[Show abstract][Hide abstract] ABSTRACT: Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores, short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications for recognition and processing of such modifications by regulatory DNA-binding proteins.
Nucleic Acids Research 11/2015; DOI:10.1093/nar/gkv1199 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA self-assembly has emerged as a new paradigm for design of biomimetic membrane channels. Several experimental groups have already demonstrated assembly and insertion of DNA channels into lipid bilayer membranes, however, the structure of the channels and their conductance mechanism have remained undetermined. Here, we report the results of molecular dynamics simulations that characterized the biophysical properties of the DNA membrane channels with atomic precision. We show that, while overall remaining stable, the local structure of the channels undergoes considerable fluctuations, departing from the idealized design. The transmembrane ionic current flows both through the central pore of the channel as well as along the DNA walls and through the gaps in the DNA structure. Surprisingly, we find the conductance of a DNA channel to depend on the membrane tension, making them potentially suitable for force sensing applications. Finally, we show that electro-osmosis governs the transport of drug-like molecules through the DNA channels.
[Show abstract][Hide abstract] ABSTRACT: Over the past decades, molecular dynamics (MD) simulations of biomolecules
have become a mainstream biophysics technique. As the length and time scales
amenable to the MD method increase, shortcomings of the empirical force
fields---which have been developed and validated using relatively short
simulations of small molecules---become apparent. One common artifact is
aggregation of water-soluble biomolecules driven by artificially strong
charge--charge interactions. Here, we report a systematic refinement of
Lennard-Jones parameters (NBFIX) describing amine--carboxylate and
amine--phosphate interactions, which brings MD simulations of basic
peptide-mediated nucleic acids assemblies and lipid bilayer membranes in better
agreement with experiment. As our refinement neither affects the existing
parameterization of bonded interaction nor does it alter the solvation free
energies, it improves realism of an MD simulation without introducing
[Show abstract][Hide abstract] ABSTRACT: With the aim of developing a DNA sequencing methodology, we theoretically examine the feasibility of using nanoplasmonics to control the translocation of a DNA molecule through a solid-state nanopore and to read off sequence information using surface enhanced Raman spectroscopy. Using molecular dynamics simulations, we show that high-intensity optical hot spots produced by a metallic nanostructure can arrest DNA translocation through a solid-state nanopore, thus providing a physical knob for controlling the DNA speed. Switching the plasmonic field on and off can displace the DNA molecule in discrete steps, sequentially exposing neighboring fragments of a DNA molecule to the pore as well as to the plasmonic hot spot. Surface enhanced Raman scattering from the exposed DNA fragments reports on their nucleotide composition, might allow for identification of the nucleotide sequence of a DNA molecule transported through the hot spot. The principles of plasmonic-nanopore sequencing can be extended to detection of DNA modifications and RNA characterization.
[Show abstract][Hide abstract] ABSTRACT: Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillararenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10(-14) cm(3)/s or 3.5(±1.0) × 10(8) water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10(8) water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10(8) water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10(7) water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10(5) pores per μm(2)) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10(3) pores per μm(2)). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.
Proceedings of the National Academy of Sciences 07/2015; 112(32). DOI:10.1073/pnas.1508575112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases.
[Show abstract][Hide abstract] ABSTRACT: Voltage-driven transport of double-stranded DNA through nanoscale pores holds much potential for applications in quantitative molecular biology and biotechnology, yet the microscopic details of translocation have proven to be challenging to decipher. Earlier experiments showed strong dependence of transport kinetics on pore size: fast regular transport in large pores (> 5 nm diameter), and slower yet heterogeneous transport time distributions in sub-5 nm pores, which imply a large positional uncertainty of the DNA in the pore as a function of the translocation time. In this work, we show that this anomalous transport is a result of DNA self-interaction, a phenomenon that is strictly pore-diameter dependent. We identify a regime in which DNA transport is regular, producing narrow and well-behaved dwell-time distributions that fit a simple drift-diffusion theory. Furthermore, a systematic study of the dependence of dwell time on DNA length reveals a single power-law scaling of 1.37 in the range of 35–20,000 bp. We highlight the resolution of our nanopore device by discriminating via single pulses 100 and 500 bp fragments in a mixture with >98% accuracy. When coupled to an appropriate sequence labeling method, our observation of smooth DNA translocation can pave the way for high-resolution DNA mapping and sizing applications in genomics.
[Show abstract][Hide abstract] ABSTRACT: Control over interactions with biomolecules holds the key to applications of graphene in biotechnology. One such application is nanopore sequencing, where a DNA molecule is electrophoretically driven through a graphene nanopore. Here we investigate how interactions of single-stranded DNA and a graphene membrane can be controlled by electrically biasing the membrane. The results of our molecular dynamics simulations suggest that electric charge on graphene can force a DNA homopolymer to adopt a range of strikingly different conformations. The conformational response is sensitive to even very subtle nucleotide modifications, such as DNA methylation. The speed of DNA motion through a graphene nanopore is strongly affected by the graphene charge: a positive charge accelerates the motion, whereas a negative charge arrests it. As a possible application of the effect, we demonstrate stop-and-go transport of DNA controlled by the charge of graphene. Such on-demand transport of DNA is essential for realizing nanopore sequencing.
