Enzyme-driven Nanorobots Walking Along Predesigned Tracks on the DNA Origami for Cargo Transport and Catalysis

In molecular engineering,designing and synthesizing molecular machines with capable of performing complex tasks,remains a formidable challenge.DNA is an excellent candidate for building molecular robots because it is highly programmable.Here,we present an artificial nanorobot,in which a DNA cube serves as the inert‘body’,and nucleic acid catalysts based on an enzymatic nicking reaction act as the‘legs’for walking.The nanorobot can execute a series of actions,such as‘start’,‘turn’,and‘stop’when it walks along a predefined track.Its performance could be confirmed and monitored by using an atomic force microscope(AFM)and fluorescence spectroscopy.Inspired by biological machines,we artificially designed a series of specialized tasks that combined walking with control of cargo transport and catalysis.Real-time fluorescence kinetics curves provide monitoring signals for cargo transport and catalytic processes.Our work can enrich the toolbox of DNA machinery and has great potential for engineering molecular nanofactories.

DNA origami nanodevice with spatial regulation of CD95 signaling for rheumatoid arthritis treatment

Recently,a work jointly studied by Ling Li and coworkers1 was published in Nature Materials,describing a reconfigurable DNA origami nanodevice designed to regulate CD95 death-inducing signaling of immune cells.The researchers utilized the DNA origami nanodevice to establish selective local immune tolerance and demonstrated its ability to alleviate rheumatoid arthritis(RA)in the inflamed synovial tissue of mice without causing any obvious side effects(Fig.1).This approach presents a novel idea for the development of drug interventions involving ligandreceptor interactions.

Nanoscale Precise Editing of Multiple Immune Stimulating Ligands on DNA Origami for T Cell Activation and Cell-Based Cancer Immunotherapy

The spatial arrangement of activating ligands is known to have great influence on T cell activation.However,independently studying each ligand’s spatial organization parameter that affects T cell activation remains a great challenge.Here,with DNA origami,we precisely organized the CD3ɛantibodies simulating T cell receptor(TCR)ligands and CD28 antibodies simulating co-stimulatory ligands to interrogate the independent role of TCR-ligand spacing and local copy numbers as well as the spacing between TCR ligands and co-stimulatory ligands on T cell activation.We found that T cell activation benefited fromlocally concentrated TCR ligands with a shorter spacing and was maximized by an∼38 nm spacing between TCR ligands and co-stimulatory ligands.The T cell expander constructed based on our findings could efficiently expand CD8+T cells for tumor immunotherapy.Thus,the DNA nanostructurebased ligands’precise arrangement can be a unique tool in studying immune cell activations and cellbased immunotherapies.

可控自组装DNA折纸结构作为药物载体的研究进展

在过去的几十年里, DNA纳米技术作为一种快速发展的可控自组装技术,使人们能构建出各种复杂的纳米结构. DNA折纸结构具备可编程性、空间可寻址性、易修饰性及良好的生物相容性等多种优越的特性,这些优异的性质使其在药物递送方面具有广阔的应用前景.本文总结了近年来可控自组装DNA折纸结构作为药物递送系统的研究进展,展望了DNA折纸纳米载体未来的发展方向,并讨论了该领域面临的挑战和可能的解决方法.

基于DNA折纸系统求解0-1整数规划问题的模型

DNA折纸术具有可编程性、动态调节能力以及精确的结构控制能力,有着广泛的研究和应用.文中将DNA折纸应用于0-1整数规划问题,建立了一个DNA折纸系统,该系统由DNA折纸基底和四种类型的辅助链自组装而成.加入输入链后,通过DNA链置换,有选择的释放折纸系统中辅助链上的金纳米颗粒(AuNPs).借助电镜观察折纸系统中金纳米颗粒被释放的情况,读取可行解.这种设计方法操作简单,读解方便,也可用于组装更复杂的系统中.

On-chip isotachophoresis separation of functional DNA origami capture nanoarrays from cell lysate

Scaffolded DNA origami, a versatile method to construct high yield self- assembled DNA nanostructures, has been investigated to develop water-soluble nanoarrays for label free RNA detection, drug delivery, molecular positioning and recognition, and spatially ordered catalysis of single molecule chemical reactions. Its attributes that facilitate these applications suggest DNA origami as a candidate platform for intracellular targeting. After the interaction with targeted proteins in cell lysate, it is critical to separate and concentrate DNA origami nanoarrays from the crude cell lysate for further analysis. The recent development of microchip isotachophoresis （ITP） provides an alternative robust sample preconcentration and electrophoretic separation method. In this study, we present online ITP for stacking, separation and identification of aptamer-functionalized DNA origami and its thrombin complex in a simple cross-channel fused silica microfluidic chip. In particular, the method achieved separation of a binding complex in less than 5 min and 150-fold signal enhancement. We successfully separated and analyzed the thrombin bound origami-aptamer spiked into cell lysate using on-chip ITP. Our results demonstrate that origami/thrombin nanostructures can be effectively separated from cell lysate using this method and that the structural integrity of the concentrated binding complex is maintained as confirmed by atomic force microscopy （AFM）. An ITP-based separation module can be easily coupled to other microchip pre- and post-processing steps to provide an integrated proteomics analysis platform for diagnostic applications.

Deterministic assembly of single optical cavity formed by gold dimensional DNA origami

Controllable strong interactions between a nanocavity and a single emitter is important to manipulating optical emission in a nanophotonic system but challenging to achieve.Herein a three-dimensional DNA origami,named as DNA rack(DR)is proposed and demonstrated to deterministically and precisely assemble single emitters within ultra-small plasmonic nanocavities formed by closely coupled gold nanorods(AuNRs).Uniquely,the DR is in a saddle shape,with two tubular grooves that geometrically allow a snug fit and linearly align two AuNRs with a bending angle <10°.It also includes a spacer at the saddle point to maintain the gap between AuNRs as small as 2-3 nm,forming a nanocavity estimated to be 20 nm^(3) and an experimentally measured O factor of 7.3.A DNA docking strand is designed at the spacer to position a single fluorescent emitter at nanometer accuracy within the cavity.Using Cy5 as a model emitter,a -30-fold fluorescence enhancement and a significantly reduced emission lifetime(from 1.6 ns to 670 ps)were experimentally verified,confirming significant emitter-cavity interactions.This DR-templated assembly method is capable of fitting AuNRs of variable length-to-width aspect ratios to form anisotropic nanocavities and deterministically incorporate different single emitters,thus enabling flexible design of both cavity resonance and emission wavelengths to tailor light-matter interactions at nanometer scale.

A DNA origami plasmonic sensor with environment-independent read-out

DNA origami is a promising technology for its reproducibility,flexibility,scalability and biocompatibility.Among the several potential applications,DNA origami has been proposed as a tool for drug delivery and as a contrast agent,since a conformational change upon specific target interaction may be used to release a drug or produce a physical signal,respectively.However,its conformation should be robust with respect to the properties of the medium in which either the recognition or the read-out take place,such as pressure,viscosity and any other unspecific interaction other than the desired target recognition.Here we report on the read-out robustness of a tetragonal DNA-origami/gold-nanoparticle hybrid structure able to change its configuration,which is transduced in a change of its plasmonic properties,upon interaction with a specific DNA target.We investigated its response when analyzed in three different media:aqueous solution,solid support and viscous gel.We show that,once a conformational variation is produced,it remains unaffected by the subsequent physical interactions with the environment.

Advances in DNA Origami Nanopores： Fabrication：Characterization and Applications

In recent years, bio-nanopore and solid-state nanopore have been greatly improved for molecule bio-sensing. Whereas, the development of this scientific field seems to have encountered a bottleneck due to their respective limitations. The small pore size of the former impedes the detection of large single molecule, and the latter is difficult to achieve similar accuracy and functional control. DNA origami plays a novel role to bring more opportuni- ties for the development of nanopore technology since it is relatively easy to synthesize and modify. This review mainly focuses on introducing the DNA origami nanopore fabrication methods, characterization and application. Meanwhile, the challenges in the present DNA origami nanopore research are also discussed.

Self-assembly of highly ordered DNA origami lattices at solid-liquid interfaces by controlling cation binding and exchange

The surface-assisted hierarchical self-assembly of DNA origami lattices represents a versatile and straightforward method for the organization of functional nanoscale objects such as proteins and nanoparticles.Here,we demonstrate that controlling the binding and exchange of different monovalent and divalent cation species at the DNA-mica interface enables the self-assembly of highly ordered DNA origami lattices on mica surfaces.The development of lattice quality and order is quantified by a detailed topological analysis of high-speed atomic force microscopy(HS-AFM)images.We find that lattice formation and quality strongly depend on the monovalent cation species.Na^(+)is more effective than Li^(+)and K^(+)in facilitating the assembly of high-quality DNA origami lattices,because it is replacing the divalent cations at their binding sites in the DNA backbone more efficiently.With regard to divalent cations,Ca^(2+)can be displaced more easily from the backbone phosphates than Mg^(2+)and is thus superior in guiding lattice assembly.By independently adjusting incubation time,DNA origami concentration,and cation species,we thus obtain a highly ordered DNA origami lattice with an unprecedented normalized correlation length of 8.2.Beyond the correlation length,we use computer vision algorithms to compute the time course of different topological observables that,overall,demonstrate that replacing MgCl_(2) by CaCl_(2) enables the synthesis of DNA origami lattices with drastically increased lattice order.

