ДНК-нанотехнологии 1 введение и основные методы / acs.chemrev.0c00294

PCR to prepare stable and condensed gene nanoparticles with multiple target gene cassettes together, which acted as transcription vectors for e cient protein expression and gene delivery in living cells.192 In 2016, Finke et al. used branched DNA that covalently linked with a cyclic peptide by click chemistry as primers of branched PCR to generate a new functional DNA-peptide network for specific cell attachment and culture.88

Psoralen was capable of stabilizing the branched DNA nanostructures. Upon UV irradiation, the compound could intercalate into the T bases and locked the double helical regions, thus enhancing the thermal stability of DNA constructions.193 Using this organic molecule, Hartman et al. developed thermostable branched DNA as modular primers for PCR, which remained intact under denaturing conditions such as high temperature and denaturing bu er (Figure 8).87 In

Figure 8. Enzymatic extension for the construction of branched DNA with ultralong arms. (A) Scheme of thermostable branched DNA as PCR primers. (B) Modular primers labeled with multiple fluorophores (left); gel electrophoresis demonstrating multiplexed PCR products with di erent combinations of FAM (green) and Cy5 (red) fluorophores. (C) Extension of Y-DNA by branched PCR (left); AFM images of branched elongated PCR products (right). Adapted with permission from ref 87. Copyright 2013, Wiley-VCH.

detail, the branched DNA contained a Y-shaped region and a single-stranded primer region. After treatment with psoralen, a high proportion of cross-linking sites formed in the doublestranded core region, which led the Y-shaped branch remain chemical stability at temperatures even higher than 95 °C. Meanwhile, the single stranded primer region remained its biological activity as a substrate for DNA polymerase (Figure 8A). By using the thermostable branched DNA as primers, branched PCR products were obtained in the process of PCR. The anisotropy branched DNA primers were modified with multiple fluorophores, resulting in a series of PCR products with specific colors (Figure 8B). Furthermore, when sticky ends of thermostable Y-shaped DNA were all designed as primer regions, elongated branched DNA products were obtained through PCR (Figure 8C). The chemically cross-linked branched DNA took advantages of both the thermostable assembly and intrinsic biological capabilities in a simple PCR process, and this technology created a versatile platform to

produce multifunctional DNA materials for a variety of applications.194 By using the branched PCR, Yang group constructed branched gene architectures as membrane-free gene compartment for studying proximity e ect in cell-free protein expression system (Figure 9).195 In single branched gene architecture, three transcription units were integrated by branched PCR, which enabled the RNA polymerase to shuttle more e ciently between neighbored units than free units (Figure 9A). The branched gene system increased local concentrations of DNA and RNA polymerase, resulting in improved gene expression yield with increasing concentration of transcription units and crowding agents (Figure 9B). The branched gene platform provided a strategy for studying the e ect of cellular crowed microenvironment on gene transcript and translation.

3.CONSTRUCTION OF ARTIFICIAL BRANCHED DNA-BASED FUNCTIONAL MATERIALS

Great progress in DNA nanotechnology has demonstrated that artificial branched DNA monomers as building-blocks exhibit the tremendous values in the fabrication of an assortment of functional DNA materials. According to the assembly principle, the methods for the construction of branched DNA-based materials were classified as tile-mediated assembly, DNA origami, dynamic assembly, and hybrid assembly.

3.1. Tile-Mediated Assembly

Tile-mediated assembly relied on branched DNA monomers as tile motifs that connected into high-level structures through the bottom-up hybridization of single-stranded overhangs. In tilemediated assembly, the structural and mechanical diversity of di erent branched DNA monomers were suitable for di erent types of structures. Branched DNA with multihelix as an arm, multipoint-star motif and DNA tensegrity triangle, had the characteristic of intermolecular tensegrity and fixed interbranch angle; consequently, they were more likely to be considered as tiles for the assembly of well-defined shape of wireframe structures. Wireframe structures containing hollow grid of geometric primitives were presented with the skillful combination of the flexible ssDNA as vertices and rigid dsDNA regions,196−198 including DNA arrays, polyhedrons and crystals. These highly ordered wireframe structures have shown merit as fundamental templates for the precise arrangement of nanoparticles and biomacromolecules due to predictable and addressable sites provided. Compared to the two branched DNA monomers, branched DNA with single-helix arm as tiles had relative flexibility and was amenable to the preparation of irregular DNA materials such as DNA dendrimers and DNA hydrogels.

