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

selective recognition and specific binding to cancer cells, as well as improved internalization e ciency.451−454 Combined with the high loading capacity of branched DNA, branched DNA/ aptamer-based nanomaterials with anticancer drugs promoted cytotoxic e ect against target cancer cells, and less side e ects on nontarget cells.455,456 Bi et al. presented a novel branched DNAbased nanosphere (NS) as drug delivery nanocarriers.457 The constituent units containing three ssDNA generated NS with high molecular weight under the action of connector DNA and dense packing. The NS owned several appealing advantages: uniform size, excellent monodispersity, dense compaction, and multifunctional overhangs. The incorporation of functional arm, such as aptamer, endowed NS with targeted delivery with high payload e cacy of anticancer drug. Chang et al. fabricated aptamer conjugated-DNA icosahedron for drug delivery and cancer therapy.458 The DNA icosahedron was composed of sixbranched DNA, with aptamers linked and Dox intercalated. The resulting DNA icosahedron showed e cient cell internalization and antitumor e cacy.

4.3.2. Gene Delivery. The e cient delivery of native nucleic acid in organism was always an enormous challenge due to nuclease degradation and multiple physiological barriers such as vascular barrier, reticulo-endothelial system uptake, and immune system removal.459 The construction of vectors based on DNA nanostructures can improve nuclease resistance and transfection e ciency.460−463 Wang and Mao groups collaborated to report a series of siRNA/miRNA-loaded delivery systems constructed from three-point-star branched DNA.464−466 The extended strands were integrated into three- point-star branched DNA for loading siRNA or miRNA, so that the three-point-star branched DNA protected these RNAs from being degraded by nuclease in complicated medium relative to naked RNA. The three-point-star branched DNA was internalized into cells via micropinocytosis and clathrinmediated endocytosis pathways, showing enhanced transfection e ciency and interference e ects of targeting mRNA and protein. The siRNA/miRNA-loaded branched DNA could also self-assembled to DNA nanotubes, which caused significant

autophagy and inhibition of cell growth.466,467 In addition, they introduced spermidine into the DNA nanostructure for isothermal and Mg2+-free self-assembly.468 The spermidineDNA complex showed better thermal stability and nuclease resistance than Mg2+-assembled DNA nanostructure. Meanwhile, the complex e ciently delivered into cancer cells via clatherin-mediated endocytosis, which exhibited higher cellular uptake and gene knockdown e cacy as well as excellent antitumor e ect. Nahar et al. incorporated multiple antimiRNAs into branched DNA nanostructures by self-assembly for downregulating oncogenic miRNAs (oncomiRNAs).469 The branched DNA nanostructures improved the resistance to nuclease of antimiRNAs in serum. The antimiRNAs selectively bound to corresponding oncomiRNAs, synergistically knocking out these miRNAs and restoring protein levels.

In general, most conventional materials for drug or gene delivery were isotropic, and thus di cult to combine di erent drugs or genes. Branched DNA with anisotropic structure and biocompatibility provided a promising solution for multiplexed drug or gene delivery. The branched DNA monomers were integrated into a modular system with “plug-and-play” feature where di erent therapeutic nucleic acids were conjugated onto peripheral DNA branches with desired ratios.74 Meanwhile, the multigene nanocarriers with size adjustability could e ectively deliver the loaded goods (including antisense oligonucleotide, mRNA, siRNA, miRNA), and be internalized into tumor cells by enhanced permeability and retention (EPR) e ect and endocytic pathway.470−472 Luo group developed a kind of multiplexed delivery vehicle named DNAsome.473 The DNAsome was a liposome-like structure assembled from amphiphilic subunits of Y-DNA building-blocks, whose size and surface charge could be precisely tuned by altering the concentration of Y-DNA amphiphiles. These Y-DNA buildingblocks contained three di erent moieties: a lipid molecule for self-assembly to form DNA-lipid amphiphilic structure, a DNA sequence for specific siRNA loading and a fluorescent dye for tracing. In addition, DNAsome was loaded with Dox, therefore successfully achieving codelivery of drugs and siRNA, and

exhibited higher potency compared to the simple mixture of individual drugs due to the synergistic e ects. The endocytosis mechanisms on DNAsome delivery to mammalian cells were explored by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression after gene knockdown, revealing that the endocytosis pathway was primarily mediated by caveolae and actin. These DNA-based materials could be further developed as e ective vectors for drug and gene codelivery by incorporating additional target moieties as well as future biomedical applications.474

