DNA-Based Self-Assembly for Functional Nanomaterials

Transcription

1 DNA-Based Self-Assembly for Functional Nanomaterials Zhen-Gang Wang, and Baoquan Ding * The unprecedented development of DNA nanotechnology has caused DNA self-assembly to attract close attention in many disciplines. In this research news article, the employment of DNA self-assembly in the fields of materials science and nanotechnology is described. DNA self-assembly can be used to prepare bulk-scale hydrogels and 3D macroscopic crystals with nanoscale internal structures, to induce the crystallization of nanoparticles, to template the fabrication of organic conductive nanomaterials, and to act as drug delivery vehicles for therapeutic agents. The properties and functions are fully tunable because of the designability and specificity of DNA assembly. Moreover, because of the intrinsic dynamics, DNA self-assembly can act as a program switch and can efficiently control stimuli responsiveness. We highlight the power of DNA self-assembly in the preparation and function regulation of materials, aiming to motivate future multidisciplinary and interdisciplinary research. Finally, we describe some of the challenges currently faced by DNA assembly that may affect the functional evolution of such materials, and we provide our insights into the future directions of several DNA self-assemblybased nanomaterials. 1. Introduction DNA is undoubtedly one of the most exciting known molecules, having evolved in nature for billions of years. Since the discovery of its double helical structure by Watson and Crick, interest in DNA has expanded beyond its central genetic role to applications in nanotechnology and materials science. The construction of DNA-based nanomaterials is based on the process of self-assembly, which sometimes involves enzymatic assistance. The simplest DNA self-assembly technique is the hybridization of two complementary single-stranded DNA sequences into double-helical strands. DNA hybridization was first proposed as an artificial strategy for complex nanoscale assembly by Seeman, who was the founder of the field of DNA nanotechnology. Double-stranded DNA can be engineered with single-stranded overhangs at the ends (also called sticky ends ) such that hybridization can occur between two double-helical strands to facilitate further assembly. The range of DNA structures was extended using DNA branched junction structures, called Holiday junctions, as building blocks to construct stable four-way junctions. [ 1 ] Thereafter, a variety of DNA crossover Dr. Z. G. Wang, Prof. B. Q. Ding National Center for Nanoscience and Technology Beijing, , PR China dingbq@nanoctr.cn tiles with greater rigidity, such as doublecrossover (DX) tiles, triple-crossover (TX) tiles, and paranemic-crossover (PX) tiles, were created to generate versatile DNA nanostructures, including well-defined periodic or aperiodic 2D lattices and 3D architectures. [ 2 4 ] Scaffolded DNA origami marked a milestone in artificial DNA assembly. In this technique, a long scaffold strand is folded by hundreds of short staple strands into an antiparallel array of helices through the arrangement of crossovers, as demonstrated by Rothemund. [ 5 ] In DNA origami, neither strict stoichiometry nor purification of the oligonucleotides is required, and welldefined shapes can be produced in nearquantitative yields. DNA origami markedly increased the versatility and complexity of DNA assembly and can be used to engineer any arbitrary 2D or 3D pattern, such as a smiley face, [ 5 ] twisted or curved shapes, [ 6 ] or 3D curved surfaces (a nanoflask, sphere, etc.). [ 7 ] Very recently, a method was developed to use hundreds of DNA short strands with distinct sequences, called DNA bricks, for self-assembly into a wide range of predesigned 2D and 3D nanoscale shapes that exhibited interior cavities and tunnels. [ 8 ] DNA-based materials can also serve as a bridge from the microcosm to the macrocosm through the fabrication of nanoscale DNA self-assembling molecules into 3D macroscopically visualized structures. Examples of such materials include hydrogels and crystals, which will be discussed in the following sections. In addition to the intermolecular spontaneous hybridization harnessed for the synthesis of nanomaterials, DNA-based selfassembly is impressive because of its ability to organize the heteroelements in a controlled fashion. This heteroelements organization can be traced back to Seeman's ideas that biomolecules, such as proteins, could be organized in the same orientation in the holes of DNA lattices, so that they could exhibit ordered structures as in a real crystal, allowing for study with X-ray crystallography. [ 9 ] Currently, DNA nanotechnology has grown beyond its initial goal of protein crystallography. Today, DNA serves as excellent scaffolding material for a wide range of applications. Many DNA superstructures, including periodic lattices, DNA origami nanostructures, and discrete 3D architectures have been used to assemble various nanoscale objects such as metal nanoparticles, [ 10 ] quantum dots, [ 11 ] carbon nanotubes, [ 12 ] small molecules, [ 13 ] or proteins, [ 14 ] for optical, biological, or transistor functions. These superstructures also facilitate the templated synthesis of metallic [ 15 ] or conductive-polymer nanomaterials, [ 16 ] 1

