Biophysical studies of Biological Nanopores used in Biopolymer Sequencing.

During evolution, various organisms developed the ability to express types of outer-membrane/secreted pores that function to translocate peptides and other large molecules. This functionality was discovered to have applications in biotechnology, specifically nanopore sequencing.
Nanopore sequencing has emerged in the past decade, as forerunner for accurate, inexpensive, long-read DNA sequencing. In 1996, Kasianowicz et al, the used a PFP, alpha-hemolysin, to sequence single stranded DNA by transferring it into a synthetic membrane, that separated a solution containing electrolytes. When a voltage was applied across the membrane, it resulted in an ionic current that flowed through the pores, which drew DNA to the pore and allowed it to translocate. As each base of DNA has a different size and charge distribution there are detectable base specific reductions in current, allowing the DNA to be sequenced. Initially, the fidelity of this DNA sequencing method was low, however the addition of a helicase enzyme to the top of the pore and manipulation of the internal charge of the pore allowed controlled translocation of the DNA which improved the signal resolved. Additionally, the use of nanopores which had a region of the pore that was narrower in diameter and small in length (constriction point) contributed to a higher level of sequencing accuracy. This increases fidelity as the shorter constriction only spans one base of DNA, meaning the reduction in current can be attributed to only one base. Despite, the many improvements to these nanopores, they still have a lower level of accuracy when sequencing a single molecule or tandem repeats, compared to current sequencing methods.
In 2005, the company Oxford Nanopore Technologies (ONT) was established with the aim to commercialise Nanopore sequencing. Their product utilises a mutant of an outer-membrane pore Escherichia coli, CsgG associated with a molecular motor, similar to Phi29 DNAP, to produce a base specific signal that allow long-read DNA sequencing. The method of improvement used by ONT to produce their new pores is largely down to random mutagenesis. Due to very little understanding of the interaction between the DNA and the pores used, the only way to develop new pores is to build upon previous mutants and test them repetitively, which is a time consuming and expensive. As previously discussed, there are constriction points within the pores which form sensing regions for the bases, but there is limited experimental or computational data on the interactions between the DNA and pore. Therefore, the further characterisation of the loop regions in CsgG that form the sensing region and the interactions with the DNA, alongside the effects of other factors such as membrane stability, could be used to generate improved mutants via rational design.
Whilst CsgG and other pores used for sequencing, for example Lysenin a pore found in Eisenia fetida, have been well characterised using crystallography and cryo-EM, very little is understood about the dynamics of them. This project will partially focus on the optimisation of the recombinant expression, purification and assembly of these outer-membrane/secreted pores for TROSY NMR experiments. Alongside this, computational studies will be performed on CsgG to elucidate dynamics within the pore and interactions with DNA to drive rational design of mutants, that can also be applied to other proteins.