Modular assembly as a representation of tile-mediated assembly opened up new avenues to implement the design and construction of 2D DNA arrays from a family of multipointstar branched DNA.199,200 The periodic DNA arrays with di erent lattice patterns was attributed to the precisely designed branched DNA with diverse rotational symmetry, for example, 2D hexagonal, quadrangular, tetragonal, and triangular DNA arrays were constructed from three-point-star, four-point-star,

five-point-star, and six-point-star branched DNA, respectively (Figure 10A).58,201−203 The central loop was designed to

prevent the base stacking, and the sticky ends could be designed as palindromic sequences or symmetry−asymmetry pairs for end elongation and simplified operation;201 meanwhile, the interconnected junctions assembled from two branched DNA

forced the DNA duplex to keep on a plane. In addition, to ensure the plane structures extended from multipoint-star branched DNA, a corrugation strategy was applied that neighboring units was linked by two-folded rotation axis (face-to-opposite sides of a plane) to eliminate the deviation of dihedral angle, determining the resulting plane structures.58 Note that the use of corrugation strategy was not amenable to all kinds of branched DNA to construct 2D arrays, which was in close correlation to the rotational symmetry. When rotational symmetry was 6-fold, which was not compatible with twofolded rotational symmetry, the neighboring units needed to face the same side to cancel existing curvatures.203 Interestingly, the subtle balance of conformational flexibility and stress, namely fine-tuning tensegrity, greatly influenced the stability, rigidity, and geometric structures of obtained assemblies, which were precisely adjusted by controlling the length of loop, sticky ends, and rigidity joints, as well as the duplex turnover number and interhelical angle.204−209 The bottom-up modular selfassembly was also amenable to the fabrication of DNA-RNA

hybrids branched motifs as programmable building-blocks and further 2D DNA arrays (Figure 10B).210 The fine and highly

ordered DNA arrays provided versatile platforms for the multiplexed and sensitive diagnostics of biomolecules, and information coding.211−213 Recently, a sca old-free “LEGO” approach to construct wireframe DNA arrays entirely from short strands was developed.214 In this approach, branched motifs were designed as two parallel duplexes with intersection at vertices instead of traditional intersection in the middle of duplexes, and three or four edges were diverged from a vertex. These subtle designs contributed to the formation of lattice-free wireframe DNA structures, including 2D arrays with honeycomb, rhombic, T-shaped, and cross-shaped patterns (Figure 10C). Furthermore, the flexible conformation could be achieved by adjusting the joint angels through the incorporation of ssDNA or dsDNA linkers. The sca old-free approach was also

employed to build fully addressable wireframe DNA architectures composed entirely of single-stranded tiles such as 2D arrays with hexagonal, chimeric, and cobweb-like tessellation patterns.72 These supramolecular 2D arrays could be covalently linked by photoactive anthracene dimerization to construct 2D polymers in solution, which exhibited solvent responsivity and structural transitions owing to the amphiphilic nature of organic molecules.185

Additionally, surface-mediated self-assembly that was first proposed in 2009 was considered as an alternative to assembly in solution (Figure 11A).215 As a demonstration, the 2D DNA arrays were synthesized directly in situ on the surface of mica. During the self-assembly process, temperature as the essentially important parameter, needed to be controlled within the appropriate range, which required a comprehensive consideration of the balancing states of individual tiles and the entire array. The selection of appropriate temperature facilitated to the interaction of DNA tiles to mica surface and e ciently circumvent kinetic barrier.216 Compared with solution assembly, surface-mediated assembly showed advantages including eliminating the process from sample preparation to characterization and preventing the formation of 3D aggregations. It was noteworthy that surface-mediated assembly provided the opportunities in the building of irregular 2D arrays by the heterogeneous assembly of discrepant branched DNA motifs. These branched DNA motifs were designed with di erent finetuned interbranch angel and precisely regulated length of multibranches, and followed by depositing on solid surface to form irregular DNA arrays via blunt-end stacking (Figure 11B).

