利用DNA折纸术构建功能纳米材料

DNA折纸术(DNA origami)作为一种精确高效的自组装技术,自2006年Rothemund发明以来在生物医药、高灵敏度检测、纳米光电子器件、等离子体光子学等领域展现出巨大的应用潜力,近年来受到广大研究者的高度关注。 利用DNA折纸术构建纳米材料是以DNA origami结构为载体,通过碱基互补配对的原则及三维结构上可程序化设计和可寻址的特点精确地组装很多功能基团如金属及半导体纳米颗粒,蛋白质和单壁碳纳米管等,并应用于研究无标记的RNA杂交检测、单分子的化学反应、检测间距对多价态的配位体-蛋白质之间键合的影响等。本文对近几年来DNA origami构建功能纳米材料的研究进展加以系统综述,并对DNA origami的发展方向和应用前景进行了展望。     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

Programmed‐Assembly System Using DNA Jigsaw Pieces

A novel method for assembling multiple DNA origami structures has been developed by using designed 2D DNA origami rectangles, so‐called “DNA jigsaw pieces” that have sequence‐programmed connectors. Shape and sequence complementarity were introduced to the concavity and convex connectors in the DNA rectangles for selective connection with the help of nonselective π‐stacking interactions between the side edges of the DNA jigsaw piece structures. Single DNA jigsaw piece units were assembled into unidirectional nanostructures with the correct alignment and uniform orientation. Three and five different DNA jigsaw pieces were assembled into predesigned and ordered nanostructures in a programmed fashion. Finally, three‐, four‐, and five‐letter words have been displayed by using this programmed DNA jigsaw piece system.     

Directed Self‐Assembly of DNA Tiles into Complex Nanocages

Tile‐based self‐assembly is a powerful method in DNA nanotechnology and has produced a wide range of well‐defined nanostructures. But the resulting structures are relatively simple. Increasing the structural complexity and the scope of the accessible structures is an outstanding challenge in molecular self‐assembly. A strategy to partially address this problem by introducing flexibility into assembling DNA tiles and employing directing agents to control the self‐assembly process is presented. To demonstrate this strategy, a range of DNA nanocages have been rationally designed and constructed. Many of them can not be assembled otherwise. All of the resulting structures have been thoroughly characterized by gel electrophoresis and cryogenic electron microscopy. This strategy greatly expands the scope of accessible DNA nanostructures and would facilitate technological applications such as nanoguest encapsulation, drug delivery, and nanoparticle organization.     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

Gold‐Nanoparticle‐Mediated Jigsaw‐Puzzle‐like Assembly of Supersized Plasmonic DNA Origami

DNA origami has rapidly emerged as a powerful and programmable method to construct functional nanostructures. However, the size limitation of approximately 100 nm in classic DNA origami hampers its plasmonic applications. Herein, we report a jigsaw‐puzzle‐like assembly strategy mediated by gold nanoparticles (AuNPs) to break the size limitation of DNA origami. We demonstrated that oligonucleotide‐functionalized AuNPs function as universal joint units for the one‐pot assembly of parent DNA origami of triangular shape to form sub‐microscale super‐origami nanostructures. AuNPs anchored at predefined positions of the super‐origami exhibited strong interparticle plasmonic coupling. This AuNP‐mediated strategy offers new opportunities to drive macroscopic self‐assembly and to fabricate well‐defined nanophotonic materials and devices.     

A Signal‐Passing DNA‐Strand‐Exchange Mechanism for Active Self‐Assembly of DNA Nanostructures

DNA nanostructured tiles play an active role in their own self‐assembly in the system described herein whereby they initiate a binding event that produces a cascading assembly process. We present DNA tiles that have a simple but powerful property: they respond to a binding event at one end of the tile by passing a signal across the tile to activate a binding site at the other end. This action allows sequential, virtually irreversible self‐assembly of tiles and enables local communication during the self‐assembly process. This localized signal‐passing mechanism provides a new element of control for autonomous self‐assembly of DNA nanostructures.     

