Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Effects of Nanopore Charge Decorations on the Translocation Dynamics of DNA.

ABSTRACT: We have investigated the dynamics of single-stranded DNA as it translocates through charge-mutated protein nanopores. Translocation of DNA is a crucial step in nanopore-based sequencing platforms, where control over translocation speed remains one of the main challenges. Taking advantage of the interactions between negatively charged DNA and positively charged amino acid residues, the translocation speed of DNA can be manipulated by deliberate charge decorations inside the nanopore. We employed coarse-grained Langevin dynamics simulations to monitor the step-by-step movement of DNA through different mutations of ?-hemolysin protein nanopores. We found that although the average translocation time per nucleotide is longer, in agreement with experiments, the DNA nucleotides do not translocate with a uniform speed. Furthermore, the location and spacing of the charge decorations can alter the translocation dynamics significantly, trapping DNA in some cases. Our findings can give insights when designing charge patterns in nanopores.

SUBMITTER: Jou I

PROVIDER: S-EPMC5647626 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Effects of Nanopore Charge Decorations on the Translocation Dynamics of DNA.

Jou Ining I Muthukumar Murugappan M

Biophysical journal 20171001 8

We have investigated the dynamics of single-stranded DNA as it translocates through charge-mutated protein nanopores. Translocation of DNA is a crucial step in nanopore-based sequencing platforms, where control over translocation speed remains one of the main challenges. Taking advantage of the interactions between negatively charged DNA and positively charged amino acid residues, the translocation speed of DNA can be manipulated by deliberate charge decorations inside the nanopore. We employed ...[more]

PMID: 29045861

Similar Datasets

DNA translocation and unzipping through a nanopore: some geometrical effects.

Project description:This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

| S-EPMC2872221 | biostudies-literature

Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers.

Project description:Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.

| S-EPMC5692567 | biostudies-literature

Effect of charge patterns along a solid-state nanopore on polyelectrolyte translocation.

Project description:We investigate the effectiveness of charge patterns along a nanopore on translocation dynamics of a flexible polyelectrolyte. We perform a three dimensional Langevin dynamics simulation of a uniformly charged flexible polyelectrolyte translocating under uniform external electric field through a solid-state nanopore. We maintain the total charge along the pore to be constant, while varying its distribution by placing alternate charged and uncharged sections of different lengths along the pore length. Longest average translocation time is observed for a pattern corresponding to an optimum section length, with a major delay in the translocation time during the pore ejection stage. This optimum section length is independent of lengths of polyelectrolyte and pore within the range studied. A theory based on the Fokker-Planck formalism is found to successfully describe the observed trends with reasonable quantitative agreement.

| S-EPMC3976468 | biostudies-other

Enzyme-modulated DNA translocation through a nanopore.

Project description:We present a Langevin dynamics simulation study of enzyme-modulated translocation of a single-stranded DNA molecule through a cylindrical nanopore. The toroidal-shaped enzyme placed along the axis of the pore, threads a DNA molecule at a constant rate. As a result of this controlled release process, the length of DNA available for translocation varies with time. We examine the effect of time-dependent conformational entropy of the DNA on the translocation process. In addition, we also examine the effects of both the separation between the exonuclease and the pore, and the rate at which DNA is released by the enzyme. Our results indicate that the separation distance primarily influences the entry of the DNA into the pore. The length of the DNA released by the exonuclease that is most likely to enter the pore is nearly equal to separation distance between the pore and the exonuclease despite the flexibility of the polymer. However, the speed at which the DNA translocates through the nanopore is solely determined by the rate at which the exonuclease releases the DNA. We find that the translocation velocity is directly proportional to the rate of release.

| S-EPMC2797554 | biostudies-literature

Measurement of DNA Translocation Dynamics in a Solid-State Nanopore at 100 ns Temporal Resolution.

