227 research outputs found
Stretching and unzipping nucleic acid hairpins using a synthetic nanopore
We have explored the electromechanical properties of DNA by using an electric field to force single hairpin molecules to translocate through a synthetic pore in a silicon nitride membrane. We observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the DNA. The threshold for a diameter 1.5 < d < 2.3 nm is V > 1.5 V, which corresponds to the force required to stretch the stem of the hairpin, according to molecular dynamics simulations. On the other hand, for 1.0 < d < 1.5 nm, the threshold voltage collapses to V < 0.5 V because the stem unzips with a lower force than required for stretching. The data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in DNA
Mesoscale simulations of surfactant dissolution and mesophase formation
The evolution of the contact zone between pure surfactant and solvent has
been studied by mesoscale simulation. It is found that mesophase formation
becomes diffusion controlled and follows the equilibrium phase diagram
adiabatically almost as soon as individual mesophases can be identified,
corresponding to times in real systems of order 10 microseconds.Comment: 4 pages, 2 figures, ReVTeX
Transport in one dimensional Coulomb gases: From ion channels to nanopores
We consider a class of systems where, due to the large mismatch of dielectric
constants, the Coulomb interaction is approximately one-dimensional. Examples
include ion channels in lipid membranes and water filled nanopores in silicon
or cellulose acetate films. Charge transport across such systems possesses the
activation behavior associated with the large electrostatic self-energy of a
charge placed inside the channel. We show here that the activation barrier
exhibits non-trivial dependence on the salt concentration in the surrounding
water solution and on the length and radius of the channel.Comment: New references are have been added and discussed. 18 pages, 8 figure
Ionic conductivity, structural deformation, and programmable anisotropy of DNA origami in electric field.
The DNA origami technique can enable functionalization of inorganic structures for single-molecule electric current recordings. Experiments have shown that several layers of DNA molecules, a DNA origami plate, placed on top of a solid-state nanopore is permeable to ions. Here, we report a comprehensive characterization of the ionic conductivity of DNA origami plates by means of all-atom molecular dynamics (MD) simulations and nanocapillary electric current recordings. Using the MD method, we characterize the ionic conductivity of several origami constructs, revealing the local distribution of ions, the distribution of the electrostatic potential and contribution of different molecular species to the current. The simulations determine the dependence of the ionic conductivity on the applied voltage, the number of DNA layers, the nucleotide content and the lattice type of the plates. We demonstrate that increasing the concentration of Mg(2+) ions makes the origami plates more compact, reducing their conductivity. The conductance of a DNA origami plate on top of a solid-state nanopore is determined by the two competing effects: bending of the DNA origami plate that reduces the current and separation of the DNA origami layers that increases the current. The latter is produced by the electro-osmotic flow and is reversible at the time scale of a hundred nanoseconds. The conductance of a DNA origami object is found to depend on its orientation, reaching maximum when the electric field aligns with the direction of the DNA helices. Our work demonstrates feasibility of programming the electrical properties of a self-assembled nanoscale object using DNA.C.Y.L., J.Y. and A.A. were supported in part by the grants from the National Science Foundation
(DMR-0955959, PHY-1430124 and ECC-1227034), and the National Institutes of Health (R01-
HG007406). E.A.H. acknowledges support from Schweizerische Studienstiftung (Swiss Study
Foundation) and Gonville & Caius College. S.H.A. acknowledges support from a Herchel Smith
postdoctoral fellowship. J.K. acknowledges support from Chinese Scholarship Council and Cambridge
Overseas Trust. UFK was supported by an ERC starting grant (PassMembrane, 261101).
The authors gladly acknowledge supercomputer time provided through XSEDE Allocation Grant
MCA05S028 and the Blue Waters Sustained Petascale Computer System (UIUC).This is the accepted manuscript. The final version is available from ACS at pubs.acs.org/doi/abs/10.1021/nn505825z
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Controlling aggregation of cholesterol-modified DNA nanostructures.
DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.This work was supported by: European Research Council (ERC) consolidator grant [DesignerPores 647144 to U.F.K.]. Winton Programme for the Physics of Sustainability; Gates Cambridge; Oppenheimer Ph.D. studentship; and the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 792270 [to K.G.]. Engineering and Physical Sciences Research Council (EPSRC); and the Cambridge Trust Vice Chancellor’s Award [to A.O.]. National Science Foundation (USA) [DMR-1827346]; National Institutes of Health [P41-GM104601]; the supercomputer time provided through XSEDE allocation grant [MCA05S028]; and the Blue Waters petascale supercomputer system (UIUC) [to A.A. and H.J.]. Winton Programme for the Physics of Sustainability; and Engineering and Physical Sciences Research Council (EPSRC) [to D.S.]. The FEI Tecnai G2-Spirit was funded by the Wellcome Trust [090932/Z/09/Z]. Funding for open access charge: ERC [DesignerPores 647144]
Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore
Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments
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Optical Voltage Sensing Using DNA Origami.
We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nanodevices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labeled with a single pair of FRET dyes. The DNA structures were reversibly immobilized on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterized voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures and highlights potential applications of dynamic DNA nanostructures as voltage sensors
Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers : Nanopore Engineering and Characterization
Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function
Nanopore surface coating delivers nanopore size and shape through conductance-based sizing
The performance of nanopore single-molecule sensing elements depends intimately on their physical dimensions and surface chemical properties. These factors underpin the dependence of the nanopore ionic conductance on electrolyte concentration, yet the measured, or modeled, dependence only partially illuminates the details of geometry and surface chemistry. Using the electrolyte-dependent conductance data before and after selective surface functionalization of solid-state nanopores, however, introduces more degrees of freedom and improves the performance of conductance-based nanopore characterizations. Sets of representative nanopore profiles were used to generate conductance data, and the nanopore shape and exact dimensions were identified, through conductance alone, by orders-of-magnitude 3 reductions in the geometry optimization metrics. The optimization framework could similarly be used to evaluate the nanopore surface coating thickness
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