Programmable Assembly of Nano‐architectures through Designing Anisotropic DNA Origami Patches

Programmable assembly of nanoparticles (NPs) into well‐defined architectures has attracted attention because of tailored properties resulting from coupling effects. However, general and precise approaches to control binding modes between NPs remain a challenge owing to the difficulty in manipulating the accurate positions of the functional patches on the surface of NPs. Here, a strategy is developed to encage spherical NPs into pre‐designed octahedral DNA origami frames (DOFs) through DNA base‐pairings. The DOFs logically define the arrangements of functional patches in three dimensions, owing to the programmability of DNA hybridization, and thus control the binding modes of the caged nanoparticle with designed anisotropy. Applying the node‐and‐spacer approach that was widely used in crystal engineering to design coordination polymers, patchy NPs could be rationally designed with lower symmetry encoded to assemble a series of nano‐architectures with high‐order geometries.     

Assembly and Purification of Enzyme‐Functionalized DNA Origami Structures

The positioning of enzymes on DNA nanostructures for the study of spatial effects in interacting biomolecular assemblies requires chemically mild immobilization procedures as well as efficient means for separating unbound proteins from the assembled constructs. We herein report the exploitation of free‐flow electrophoresis (FFE) for the purification of DNA origami structures decorated with biotechnologically relevant recombinant enzymes: the S‐selective NADP⁺/NADPH‐dependent oxidoreductase Gre2 from S. Cerevisiae and the reductase domain of the monooxygenase P450 BM3 from B. megaterium. The enzymes were fused with orthogonal tags to facilitate site‐selective immobilization. FFE purification yielded enzyme–origami constructs whose specific activity was quantitatively analyzed. All origami‐tethered enzymes were significantly more active than the free enzymes, thereby suggesting a protective influence of the large, highly charged DNA nanostructure on the stability of the proteins.     

Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions

DNA has become a prime material for assembling complex three‐dimensional objects that promise utility in various areas of application. However, achieving user‐defined goals with DNA objects has been hampered by the difficulty to prepare them at arbitrary concentrations and in user‐defined solution conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)‐induced depletion of species with high molecular weight. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient separation from non‐integrated DNA strands. DNA objects may be prepared at concentrations up to the limit of solubility, including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self‐assembled DNA objects.     

Synchronization of Two Assembly Processes To Build Responsive DNA Nanostructures

Herein, we report a strategy for the synchronization of two self‐assembly processes to assemble stimulus‐responsive DNA nanostructures under isothermal conditions. We hypothesized that two independent assembly processes, when brought into proximity in space, could be synchronized and would exhibit positive synergy. To demonstrate this strategy, we assembled a ladderlike DNA nanostructure and a ringlike DNA nanostructure through two hybridization chain reactions (HCRs) and an HCR in combination with T‐junction cohesion, respectively. Such proximity‐induced synchronization adds a new element to the tool box of DNA nanotechnology. We believe that it will be a useful approach for the assembly of complex and responsive nanostructures.     

Icosahedral DNA Nanocapsules by Modular Assembly

It's a trap! DNA polyhedra formed through molecular self‐assembly may function as nanocapsules for the targeted delivery of encapsulated entities. This functional aspect was demonstrated for the most complex DNA‐based platonic solid: During the stepwise amalgamation of discrete polyhedra to form icosahedra, gold nanoparticles (GNPs) were encapsulated from solution (see illustration and TEM image of icosahedral cages containing GNPs).

     

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K⁺ channel proteins are assembled through zinc‐finger protein (ZFP)‐adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP‐fused Kir3 channels and ZFP‐based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K⁺ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.     

Surface Assembly of DNA Origami on a Lipid Bilayer Observed Using High-Speed Atomic Force Microscopy

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice–tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture.