Optimising nanopores for DNA sequencing: A computational perspective

Abstract

Nanopore DNA sequencing is a well-established technology that has accelerated advancements in many fields, including medical research. Over the years, research has focussed on optimising protein nanopores for DNA sequencing. Optimisation strategies broadly focus on (1) slowing the translocation of DNA to increase the time available for base recognition and (2) improving the resolution of detection to attain single-base sensing. Molecular dynamics (MD) simulations have been invaluable in obtaining molecular-level insights to pave the way for informed nanopore optimisation. In this thesis, MD simulations were used to study DNA translocation through nanopores and elucidate the design principles for optimising nanopores for DNA sequencing.

In the first chapter, the translocation of short and longer single-stranded (ss)DNAs was studied through protein-inspired hydrophobic nanopores with dual-constrictions. It was found that DNA translocation is slowed down by aromatic residues, and when combined with a narrow geometry, DNA retains a largely linear conformation during translocation and without forming secondary structures that can impede DNA sequencing. Following this, the proteins CsgG and the CsgG-CsgF complex were characterised in terms of their conformational dynamics and ability to allow DNA translocation. Eyelet loops forming the CsgG constriction were found to exhibit large variations in their mobility, with at least one loop moving upwards into the vestibule under an applied electric field. CsgF was found to stabilise CsgG and the eyelet loop region. Subsequently, the translocation of short ssDNA through CsgG and the CsgG-CsgF complex was studied. The speed of DNA translocation was found to be primarily influenced by DNA interacting with key residues in the CsgG constriction region. DNA is retained in a more linear conformation during translocation through the dual-constriction hydrophobic channel formed by the CsgG-CsgF complex compared to CsgG. Next, the translocation of longer ssDNA in an applied electric field was studied through these proteins/protein complex. These simulations revealed that the eyelet loops of the CsgG constriction region are mobile during DNA translocation, and the stochastic nature of their mobility perturbs the pore geometry which may give rise to noise in the ionic current through the nanopores. In the last chapter, Markov State Model methodology was employed to characterise the kinetics of the mobility of the CsgG eyelet loops under an applied electric field. The model construction was limited by the duration of the MD simulations. The data and analyses presented in this, and previous, chapters emphasise the need for a model that describes the complex conformational dynamics of the CsgG eyelet loops.

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Identifiers

ORCID for Syma Khalid: orcid.org/0000-0002-3694-5044

ORCID for Chris-Kriton Skylaris: orcid.org/0000-0003-0258-3433

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