共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
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
3.
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
4.
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
5.
Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided. 相似文献
6.
Advances in nanomanipulation techniques have made it possible to measure the response of an individual biomolecule to a force applied in the laboratory. Experiments that stretch a single molecule of duplex DNA have been difficult to interpret theoretically, particularly as the major changes in molecular structure caused by the force cannot be measured. In principle, computer simulation can calculate these conformational changes in atomic level detail, but to date such studies have failed to reproduce the experimental data due to the computational expense of the calculations. Here we show that a combination of molecular modeling and statistical physics can be used successfully to understand the stretching behavior of DNA. Our simulations provide new information about the dynamics of DNA denaturation under force in atomic level detail and also show the importance of entropy in determining biomechanical properties in general. 相似文献
7.
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
8.
Alexey V. Shvetsov Dmitry V. Lebedev Daria B. Chervyakova Irina V. Bakhlanova Igor A. Yung Aurel Radulescu Aleksandr I. Kuklin Dmitry M. Baitin Vladimir V. Isaev-Ivanov 《FEBS letters》2014
Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations. 相似文献
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10.
Trabuco LG Schreiner E Gumbart J Hsin J Villa E Schulten K 《Journal of structural biology》2011,173(3):420-427
In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex. 相似文献
11.
For the purpose of molecular dynamics simulations of large biopolymers we have built a parallel computer with a systolic loop architecture, based on Transputers as computational units, and have programmed it in occam II. The computational nodes of the computer are linked together in a systolic ring. The program based on this topology for large biopolymers increases its computational throughput nearly linearly with the number of computational nodes. The program developed is closely related to the simulation programs CHARMM and XPLOR, the input files required (force field, protein structure file, coordinates) and output files generated (sets of atomic coordinates representing dynamic trajectories and energies) are compatible with the corresponding files of these programs. Benchmark results of simulations of biopolymers comprising 66, 568, 3 634, 5 797 and 12 637 atoms are compared with XPLOR simulations on conventional computers (Cray, Convex, Vax). These results demonstrate that the software and hardware developed provide extremely cost effective biopolymer simulations. We present also a simulation (equilibrium of X-ray structure) of the complete photosynthetic reaction center of Rhodopseudomonas viridis (12 637 atoms). The simulation accounts for the Coulomb forces exactly, i.e. no cut-off had been assumed. 相似文献
12.
Programmed self-assembly of DNA enables the rational design of megadalton-scale macromolecular assemblies with sub-nanometer scale precision. These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient in silico evaluation of nucleic acid nanostructures for diverse light-harvesting and photonic applications. 相似文献
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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
15.
Arivazhagan Rajendran Masayuki Endo Kumi Hidaka Phong Lan Thao Tran Jean-Louis Mergny Hiroshi Sugiyama 《Nucleic acids research》2013,41(18):8738-8747
Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. 相似文献
16.
《Journal of molecular biology》2022,434(2):167351
Building structural models of entire cells has been a long-standing cross-discipline challenge for the research community, as it requires an unprecedented level of integration between multiple sources of biological data and enhanced methods for computational modeling and visualization. Here, we present the first 3D structural models of an entire Mycoplasma genitalium (MG) cell, built using the CellPACK suite of computational modeling tools. Our model recapitulates the data described in recent whole-cell system biology simulations and provides a structural representation for all MG proteins, DNA and RNA molecules, obtained by combining experimental and homology-modeled structures and lattice-based models of the genome. We establish a framework for gathering, curating and evaluating these structures, exposing current weaknesses of modeling methods and the boundaries of MG structural knowledge, and visualization methods to explore functional characteristics of the genome and proteome. We compare two approaches for data gathering, a manually-curated workflow and an automated workflow that uses homologous structures, both of which are appropriate for the analysis of mesoscale properties such as crowding and volume occupancy. Analysis of model quality provides estimates of the regularization that will be required when these models are used as starting points for atomic molecular dynamics simulations. 相似文献
17.
Mahmoud Moradi Volodymyr Babin Christopher Roland Celeste Sagui 《Nucleic acids research》2013,41(1):33-43
Since its discovery in 1979, left-handed Z-DNA has evolved from an in vitro curiosity to a challenging DNA structure with crucial roles in gene expression, regulation and recombination. A fundamental question that has puzzled researchers for decades is how the transition from B-DNA, the prevalent right-handed form of DNA, to Z-DNA is accomplished. Due to the complexity of the B–Z-DNA transition, experimental and computational studies have resulted in several different, apparently contradictory models. Here, we use molecular dynamics simulations coupled with state-of-the-art enhanced sampling techniques operating through non-conventional reaction coordinates, to investigate the B–Z-DNA transition at the atomic level. Our results show a complex free energy landscape, where several phenomena such as over-stretching, unpeeling, base pair extrusion and base pair flipping are observed resulting in interconversions between different DNA conformations such as B-DNA, Z-DNA and S-DNA. In particular, different minimum free energy paths allow for the coexistence of different mechanisms (such as zipper and stretch–collapse mechanisms) that previously had been proposed as independent, disconnected models. We find that the B–Z-DNA transition—in absence of other molecular partners—can encompass more than one mechanism of comparable free energy, and is therefore better described in terms of a reaction path ensemble. 相似文献
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《Biochimica et Biophysica Acta (BBA)/General Subjects》2022,1866(12):130252
BackgroundNon-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome.MethodsTo better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated.ResultsOur results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages.ConclusionsWe showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations.General significanceThe fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms. 相似文献
20.
《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