2 Figure 1. Several DNA-based hydrogels: A) X-shaped DNA building blocks self-assemble into hydrogels, which can form several patterns and exhibit swelling behaviors. Adapted with permission. [ 22 ] Copyright 2006, Macmillan Publishers Ltd. B) Y-shaped DNA monomers and linkers crosslink by sticky-end (marked by the circles) hybridization to form hydrogels, which show thermal or enzymatic responses depending on the sequence of the sticky region. Reproduced with permission. [ 26 ] C) The reversible volume change of DNA-hybridization-crosslinked polyacrylamide hydrogel, which is regulated by UV and visible light. The inset is light-sensitive azobenzene, which is tethered to the DNA backbone; its isomerization can tailor the stability of the DNA duplex [ 29 ] many of which can be applied in electronics. As a naturally occurring biocompatible molecule, DNA molecules have also been integrated with bioactive molecules, such as folic acid, [ 17 ] antibiotics, [ 18 ] or antibody fragments, [ 19 ] for self-assembly into multifunctional hybrids that act effectively as nanomedical materials. In addition to serving as a structured scaffolding, DNA can behave as a double-helical linker that can be used to organize nanoparticles into an ordered lattice and to realize 3D nanoparticle crystallization with controlled structures. [ 20 ] DNA-based self-assembly provides great opportunities for the synthesis of functional materials that exhibit special properties. Many excellent reviews have addressed the different facets of DNA nanotechnology. [ 21 ] This review discusses the advances in materials science, a field that has fully incorporated both the power of DNA organization and the intriguing functionalities of self-assembled DNA. We concentrate on the designs and emerging properties that are unique to DNA self-assembly. The scope of this review mainly covers hydrogels formed by DNAinduced crosslinking, crystals induced by DNA hybridization, conductive polymer nanomaterials developed from nucleictemplated synthesis, and nanomedical materials based on functionalized DNA assemblies. We provide our insights into preparation strategies and regulation of the functionality of the materials for practical applications. 2. DNA-based Hydrogels Hydrogels are three-dimensional crosslinked networks of hydrophilic polymeric chains. They have received much attention in the biomedical field for use as drug delivery vehicles or tissue-engineering scaffolds. Hydrogels have also found wide application in chemical separation and analysis, and are used in the fabrication of contact lenses and microfluidic devices. Using DNA nanotechnology to form DNA molecules into self-assembled hydrogels has several advantages. These include i) the flexibility of DNA hybridization, which provides a rich library of building blocks for hydrogels, ii) programmable crosslinking through sequence design, iii) stimuli-responsive DNA conformations that bring about switchable behaviors, and, iv) the technique of nucleic base modification and controlled association/dissociation of targets, which is capable of introducing multiple functionalities into the hydrogels. DNA self-assembly can be used for hydrogels in two ways, either the bottom-up construction of hydrogels from the assembly of branched DNA monomers or the crosslinking of polymeric chains (e.g., polyacrylamide) by DNA hybridization or DNA-substrate complexing. In the bottom-up approach, DNA monomers have been engineered with palindromic sticky ends, which self-associate to crosslink monomers into large-scale, three-dimensional structures. [ 21j ] Luo and coworkers reported the use of several DNA monomers, which were X-, Y-, and T-shaped, [ 22 ] to form bulk-scale hydrogels through sticky-end hybridization and enzymatic ligation. The enzymatic ligation acted as the crosslinker, as shown in Figure 1 A. The hydrogel properties, such as the swelling behavior, mechanics, size, and shape, were finely tuned based on the initial monomer concentration and type. These physical and chemical factors could also controll the drug (e.g., insulin, camptothecin) release properties through tuning the degradability of the hydrogels. The hydrogels were capable of encapsulating a variety of nanoscale components and molecules during gelation, protecting the bioactive elements [ 22 ] and increasing the localized concentration. These factors resulted in the efficient production of several protein species through the enhancement of the transcription of the hydrogel-incorporated free genes (linear plasmids) into mrna. [ 23 ] DNA hydrogels have also been engineered with shape-memory features; the liquid-to-solid transition could return the hydrogels to their original shapes. [ 24 ] This newly identified feature of hydrogels that were prepared by non-covalent weaving of DNA chains elongated by polymerase reaction was correlated to the birdnested internal structures and thought to originate from the ultralow elastic modulus of the DNA hydrogel. Such shapememory DNA hydrogels have been used to fabricate a switchable hydrogel-based electric circuit; [ 24 ] in this case, water acted 2

