| Deoxyribonucleic acid(DNA)is a natural biological macromolecule and the carrier of genetic information for most organisms.Meanwhile,DNA molecules can also be used as the units for precise construction of nucleic acid nanostructures with controllable scales and morphologies.As one of the representative DNA nanostructures,DNA origami is usually created by the co-assembly of a long single-stranded circular DNA scaffold and multiple short DNA staple strands through complementary base pairing.DNA origami is programmable,addressable,and biocompatible,and can be folded into controllable size and shape,so that it has been developed to precisely organize multiple components and widely employed in numerous research fields,such as information storage,biosensor,and drug delivery.Because of the limited length of available DNA scaffold(a single-stranded genome from bacteriophage M13mp18,7249 nt),the diameter of conventional DNA origami is 100 nm.To enlarge the DNA origami,much attention has been devoted to constructing the larger DNA origami nanostructures depending on sticky end association,base stacking,and shape complementarity of the DNA origami tiles.Based on the designed specific base sequence and/or geometric shape at the edges,DNA origami is able to be interconnected by geometric edges or faces to form two-dimensional and three-dimensional DNA nanostructures.Meanwhile,DNA-modified non-nucleic acid were used as connectors to organize multiple DNA origami tiles,which greatly simplifies the design of DNA origami tiles.At present,various-connectors have been constructed,such as peptides,cyclodextrin/adamantane and inorganic materials(gold nanoparticles,diamond,quantum dots).DNA-modified gold nanospheres with controlled the overall sizes have been widely studied.However,the number of organized DNA origami tiles is uncertain in these designs and the malposition behavior in the center is unavoidable.Thus,we report a general strategy to efficiently organize multiple DNA origami tiles to form super-DNA origami using a flexible and covalent-bound branched DNA structure.In our design,equilateral triangular DNA origamis with a capture strand in the vertex were co-assembled with the flexible and covalent-bound branched DNA,resulting in the formation of predesigned flower-like nanostructures.After hybridization with the branched DNA structures,sophisticated and larger higher-order DNA origamis can be efficiently ordered in the predesigned patterns through hierarchical assembly.The flower-like DNA origamis can further precisely organize gold nanoparticles into different patterns.This rationally developed DNA origami ordering strategy based on the flexible and covalent-bound branched DNA structure presented a new avenue for the construction of sophisticated DNA architectures with larger molecular weights and more nanomaterials with multiple functionalities.The specific research contents are as follows:1.Synthesis of the flexible and covalent-bound branched DNA structure.To efficiently and controllably assemble the dense super-DNA origami structure,small organic molecules were utilized as the building block cores of branched DNA structures throuth cross-linking reactions with single-stranded DNA as the arms.As a proof of concept,we chose dipentaerythritol(Di-PE)as the cores,which had a small molecular weight and symmetrical shape.The flexible and covalent-bound branched DNA structures(Bn)were synthesized by a copper-free click reaction between azide-modified dipentaerythritol and DBCO-modified single-stranded DNA.According to the images of 8%native polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS),it proved that we have successfully obtained a series of covalent-bound branched DNA structures(Bn:B2-B6)with a certain number of 2-6 branches.This flexible and covalent-bound branched DNA structure had some advantages:First,the number of DNA strands is well-defined,resulting in a controllable number of the DNA origami tiles.Second,the small molecular weight and high flexibility of Di-PE can reduce the malposition behavior to improve the assembly yield and structural density.Third,the covalent bonding of the Bn made it stable under certain conditions,such as high temperatures.So that it will show great potential in organizing a wider variety of DNA nanostructure tiles and their applications.2.Construction and characterization of flower-like super-DNA origami structures by a covalent-bound branched DNA structure.Using flexible and covalent-bound branched DNA as linkers,we achieved efficient and controllable assembly of multiple DNA origami tiles and formed dense flower-like higher-order DNA origami structures.At the same time,we also explored the effect of two kinds of branched DNA on the co-assembled higher-order DNA origami structures.The purified triangular DNA origami with modification of a capture strand in the vertex(T1)as the building tiles for co-assembly with branched DNA(Bn).The products were analyzed by 0.6%agarose gel electrophoresis and an atomic force microscope(AFM).The results proved that we obtained five co-assembled DNA origami structures(BnT1)dependent on the certain numbers of arms in branched DNA structures,resulting in the formation of flower-like nanostructures with 2-6 triangular origami“petals”,respectively.As a control,we designed another branched DNA structure,a rigid and non-covalently linked DNA junction(Jn,n=2-6),and co-assembled with T1 through the same assembly condition as Bn.Compared with rigid DNA junction(JnT1),the higher co-assembly yields(P<0.01)and more compact structures(P<0.001)were obtained by the flexible branched DNA structures(BnT1).These demonstrate the feasibility of our strategy and provide a way for the controllable assembly of other DNA nanostructure modules.3.Construction and characterization of sophisticated and larger higher-order DNA origami structures based on the covalent-bound branched DNA structure.After efficiently ordering the triangular DNA origami tile(T1)by branched DNA structures(Bn),we subsequently employed the branched DNA structure as a connector to construct a higher-order DNA origami structure with more complex shape and larger molecular weight by a hierarchical model step by step.The branched DNA structure prehybridized triangular DNA origami tiles(T2 and T3)as the cores were co-assembled with T1.The assembled higher-order DNA origami nanostructures were then purified by 0.6%agarose gel electrophoresis and visualized by the AFM imaging.The results show that we successfully construct 9 predesigned higher-order DNA origami nanostructures with T2or T3 as the core(BnT2T1 and BnT3T1).Meanwhile,the flexible and covalent-bound branched DNA structures can function well as the connector to organize 13 DNA origami tiles into sophisticated higher-order DNA origami nanostructures.Our strategy provides an effective reference for obtaining large-scale DNA origami structures with various shapes in the future.4.Precisely organizing Au nanoparticles on higher-order DNA Origami nanostructures.We employed the conventional Au nanoparticles as the evaluation model to investigate the addressability of these constructed DNA origami nanostructures and the ability to precisely arrange functional materials.We next efficiently synthesized an Au nanosphere(Au NS)and nanorod(Au NR),and they were modified by the thiolated ss DNAs.After obtaining the capture strands anchored DNA origami tiles,they were organized by branched DNA(Bn)to co-assemble flower-like higher-order DNA origami nanostructures.The DNA modified Au NPs were directly hybridized with the super-DNA origami structures and visualized by the AFM imaging.We achieved the controllable arrangement of Au NSs and larger-sized Au NRs on five co-assembled DNA origami structures,and obtained a series of regular gold nanoparticle patterns with different combinations.These results demonstrated that the well-ordered DNA origami structure based on the flexible and covalent-bound branched DNA structure also possessed the excellent addressability to precisely organize multiple nanoparticles,opening a new avenue for the development of multifunctional DNA nanostructures. |
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