Engineering a membrane-adapted viral helicase nanopore

工程膜适应病毒解旋酶纳米孔

• 批准号: 2764016
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2764016
• 关键词: Engineering; membrane; adapted; viral; helicase

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.

背景纳米孔测序设备使用嵌入在抗电膜中的蛋白质纳米孔读取转移单链DNA分子中的碱基，DNA碱基通过跨膜电流的变化来识别。目前的装置将DNA易位解旋酶与单独的孔蛋白偶联。虽然该技术能够进行长读取（兆字节）和高准确度（~99%），但这些都可以改进。关键参数包括转运速度、膜-蛋白质-DNA复合物的稳定性和孔径，这些参数决定了核苷酸的分辨率。六聚体复制解旋酶的直接膜插入，本质上是进行性的，并且具有为ssDNA移位而优化的中心孔，可以导致改进的装置。具有单个孔/移位酶还可以简化样品DNA制备，避免了如在当前方案中那样将DNA预先附着到解旋酶然后附着到孔的需要。本项目将利用分子生物学和生物化学方法，将来自乳头瘤病毒的六聚体复制解旋酶E1重新设计为适合DNA测序的膜结合纳米孔。