In 2015, Liu et al. constructed condensed gene nanoparticles through the cross-linking process of reconstructed target gene and flexible branched DNA.475 The gene was manipulated by DNA restriction endonuclease to obtain the desired sticky ends for self-assembled hybridization. The resulting nanoparticle-like structures showed enhanced stability against enzyme degradation. In addition, these condensed gene nanoparticles acted as cross-linked gene cargos for in vivo gene delivery and excellent target protein expression in living cells (Figure 43A). This construction approach o ered a universal strategy for target gene delivery. Tan group constructed the conjugation of aptamer-based DNA dendrimers and viral vector (AAV2) for high-e cient gene (eGFP) delivery.253 The outer layer of DNA dendrimers was chemically hybridized with sgc8 aptamers to generate multivalent G-sgc8, followed by the linkage of AAV2 capsid through the modification with disulfide-containing crosslinker dithiobis (succinimidyl propionate) (DSP) (Figure 43B). The resulting G-apt-AAV2 vector conjugates not only protected sgc8 aptamers from degradation by nuclease but also specifically released AAV2 with the action of intracellular reductive environment. Owing to the existence of multivalent aptamers, the G-apt-AAV2 vectors showed high-specific recognition ability and an enhanced e ciency of gene transduction.

4.4.Therapeutics

4.4.1.Chemo-Gene Therapy. As excellent codelivery vehicles of chemotherapeutic drugs and therapeutic nucleic acids, branched DNA-based functional materials have shown prominent advantages, such as size adjustability, targeted

delivery, excellent endocytosis, and responsive release in the treatment of cancer and other diseases.476

Tan group constructed a multifunctional DNA nanoassembly with targeting aptamers and therapeutic oligonucleotides for the

treatment of certain types of cancer (Figure 44A).477 The aptamer-based DNA nanoassembly was formed by the combination of prehybridized branched DNA with acrydite modification through photo-cross-linking. The incorporation of di erent functional elements made the DNA nanoassembly a promising and instructive system for e ective cancer treatment including aptamers capable of specific recognizing target cancer cell, therapeutic oligonucleotides, and chemotherapeutic drugs capable of suppressing the expression of specific protein and proliferation of tumor cells. On the basis of DNA nanoassembly system, Tan group further introduced stimulus-responsive elements in the size-controllable and stimuli-responsive DNA nanohydrogels for combined gene therapy (Figure 44B).66 The DNA nanohydrogels were assembled from Y-shaped monomers A with DNAzyme, Y-shaped monomers B with aptamer, and dsDNA linking units with antisense DNA. In comparison with DNA nanoassembly system, the formation of DNA nanohydrogel depended on the self-assembly of complementary bases without photo initiation. Intramolecular disulfide linkages were cleaved by reductive GSH, thereby releasing therapeutic genes into the cytoplasm of cancer cells. DNAzyme and antisense DNA had the capacity of inhibiting cell proliferation and migration, and their synergism had an improved therapeutic e ect on target cancer cells.

Zhang et al. designed a smart nanocarrier composed of stimuli-responsive Y-DNA motifs, gold nanorods and thermosensitive polymers for codelivery of siRNA and Dox.478 The Y- DNA motifs acted as vehicles to coload of siRNA and Dox, and then disassociated for controlled release of siRNA and Dox with the trigger of endogenous miRNA. To amplify the low abundance of intracellular miRNA, they introduced ATP aptamers into Y-DNA motifs and took use of toehold-mediated strand displacement reaction to achieve the recycling of miRNA. In addition, two thermosensitive polymers underwent reversible hydrophilic−hydrophobic transition at di erent LCST to expose arginine-glycine-aspartic acid (RGD) and Y-DNA motifs in a successive manner. Gold nanorods were used to heat the surroundings to the LCST under near-infrared (NIR) irradiation. The exposed RGD targeted integrins overexpressed on the surface of tumor cells, and the exposed Y-DNA motifs released siRNA and Dox for the synergistic induction of cell apoptosis and enhanced therapy e cacies. Park and co-workers also used the photothermal e ect of gold nanomaterials to

disassemble X-DNA-based nanohydrogel and release interca-

lated drugs, realizing the light-responsive and synergistic cancer therapy.479,480