Numerous attempts have been made to demonstrate that tilemediated assembly has great potential in building custom 3D DNA polyhedrons from branched DNA, in particularly multipoint-star branched DNA.125,218 Because the entirety of a vertex and adjacent edges in DNA polyhedron could be regarded as branched DNA monomer, DNA polyhedron was the

connectome of dendritic DNA by advantage of hierarchical assembly concept. The diversities of branched DNA in sequences, length, angles, and directions made them have a probability to assemble polyhedrons with multiple combinations (Figure 12) including same polyhedron from di erent types of motifs, individual polyhedron from copies of identical branched DNA, and single polyhedron from coassembly of multiple branched DNA.219,220 Typically, the branched points of branched DNA served as vertexes interconnected by sticky ends on edges, resulting in well-defined DNA polyhedron. The length and curvatures of DNA duplex needed to be adjusted to satisfy the requirement that all component branched DNA faced toward the same direction to form polyhedrons instead of planar structures. Meanwhile, lower branched DNA concentrations and more flexibility of loops by increasing bases number were

both prone to the formation of polyhedron.60 Assembled morphologies of DNA polyhedrons were tightly in association with the concentrations, flexibilities, and rigidities of the corresponding branched DNA monomers.202,221 By slightly changing the length of central loop, the relative inclination between the center and tile plane could be controlled, eventually forming the isomeric DNA polyhedron with inward or outward bending.222 The formation of chiral DNA structures also could be achieved by removing the rotation symmetry of DNA motifs on purpose.126 In addition, exterior modifications, such as hairpin, could be decorated at the addressable sites on DNA polyhedron for easily introducing appendage groups (i.e., component proteins).223 As an alternative of traditionally used sticky ends, T-junctions could also play a role in connection with neighborhood branched DNA, facilitating the formation of

DNA polyhedrons with hairpins or aptamers for wider applications.224

Additionally, a strategy of hierarchical assembly was exploited to construct fully addressable DNA arrays. In hierarchical subtile strategy, branched DNA tiles were first self-assembled from tweezer-like subtiles with flexible loop in two steps, and then assembled into a family of programmable superstructures in a more facile and controllable step, which provided steric crowding and reduced the undesired pairing in one-pot method.226 In addition, a stepwise hierarchical assembly was developed to construct DNA arrays with finite boundary (Figure 13A), which addressed the issue that spatial positioning was inaccurate in DNA array without preset boundaries. The stepwise hierarchical assembly began with the branched DNA tiles as monomers linked by sticky ends, and preformed smallsized lattices and subsequent enlarged arrays in sequential steps. The obtained DNA arrays as molecular pegboard allowed for the specific locations of biomolecules with nanoscale accuracy (Figure 13B).229 There were two extreme cases in hierarchical self-assembly: the minimum number of assembly units and the minimum number of assembly steps (also called depth). Taking a 4 (row) × 4 (column) DNA lattice as an example, eight sequence sets each consisting of two motifs, and four sequential steps were required in the case of minimum number of assembly units. When the case of minimum depth was applied, a sequence set containing 16 motifs with unique sticky ends and one-step equimolar mixing were needed. The hierarchical assembly technique reliably controlled each step of assembly at moleculelevel, so that arbitrary finite-size DNA arrays were guaranteed to obtained.226 The production yield was increased by the compromise between two extreme cases. In addition, the incorporation of dsDNA nanobridges joining adjacent tiles was used for regulating the length and directionality of DNA arrays (Figure 13B).230 The fully addressable and finite-size DNA

arrays allowed the accurate positioning of specific biomolecules in nanosized space.229

Contrary to the assembly order of traditional hierarchical assembly, inverse hierarchical assembly performed with joint wires as beginning modules to form branched junctions (Figure 13C).231 The number of composition chains involved in inverse hierarchical assembly was significantly less than that in hierarchical assembly; moreover, the strategy of inverse hierarchical assembly was conducive to the formation of ultrabranched junction with centrosymmetric topology. In addition, to prove the universality of the inverse strategy in constructing complex DNA nanostructures, two kinds of connection wires were in the attempt designed at first, including half double-stranded connection wires and double-stranded connection wires. Following the inverse hierarchical assembly, stable zero dimensional (0D), one-dimensional (1D), 2D, and 3D DNA arrays could be assembled by the ingenious combination of two kinds of connection wires (Figure 13D).

