Aptamer biosensors to measure PAR dynamics in living cells

聚ADP-核糖基化（PAR化）是一种可逆的翻译后修饰，其中聚ADP-核糖（PAR）链组装到受体蛋白上。PAR化调节各种基本细胞过程，包括染色质组织、DNA修复、转录和复制。PAR链由PAR聚合酶（PARP）合成，并由PAR糖水解酶（PARG）分解代谢;因此PAR动力学受到两种酶之间平衡的仔细调节。然而，我们目前缺乏在活细胞中测量PAR动态的能力。该项目的目的是探索荧光或其他化学修饰和可测量的适体作为“PAR生物传感器”，能够与活细胞中的PAR链结合;并与工业合作伙伴Aptamer Group一起产生新的工具来研究PAR化及其在正常细胞生理和病理状态（如卵巢癌和前列腺癌）中的DNA损伤，修复和复制中的作用。学生将生成合成PAR化肽;创建适体信标并在活细胞中评估PAR生物传感器。学生将接受广泛的跨学科技能培训，包括最先进的适体技术，用于开发新型生物传感器，体外分析技术和细胞生物学方法（细胞培养，延时和免疫荧光显微镜）以及功能测定（例如凋亡，复制应力）。他们还将获得相关背景（细胞周期，DNA复制和癌症）以及实验设计和战略多学科方法的强大知识培训。

PolyADP-ribosylation (PARylation) is a reversible post-translational modification where poly ADP-ribose (PAR) chains are assembled onto acceptor proteins. PARylation regulates various fundamental cellular processes including chromatin organisation, DNA repair, transcription and replication. PAR chains are synthesised by PAR polymerases (PARPs), and catabolised by PAR glycohydrolases (PARGs); so PAR dynamics is carefully regulated by the balance between both enzyme classes. However, we currently lack the ability to measure PAR dynamics in living cells. The aim of the project is to explore fluorescent or other chemically modified and measurable aptamers as 'PAR biosensors' capable of binding to PAR chains in living cells; and to generate new tools to study PARylation and its role in DNA damage, repair and replication in normal cellular physiology and pathological states such as ovarian and prostate cancers, with the industrial partner, Aptamer Group. The student will generate synthetic PARylated peptides; create aptamer beacons and evaluate the PAR biosensor(s) in living cells. The student will receive a broad interdisciplinary skills training in state-of-the-art aptamer technology for developing novel biosensors, in vitro analytical techniques and cell biology methods (cell culture, time-lapse and immunofluorescence microscopy), and functional assays (e.g. apoptosis, replication stress). They will also gain a strong intellectual training in the relevant background (cell cycle, DNA replication and cancer), and in experimental design and strategic multidisciplinary approaches.