Biophysical studies of Biological Nanopores used in Biopolymer Sequencing.

在进化过程中，各种生物发展出表达各种外膜/分泌孔的能力，这些孔的功能是转运肽和其他大分子。这种功能被发现在生物技术中有应用，特别是纳米孔测序。纳米孔测序在过去十年中出现，作为精确、廉价、长读DNA测序的先驱。1996年，Kasianowicz等人使用PFP （α -溶血素）将单链DNA转移到一种合成膜中，分离含有电解质的溶液，从而对单链DNA进行测序。当在膜上施加电压时，就会产生离子电流流过孔，将DNA吸引到孔中并使其转移。由于DNA的每个碱基具有不同的大小和电荷分布，因此存在可检测到的碱基特异性电流减少，从而允许对DNA进行测序。最初，这种DNA测序方法的保真度很低，但是在孔顶部添加解旋酶和操纵孔的内部电荷允许DNA的可控易位，从而提高了信号的分辨率。此外，纳米孔的使用具有直径较窄且长度较小的孔区域（收缩点）有助于提高测序精度。这增加了保真度，因为较短的收缩只跨越DNA的一个碱基，这意味着电流的减少可以归因于只有一个碱基。尽管对这些纳米孔进行了许多改进，但与目前的测序方法相比，它们在测序单个分子或串联重复序列时的准确性仍然较低。2005年，牛津纳米孔技术公司（ONT）成立，其目标是将纳米孔测序商业化。他们的产品利用大肠杆菌外膜孔的突变体CsgG与分子马达相关，类似于Phi29 DNAP，产生碱基特异性信号，允许长读DNA测序。ONT用于产生新孔隙的改进方法主要是随机诱变。由于对DNA和所用孔之间的相互作用了解甚少，开发新孔的唯一方法是在先前的突变体基础上进行重复测试，这既耗时又昂贵。如前所述，在形成碱基感应区域的孔内存在收缩点，但关于DNA与孔之间相互作用的实验或计算数据有限。因此，进一步表征CsgG中形成感应区和与DNA相互作用的环区，以及膜稳定性等其他因素的影响，可以通过合理的设计来产生改进的突变体。虽然CsgG和其他用于测序的孔隙，例如在Eisenia fetida中发现的Lysenin孔，已经通过晶体学和低温电镜（cryo-EM）进行了很好的表征，但对它们的动力学知之甚少。该项目将部分集中于优化这些外膜/分泌孔的重组表达、纯化和组装，用于TROSY NMR实验。除此之外，还将对CsgG进行计算研究，以阐明孔隙内的动力学以及与DNA的相互作用，从而驱动突变体的合理设计，这也可以应用于其他蛋白质。

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.