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

使用基于纳米孔的单分子测定法对 DNA 解旋酶进行机理分析

• 批准号: 2885493
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2885493
• 关键词: Mechanistic; analysis; DNA; helicases; using

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

下一代DNA测序方法，如牛津纳米孔技术公司（ONT）开创的方法，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领域，使生物学发生了革命性的变化。ONT技术通过将依赖ATP的DNA马达蛋白耦合到生物膜中的纳米级孔来工作。当马达将DNA输送通过孔时，碱基组成由跨膜电流的变化决定，最终产生准确的长读段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，基于纳米孔的测定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测序受马达活性的速率限制，因此测序迹线还包含关于DNA易位动力学的丰富信息，从而提供对必需酶如解旋酶的机制的独特见解。在这个项目中，你将把新的DNA解旋酶整合到基于纳米孔的DNA测序设备中，主要有两个目标。首先，我们的目标是通过使用具有新的和理想的生化特性的DNA解旋酶来提高纳米孔DNA测序的速度，准确性，简单性和鲁棒性。其次，我们的目标是研究医学上感兴趣的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，我们预计纳米孔痕迹将提供有关DNA中各个步骤的性质及其与ATP水解的化学机械耦合的前所未有的细节。您的项目将基于MarkDillingham教授的实验室在DNA：蛋白质相互作用单位在布里斯托大学，但也将涉及密切合作与牛津纳米孔技术（1），包括在他们的牛津总部实习。迪灵厄姆实验室（2）正在研究与修复断裂DNA有关的解旋酶，包括被认为是抗生素有吸引力的靶标的细菌酶和与包括癌症在内的遗传疾病有关的人类酶。有关我们最近工作的更多细节和示例，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