inorganic hybrid could be used as a hard mask to achieve deep etching of a Si wafer for antireflection applications ( Figure 21B). Tiron et al. also promoted the rapid development of the lithographic method by using DNA origami as a mask and further explored the fabrication of inorganic nonmetallic materials. [122] They first investigated the effect of etching time on pattern transfer from DNA origami to SiO 2 layers with resolution less than 10 nm using anhydrating HF steam in a semiconductor etching machine. [122a] The results showed that at the etching rate of 0.2 nm s À1 , the obtained SiO 2 pattern inherited the shape of DNA origami from 30 to 60 s. At 600 s of etching, the SiO 2 pattern encountered corrosion and the entire etching reaction was blocked. Subsequently, the same group reported a fast and simple lithographic method for silicon (Si) nanopatterning. [122b] As shown in Figure 21C, first, DNA origami mask was transferred to the SiO 2 substrate by a high-frequency steam etching process. The Si substrate was then etched using HBr/O 2 plasma. Each hole was transferred in the SiO 2 layer and the 20 nm size hole was transferred in the final stack. Strano and coworkers reported a metallized DNA nanolithography to realize transfer of spatial information to pattern 2D graphene nanomaterials capable of plasma etching. [89] DNA origami treated with glutaraldehyde was seeded with Ag and then coated with Au to metallize them. The metallization ensured DNA origami to survive the subsequent etching process and to function as a positive lithographic gold mask. Thus, the metallized DNA origami template allowed transfer of the spatial information from the metallized DNA origami template to the final etched graphene products after removing the unprotected region of graphene via Ar/O 2 plasma-reactive ion etching. The works reported earlier confirmed the great potential of DNA origami as nanopattern masks on silicon substrates, opening the way for their future integration into micro-electronic compatible, simple, and low-cost lithography processes.

Conclusion
In the past decades, DNA origami technology has made great strides. The construction of DNA origami has gone from the original design to the assembly of 2D, 3D, and even more complex structures. A wide variety of strategies have been developed for assembling DNA origami. Meanwhile, DNA origami has unique advantages in regulating the morphology of nanomaterials due to its excellent programmability and addressing ability, so it is widely used for the fine manufacturing of various nanomaterials, which opens a unique path for the preparation of highprecision nanomaterials.
Although several strategies have been developed to fabricate nanomaterials with specific morphologies by using DNA origami templates, challenges remain as follows: 1) it is worth noting that the fabrication of nanomaterial by DNA origami template is still unable to achieve large-scale production and practical application, which is mainly related to the high cost of DNA origami and the low yield of nanomaterials. Therefore, how to reduce the production cost of DNA origami to achieve the large-scale production and improve the preparation efficiency of nanomaterials has become the key problem to be solved in the next step.
2) Due to the lack of relevant research work, the mechanism of regulating the growth of polymer and inorganic metal materials by DNA origami templates remains unknown and needs to be further explored to establish relevant theoretical systems and provides a theoretical basis for the development of new materials in the future. 3) For some nanomaterials, more complex chemical environments (such as extremely acidic or alkaline conditions) need to be provided to form relevant nanomaterials, while DNA origami needs to be stable under mild conditions. Therefore, limited by the harsh reaction conditions, there are currently limited types of nanomaterials generated using DNA origami templates. 4) Although a certain number of nanomaterials have been achieved by DNA origami templates, the potential application value of some nanomaterials still cannot be explored due to the lack of relevant research. In addition, the influence of the shape and size of nanomaterials on their specific functions is also lacking in theoretical research.
To sum up, the accurate fabrication of nanomaterials by using the addressable properties of DNA origami is still the focus of attention in related areas. The variability of DNA origami and its ability to be precisely customized provide a powerful tool for the high-precision fabrication of nanomaterials, especially in the manufacturing of functional nanomaterials, which show broad application prospects in biomedicine, fine electronics, flexible materials, and other fields. Therefore, combining DNA origami technology with other nanomanufacturing techniques, such as photolithography or chemical catalysis, will yield ideal materials with reasonable structural design from the micro to macro level. As the understanding of structure-function relationships of nanomaterials continues to increase, it is clear that there is a great need to improve the accuracy of nanomaterials for advanced material synthesis. Overall, the development of DNA origami-programmed nanomaterial synthesis is just beginning, and a lot of effort is still needed. We believe that with the continuous development of DNA origami technology, there will be greater breakthroughs in the application of DNA origami in the field of nanomaterials preparation in the future. In addition, due to its good biocompatibility, DNA origami also shows a broad application prospect in the field of drug delivery.