Inspired by converse thinking, retrosynthetic strategy was first reported by Krishnan group for the nanofabrication of DNA icosahedrons.63 Retrosynthetic strategy was by definition to start with the desired assembly, which was divided into basic modules in the top-down principle. These modules as building units could assemble into expected nanostructures in a predesigned manner. The construction strategy minimized the trial and error process, greatly shortening the experimental period. Taking DNA icosahedron as an example, it was split into two halficosahedra on the basis of retrosynthetic strategy, and subsequently the half-icosahedra was divided into two kinds of five-armed branched DNA in a molar ratio of 1:5. The programmable overhangs on two half-icosahedra were complementary to each other to fabricate an integrated icosahedron (Figure 14A). The obtained DNA icosahedron had a large volume of cavity and a dense surface, which thus had great potential as a “smart” nanocapsule in the encapsulation and

controlled release of cargoes, especially biomacromolecules possessing relatively large molecular size.232 For example, a kind of fluorescent biopolymer as model cargoes was encapsulated into the nanocapsules during nanofabrication. Moreover, responsive modules enabled the controlled opening of nanocapsules in the presence of corresponding external triggers, finally generating fluorescence signals with enhanced intensity.233 This platform based on icosahedral DNA nanocapsules was available to study the release kinetics of signal conducting molecules under chemical trigger. In addition, by combining with other functional components, such as target modules, quantum dots, and endocytic ligands, multifunctionalized DNA nanocapsules enabled to accomplish the targeted delivery, stimuli-triggered cargo release with spatiotemporal precision, spatial pH mapping of endosomal maturation, and real-time tracking of endocytic pathways.234−237 These researches showed enormous values of “smart” DNA icosahedra-based nanocapsules in the territory of biomedicine.

Another advantage of retrosynthetic strategy was the greater tolerance for the symmetry of branched DNA, resulting in a more flexible configuration of branched DNA. The proposal of

retrosynthetic strategy broke intrinsic constraint of using highly symmetric branched DNA in conventionally used approaches. Wang et al. made use of the strategy to guide the assembly of complex DNA polyhedrons (Figure 14B).128 Three types of DNA motifs with di erent combinations of helical number were designed including branches all with odd-numbered helixes, branches all with even-numbered helixes, and chimeric branches with oddor even-numbered helixes. Accordingly, four rules for the assembly based on asymmetry tiles were proposed: (1) oddnumbered helixes as edges intended to pattern corrugated assemblies instead of closed rings, while edges containing evennumbered helixes promoted cyclization; (2) central loops extension contributed to the out-of-plane flexibility of DNA motifs with high degree; (3) the loop length showed a reverse correlation with interbranch angle; that is, the long loop favored a small angle between flanking branches; (4) higher concentrations of DNA motifs preferred to larger size of DNA structures. These fundamental rules were expected to be promising guidelines for the synthesis of complex DNA polyhedrons from various branched DNA.

Under the guidance of tile-mediated assembly, the rigid and well-structured branched DNA as building units could construct macroscopic 3D DNA crystals, breaking through the limitation of the nanoscale. Zheng et al. constructed macroscopic DNA crystals from DNA tensegrity triangle as building units.62 The tensegrity triangle consisted of three helical domains with three separated branch points, resulting in sti and periodic 3D crystals when extending from six directions (Figure 15A). Both the constitution of the building units and the interunit interactions were adjustable: the units could be functionalized by a triplex-forming oligonucleotide on each of its helical edges;111 the interunit interactions within the 3D DNA crystals could be enhanced by introducing a triplex-forming oligonucleotide at the interunit cohesion region.129 The weak lattice interactions and high concentrations were decisive factors for achieving the crystallization, while sample heterogeneity and impurities played a minor role.238 Besides, the crystallization process responded to temperature, which allowed the control of the system.239 The self-assembled crystal could in turn help to determine the structure of the unit such as incorporated stressed DNA within the crystal lattice.240 Wang et al. demonstrated the feasibility of using sticky-end directed self-assembly to control

the contents of a macromolecular 3D crystal.241 Two di erent triangle molecules were designed, and the only intermolecular interactions between the molecules were programmed with sticky ends; as the asymmetric units, the triangle molecules alternated through the lattice within the crystal (Figure 15B). In addition, the covalent attachment of dye molecules to the DNA units resulted in di erent colors of the crystals. Energy transfer in the network of dye-attached DNA crystal could be studied.242 Great attempt have been made to control the crystallization kinetics and improve the quality of engineered 3D DNA crystals. The e ect of varying sticky end lengths and sequences as well as the impact of 5′- and 3′-phosphates on the improvement of crystal formation and resolution were investigated.243,244 Another e ective strategy was introducing a rationally designed agent to modulate the crystallization process, by which means, larger crystals with higher resolution were produced.245 To overcome the constraint of stability of DNA crystals, an approach to prepare robust 3D DNA crystals by postassembly ligation was recently developed (Figure 15C).246 Sticky ends of DNA fragments at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability (Figure 15D).

