Engineering Biological Nanopore MspA for Sequencing DNA

Abstract

The sequence of a long DNA molecule can be determined, in principle, by measuring the ionic current blockade the molecule produces as it permeates a nanometer-diameter pore in a thin insulating membrane. The difficulties in realizing this idea in practice are common to both biological and synthetic nanopores: the pore geometry does not permit isolation of a single nucleotide and the DNA molecule moves too fast through the nanopore for its sequence to be determined by the ionic current measurement. Here, we report our progress in engineering biological pore MspA for sequencing applications. It has been experimentally shown that DNA strands immobilized inside the MspA pore produce ionic current blockades that permit identification of a single nucleotide substitution in the DNA sequence [doi:10.1073/pnas.1001831107]. Through all-atom molecular dynamics simulations we investigate the molecular origin of such extreme sensitivity of the ionic current to the sequence and orientation of DNA strands. Furthermore, we demonstrate the feasibility of reducing the rate of DNA transport by introducing point mutations in the MspA structure. Spanning tens of microseconds, our simulations provide the most detailed account of the atomic-scale mechanics of DNA and ion transport through biological nanopores.