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

牛津纳米孔技术 (ONT) 首创的下一代 DNA 测序方法通过在整个生物体水平上进行快速 DNA 测序，彻底改变了生物学，并影响了进化生物学、结构和分子生物学、农业、诊断和个性化医疗等多种领域。 ONT 技术的工作原理是将 ATP 依赖性 DNA 马达蛋白耦合到生物膜的纳米级孔上。当电机通过孔输送 DNA 时，碱基组成由跨膜电流的变化决定，最终产生准确的长读 DNA 测序数据。除了长读长 DNA 测序和快速诊断的核心用途之外，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 测序受到电机活动的速率限制，因此测序痕迹还包含有关 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，您将把新的 DNA 解旋酶集成到基于纳米孔的 DNA 测序设备中，主要目标有两个。首先，我们的目标是通过使用具有新颖且理想的生化特性的 DNA 解旋酶来提高纳米孔 DNA 测序的速度、准确性、简单性和稳健性。其次，我们的目标是研究具有医学意义的DNA螺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产生前所未有的详细信息，了解沿 DNA 进行的各个步骤的性质及其与 ATP 水解的化学机械耦合。您的项目将在布里斯托大学 DNA：蛋白质相互作用部门的 MarkDillingham 教授的实验室进行，但也将涉及与 Oxford Nanopore Technologies (1) 的密切合作，包括在其牛津总部进行的实习。 Dillingham 实验室 (2) 正在研究参与修复断裂 DNA 的解旋酶，包括被认为是抗生素有吸引力的靶标的细菌酶，以及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详细信息和示例，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