Engineering a membrane-adapted viral helicase nanopore

背景资料。纳米孔测序设备使用嵌入在耐电膜中的纳米蛋白质来读取转移单链DNA分子的碱基，DNA碱基通过膜上电流的变化来识别。目前的设备将DNA转位解旋酶与单独的孔蛋白偶联。虽然这项技术能够长时间读取(兆基)和高精度(~99%)，但这些都是可以改进的。关键参数包括转位速度、膜-蛋白质-DNA复合体的稳定性和孔大小，它们决定了核苷酸的拆分。六聚体复制解旋酶的直接膜插入，本质上是过程的，具有为单链DNA易位优化的中心孔，可能导致改进的装置。只有一个孔/转位酶也可以简化样品DNA的制备，不需要像目前的方案那样，先将DNA预先连接到螺旋酶上，然后再连接到孔上。这个项目将使用分子生物学和生化方法重新设计来自乳头瘤病毒的六聚体复制解旋酶E1，使其成为适合DNA测序的膜结合纳米孔。

Background. Nanopore sequencing devices read the bases in atranslocating single-stranded DNA molecule using a protein nanoporeembedded in an electro-resistant membrane, with the DNA basesidentified by changes in the current across the membrane. Presentday devices couple a DNA translocating helicase with a separate poreprotein. Although the technology is capable of long reads (megabases) and high accuracy (~99%) these could be improved. Thecritical parameters include translocation speed, stability of themembrane-protein-DNA complex and pore size, which determinesnucleotide resolution. The direct membrane-insertion of hexamericreplicative helicases, that are by nature processive and have a centralpore optimised for ssDNA translocation, could lead to improveddevices. Having a single pore/translocase could also simplify sampleDNA preparation, obviating the need to pre-attach DNA to the helicaseand then the pore, as in current protocols. This project will usemolecular biology and biochemical approaches to re-engineer thehexameric replicative helicase E1 from papillomavirus, to a membrane-bound nanopore suitable for DNA sequencing.