3 as the stimulus that switched the hydrogel shapes, formed by the counteractive forces applied to the bulk materials. In terms of their mechanical behaviors, DNA hydrogels can be triggered by a variety of stimuli because of the inherent dynamics of the sticky ends. A common driving force is temperature because all types of sticky ends can be thermally melted. Liu and coworkers introduced the i-motif sequence with a ph-sensitive conformation to the sticky ends of the monomers so that acidifying the solution would transform the fluidic solution into a gel and vice versa. [ 25 ] In another study, the restriction site was incorporated into the sticky-end duplex, and the hydrogel became digestible by specific restriction enzymes, indicating enzymatic responsiveness. [ 26 ] The thermal and enzymatic responses of the hydrogels are shown in Figure 1 B. In the crosslinking approach, only a small portion of DNA is required for the DNA polymer hybrid hydrogels, in which self-assembled DNA acts as both the crosslinks and switchable elements for the acquired response. The current hybrid hydrogels chiefly use polyacrylamide, and DNA is incorporated into the chains by the copolymerization of acrydite-modified DNA with acrylamide. Nagahara and Matsuda reported the first DNA polyacrylamide hydrogel, prepared by the hybridization of DNA strands grafted on polyacrylamide chains. [ 27 ] The DNA-induced crosslinking was reversible by changing the temperature [ 27 ] and was triggered by many competitive recognition events, which resulted in versatile functionalities. Tan and coworkers engineered the complementary region with an aptamer sequence that could recognize adenosine or thrombin with a higher affinity than the duplex. [ 28 ] The addition of adenosine or thrombin was able to separate the side-chains of the polyacrylamide, resulting in the gel-to-sol transition. Incorporating photoisomerizing molecules, such as azobenzene, into the crosslinking regions was able to bring about photoswitchable swelling behaviors in the hybrid gels, [ 29 ] as shown in Figure 1 C. Briefly, one type of polyacrylamide chain was permanently crosslinked into the hydrogels and contained ssdna strands. The other type bore the azobenzene-modified complementary strands and was entrapped in the hydrogel network. A lightwavelength switch induced the isomerization of azobenzene and thus the stability of the DNA duplex, resulting in the controlled change of the macroscopic volume. Various mechanisms, such as DNAzyme-catalyzed cleavage [ 30 ] and strand displacement, [ 31 ] have also been reported for crosslinking dissociation. The hybrid hydrogels responsively trapped and released quantum dots, [ 32 ] gold nanoparticles, [ 28, 33 ] and enzyme molecules. [ 33 ] Furthermore, the target-responsive release was used for visual detection because the released gold nanoparticles could be monitored spectroscopically or the released enzyme molecules could catalyze the transformation of the substrates into colored products. [ 33 ] The bulk scales and the transparency of the hybrid hydrogels can be employed for sensitive and specific visual detection. Liu and coworkers reported a DNA polyacrylamide hydrogel in which the polyacrylamide chains bore DNA strands rich in thymine bases that were used to specifically bind Hg 2 + ions. [ 34 ] Upon recognizing the Hg 2 +, the DNA strands folded into hairpin structures and emitted green fluorescence upon binding SYBR green I dye. The strong absorption of acrylamide to Hg 2 + was used to remove Hg 2 + efficiently from environmental water and allowed regeneration for recycled use. A similar strategy was applied for the label-free fluorescent detection of Pb 2 + ions [ 35 ] and the colorimetric detection of target DNA. [ 36 ] As alternatives to polyacrylamide, graphene, [ 37 ] SWCNT, [ 38 ] and polypeptides [ 39 ] have also been used in the formation of hybrid hydrogels with DNA self-assembly as the crosslinkers. A responsive gel-to-sol transition was realized, [ 38 ] and interesting properties were exhibited, such as a self-healing capability. [ 37 ] 3. DNA-based Crystals A crystal structure is defined as the periodic arrangement of atoms (or ions or molecules) in a particular way, which coincides with the initial goal of structural DNA nanotechnology to produce designed periodic matter. [ 40 ] The appearance of 3D DNA crystal materials represented a landmark in periodic DNA self-assembly, which relies on the use of a robust 3D motif. [ 41 ] The 3D crystal was composed of a rigid tensegrity triangle consisting of three double cohesive