Platinum-based analogs, as a common clinical chemo-drug, have been widely employed in the treatment of cancer. However, the severe side-e ects and systemic toxicity caused by the nontargeting of platinum-based drugs needed to be urgently addressed. With the aid of the noncovalent interaction of platinum drugs and DNA base pairs, Wu et al. developed a platinum drug-intercalated programmable DNA nanoplatform as a delivery carrier.476 The platinum drug (56MESS) was intercalated into double-bundle DNA tetrahedron, which was modified a nanobody for selective target of epidermal growth factor receptor (EGFR) that was overexpressed on tumor cells. The nanobody-conjugated DNA nanocarrier exhibited specific recognition of tumor cells and low systemic toxicity in the delivery of platinum drug. Ma et al. reported a telomeraseresponsive DNA icosahedron for controlled release of platinum nanodrug to overcome cisplatin resistance.481 After platinum nanoparticles (PtNPs) nanodrugs were encapsulated, the DNA icosahedron was self-assembled, and the connectors were designed as the telomerase recognition primer and telomerase repeat sequences. Human telomerase, abnormally expressed in most cancer cells, had the functions of increasing the number of telomere repeats and maintaining the continuous proliferation of cancer cells. Because of the extremely low activity of telomerase in normal cells, it can be a specific stimulus to selectively activate the response in tumor cells instead of normal cells. The telomerase primer extended under the action of telomerase, resulting in the dissociation of DNA icosahedron and the burst release of encapsulated platinum nanodrugs. The

selective release avoided the systemic toxicity and alleviated cisplatin resistant caused by drug e ux.

Other than common chemotherapeutic drugs, nucleoside analogue therapeutics floxuridine (F) was easier to incorporate into DNA nanostructures owing to the structural similarity with natural nucleoside thymidine (T). Zhang group integrated F into a series of well-defined DNA polyhedral nanostructures through solid-phase synthesis, which was functioned as the Trojan Horse to deliver therapeutic agents (Figure 45A).482 Compared with DNA tetrahedra and dodecahedra, the F- containing DNA buckyball exhibited enhanced antitumor performance according to in vivo experiments. Moreover, the body weight of tumor-bearing mice treated with F-containing DNA buckyball demonstrated that the DNA Trojan horses possessed relatively low side e ects (Figure 45B). They further incorporated F into self-assembled Y-shaped motifs and formed DNA nanogels with F-containing Y-DNA as repetitive units.276 The DNA nanogels had uniform size and good stability in 10% fetal bovine serum. As delivery vehicles, the DNA nanogels showed great cellular uptake behaviors, DNase-activated drug release and enhanced cellular apoptosis. Similarly, they introduced another nucleotide analogs-gemcitabine (Ge) as model chemodrug into DNA nanogel to construct a pHresponsive drug delivery system.277 The DNA nanogel was selfassembled from Y-shaped with Ge-rich sticky ends. The Ge-rich sticky ends hybridized with complementary sequences at neutral or basic environment, while converted into intramolecular i- motif-like structures at acidic environment. The Ge-containing nanogel realized the intracellular drug delivery and DNasepromoted release.

