Free-Energy Calculations Reveal the Subtle Differences in the Interactions of DNA Bases with α-Hemolysin

Abstract

Next generation DNA sequencing methods that utilize protein nanopores have the potential to revolutionize this area of biotechnology. While the technique is underpinned by simple physics, the wild-type protein pores do not have all of the desired properties for efficient and accurate DNA sequencing. Much of the research efforts have focused on protein nanopores, such as α-hemolysin from Staphylococcus aureus. However, the speed of DNA translocation has historically been an issue, hampered in part by incomplete knowledge of the energetics of translocation. Here we have utilized atomistic molecular dynamics simulations of nucleotide fragments in order to calculate the potential of mean force (PMF) through α-hemolysin. Our results reveal specific regions within the pore that play a key role in the interaction with DNA. In particular, charged residues such as D127 and K131 provide stabilizing interactions with the anionic DNA and therefore are likely to reduce the speed of translocation. These regions provide rational targets for pore optimization. Furthermore, we show that the energetic contributions to the protein-DNA interactions are a complex combination of electrostatics and short-range interactions, often mediated by water molecules.

DNA sequencing with MspA: Molecular Dynamics simulations reveal free-energy differences between sequencing and non-sequencing mutants

“…Hence, this reduction in the system size leads to a decrease in the required simulation time, rendering our extensive free energy calculations feasible. As the protein is known to be conformationally stable, we do not expect the use of restraints on the protein to influence the PMF, indeed we have previously used a similar approach to calculate PMF profiles for DNA bases through alpha hemolysin 10 . Examples of sampling and convergence testing are provided in figures S1-S5 of the supplementary information .…”

“…Specifically, umbrella sampling calculations and WHAM analysis have been used to construct potential of mean force profiles. We have previously used a similar approach to study DNA base and phosphate group permeation through alpha hemolysin 10 .…”

Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

“…Atomistic simulations have provided vital details to improve the design of the nanopores used for sequencing. For example, the energetic barriers to translocation for the different nucleotides have been calculated for different protein pores, the mechanism of ssDNA translocation has been predicted, and the importance of the directionality of the DNA has been determined …”

Kulturimmobilien – Erfolgsfaktor Projektmanagement

“…The former was the first to use MD to provide a complete 'image' of αHL in terms of electrostatic potential distribution, ion concentrations, ionic conductance, and electro-osmotic permeability [151]. Since then, many more MD studies have been performed on αHL, including the translocation of nucleic acids [272], and unfolded proteins [273], the cation-type dependence of the ionic current rectification [152], the magnitude of the electro-osmotic flow [274], and the recognition mechanisms of nucleobases [275,271] and amino acids [276].…”