More and more e orts have been made to create new types of branched DNA for constructing DNA crystals in tile-mediated assembly. The layered-crossover motif, which has been previously used to construct 3D framework DNA origami structures, was implemented to construct a set of rhombus-like layered-crossover DNA tiles with precisely tunable orientation angles, which further assembled into 3D crystals with dimensions up to hundreds of micrometers (Figure 15E).247 Simmons et al. demonstrated the structure of a new crystal system inspired by DNA tensegrity triangle. The motif consisted of three component strands self-assembled into designed crystalline arrays for each form, with a layered lattice mediated via a central strand that contained a sequence of 4 sequential repeats of 6 bases (Figure 15F).248 The left-handed enantiomer of this motif was crystallized to obtain two self-assembling DNA crystal systems. They further designed and crystallized the first example of a 3D DNA array with a layered hexagonal lattice (Figure 15G).249 Their approach provided a new avenue to create self-assembled macromolecular 3D crystalline lattices with atomic precision, which contained a highly organized array of well-defined cavities.

By adapting multistage hierarchical assembly, branched DNA with single-helix as an arm was used to construct DNA dendrimers. Generally, the structure of DNA dendrimers could be divided into three regions: the branched core, the repetitive units, and the end groups.162,250 Branched DNA monomers could act not only as the branched core but also as repetitive units to generate multiple layered DNA dendrimers.251,252 In general, the resulting DNA dendrimers had high synthetic productivity and excellent monodispersity, whose controllable sizes were regulated by monomers types and

concentrations, facilitating to size-dependent endocytosis. Relative to branched DNA monomers, DNA dendrimers possessed numerous prominent advantages such as larger surface area, more binding sites, and improved robustness in cellular microenvironment. In addition, the multiple end groups endowed DNA dendrimers with inherent properties of multivalency, modification, and functionality.253,254 These incomparable advantages made it widely applied in biomedical fields including multiplexed detection of biomolecules, drug delivery, and therapeutics.255

Relying on the complementary pairing, Li et al. achieved the controlled assembly of multigeneration DL-DNA structures with Y-shaped DNA as the branched core and repetitive units (Figure 16A).57 The sticky ends on Y-DNA were specially designed as nonpalindromic sequences to avoid self-ligation. And the design of sticky ends of Y-DNA followed the principles of “intra-layer consistency, inter-layer di erence”, to ensure the ordered assembly of DNA dendrimers in multistage hierarchical assembly. The resulting DL-DNA was obtained through repeatedly ligating peripheral Y-DNA to the sticky ends of the previous generation. The resulting dendrimer DNA showed stable and monodisperse structures, and di erent di usion behavior relative to linear DNA structures.94 The multiplex and anisotropic DL-DNA were developed as a fluorescent nano- barcodes-based platform for the multiplex detection such as pathogens DNA, ribonucleic acid (RNA), and protein.251 The size of DL-DNA structures was tunable not only by controlling the number of generations but also by introducing adjustable modules in response to corresponding stimulus. In addition, the introduction of pH-responsive modules (i.e., i-motif) into DNA dendrimers in a stepwise assembly caused DNA dendrimers to

possess the characteristics of stimulus responsiveness and sizeadjustability (Figure 16B).97 Under weak acidic environment (pH 5.0), the i-motif conformation changed from linear to fourstranded structures, leading to a reduction in size. In particular, DNA dendrimers could e ectively protect single-stranded nucleic acid from degradation, which usually occurs in the cellular or plasma environments. Therefore, DNA dendrimers could be used as highly e cient and intracellular delivery nanocarriers of single-stranded nucleic acid.

Tile-mediated assembly has drawn great attentions as an outstanding strategy not only in the construction of nanoor microscale materials but also at macroscale, typically DNA hydrogels. The multidirection extension of DNA strands from branched DNA was used for the formation of cross-linked network in hydrogels. Meanwhile, the tunable rigidity and flexibly of branched DNA dominated the mechanical strength of DNA hydrogel, accompanied with shape plasticity or shape adaptability. DNA hydrogels have exhibited unique advantages such as mimicking extracellular matrix (ECM) environment and possessing appropriate mechanical strength; DNA hydrogels

therefore had prospective biological applications including sca olds for tissue engineering, wound dressing, 3D cell culture, capture and delivery, and protein production.256−263 Implementing enzymatic reactions was a general approach to synthesize branched DNA-based hydrogels,264 and enzymefree self-assembly was also adopted.265