Recently, clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas)-based genome editing tools have drawn widespread attentions because of the ability of facile, precise and highly e cient gene knockout.483 Zhu et al. reported a kind of protein-sca olded Y-DNA conjugation for Cas9/single guide RNA (sgRNA) loading and DNAzyme-controlled release.484 Two strands of Y- DNA were ligated with a DNAzyme component and Cas9/ sgRNA complex respectively, and the third one of which was functionalized with biotin for immobilization on SA. The substrate strand of DNAzyme was designed as a DNA-RNA chimeric strand, which could be cleaved to release Cas9/sgRNA complex when DNAzyme was activated in the presence of cofactor Mn2+. The released Cas9/sgRNA complex was transported to nucleus by the assistance of nuclear localization sequence (NLS), leading to the e cient gene editing. Liu et al. constructed a branched DNA-based platform for codelivery of Cas9/sgRNA gene editing tool and antisense for targeting DNA in the nucleus and mRNA in the cytoplasm, respectively, resulting in synergistic tumor therapy (Figure 46).89 The branched DNA with seven arms was fabricated by azidemodified β-cyclodextrin and diphenylcyclooctyne (DBCO)- conjugated DNA through copper-free click chemistry. To enhance the transfection e ciency of the branched-DNA nanoassembly, adamantine (Ad)-functionalized aptamer for targeted delivery and Ad-functionalized influenza hemagglutinin (HA) peptide for endosomal escape were separately incorporated into branched DNA by host−guest interaction. In addition, 3′ terminal extended sgRNA in Cas9/sgRNA complex was linked with reductant-responsive DNA linkers containing

antisense oligonucleotide, followed by which DNA linkers was used to connect two types of branched DNA. The resultant sgRNA/Cas9/antisense coassembly released sgRNA/Cas9 complex and antisense with the action of intracellular GSH and RNase H digestion, ultimately leading to simultaneous gene editing and gene silencing and remarkable antitumor e ect (Figure 46). The fluorescent intensity and tumor volume obviously decreased in a MCF7-eGFP tumor-bearing and MCF7 tumor-bearing mice, respectively, after the treatment of corresponding Cas9/sgRNA complex. At the same time, the relative mRNA levels of targeting gene evaluated by RT-qPCR analysis occurred potent downregulation. The excellent antitumor e ects were attributed to the synergistic gene editing and gene silencing therapy.

4.4.2. Immunotherapy. By incorporating special motifs with immunomodulatory activity, functional materials could elicit specific immunological responses for therapy. The e ectivity of immunotherapy depended largely on the dose of the immunomodulatory sequences, and increasing the dose of agent in a single carrier was an urgent problem to be solved. To address this issue, Nishikawa group has made lots of outstanding contributions by advantage of branched DNA-based nanostructures for immunostimulatory and immunotherapy. They first demonstrated that Y-shaped DNA containing potent cytosine- phosphate-guanosine (CpG) motifs significantly increased the secretion of cytokines from macrophage cells (RAW264.7) compared to conventional dsDNA and ssDNA, mainly due to the higher uptake of Y-shaped DNA by macrophage cells.485,486 They further prepared multibranched DNA nanostructures containing CpG motifs by increasing the arm number, and

investigated their immunological activities.64 The secretion amount of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from RAW264.7 cells greatly increased with the increasing of arm numbers.487 These results proved that the immunological properties of these multibranched DNA were closely related to their intrinsic structures, of which branchedlike DNA with six or more arms had a better biodegradability and higher immunostimulatory activity. Inspired by the above findings, Nishikawa group successfully took advantage of DLDNA to further enhance immune-potency.488 They designed Y-

DNA as building-blocks to form DL-DNA containing potent CpG motifs, which specifically promoted the cellular uptake, activated murine immune cell secretion of various cytokines and promoted the production of immunoglobulin. The highly branched structures significantly increased CpG dosage and showed much stronger immunostimulatory activity than individual Y-DNA with the same motif. Yang et al. also validated that Y-DNA with multiple cross-linking sites could trigger the higher immune activation in dendritic cells and macrophages.489 These results indicated that the branched DNA structure

contributed to designing enhanced immunostimulatory drugs. In 2015, Nishikawa group reported that DNA dendrimers with high immunostimulatory potency could be prepared by selfassembly of a series of branched DNA structures (Figure 47A).490 Similarly, their cellular uptake and subsequent cytokine release induced by immune response were greatly dependent on the structural complexity of the branched DNA (Figure 47B).

