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，例如牛津纳米孔技术（ONT）Haverolution的生物学率先使用的DNA测序方法，通过允许在整个生物体水平上快速DNA测序，并以多样化的进化生物学，结构和分子生物学，农业，农业，诊断和个性化医学为影响领域。 Onttechnology通过将ATP依赖性DNA运动蛋白与生物膜中的纳米级孔耦合来起作用。当电动机通过孔传递DNA时，碱基组成是由跨膜的电流变化确定的，最终产生准确的长读取DNA测序数据。除了对长阅读DNaseQuencing和快速诊断的核心用途外，基于纳米孔的测定法还发现了基础科学中意外的应用。例如，由于纳米孔DNA测序是由电动机的活性限制的，因此测序痕迹trace trace trace trace aclsocontain关于DNA易位动力学的富含信息，从而为诸如Helicases之类的机制提供了独特的见解。在这个项目中，您将使用两个主要目标将新的DNA解放酶整合到基于纳米孔的DNA序列设备中。首先，我们旨在通过将DNAHELICases使用具有新颖和可取的生物学特性的DNAHELICase进行纳米孔DNA测序的速度，准确性，简单和鲁棒性。其次，我们旨在研究医疗兴趣的dnahelicases的作用机理，以及它们如何受到小分子药物或遗传突变的影响。重要的是，纳米孔痕迹将产生有关沿着DNA及其化学机械耦合到ATP水解的单个步骤的性质的前所未有的细节。您的项目将基于DNA的Markdillingham教授的实验室：布里斯托大学的蛋白质相互作用部门，但还将与Oxford Nanopore Technologies（1）进行密切合作，包括在牛津总部所花费的实习。 Dillingham Lab（2）是涉及破裂DNA的涉及的解旋酶，包括被认为是抗生素和人类酶的吸引人的细菌酶，这些酶与遗传性疾病有关。有关我们最近工作的更多详细信息，请参见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