Hetero-assembly of gold nanoparticles on a DNA origami template

Hetero-assembling of spherical building blocks with well-defined spatial distribution holds great significance in developing chiral nanostructures. Herein, a strategy for hetero-assembling of gold nanoparticles(Au NPs) was demonstrated using rigid bifacial DNA origami as templates. By tuning the sizes and the fixed location of Au NPs on DNA origami, right-handed and left-handed Au NPs nanostructures were respectively constructed. Gel electrophoresis indicated the formation of the DNA origami-Au NPs complex and transmission electron microscopy(TEM) visually displayed the arrangement of Au NPs in these two chiral structures. The spatial configuration and 3D geometry of Au NPs were further illustrated by the stereographic TEM with tilting angles from ?30° to 30°. This strategy provides a universal approach to construct the asymmetrical 3D geometries, which may have potential applications in biomimicking and nanophotonics.

Self-assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to create self-assembling structures at the nanometer scale exhibiting the addressable character.However,the assembly of DNA origami is disorderly and unpredictable.Herein,we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers.Firstly,long single-stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs.Subsequently,the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons.By mixing them up,we illustrate the one-dimensional even two-dimensional assembly of DNA origami with good orientation.

Catalytic DNA Origami-based Chiral Plasmonic Biosensor

Plasmonic circular dichroism(CD) has been emerged as a promising signal for building biosensors due to its high sensitivity and specificity. In the past years, DNA nanotechnology enabled diverse chiral plasmonic devices, which can response biomolecules and then generate dynamic plasmonic CD signals at the visible range. Although some of them have been successfully employed as biosensors, the detection sensitivity is still relatively low. Herein we report a chiral plasmonic sensor with an improved detection sensitivity by integrating catalytic hairpin assembly circuits into DNA origami structures. We tested two kinds of tumor marker RNA sequences as detection targets and it turns out that the detection limit is below 10 pmol/L, improving one order of magnitude compared to previous work. The chiral plasmonic sensor with internal signal amplification circuits can stimulate a variety of smart nano-sensors for biological detection and offer a promising strategy for pathogenic RNA detection with plasmonic CD output.

High-fidelity transfer of area-selective atomic layer deposition grown HfO_(2)through DNA origami-assisted nanolithography

DNA origami-assisted nanolithography(DOANL)for fabricating custom-designed nanomaterials through pattern transfer from DNA origami to different substrates materials are presented.However,the pattern's integrity and resolution face considerable challenges due to the uncontrollable growth of the nanomaterials during transformation and the unclear mechanism of DOANL.Herein,we report a DOANL combined with area-selective atomic layer deposition(ALD)strategy for fabricating custom shapes hafnium oxide(HfO2)with the high-fidelity and high-throughput.We find that the HfO_(2)selectively grows on DNA origami substrates in a hydroxyl-rich area instead of a methyl-rich protective layer.Combined with the merit of the area-selective ALD method,theHfO_(2)atom selectively coated on the DNA origami surface,thus,precisely modeling the shapes with high-precision in our study based on the surface groups difference of DNA origami and the naked hexamethyldisilane(HMDS)-treated substrates,which reveal the mechanical of high-fidelity pattern transfer based on DOANL.As a result,DNA origami structures can program the shape ofHfO_(2)nanostructures.The DOANL that is based on the principle of"bottom-up"precision assembly breaks through the shape complexity and high-throughput fabrication limitation of theHfO_(2)nanostructures,including two-and three-dimensional structures,plane and curved structures,monolithic and hollow structures.Based on the"top-down"accurate fabrication principle,the area-selective ALD on methyl-rich protective layer substrates improves the integrity and resolution of the pattern transfer process.Overall,this work provides a general technology for nanofabrication strategy.

Patterning with Organized Molecules

Decades of progress in the semiconductor industry has led to lithographically printed dimensions that are small enough that the positions of individual molecules and the stochastic variation in the number of photons have a significant effect on the quality of photoresist patterns.These effects scale badly and will be more important as feature sizes continue to shrink.Selforganizing materials can provide regular patterns of molecules that have the potential to minimize stochastic effects.Some such reported materials are block copolymers,bottle brush polymers and DNA,all of which have been used as part of lithographic patterning.A key challenge for selforganizing materials is defect levels.The energy to rearrange has to be high enough that random defects aren’t created thermally but low enough that rearrangement into preferred domains can occur.All of the methods can generate accurate CDs based on the chemical composition of the material,but they all need some way to control the positions of the feature edges.There are methods for guiding the self-organization,but the final position is the sum of the guide pattern misalignment and the intrinsic alignment error of the self-organizing materials.Thus it can be worse than the positioning of the guide structures.Alignment and defect levels are thus two big challenges for manufacturing introduction of self-organizing materials.