These studies provided a promising strategy for developing enhanced immunostimulatory activity via structure/size-de- pendent DNA dendrimers. In addition to branched DNA and DNA dendrimers, their group further developed many DNA hydrogels systems that contained specially designed branched DNA with immunoregulatory factors, including CpG/antigen (ovalbumin, OVA) motifs for stimulating the immune response or immunoinhibitory oligodeoxynucleotides (INH-ODNs) for

inhibiting the overexpression of proinflammatory cytokines.264,491,492 The CpG motifs or INH-ODNs could regulate

immune response by a ecting a class of specialized immune cells (TLR9-positive cells). CpG/OVA hydrogels with cationization achieved the sustained release of antigen and significantly delayed the growth of tumor cells through immunotherapy.493 Injection of CpG/Dox DNA hydrogels into tumor-bearing mice e ectively inhibited tumor growth due to the synergistic e ect between anticancer agents and immunostimulatory signals.264

In 2017, Ding and co-workers reported the construction of functional DNA dendrimer decorated with CpG sequences, which could be used as an e ective tool for activating immune response.494 Loop DNA structures were designed as the outermost layer of DNA dendrimer to enhance the stability of CpG motifs. Furthermore, the TAT peptide (a typical cellpenetrating peptide) modified DNA strands was introduced to improve the cell permeability (Figure 47C). The obtained functional DNA structure showed excellent biocompatibility, enhanced cell internalization, and stronger immune response (Figure 47D). Liu group further constructed a DNA supramolecular hydrogel vaccine for antitumor immune regulation (Figure 48A).495 The hydrogel was formed by the self-assembly and electrostatic interaction of Y-DNA, CpG sequences as linkers and antigen P1, which functioned as lymph node to e ciently recruit and activate antigen-presenting cells (APCs). Owing to the local enrichment of CpG sequences and the specific binding of antigen-antibodies, the hydrogel vaccine showed strong immune response and remarkable antitumor e ect (Figure 48B).

Taken together, these studies demonstrated that branched DNA structures can be hybridized to specific therapeutic and immunomodulatory sequences in desired sites following the base-pairing principle, endowing structures with the capability of tumor therapy and immune response.496 Meanwhile, the multibranched structures exhibited an increasing therapeutic e cacy and immunostimulatory activity compared to simple double helix. In addition, branched DNA could both serve as ideal carriers for a variety of therapeutic agents and as stable vehicles for the target-delivery to immune cells or tumor cells. In all, branched DNA could be engineered into multifunctional materials for tumor therapy.

4.5. Cell Engineering

Hydrogels have been widely used for 3D cell culture and cell encapsulation owing to the ECM-like environment. Hydrogel was a kind of highly cross-linked 3D network with porous structures and high water retention, which enabled e cient transport of nutrients, wastes and gases. Several di erent types of

hydrogels have been employed for 3D cell culture, including animal ECM extract hydrogels, protein/peptide hydrogels and polymer hydrogels.256 However, the gelation conditions of some polymer hydrogels were not best-suited for 3D cell culture due to the limitation of nonphysiological conditions, such as high salt, extreme pH, and toxic molecules. To this end, a myriad of DNA-based hydrogel systems have been developed to overcome this limitation.96,497 A class of DNA hydrogels made of branched DNA including X-, Y-, and T-DNA was reported.59 Because of the involvement of enzymatic ligation, these DNA hydrogels could be formed under physiological conditions. As a result, DNA hydrogels have been successfully employed to in situ encapsulate live mammalian cells only in a single step, providing opportunities for in vivo gelation at the same time.

Jin et al. created an enzyme-stimulus DNA hydrogel for the encapsulation and release of single cells in microwells.498 The DNA hydrogel formed by Y-sca olds and linkers served as the cover to seal the trapped cells, as a closed state. The permeable DNA hydrogel allowed nutrients and wastes to pass through the cover, providing a favorable environment for cell growth and proliferation. Using the restriction enzyme, the DNA hydrogel could be specifically degraded to release the trapped cells, namely an open state. On the basis of the DNA supramolecular hydrogel with self-healing, a technology of “brick-to-wall” was proposed to fabricate 3D tissue-like matrix, which allowed to encapsulate multiple cell types, and study the cell-to-cell communication and migration behaviors.499 Their design provided a promising strategy for 3D cell encapsulation and cell culture.

