Mechanistic analysis of DNA helicases using nanopore-based single molecule assays

由牛津纳米孔技术公司（ONT）开创的下一代DNA测序方法，通过允许在整个生物体水平上进行快速DNA测序，并影响进化生物学、结构和分子生物学、农业、诊断和个性化医学等多种领域，使生物学发生了进化。ont技术的工作原理是将atp依赖的DNA马达蛋白偶联到生物膜的纳米级孔上。当马达通过孔传递DNA时，碱基组成由膜上电流的变化决定，最终产生准确的长读DNA测序数据。除了用于长读dna测序和快速诊断的核心用途外，基于纳米孔的检测还在基础科学中发现了意想不到的应用。例如，由于纳米孔DNA测序受到马达活性的速率限制，测序痕迹也包含了DNA易位动力学的丰富信息，为解旋酶等必需酶的机制提供了独特的见解。在这个项目中，你将整合新的DNA解旋酶到纳米孔DNA序列设备中，有两个主要目标。首先，我们的目标是通过使用具有新颖和理想生化特性的DNA解旋酶来提高纳米孔DNA测序的速度、准确性、简单性和稳健性。其次，我们的目标是研究具有医学意义的dna解旋酶的作用机制，以及它们如何受到小分子药物或基因突变的影响。重要的是，我们预计纳米孔的痕迹将提供前所未有的细节，以了解与dna一起产生的单个步骤的性质以及它们与ATP水解的化学-机械耦合。你的项目将在布里斯托尔大学DNA：蛋白质相互作用单元的MarkDillingham教授的实验室进行，但也将涉及与牛津纳米孔技术公司的密切合作，包括在牛津总部实习。迪林厄姆实验室(2)正在研究参与DNA断裂修复的解旋酶，包括被认为是抗生素有吸引力靶点的细菌酶，以及涉及遗传疾病（包括癌症）的人类酶。有关我们最近工作的进一步细节和示例，请参阅我们的网站或联系实验室(2)。(1)https://nanoporetech.com/ (2) https://research-information.bris.ac.uk/en/persons/mark-s-dillingham

Next generation DNA sequencing methods such as those pioneered by Oxford Nanopore Technologies (ONT) haverevolutionised biology by allowing rapid DNA sequencing at the whole organism level and impacting fields as diverse asevolutionary biology, structural and molecular biology, agriculture, diagnostics and personalised medicine. The ONTtechnology works by coupling an ATP-dependent DNA motor protein to a nanoscale pore in a biological membrane. Asthe motor delivers DNA through the pore, the base composition is determined by changes in the current across themembrane ultimately yielding accurate long read DNA sequencing data. In addition to their core uses for long-read DNAsequencing and rapid diagnostics, nanopore-based assays have also found unexpected applications in basic science. Forexample, because nanopore DNA sequencing is rate-limited by the activity of the motor, the sequencing traces alsocontain rich information on the kinetics of DNA translocation providing unique insights into the mechanism(s) ofessential enzymes such as helicases. In this project, you will integrate new DNA helicases into nanopore-based DNA sequence devices with two major goals.Firstly, we aim to improve the speed, accuracy, simplicity and robustness of nanopore DNA sequencing by using DNAhelicases with novel and desirable biochemical properties. Secondly, we aim to study the mechanism of action of DNAhelicases of medical interest and how they are affected by small molecule drugs or genetic mutation. Importantly, weanticipate that nanopore traces will yield unprecedented detail on the nature of the individual steps that are made alongDNA and their chemo-mechanical coupling to ATP hydrolysis. Your project will be based in the laboratory of Prof. MarkDillingham in the DNA:protein interactions Unit at the University of Bristol but will also involve close collaboration withOxford Nanopore Technologies (1) including internships spent at their Oxford headquarters. The Dillingham lab (2) isstudying helicases involved in the repair of broken DNA, including bacterial enzymes that are considered attractivetargets for antibiotics and human enzymes implicated in genetic diseases including cancer. For further details andexamples of our recent work see our website or contact the laboratory (2). (1) https://nanoporetech.com/ (2) https://research-information.bris.ac.uk/en/persons/mark-s-dillingham