Programmed co-assembly of DNA-peptide hybrid microdroplets by phase separation

Biopolymers, including DNA and peptides have been used as excellent self-assembling building blocks for programmable single-component or hybrid materials, due to their controlled molecular interactions.However, combining two assembling principles of DNA-based programmability and peptide-based specific molecular interactions for hybrid structures to microscale has not yet been achieved. In this study,we describe a hybrid microsystem that emerges from the co-assembly of DNA origami structure and short elastin-like polypeptide conjugated oligonucleotides, and initiates liquid-liquid phase separation to generate microdroplets upon heating above the transition temperature. Moreover, the hybrid microdroplets are capable for guest molecule trapping and perform bi-/tri-enzymatic cascades with rate enhancements as open “microreactors”. Our programmed assembled DNA-peptide microsystem represents a new combination of DNA nanotechnology and peptide science and opens potential application routes toward lifeinspired biomaterials.

基于DNA折纸超分子体系的自组装金属纳米结构及其光学性能研究

DNA分子是生物体内主要的遗传物质,其通过碱基互补配对作用形成稳定的双螺旋结构.而碱基互补配对的特异性和强相互作用使DNA分子在制造纳米结构和材料方面也发挥着重要作用.在几种基于DNA分子构建纳米结构的方法中,DNA折纸术凭借其高产率、稳定性和构建复杂形状的优异能力,能够对数百纳米尺寸的复杂、任意形状的结构进行可编程的自组装,这是其他纳米制造技术难以达到的.在本文中,我们主要介绍了基于DNA折纸术的自组装金属纳米复合结构及其光学性能的研究进展.首先,介绍了DNA折纸结构设计和构建的基本原理.随后,综述了基于DNA折纸超分子体系构建的金属纳米复合结构在手性效应、表面增强拉曼散射效应和表面增强荧光效应中的最新应用进展.最后,对现有体系中需要解决的问题以及未来的发展方向进行了展望.

Applications of DNA Nanotechnology in Synthesis and Assembly of Inorganic Nanomaterials

In addition to its inherited genetic function, DNA is one of the smartest and most flexible self-assembling na- nomaterials with programmable and predictable features, for which, more and more scientists combine DNA with nanomaterials and put them into designing, synthesizing and assembling. In this review, four modes of action of DNA molecules are introduced in a figurative and intuitive way, based on the four different roles it plays in synthe- sis and assembly of nanomaterials： （a） smart linkers to guide nanoparticle assembly, （b） 2D or 3D scaffold with well-designed binding sites, （c） nucleation sites to directly facilitate Au/Pd/Ag/Cu nanowires, nanoparticles, nano- arrays and （d） serving as capping agents to prevent crystal growth, and control size and morphology. To be sure, this state-of-the-art combination of functional DNA molecules and inorganic nanomaterials greatly encouraged step towards the development of analytical science, life science, environmental science, and other promising field they can address. DNA-guided nanofabrication will eventually exceed expectations far beyond our scope in the near fu- ture.

Design and Fabrication of Plasmonic Nanostructures with DNA for Surface-Enhanced Raman Spectroscopy Applications

Plasmonic nanostructures display unique and strongly enhanced optical properties, therefore hold great promise for a wide range of spectroscopic applications, particularly surface-enhanced Raman spectroscopy （SERS）. It is well acknowledged that the major contributions to SERS arise from molecules positioned in nanojunctions where the op- tical field is intensively concentrated due to localized surface plasmon excitations. One of the key challenges in SERS therefore lies in the design and fabrication of plasmonic nanostructures with controllable nanojunctions. In recent years, by exploiting the unparalleled base-pairing self-recognition properties, DNA-mediated assembly has emerged as a powerful and programmable tool for the accurate construction of complex and hierarchical plasmonic nanostructures with well-defined geometry and topology. In this review, we will summarize recent advances on de- sign and fabrication of a rich variety of plasmonic nanostructures by virtue of DNA nanotechnology, and discuss their optical properties as well as applications in SERS.

Self-assembly of Micrometer-long DNA Nanoribbons with Four Oligonucleotides

DNA nanostructures have found widespread applications in areas including nanoelectronics and biomedicine.However,traditional DNA origami needs a long single-stranded virus DNA and hundreds of short DNA strands,which make this method complicated and money-consuming.Here,we present a protocol for the assembly of DNA nanoribbons with only four oligonucleotides.DNA nanoribbons with different dimensions were successfully assembled with a 96-base scafford strand and three short staples.These biotinylated nanoribbons could also be decorated with streptavidins.This approach suggests that there exist great design spaces for the creation of simple nucleic acid nanostructures which could facilitate their application in plasmonic or drug delivery.