共查询到20条相似文献,搜索用时 15 毫秒
1.
Anastasia Shapiro Avital Hozeh Olga Girshevitz Almogit Abu-Horowitz Ido Bachelet 《Nucleic acids research》2015,43(13):6587-6595
DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations. 相似文献
2.
《Biophysical journal》2022,121(24):4882-4891
DNA nanotechnology has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resolution. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equivalent charge surplus as a discriminant. We compare the assembly yield of four supramolecular DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and atomic force microscopy imaging. We demonstrate that the nanopore system can provide analytical quantification of the complex supramolecular nanostructures within minutes, without any need for labeling and with single-molecule resolution. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the analysis and manipulation of large DNA assemblies in real time. 相似文献
3.
Castro CE Kilchherr F Kim DN Shiao EL Wauer T Wortmann P Bathe M Dietz H 《Nature methods》2011,8(3):221-229
Molecular self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA origami objects. We also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure. 相似文献
4.
DNA is a useful material for constructing nanoscale structures in nearly any three-dimensional (3D) shape desired. The DNA nanostructure can also be equipped with specific docking sites for proteins. Cellular processes and chemical transformations take place in several reaction steps. Multiple enzymes cooperate in specific fashion to catalyze the sequential chemical transformation steps. Such natural systems are effectively reconstructed in vitro if the individual enzymes locate in the correct relative orientations. DNA-origami structures can be used as “molecular switchboards” to arrange enzymes and other proteins with nanometer-scale precision. A new method was developed for locating the proteins by means of special “adapters” known as zinc-finger proteins based only on proteins. Zinc fingers are suitable site-selective adapters for targeting specific locations within DNA-origami structures. Several different adapters carrying different proteins can independently bind at defined locations on this type of nanostructure. A basic leucine zipper (bZIP) protein is also a candidate for the site-selective adaptor. A well-characterized bZIP protein GCN4 was chosen as an adaptor for specific addresses. Analyses by atomic force microscopy and gel electrophoreses demonstrate specific binding of GCN4 adaptor to the addresses containing the GCN4 binding sites on DNA origami. The adaptor derived from GCN4 and that form a zinc-finger protein zif268, for which we have reported previously, acted as orthogonal adaptors to the respective addresses on DNA origami. Therefore, these orthogonal adaptors would be useful to place multiple engineered proteins at different addresses on DNA origami. Especially, the homodimeric nature of GCN4 adaptor is indispensable for constructing the assembly of the naturally abundant dimeric proteins and/or enzymes to efficiently carry out chemical reactions and signal transductions in vitro on DNA origami. 相似文献
5.
Na Wu Qi Wang Xingfei Zhou Si Si Jia Youjie Fan Jun Hu Bin Li 《Journal of molecular recognition : JMR》2013,26(12):700-704
DNA origami shows tremendous promise as templates for the assembly of nano‐components and detection of molecular recognition events. So far, the method of choice for evaluating these structures has been atomic force microscopy (AFM), a powerful tool for imaging nanoscale objects. In most cases, tethered targets on DNA origami have proven to be highly effective samples for investigation. Still, while maximal assembly of the nanostructures might benefit from the greatest flexibility in the tether, AFM imaging requires a sufficient stability of the adsorbed components. The balance between the tether flexibility and sample stability is a major, poorly understood, concern in such studies. Here, we investigated the dependence of the tethering length on molecular capture events monitored by AFM. In our experiments, single biotin molecules were attached to DNA origami templates with various linker lengths of thymidine nucleotides, and their interaction with streptavidin was observed with AFM. Our results show that the streptavidin‐biotin complexes are easily detected with short tethered lengths, and that their morphological features clearly change with the tethering length. We identify the functionally useful tether lengths for these investigations, which are also expected to prove useful in the construction and further application of DNA origami in bio‐nanotechnology studies. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
6.
DNA纳米技术是基于沃森克里克碱基配对原则产生可编程核酸结构的技术。因其具有高精度的工程设计、前所未有的可编程性和内在的生物相容性等特点,运用该技术合成的纳米结构不仅可以与小分子、核酸、蛋白质、病毒和癌细胞相互作用,还可以作为纳米载体,递送不同的治疗药物。DNA折纸作为一种有效的、多功能的方法来构建二维和三维可编程的纳米结构,是DNA纳米技术发展的一个里程碑。由于其高度可控的几何形状、空间寻址性、易于化学修饰,DNA折纸在许多领域具有巨大的应用潜力。本文通过介绍DNA折纸的起源、基本原理和目前进展,归纳总结了运用DNA折纸进行药物装载和释放的方式,并基于此技术,展望了今后的发展趋势以及所面临的机遇和挑战。 相似文献
7.
《Biophysical journal》2022,121(24):4810-4818
DNA nanotechnology facilitates the synthesis of biomimetic models for studying biological systems. This work uses lipid bilayers as platforms for two-dimensional single-particle tracking of the dynamics of DNA nanostructures. Three different DNA origami structures adhere to the membrane through hybridization with cholesterol-modified strands. Their two-dimensional diffusion coefficient is modulated by changing the concentration of monovalent and divalent salts and the number of anchors. In addition, the diffusion coefficient is tuned by targeting cholesterol-modified anchor strands with strand-displacement reactions. We demonstrate a responsive system with changing diffusivity by selectively displacing membrane-bound anchor strands. We also show the programmed release of origami structures from the lipid membranes. 相似文献
8.