5. CONCLUSION AND OUTLOOK

Through a period of development and continuous innovation, branched DNA-based materials have proved to achieve precise design, controllable construction, and diverse functionalization. This review systematically summarizes the advances in the constructions and biological applications of branched DNAbased materials. Two methods for the assembling of branched DNA includes base-pairing and chemical bonding. On the one hand, because of the sequence programmability and specific recognition, branched DNA with desired size and number of branches was successfully assembled by base-pairing method in a controlled manner. On the other hand, the operability and modification of DNA enabled it to bond with chemical molecules to fabricate branched DNA with enhanced flexibility and chemical stability. From the material point of view, the construction of functional materials based on branched DNA as building units can span the range from nanoscale (i.e., DNA dendrimers) to macro-scale (i.e., DNA macro-hydrogels), which provided a wide range of options for applications in various occasions. Owing to unique physical and chemical properties of DNA, some precisely designed sequences (i.e., i-motif) embedded into branched DNA endowed materials with specific functions, such as stimulus-responsiveness and reversible morphological transition. In addition, anisotropic multibranches of branched DNA possessed multiple modified sites for conjugating with di erent functional components, such as fluorescent groups and photo-cross-linking groups, enriching the functionality of DNA materials. Moreover, the incorporation of nanoparticles and other molecules represented combined advantages in optics, magnetics and electricity. From the application point of view, the engineered DNA materials had tremendous advantages in biological and biomedical fields due to their inherent physiological e ects, biocompatibility, and

biodegradability. The introduction of branched DNA promoted the advantages due to its unique isomeric modification, sizeadjustability, and multiarm topology. By using isomeric modification, branched DNA modified with di erent moieties served as simultaneous signal probes and amplifiers for highsensitive, high-specific, and multiplex diagnosis. By taking advantage of size-adjustability, branched DNA-based carriers realized e ective cell internalization, intracellular RNA imaging, and drug/gene codelivery. By using multiarm topology, branched DNA was coloaded with biotherapeutic oligonucleotide fragments (i.e., CpG oligonucleotides and antisense DNA) and chemotherapeutic drugs, reaching high loading capacity and enhancing therapeutic e cacy. In a word, branched DNA-based functional materials with precise design and multiple functions have successfully proven their great potential in the fields of diagnostics, protein engineering, drug and gene delivery, therapeutics, and cell engineering.

Despite these exciting achievements, there is still much room for further development of branched DNA in the perspective of physical and chemical nature, adaptability, dynamics, biomedicine, life-like systems, out-of-equilibrium, and real-world applications.

(1)The physical and chemical nature of branched DNA needs further exploration. Research studies have shown that branched DNA and its derived network had the

properties of colloidal particles and could undergo a phase transition within a certain temperature range.120 This discovery drove people to conduct more in-depth research on the physical and chemical properties of branched DNA that have not been concerned before. For example, what factors would a ect this dynamic unstable state and thermodynamic stable state and how to balance the two states? This apparent population behavior of branched DNA would urge researches to continuously explore its actual microstructure. There were experimen-

tal results showing the global structure of branched DNA junction in solution.118,500 Accordingly, what was the configuration of other types of branched DNA in solution? In addition, how does the existence state of covalently linked branched DNA with organic molecules as branched points? And how could the flexibility of central organic molecule a ect the mobility and di usion of branched DNA in solution? To depict this process,

many computer-assisted simulation methods were developed.501 Computer simulations were also used for quantifying the role of cations character and strength on

the enthalpic stability and topological conformation of branched DNA.116,444,502 Although computational procedure acted an assistant role in the design and prediction of branched DNA, the experimental results in accordance with simulation would be more convincing verification. We believe that these explorations in physical and chemical nature, and actual conformation of branched DNA will provide better guidance for structural construction and material assembly in a more predictable fashion.

(2)The adaptability of branched DNA needs further exploration on the mechanisms and applications. The adjustable flexibility and rigidity of branched DNA were easily used to tune the mechanical strength and viscoelasticity of resulting materials, manifested as tunable shape plasticity and adaptability. This feature was

expected to dealing with di erent scenes in a more suitable way. For example, passing through a narrow gap required a supersoft DNA hydrogel; resuming the initial state after passing required a superelastic hydrogel;503 adhesion to the surface of a substrate, such as biological tissue, required high-viscous hydrogels. In addition, for the sake of materials with certain shapes, some rigid branched DNA building-blocks, such as three-point-star motifs and DNA tensegrity triangle, could be considered to introduce, which is hopeful to improve the strength of the hydrogels and obtain predesigned shapes in molds. Furthermore, in pursuit of wider adaptability, a kind of environmental adaptability should also be considered, specifically the color changes varying with surroundings resembling chameleon. Some works that reported bioinspired structural color hydrogels could be used for