[Show abstract][Hide abstract] ABSTRACT: Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.
[Show abstract][Hide abstract] ABSTRACT: A simple coarse-grained model of single-stranded DNA (ssDNA) was developed, featuring only two sites per nucleotide that represent the centers of mass of the backbone and sugar/base groups. In the model, the interactions between sites are described using tabulated bonded potentials optimized to reproduce the solution structure of DNA observed in atomistic molecular dynamics simulations. Isotropic potentials describe nonbonded interactions, implicitly taking into account the solvent conditions to match the experimentally determined radius of gyration of ssDNA. The model reproduces experimentally measured force-extension dependence of an unstructured DNA strand across 2 orders of magnitude of the applied force. The accuracy of the model was confirmed by measuring the end-to-end distance of a dT14 fragment via FRET while stretching the molecules using optical tweezers. The model offers straightforward generalization to systems containing double-stranded DNA and DNA binding proteins.
Journal of Chemical Theory and Computation 08/2014; 10(8):2891-2896. DOI:10.1021/ct500193u · 5.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Local modulation of temperature has emerged as a new mechanism for regulation of molecular transport through nanopores. Predicting the effect of such modulations on nanopore transport requires simulation protocols capable of reproducing non-uniform temperature gradients observed in experiment. Conventional molecular dynamics (MD) method typically employs a single thermostat for maintaining a uniform distribution of temperature in the entire simulation domain, and, therefore, can not model local temperature variations. In this article, we describe a set of simulation protocols that enable modeling of nanopore systems featuring non-uniform distributions of temperature. First, we describe a method to impose a temperature gradient in all-atom MD simulations based on a boundary-driven non-equilibrium MD protocol. Then, we use this method to study the effect of temperature gradient on the distribution of ions in bulk solution (the thermophoretic effect). We show that DNA nucleotides exhibit differential response to the same temperature gradient. Next, we describe a method to directly compute the effective force of a thermal gradient on a prototypical biomolecule—a fragment of double-stranded DNA. Following that, we demonstrate an all-atom MD protocol for modeling thermophoretic effects in solid-state nanopores. We show that local heating of a nanopore volume can be used to regulate the nanopore ionic current. Finally, we show how continuum calculations can be coupled to a coarse-grained model of DNA to study the effect of local temperature modulation on electrophoretic motion of DNA through plasmonic nanopores. The computational methods described in this article are expected to find applications in rational design of temperature-responsive nanopore systems.
[Show abstract][Hide abstract] ABSTRACT: PEGylation, or addition of poly(ethylene glycol) chains to proteins, is widely used to improve delivery in pharmaceutical applications. Recent studies suggest that stabilization of a protein by PEG, and hence its proteolytic degradability, are sequence-dependent and require only short PEG chains. Here we connect stabilization by short PEG chains directly to the structural dynamics of the protein and PEG chain. We measured the stability of human Pin1 WW domain with PEG-4 at asparagine 19 for a full mutant cycle at two positions thought to influence PEG-protein interaction: Ser16Ala and Tyr23Phe. We then performed explicit solvent molecular dynamics simulations on all PEGylated and PEG-free mutants. The mutant cycle yields a non-additive stabilization effect where the pseudo wild type and double mutant are more stabilized relative to unPEGylated proteins than are the two single mutants. The simulation reveals why: the double mutant suffers loss of beta sheet structure, which PEGylation restores even though the PEG extends as a coil into the solvent. In contrast, in one of the single mutants PEG preferentially interacts with the protein surface while disrupting the interactions of its asparagine host with a nearby methionine side chain. Thus PEG attachment can stabilize a protein differentially depending on the local sequence, and either by interacting with the surface, or by extending into the solvent. A simulation with PEG-45 attached to asparagine 19 shows that PEG even can do both in the same context.
The Journal of Physical Chemistry B 05/2014; 118(28). DOI:10.1021/jp502234s · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rectifying nanopores feature ion currents that are higher for voltages of one polarity compared to the currents recorded for corresponding voltages of the opposite polarity. Rectification of nanopores has been found to depend on the pore opening diameter and distribution of surface charges on the pore walls as well as pore geometry. Very little is known, however, on the dependence of ionic rectification on the type of transported ions of the same charge. We performed experiments with single conically shaped nanopores in a polymer film and recorded current–voltage curves in three electrolytes: LiCl, NaCl, and KCl. Rectification degrees of the pores, quantified as the ratio of currents recorded for voltages of opposite polarities, were the highest for KCl and the lowest for LiCl. The experimental observations could not be explained by a continuum modeling based on the Poisson–Nernst–Planck equations. All-atom molecular dynamics simulations revealed differential binding between Li+, Na+, and K+ ions and carboxyl groups on the pore walls, resulting in changes to both the effective surface charge of the nanopore and cation mobility within the pore.
The Journal of Physical Chemistry C 04/2014; 118(18):9809–9819. DOI:10.1021/jp501492g · 4.77 Impact Factor