Vikash Verma Leena Mallik Rizal F. Hariadi Sivaraj Sivaramakrishnan Georgios Skiniotis Ajit P. Joglekar 《PloS one》2015,10(9)
DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. 相似文献
9.
The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain. 相似文献
10.
《Biophysical journal》2022,121(24):4840-4848
The correlation between genetic information and characteristics of a living cell—its genotype and its phenotype—constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a “1” and its absence to a “0.” The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information (“genotype”) and cluster morphology (“phenotype”) thus reproduces key properties of genotype-phenotype maps known from living systems. 相似文献
11.
Arivazhagan Rajendran Kirankumar Krishnamurthy Amulya Giridasappa Eiji Nakata Takashi Morii 《Nucleic acids research》2021,49(14):7884
The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner. 相似文献
12.
Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array–array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails. 相似文献
13.
Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. 相似文献
14.
Jahn K Tørring T Voigt NV Sørensen RS Bank Kodal AL Andersen ES Gothelf KV Kjems J 《Bioconjugate chemistry》2011,22(4):819-823
We demonstrate here a rapid and cost-effective technique for nanoscale patterning of functional molecules on the surface of a DNA origami. The pattern is created enzymatically by transferring a functionalized dideoxynucleotide to the 3'-end of an arbitrary selected set of synthetic DNA oligonucleotides positioned approximately 6 nm apart in a 70 × 100 nm(2) rectangular DNA origami. The modifications, which are performed in a single-tube reaction, provide an origami surface modified with a variety of functional groups including chemical handles, fluorescent dyes, or ligands for subsequent binding of proteins. Efficient labeling and patterning was demonstrated by gel electrophoresis shift assays, reverse-phase HPLC, mass spectrometry, atomic force microscopy (AFM) analysis, and fluorescence measurements. The results show a very high yield of oligonucleotide labeling and incorporation in the DNA origami. This method expands the toolbox for constructing several different modified DNA origami from the same set of staple strands. 相似文献
15.
Multicomponent reactions are difficult synthetic transformations. For DNA, there is a special opportunity to align multiple strands in a folded nanostructure, so that they are preorganized to give a specific sequence. Multistrand reactions in DNA origami structures have previously been performed using photochemical crosslinking, 1,3‐diploar cycloadditions or phosphoramidate‐forming reactions. Here we report carbodiimide‐driven phosphodiester formation in a small origami sheet that produces DNA strands up to 600 nucleotides in length in a single step. The method uses otherwise unmodified oligodeoxynucleotides with a 5′‐terminal phosphate as starting materials. Compared to an enzymatic multistrand ligation involving linear duplexes, the carbodiimide‐driven ligation gave fewer side products, as detected by gel electrophoresis. The full‐length 600mer product was successfully amplified by polymerase chain reaction. 相似文献
16.
17.
《Biophysical journal》2022,121(11):2127-2134
Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25–75% loop formation, we found the persistence length to be 1.9–4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA. 相似文献
18.
Interlock is a structural element in DNA G-quadruplexes that can be compared with the commonly used complementary binding of ‘sticky ends’ in DNA duplexes. G-quadruplex interlocking can be a basis for the assembly of higher-order structures. In this study, we formulated a rule to engineer (3 + 1) interlocked dimeric G-quadruplexes and established the folding topology of the designed DNA sequences by nuclear magnetic resonance spectroscopy. These interlocked G-quadruplexes are very stable and can serve as compact robust scaffolds for various applications. Different structural elements can be engineered in these robust scaffolds. We demonstrated the anti-HIV inhibition activity of the newly designed DNA sequences. 相似文献
19.
The molecular chaperone concept 总被引:29,自引:0,他引:29
R J Ellis 《Seminars in cell biology》1990,1(1):1-9
Molecular chaperones are a ubiquitous family of cellular proteins which mediate the correct folding of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures. Known chaperones do not possess steric information for protein folding but inhibit unproductive folding and assembly pathways which would otherwise act as dead-end kinetic traps and produce incorrect structures. Chaperones function by binding specifically and non-covalently to interactive protein surfaces that are exposed transiently during cellular processes such as protein synthesis, protein transport across membranes, DNA synthesis, the recycling of clathrin cages, the assembly of organellar complexes from imported subunits, and stress responses. This binding is reversed under circumstances which favour correct interactions and in some cases ATP hydrolysis is involved in this reversal. Some chaperones bind specifically to a structural feature present in a wide range of unrelated proteins that is accessible only during the early stages of folding. The nature of this structural feature is unknown, but its identification is an important goal of current research. Knowledge of chaperone function may be important for the production of proteins for biotechnological purposes since in some cases chaperones may improve the yield of functional product. It is likely that chaperone diseases exist which result from the failure of certain proteins to fold correctly due to changes in chaperone structure. 相似文献
20.
Eldad Ben-Ishay Almogit Abu-Horowitz Ido Bachelet 《Journal of visualized experiments : JoVE》2013,(77)
Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (>7,000 bases) single-stranded DNA ''scaffold'' is directed to a desired shape by hundreds of short complementary strands termed ''staples''. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is ''robotic'' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings. 相似文献