reference to develop DNA materials with high-level adaptability.504,505

(3)At present, the assembled dynamic DNA materials based

on branched DNA are far from meeting the requirements of “smart” feature. Making a more refined design of the structure and introducing more kinds of stimulating components (such as light-sensitive, heat-sensitive, redoxsensitive, metal-sensitive, and magnetic-sensitive compo-

nents) are needed to expand the structural and functional diversity.506−509 It is necessary to consider the e ects of external stimuli on the structure, morphology, and properties of branched DNA-based materials to cope with a wider range of applications, ultimately realizing automation and intellectualization of DNA materials.

(4)The contributions of branched DNA in the field of biomedicine needs further development. The size of branched DNA-based materials could be precisely controlled within nanoscale by virtue of concentrationdependent tunability. This characteristic exhibited outstanding superiority in biomedical applications, especially diagnostics, drug delivery, and therapeutics. However, to improve performance of these biomedical applications, several issues need to be addressed: (i) Making full use of analytes-triggered dynamic formation of branched DNA or DNA dendrimers, coupled with the function of signal amplification, diagnostics needs to be improved with better sensitivity, and accuracy in noninvasive diagnostics to avoid false-positive results and the damage to organism. (ii) Drug nanocarriers need to meet the requirements for nanomedicine such as long circulating time in vivo, specific targeting, and e cient cellular internalization. Although multibranched structures resisted the nuclease degradation in serum or endosomes to some extent, its internalization utilization needs to be further improved in other aspects: compact structures by reasonable branched interval; multiple targeting molecules be simultaneously connected to the branches; molecular designs for accelerating lysosomal escape. (iii) As a genetic molecular, the inherent genetic function and regulatory role of branched DNA need to be further exploited and utilized. For instance, integration of a variety of endogenous therapeutic or regulatory fragments into branched DNA is a promising strategy. (iv) By taking full advantage of the anisotropic structure and the function of intercalating various active molecules, branched DNA is expected to integrate many kinds of therapeutic methods together for realizing all-in-one combined therapy to

synergistically enhance the therapeutic e cacy. In addition, as a promising biomedical field, personalized medicine is an advanced method based on patient genome information and o ering optimal solutions. By using the sequential programmability of DNA, branched DNA-based materials can be designed specifically to aim at di erent patient characteristics, which are expected to exhibit unique advantages in personalized diagnosis and treatment.

(5)Branched DNA can be considered for simulating life-like systems. Living systems are extremely complicated and elusive, so simplifying and simulating the systems will be a feasible way to elaborate the molecular mechanism. Because of the inherent biological properties, branched DNA had great potential in the construction of bionic and life-like system. For example, network or nano/microgels systems extended from branched DNA were closely similar to the authentic environments of cytoplasm of living cells. Studies have shown that liposome-capped

DNA networks acted as cytoskeleton for stabilizing artificial cells.510 Inspired by the study, other components, such as functional proteins and cell membrane, could be incorporated into DNA networks to prepare biomimetic cells, membranous organelles, and membrane-free organelles. The development of some technologies, such as microfluidics and bioimaging technology, also assisted the establishment and research of bionic systems. These simplified systems were greatly beneficial to clarify cell-to- cell communication behaviors, molecular pathways in organelles and protein production mechanism. In addition, highly condensed DNA nanoparticles derived from branched DNA could mimic the nuclear compart-

mentalization, providing a simplified model to study the proximity and concentrated e ect.262,442

(6)Branched DNA is expected to have application prospects in the construction of out-of-equilibrium systems. One of the unique features of living systems is out-of-equilibrium,

meaning that life exists far from equilibrium and keeps in a continuously thermodynamic instability state.511,512 This is a more advanced manifestation of stimulus responsiveness and adaptability. Some motion, oscillations, sensing, cycling-feedback, and communication devices could be integrated, and energy absorption, transformation, and dissipation are considered. Therefore, it is expected to develop fully automatic life-like machines fueled by selfregulating chemistry. The autonomous molecular systems are believed to bridge the gap between materials science and life science, and further facilitate to explore the nature of living activities. Branched DNA will be highly promising as building-blocks to construct artificial out- of-equilibrium systems.