helical domains that pointed in independent directions, as shown in Figure 2A. Assembling the helices with six other molecules in six directions yielded a 3D periodic rhombohedral lattice that diffracted X-rays to 4 Å resolution. The size of the crystal materials could reach at least 250 μ m, in comparison with only a few micrometers for 2D lattices. Such 3D nucleic crystals have the potential to be used as scaffold biomolecules for crystallographic structure determination and the organization of nanoelectronics. Instead of a single species of tensegrity triangle per unit cell, Seeman s group further used two different tensegrity triangles to crystallize an alternating motif, [ 42 ] with the crystals showing different colors through the attachment of dyes. In addition to the tensegrity triangle, three-way junctions were used to grow 3D crystal lattices through the recognition of helical coordination compounds. [ 43 ] The production of DNA crystals is not where this story ends. The ordered organization of other molecules in two or three dimensions was enabled, such as 2D DNA protein crystals [ 44 ] and 3D colloidal crystal structures. [ 45 ] Gold nanoparticles, serving as building blocks, have been studied extensively for 3D nanoparticle crystallization, driven by the programmable recognition and hybridization between DNA strands. This was first reported concurrently by Mirkin s group [ 20 ] and Gang s group. [ 46 ] It was revealed that the polyvalent nanoparticle crystals grow via three key steps, including: i) an initial random binding phase, ii) localized reorganization, and, iii) growth of the crystalline domain size. [ 47 ] Unquestionably, DNA-assembly factors have played very significant roles in the control of crystal structures. The Mirkin group found that either the single- or binary-component assembly system, depending on the choice of the DNA sequences conjugated to the nanoparticle building blocks, could generate close-packed face-centered-cubic or non-close-packed body-centered structures [ 20 ] (Figure 2 B). The flexibility of the DNA linkers, which was dependent on the length of the uncomplementary region, and the aspect ratio between the effective radii of the binary nanoparticles were used to enhance the stability of the crystal structures, producing well-defined structures. In parallel studies, Gang and coworkers demonstrated that greater linker flexibility 3

4 Figure 2. Several DNA-based crystalline materials: A) A tensegrity triangle hybridizes with six other molecules in six different directions to form 3D visible crystals with a rhombohedral shape. Reproduced with permission. [ 41 ] Copyright 2009, Macmillan Publishers Ltd. B) a programmed assembly of gold nanoparticle DNA conjugates into different crystallographic arrangements nanoparticles capped by one DNA sequence (green) assemble into a face-centered cube, and, in contrast, a binary DNA sequence (blue and red) yields a body-centered cube; the corresponding SAXS pattern is shown at right. Reproduced with permission. [ 20 ] Copyright 2008, Macmillan Publishers Ltd. C) switchable interparticle spacing of DNA-linked nanoparticles crystals triggered by the addition of DNA strands, based on reversible strand displacement and hybridization. Adapted with permission. [ 59 ] Copyright 2010, Macmillan Publishers Ltd. and increased numbers of linkers per nanoparticle could result in spontaneous crystalline organization with enhanced degrees of long-range order. [ 46, 48 ] The enhancement of long-range order caused the material to transform from the amorphous state to an ordered structure. [ 46, 48 ] Additionally, the number of linkers per nanoparticle was able to control the onset of system crystallization. [ 49 ] The linker-length effect on the crystallization was further studied by Mirkin s group and was found to control the interparticle spacing (one of the unit cell parameters) through the length of the complementary region of the linkers. [ 50 ] Essentially, the effect of DNA linkage was related to the fine-tuning of the interparticle attraction and repulsion energy. To fully engineer the lattice using DNA, Mirkin and coworkers presented a design strategy that provided a set of rules for addressing the independent thermodynamic and kinetic control of the crystal assembly. [ 51 ] The nanoparticle sizes, interparticle spacing, and crystallographic symmetries were independently controlled by the design rules, resulting in a wide range of crystal types. [ 51, 52 ] In addition to spherical gold nanoparticles, the 3D crystallization of anisotropic building blocks was induced by DNA self-assembly, forming five distinct symmetries. [ 53 ] Other crystal building blocks include AuNPs/protein particles that self-assemble into NaTI-type crystalline structures, [ 54 ] AuNPs/ spherical nucleic acid (SNA; spherical structures with a layer of densely packed, highly oriented nucleic acids) binary lattices within which the SNA acts as a 3D spacer replacing some of the nanoparticles, [ 55 ] AuNPs/quantum-dot heterogeneous superlattices with AuNPs positioned at the corners of the unit cell, [ 56 ] and SNA binary crystals in which the structure types can be tailored according to the theoretical simulation. [ 57 ] This indicates the great potential of extending the programmability of the crystal systems through the nanoparticle shape or species to facilitate the synthesis of novel crystalline structures. The above-mentioned crystals were all produced in aqueous solution for stability; Mirkin and coworkers encapsulated the superlattices in silica, realizing the stable existence of crystal structures while in the solid state. [ 58 ] This observation may improve the resistance of these materials with respect to harsh environments. The nanoparticle crystals not only exhibited static tenability but also showed mechanical behaviors based on the stimuliresponsiveness of DNA self-assembly, which could result in dynamic interparticle distances. The most common situation is the temperature-induced dehybridization and rehybridization 4

5 of duplex DNA, which allows reversible crystallization of the nanoparticles during heating and cooling cycles. [ 46 ] Additionally, the lattice structure could be tuned by temperature. [ 46 ] The stimuli species used to modulate the unit cell parameters are dependent on the design of the interparticle DNA linkage. Gang and coworkers used DNA strands to switch the interparticle spacings through reversible strand displacement and hybridization (Figure 2 C)). [ 59 ] They also altered the ionic strength to change the persistence strength of ssdna, leading to dynamic tuning of the fluorescence response of the chromophore-containing superlattices. [ 60 ] Although not yet reported, it is expected that the response of the DNA-induced crystal structures can be triggered by a wide range of stimuli, such as light, metal ions, and ph. 4. Conductive Polymers Based on DNA As noted in the introduction, DNA self-assembled structures act as important templates. This is not only true for the organization of nanoscale elements but also in the templated synthesis of polymers, especially that of unnatural macro molecules. [ 61 ] DNA can direct sequence-defined polymerization behavior, [ 61, 62 ] such as the synthesis of various nucleic acids (e.g., DNA, peptide nucleic acid, and altritol nucleic acid) through DNAtemplated amplification and template-hybridization-induced oligomolecule conjugation. Additionally, the negative charges from the phosphodiester backbone of the DNA template provide requisite counterions for the synthesis of conductive polymers with preferred molecular structures. Moreover, the versatility of the DNA nanostructures enables the formation of conductive polymers with well-defined morphologies, which grow around the anionic double-helical strands. The intrinsic charges of DNA also explain why conductive polymers can be self-doped in slightly acidic solutions. The self-doping property of the conductive polymers indicates that such soft templates require no depletion from the complex compared with hard templates. Currently, 1D nanowires of conductive polymers, such as polyaniline, polypyrrole, or their derivatives, have primarily been prepared by using duplex templates. Samuelson and coworkers explored the enzymatic synthesis of polyaniline in the presence of calf thymus DNA, in which horseradish peroxidase was used as the catalyst and H 2 O 2 as the oxidant. [ 63 ] At a ph lower than the p K a of aniline, the emeraldine form of polyaniline was generated on the negatively charged template, and polyaniline showed reversible redox behavior when doped and dedoped with acid and base. Preferred handedness was observed for polyaniline, revealing the tight DNA polyaniline interaction that transferred chirality from the duplex to the polyaniline. Manning's theory described the counterion condensation phenomenon, indicating that the surface ph of a negatively charged polyelectrolyte was lower than that of the environment. [ 64 ] This factor causes the aniline monomers to align preferentially and promotes a para-directed reaction, while leading to the extended conjugation of the resulting polyaniline chains with limited parasitic branching. The enzymatic approach offers a high degree of control over the kinetics of the reaction, making it potentially possible to fabricate the product with a high yield. [ 65 ] Subsequently, polyaniline nanowires were synthesized successfully on calf thymus DNA templates, immobilized, and stretched across the treated Si surface. [ 16 ] Based on natural DNA, conductive polypyrrole, [ 66 ] polyindole, [ 67 ] and polythiophene derivative [ 68 ] nanomaterials were also synthesized by chemical oxidization. The natural DNA assembly-templated formation of conductive nanowires paved the way for the DNA self-assemblydirected synthesis of conductive polymers, which may enrich the functionalities and versatility of the conductive nanomaterials. Schuster and coworkers reported a strategy to prepare aniline oligomers