Project description:Despite the potential for nanopores to be a platform for high-bandwidth study of single-molecule systems, ionic current measurements through nanopores have been limited in their temporal resolution by noise arising from poorly optimized measurement electronics and large parasitic capacitances in the nanopore membranes. Here, we present a complementary metal-oxide-semiconductor (CMOS) nanopore (CNP) amplifier capable of low noise recordings at an unprecedented 10 MHz bandwidth. When integrated with state-of-the-art solid-state nanopores in silicon nitride membranes, we achieve an SNR of greater than 10 for ssDNA translocations at a measurement bandwidth of 5 MHz, which represents the fastest ion current recordings through nanopores reported to date. We observe transient features in ssDNA translocation events that are as short as 200 ns, which are hidden even at bandwidths as high as 1 MHz. These features offer further insights into the translocation kinetics of molecules entering and exiting the pore. This platform highlights the advantages of high-bandwidth translocation measurements made possible by integrating nanopores and custom-designed electronics.

| S-EPMC5312517 | biostudies-literature

Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers.

Project description:Motor enzymes that process nucleic-acid substrates play vital roles in all aspects of genome replication, expression, and repair. The DNA and RNA nucleobases are known to affect the kinetics of these systems in biologically meaningful ways. Recently, it was shown that DNA bases control the translocation speed of helicases on single-stranded DNA, however the cause of these effects remains unclear. We use single-molecule picometer-resolution nanopore tweezers (SPRNT) to measure the kinetics of translocation along single-stranded DNA by the helicase Hel308 from Thermococcus gammatolerans. SPRNT can measure enzyme steps with subangstrom resolution on millisecond timescales while simultaneously measuring the absolute position of the enzyme along the DNA substrate. Previous experiments with SPRNT revealed the presence of two distinct substates within the Hel308 ATP hydrolysis cycle, one [ATP]-dependent and the other [ATP]-independent. Here, we analyze in-depth the apparent sequence dependent behavior of the [ATP]-independent step. We find that DNA bases at two sites within Hel308 control sequence-specific kinetics of the [ATP]-independent step. We suggest mechanisms for the observed sequence-specific translocation kinetics. Similar SPRNT measurements and methods can be applied to other nucleic-acid-processing motor enzymes.

| S-EPMC6412116 | biostudies-literature

A nanopore-nanofiber mesh biosensor to control DNA translocation.

Project description:Solid-state nanopores show promise as single-molecule sensors for biomedical applications, but to increase their resolution and efficiency, analyte molecules must remain longer in the nanopore sensing volume. Here we demonstrate a novel, facile, and customizable nanopore sensor modification that reduces the double-stranded DNA translocation velocity by 2 orders of magnitude or more via interactions outside the nanopore. This is achieved by electrospinning a copolymer nanofiber mesh (NFM) directly onto a solid-state nanopore (NP) chip. The effect of NFMs on dsDNA translocation through an NP is highlighted using a set of NFMs of varying mesh composition that reduce the translocation speed relative to a bare pore from 1- to >100-fold. A representative NFM from this set is effective on DNA as long as 20 kbp, improves the nanopore resolution, and allows discrimination among different DNA lengths.

| S-EPMC4039743 | biostudies-literature

DNA translocation through hydrophilic nanopore in hexagonal boron nitride.

Project description:Ultra-thin solid-state nanopore with good wetting property is strongly desired to achieve high spatial resolution for DNA sequencing applications. Atomic thick hexagonal boron nitride (h-BN) layer provides a promising two-dimensional material for fabricating solid-state nanopores. Due to its good oxidation resistance, the hydrophilicity of h-BN nanopore device can be significantly improved by UV-Ozone treatment. The contact angle of a KCl-TE droplet on h-BN layer can be reduced from 57° to 26° after the treatment. Abundant DNA translocation events have been observed in such devices, and strong DNA-nanopore interaction has been revealed in pores smaller than 10 nm in diameter. The 1/f noise level is closely related to the area of suspended h-BN layer, and it is significantly reduced in smaller supporting window. The demonstrated performance in h-BN nanopore paves the way towards base discrimination in a single DNA molecule.

| S-EPMC3836030 | biostudies-literature

Quantitative analysis of the nanopore translocation dynamics of simple structured polynucleotides.

Project description:Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using ?-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through ?-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding.

| S-EPMC3250678 | biostudies-literature

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation.

Project description:Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.

| S-EPMC3390175 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data