(7)To meet the requirements of wider applications, the branched DNA-based materials need to be transformed from laboratory setting to the real-world. It is also necessary to satisfy scale-up preparation of fine structures, especially for macro-materials. Although considerable progress has been made to prepare branched DNA-based macroscopic DNA materials, the high cost is still one of the major challenges. There is a possible way that combines branched DNA with PCR, which has been proven feasible in a test tube.

Branched DNA is the original point of DNA nanotechnology that was coined by Professor Nadrian Seeman. Until now,

branched DNA derived nanotechnology and materials have aroused the attention of researchers with backgrounds in chemistry, biology, material science, and engineering. The collaboration between di erent fields would be promoted by interdisciplinary research; the achievements of cross-disciplines in turn would accelerate the development of branched DNA by creating new strategies and technologies including molecular design, assembly, and applications. We envision that branched DNA-based materials with more functions and more diversity would be useful tools to help us to look into the molecular-level mechanisms of life as well as serve in the broad fields of material fabrication, human healthcare, life-like systems, energy sources, ecological environment, and artificial intelligence.

AUTHOR INFORMATION

Corresponding Author

Dayong Yang − Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China; orcid.org/0000-0002-2634- 9281; Email: dayong.yang@tju.edu.cn

Authors

Yuhang Dong − Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China

Chi Yao − Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China

Yi Zhu − Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China

Lu Yang − Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China

Dan Luo − Department of Biological & Environmental Engineering, Cornell University, Ithaca, New York 14853, United States; orcid.org/0000-0003-2628-8391

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.chemrev.0c00294

Author Contributions

§Y.D. and C.Y. equally contributed to the manuscript.

Notes

The authors declare no competing financial interest.

Biographies

Yuhang Dong received her B.S. degree at School of Chemical Engineering and Technology in Tianjin University in 2017. She is currently a Ph.D. candidate in Prof. Dayong Yang’s Lab. Her current research focuses on DNA-based materials and their applications in biomedicine.

Chi Yao obtained her Ph.D. degree from Fudan University in 2017. She joined in the School of Chemical Engineering and Technology in Tianjin University in 2017. Her current research focuses on DNA materials with biological functions.

Yi Zhu received his B.S. degree at School of Chemical Engineering and Technology in Tianjin University in 2018. He is currently a graduate student in Prof. Dayong Yang’s Lab. He is interested in the design of DNA functional materials and novel drug delivery carriers.

Lu Yang received a M.S. degree at School of Chemical Engineering and Technology in Tianjin University in 2018.

Dan Luo obtained his B.S. from the University of Science and Technology of China in 1989 and Ph.D. from the Ohio State University in 1997. After his postdoctoral training in the School of Chemical Engineering at Cornell University, he joined Cornell faculty as an assistant professor in 2001 and was promoted to the position of full professor in 2011. His research interests focus on engineering DNA as both a generic and a genetic material for real-world applications.

Dayong Yang is a professor in the School of Chemical Engineering and Technology of Tianjin University. He received his B.S. and M.S. degrees with Professor Xiaolin Xie at Huazhong University of Science and Technology of China in 2002 and 2005, respectively. In 2008, he received his Ph.D. degree with Professor Xingyu Jiang at the National Center for Nanoscience and Technology, Chinese Academy of Sciences. He did postdoctoral training at Cornell University in the USA with Professor Dan Luo and Radboud Universiterit Nijmegen in The Netherlands with Professor Wilhelm Huck. The themes of his research include DNA-based materials, biofunctional polymers, and synthetic biology.

ACKNOWLEDGMENTS

This work was supported in part by National Natural Science Foundation of China (Grant Nos. 21622404 and 21621004) and Ministry of Science and Technology of China (National Key Technology Research and Development Program, Grant Nos. 2019YFA09005800 and 2018YFA0902300). We thank Dr. Feng Li, Wenting Yu, Weijian Wu, Xiaocui Guo, and Xihan Xu for discussion and proofreading.

ABBREVIATIONS

REFERENCES

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