in which one strand of the self-assembled duplex included the controlled number of contiguous anilinemodified cytosines. [ 69 ] As the amount of modified cytosine increased, the spectral characteristic of the emeraldine form became stronger, indicating the well-defined number of aniline groups in the oligomers, the DNA-doping behavior, and the transfer of sequence programmability. A similar strategy was used for the preparation of 4-aminobiphenyl [ 70 ] and 2,5-bis(2- thienyl)pyrrole [ 71 ] oligomer nanowires. The same group also attached 2,5-bis(2-thienyl)pyrrole to the DNA modules, which self-assembled to form closed-cycle or linear arrays of aligned conductive monomers. [ 72 ] As a result, cyclic or linear conductive polymers were synthesized by enzymatic oxidization and exhibited the chemical and optical properties of thiophene-like polymers. Despite the intimate interaction between DNA and aniline monomers, which favors the para -coupling of polyaniline, the chemical modification of cytosine requires complicated procedures relative to the enzymatic oxidization of aniline adsorbed on the DNA template. To achieve the efficient and simple templated synthesis of the para -coupling polyaniline nanomaterials, Wang, Ding and coworkers developed a method that associated the self-assembled DNA template with a natural enzyme-mimicking DNA sequence. [ 73 ] The artificial enzyme interacted efficiently with the template, so that the enzyme-generated aniline radicals could diffuse to the charged surface quickly, facilitating the formation of the para-directed polyaniline. The molecular structure and doping/dedoping behaviors exhibited dependence on the charge density of the template, which was affected by the template configuration. Moreover, the polyaniline was found to grow preferentially around the enzyme site, expanding over the overall template surface, which indicated the site-selective growth of the conductive polymers, as shown in Figure 3 A. This provided the potential for the fabrication of complex polyaniline patterns on 2D or 3D template surfaces, with the rational arrangement of the enzyme sites. DNA has brought novel electrical properties, such as Schottky emission-dominated conduction and a rectification effect, [ 74 ] and novel functions, such as employment in supercapacitors, because of the porous pattern of the nanomaterials, which favored ion diffusion. [ 68 ] Gao and coworkers demonstrated the unique application of the nucleic-templated formation of polyaniline in the detection of nucleic acids, including DNA [ 75 ] and microrna. [ 76 ] The nucleic capture probes were immobilized on the electrodes, and upon hybridization with the target, the polyaniline was enzymatically catalyzed to deposit on the probetarget self-assemblies, as shown in Figure 3 B. The conductance was directly correlated with the number of target nucleic acid molecules, allowing ultrasensitive electrical sensing. 5

6 Figure 3. Two examples of DNA self-assembly-templated synthesis of conductive polyaniline: A) the catalytic synthesis of para -coupling polyaniline by the HRP (horseradish peroxidase)-mimicking DNAzymes assembled on the linear template and the site-selective growth of polyaniline; time-dependent AFM images recording the growth of polyaniline are presented at the bottom (red indicates the DNA and white, the polyaniline). Reproduced with permission. [ 73 ] Copyright 2013, American Chemical Society. B) HRP catalyzes the synthesis of conductive polyaniline due to the assembly of HRPconjugated DNA strands to the electrode surface through hybridization to the target sequence; the electroactivity of the deposited polyaniline allows ultrasensitive electrochemical quantifi cation of the target nucleic acids. [ 75 ] 5. DNA-based Biomedical Materials The development of efficient medical approaches is a scientific issue that is related directly to human health. DNA is a naturally existing biopolymer, exhibiting excellent biocompatibility within the cellular environment, which is a prerequisite for medical materials. Several features enable DNA self-assembled materials to be highly promising for medical applications, including i) the sequence specificity of DNA, the capability of encapsulating molecular cargos, and the stimuli-triggered release of cargo, which offer the possibility of ultrasensitive detection, drug loading, and targeted delivery; [ 77 ] ii) the ease of DNA modification, which can enhance the targeting effect and imaging applications of DNA; [ 21e, 78 ] iii) the controllability of DNA self-assembly, including the controlled structural parameters and morphology programmability, which may facilitate versatile biological functions; and, iv) the structural resistance of the DNA-based rigid self-assemblies to cellular degradation, such as 3D nanoarchitecture and DNA origami, intended for applications in vivo. [ 79 ] These DNA construct features meet the important criteria (stable, tolerated, nontoxic) required by molecular carrier systems in mammals. [ 