The pure DNA hydrogel was first demonstrated by Um et al. based on branched DNA via enzymatic reactions (Figure 17A).59 Branched DNA termed as X-, Y-, and T-shaped branched DNA (namely X-, Y-, and T-DNA, respectively) hybridized and ligated with each other through covalent linkages among palindromic sticky ends, serving as both monomers and cross-linkers. In addition, the mechanical property of DNA hydrogels was controlled by the initial concentration and types of branched DNA monomers. Remarkably, the swollen DNA hydrogel constructed from X-DNA monomers showed better resistance to degradation and long-term drug release. The swollen degree and phase behaviors of DNA hydrogel were influenced by binding energy or hybridization strength, including the unpaired bases located in the sticky ends of X-

DNA monomers, salt ion types, and temperature.266 Production of sticky ends could be achieved by enzymatic treatment, mainly by terminal deoxynucleotidyl transferase (TdT), which was capable to extend the poly(A) or poly(T) tails.267 Taking advantage of the hybridization of poly(A) and poly(T), branched DNA were interconnected into DNA hydrogel with high mechanical strength. Branched DNA integrated with forward and reverse primers was utilized to create ultralong chains in networks and further hydrogels implemented by the amplification of branched PCR, which encapsulated fluorescence protein gene and was useful in some biotechnological applications such as protein production (Figure 17B).87 By encapsulating lipid on the surface of enzyme-linked DNA

network with gene-knitted X-DNA as a monomer, a shell−core structure was prepared, which was speculated to be useful for in vitro protein production.268 By introducing conductive carbon nanotubes (CNTs) and polyaniline coating into the enzymelinked DNA hydrogel template, the functional DNA hybrid hydrogel was constructed as a supercapacitor with ultralow cytotoxicity.269

In addition to employing enzyme-assisted ligation to connect branched DNA together, enzyme-free self-assembly was also used to construct branched DNA-based hydrogels. As a tile, branched DNA was networked merely by DNA linkers to form hydrogels in tile-mediated assembly. On the one hand, enzymefree and tile-mediated assembly relying on hydrogen bonds

showed irreplaceable advantages in reversible dynamic assembly, evading the extremely stable performance of covalent bonds in enzyme-assisted ligation. This enzyme-free hydrogel was more conducive to regulating its thermodynamic properties.122,270 Meanwhile, the design of branched DNA as pieces of mosaic tiles facilitated the overall dissociation of hydrogels and the incorporation of functional elements. Some responsive elements embedded into sticky ends of branched DNA endowed the adjustable mechanical strength and repetitive gel-to-sol transition of hydrogels under the action of corresponding stimulus. For example, the hybridization of aptamer and complementary sequences induced the disintegration of DNA hydrogel with aptamer functionalized.271,272 A photoresponsive base had the capability of controlled disassembly of hydrogel under UV irradiation.273 The characteristic of this dynamic controlled disassembly, coupled with excellent biocompatibility, made DNA hydrogel suitable for in situ encapsulation and controlled release of proteins, while maintaining their activities.274,275 On the other hand, the enzyme-free process was advantageous to the adjustment of size of hydrogel, from macroscale to nanoscale, in a more controlled fashion, such as adding blocking chains. Regulating the concentration ration of branched DNA tiles, DNA linkers, and blocking chains

e ectively controlled the size of nanohydrogel. The nanosized DNA hydrogels occupied a dominant position in biological applications, and have drawn ever-increasing attention of researchers to develop new preparation strategies. In 2015, Tan and co-workers prepared the size-controllable and stimuliresponsive nanohydrogels based on the DNA self-assembly of Y- shaped DNA as monomers and dsDNA as linkers (Figure 18A).66 One Y-shaped monomer contained three sticky ends, while the other Y-shaped monomer contained a sticky end for extension, a blunt end and an aptamer for blocking. The size of DNA nanohydrogels was controlled by varying the ratio of two kinds of Y-shaped monomers (Figure 18B). The incorporation of other functional components, such as aptamers, therapeutic agents, and disulfide linkages, endowed the nanohydrogel with multiple performances, including the specific targeting mediated by aptamers and stimuli-responsive degradation to reductive microenvironment (i.e., endogenous glutathione) that was provided by disulfide linkages. In addition, by conjugating therapeutic antisense DNA and DNAzyme oligonucleotides, the DNA nanohydrogels showed e ective inhibition of cell proliferation and migration. Some nucleoside analogues, such as floxuridine (F) similar to thymidine (T) and gemcitabine (Ge) similar to deoxycytidine (dC), could be easily integrated