What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study.

The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).

BROMOC suite: Monte Carlo/Brownian dynamics suite for studies of ion permeation and DNA transport in biological and artificial pores with effective potentials.

The transport of ions and solutes by biological pores is central for cellular processes and has a variety of applications in modern biotechnology. The time scale involved in the polymer transport across a nanopore is beyond the accessibility of conventional MD simulations. Moreover, experimental studies lack sufficient resolution to provide details on the molecular underpinning of the transport mechanisms. BROMOC, the code presented herein, performs Brownian dynamics simulations, both serial and parallel, up to several milliseconds long. BROMOC can be used to model large biological systems. IMC-MACRO software allows for the development of effective potentials for solute-ion interactions based on radial distribution function from all-atom MD. BROMOC Suite also provides a versatile set of tools to do a wide variety of preprocessing and postsimulation analysis. We illustrate a potential application with ion and ssDNA transport in MspA nanopore.

Effect of confinement on DNA, solvent and counterion dynamics in a model biological nanopore.

The application of recent advances in nanopore technology to high-throughput DNA sequencing requires a more detailed understanding of solvent, ion and DNA interactions occurring within these pores. Here we present a combination of atomistic and coarse-grained modeling studies of the dynamics of short single-stranded DNA (ssDNA) homopolymers within the alpha-hemolysin pore, for the two single-stranded homopolymers poly(dA)40 and poly(dC)40. Analysis of atomistic simulations along with the per-residue decomposition of protein-DNA interactions in these simulations gives new insight into the very complex issues that have yet to be fully addressed with detailed MD simulations. We discuss a modification of the solvent properties and ion distribution around DNA within nanopore confinement and put it into the general framework of counterion condensation theory. There is a reasonable agreement in computed properties from our all-atom simulations and the resulting predictions from analytical theories with experimental data, and our equilibrium results here support the conclusions from our previous non-equilibrium Brownian dynamics studies with a recently developed BROMOC protocol that cations are the primary charge carriers through alpha-hemolysin nanopores under an applied voltage in the presence of ssDNA. Clustering analysis led to an identification of distinct conformational states of captured polymer and depth of the current blockade. Therefore, our data suggest that confined polymer may act as a flickering gate, thus contributing to excess noise phenomena. We also discuss the extent of water structuring due to nanopore confinement and the relationship between the conformational dynamics of a captured polymer and the distribution of blocked current.

Microsecond simulations of DNA and ion transport in nanopores with novel ion-ion and ion-nucleotides effective potentials.

We developed a novel scheme based on the grand-canonical Monte Carlo/Brownian dynamics simulations and have extended it to studies of ion currents across three nanopores with the potential for single-stranded DNA (ssDNA) sequencing: solid-state nanopore Si3N4, α-hemolysin, and E111N/M113Y/K147N mutant. To describe nucleotide-specific ion dynamics compatible with ssDNA coarse-grained model, we used the inverse Monte Carlo protocol, which maps the relevant ion-nucleotide distribution functions from all-atom molecular dynamics (MD) simulations. Combined with the previously developed simulation platform for Brownian dynamics simulations of ion transport, it allows for microsecond- and millisecond-long simulations of ssDNA dynamics in the nanopore with a conductance computation accuracy that equals or exceeds that of all-atom MD simulations. In spite of the simplifications, the protocol produces results that agree with the results of previous studies on ion conductance across open channels and provide direct correlations with experimentally measured blockade currents and ion conductances that have been estimated from all-atom MD simulations.

BROMOC-D: Brownian Dynamics/Monte-Carlo Program Suite to Study Ion and DNA Permeation in Nanopores.

A theoretical framework is presented to model ion and DNA translocation across a nanopore confinement under an applied electric field. A combined Grand Canonical Monte Carlo Brownian Dynamics (GCMC/BD) algorithm offers a general approach to study ion permeation through wide molecular pores with a direct account of ion-ion and ion-DNA correlations. This work extends previously developed theory by incorporating the recently developed coarse-grain polymer model of DNA by de Pablo and colleagues [Knotts, T. A.; Rathore, N.; Schwartz, D. C.; de Pablo, J. J. J. Chem. Phys. 2007, 126] with explicit ions for simulations of polymer dynamics. Atomistic MD simulations were used to guide model developments. The power of the developed scheme is illustrated with studies of single-stranded DNA (ss-DNA) oligomer translocation in two model cases: a cylindrical pore with a varying radius and a well-studied experimental system, the staphylococcal α-hemolysin channel. The developed model shows good agreement with experimental data for model studies of two homopolymers: ss-poly(dA)(n) and ss-poly(dC)(n). The developed protocol allows for direct evaluation of different factors (charge distribution and pore shape and size) controlling DNA translocation in a variety of nanopores.