80 ] For use in medical applications, self-assembled DNA nanostructures are usually explored as potential delivery vehicles and are applied for cancer-cell killing. Li and coworkers appended unmethylated CpG motifs to DNA tetrahedrons, which efficiently entered macrophage-like RAW264.7 cells without the aid of transfection agents. [ 81 ] Unmethylated CpG oligodeoxynucleotides are immune stimulants that stimulate the immune system by inducing activation or increasing the activity of any of its components. Therefore, such DNA tetrahedra were recognized by Toll-like receptor 9 to induce immunostimulatory effects, which produced the high-level secretion of various proinflammatory cytokines, including tumor necrosis factors; a similar immune response in spleen cells was also triggered by CpG-bearing DNA origami. [ 80 ] CpG motifs were also integrated with Y-shaped DNA, [ 82 ] DNA-based dendrimers [ 83 ] and DNA hydrogels [ 84 ] to improve the activity of Toll-like receptor 9. Anderson and coworkers designed a DNA tetrahedron bearing cancer-targeting folate molecules to deliver small interfering 6

7 Figure 4. DNA self-assemblies for targeted delivery and therapeutic applications: A) targeted sirna delivery using DNA nanostructures as the carrier the middle is the tumor-specifi c accumulation of nanostructures, imaged by the 3D FMT-CT in vivo, and the right depicts the dose-response accumulation of nanostructures in KB tumors, imaged by in vivo fluorescence. Adapted with permission. [ 85 ] Copyright 2012, Macmillan Publishers Ltd. B) DNA origami triangles and tubes intercalated with doxorubicin (Dox) (left) the drug-loaded origami shows enhanced cytotoxicity to the Doxresistant cancer cells compared with free Dox and the Dox-double stranded plasmid DNA complex (middle); resistant cells treated with Dox origami nanostructures showed the co-localization of drug and nuclei, as imaged by confocal microscopy (right). Dox-loaded DNA origami structures are more effective at killing resistant cells. Reproduced with permission. [ 18 ] Copyright 2012, American Chemical Society. RNA (sirna) molecules into cells and silence the target genes in tumors. [ 85 ] Figure 4 A presents the multifunctional tetrahedron and its accumulation in vivo. The optimal delivery of the sirna into cells required at least three folate molecules on each nanostructure, and only the appropriate spatial orientation of the folate resulted in the occurrence of gene silencing. A longer blood circulation time of the tetrahedra in comparison to the parent sirna was indicated in vivo. These intracellular delivery studies established the basis for the chemotherapeutic applications of DNA-based self-assembled structures, which are capable of binding anticancer drugs, bearing targeting groups, and offering structural designability and stimuli response. These properties enable the drug to be released in a controlled manner. Through such a targeting strategy, the drugs can be readily delivered to the pathological area, improving the therapeutic effects while minimizing the side effects that originate from the random diffusion of drugs. Huang and coworkers developed aptamer-conjugated DNA icosahedra, which acted as a nanocarrier for doxorubicin intercalation. [ 86 ] The aptamer sequence is able to recognize MUC1, an important class of tumor surface marker, so that aptamerconjugated doxorubicin-intercalated DNA icosahedra showed an efficient and specific internalization for killing epithelial cancer cells. Compared with small-sized self-assembled nanoconstructs, DNA origami can load a high amount of anticancer drugs and can bear multiple targeting groups, and the shapes are programmable. These advantages not only enable the drug release kinetics to be controlled by adjusting the structural parameters, such as the twisting degree, [ 87 ] but also facilitate the logic-gated delivery of molecular payloads to inhibit the growth of target cells [ 19 ] in which the delivery system was activated by cooperative recognition events between aptamers and cellexpressed protein markers. Ding's group observed a remarkable reversal in the drug resistance of cancer cells, induced by doxorubicin-loaded DNA 2D and 3D origami, due to the increased cellular internalization of loaded doxorubicin, [ 18 ] as shown in Figure 4 B. It was also found that the cytotoxicity to resistant cells was related to the effective inhibition of lysosomal acidification by the delivery vehicles, resulting in the cellular redistribution of the drug to active sites. This result suggests that DNA-based nanocarriers have the potential to circumvent drug resistance, which remains a major clinical obstacle and is of great interest for medical research and commercialization. To clarify how the origami delivered the anticancer drugs, Ding s group implemented a further study to visualize the intracellular location and stability of the DNA origami in live cells with the help of carbazole-based cyanine dye, which is sensitive to DNA conformation; [ 88 ] it was found that most DNA nanostructures were maintained and localized in the lysosome after 12 h of incubation, whereas most were dissociated after an additional 60 h of culture time, which may help elucidate the process of intracellular drug delivery. Luo s group incorporated CpG motifs and doxorubicin into DNA-based hydrogel and demonstrated the synergistic cytotoxicity of these two bioactive components. [ 84 ] This indicated the effective inhibition of tumor growth by the 7

8 immunostimulatory CpG motif-mediated production of antitumor cytokines. Tan and coworkers reported the photocontrollable drug delivery from a DNA-crosslinked polyacrylamide hydrogel for wavelength-guided cancer killing. In this hydrogel, the stability of the DNA duplex was sensitive to the photoisomerizing azobenzene groups. [ 89 ] The photoresponsive DNA also acted as a dynamic gate-keeper of mesoporous silica pores, and a light switch was used to uncap the pores to release the anticancer drug. [ 90 ] 6. Concluding Remarks We have reviewed the major progress that has been made in the use of DNA self-assembly to produce functional materials, such as hydrogels, crystal materials, conductive polymer materials, and biomedical materials. DNA self-assembly is quite useful for preparing bulk-scale materials that exhibit unusual physical properties. DNA self-assembled structures can also act as key elements for controlling the functions of heterogeneous materials, can serve as a template for the synthesis of novel nanomaterials from traditional molecules, and can be used to incorporate functional components to trigger important events. It should be noted that the properties of assembled macroscopically visualized materials rely on nanoscale internal structures composed of DNA molecules or motifs and can be tuned by the DNA nanostructures, indicating the connection of the molecular structures to the macroscopic world. These exciting achievements show that interest in DNA has expanded far beyond its role in genetics. DNA is now being used to bridge the gaps between different fields. It is also evident that there is much room for improvement. The stability of DNA selfassembly is a considerable challenge, particularly with respect to DNA's resistance to harsh environments or organic solvents, which may limit the applications of DNA-based hydrogels and DNA-induced nanoparticle crystals. Such a challenge is also faced in the DNA self-assembly templated-synthesis of conductive materials. For example, the synthesis of para-coupled polyaniline usually requires a low ph, which could damage the hydrogen bonding in the DNA duplex, especially in complex DNA self-assemblies, such as origami. This restriction limits the DNA-directed formation of nanoscale conductive patterns. The second challenge involves size enlargement and controllability, which could hinder the complexity of the scaffolding organization or synthesis. Achieving high-yield fabrication, reducing cost, and producing new DNA-based hybrid molecules with various functions remain as challenges for DNA self-assembly-based nanomaterials. From the standpoint of functions, many studies are still in the very preliminary stage. Further investigations are necessary in many fields, including the following areas: producing heteroelement lattices using 3D DNA crystals, enhancing the switchable behaviors, clarifying the mechanism of accumulating drug carriers in the lysosome, and elevating the targeting specificity and the anti-interference ability in vivo of the drug vehicles. Further development of DNA self-assembly techniques and the rational design of assembly units with desired properties will guide the improvement of DNA assembly-based functional materials. With its variety of unique features, DNA self-assembly will continue to contribute significantly to the development of materials science and may open up new avenues of function regulation for multidisciplinary and interdisciplinary research. Acknowledgements The authors are grateful for fi nancial support from the National Science Foundation China ( , , , , ), the National Basic Research Program of China (973 Program, 2012CB934000), the100-talent Program of the Chinese Academy of Sciences (B.Q.D), and the Beijing Natural Science Foundation ( ). Received: March 31, 2013 Revised: April 15, 2013 Published online: [1 ] N. C. Seeman, Nature 2003, 421, 427. [2 ] a) E. Winfree, F. R. Liu, L. A. Wenzler, N. C. Seeman, Nature 1998, 394, 539 ; b) J. C. Mitchell, J. R. Harris, J. Malo, J. Bath, A. J. Turberfi eld, J. Am. Chem. Soc. 2004, 126, ; c) M. Endo, N. C. Seeman, T. Majima, Angew. Chem., Int. Ed. 2005, 44, [3 ] a) T. H. LaBean, H. Yan, J. Kopatsch, F. R. Liu, E. Winfree, J. H. Reif, N. C. Seeman, J. Am. Chem. Soc. 2000, 122, 1848 ; b) D. G. Liu, S. H. Park, J. H. Reif, T. H. Labean, Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 717. [4 ] a) W. Y. Liu, X. Wang, T. Wang, R. J. Sha, N. C. Seeman, Nano Lett. 2008, 8, 317 ; b) Y. 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