Developing optical probes for cellular imaging of G-quadruplexes.

开发用于 G-四链体细胞成像的光学探针。

• 批准号: 2454110
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2454110
• 关键词: Developing; optical; probes; cellular; imaging

G-quadruplex (G4) DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. We propose to use small molecule probes to carry out live cell imaging studies to detect G4s that currently are simply not possible. We will first perform imaging of G4s formed in the telomeric region of chromosomes using Fluorescence Lifetime Imaging Microscopy (FLIM). Given high stability of our probes over time and the bright emission of the probes, we should be able to detect the chromosome tips even with diffraction limited resolution. Once we are able to visualize G4s in the telomeric region (which would be the first time this is done with a small molecule rather than an antibody), we will explore a number of cellular processes. We will first investigate the ability of increasing amounts of well-established G-quadruplex binders (e.g. pyridostatine, telomestatine and metal salphens) to displace the probe from the telomeric regions. This will provide unique insights into the binding dynamics of small molecules to G-quadruplexes in live cells and eventually could be adapted as a technique to screen non-fluorescent molecules that target G-quadruplexes in telomeres as potential anticancer drugs. Next, we will use our probes to study in cellulo, the interaction and function of DNA helicases on G-quadruplexes biology. In vitro studies have proposed that some histone chaperones including ATRX have the ability to bind G-quadruplexes. However, it is unclear if this factor recognises and act at G-quadruplexes in vivo and during the cell cycle. In this project we will attempt to visualise the role of ATRX in G4 maintenance directly, for the first time.

G-四链体（G4）DNA是在基因组的富含鸟嘌呤的区域中形成的非典型结构。越来越多的证据表明G-四链体具有重要的生物学功能，因此可视化这些结构的分子工具非常重要。我们建议使用小分子探针进行活细胞成像研究，以检测目前根本不可能的G4。我们将首先使用荧光寿命成像显微镜（FLIM）对染色体端粒区域形成的G4进行成像。考虑到我们的探针随时间的高稳定性和探针的明亮发射，我们应该能够检测染色体尖端，即使具有衍射极限分辨率。一旦我们能够可视化端粒区域中的G4（这将是第一次用小分子而不是抗体完成），我们将探索许多细胞过程。我们将首先研究增加量的完善的G-四链体结合剂（例如吡啶司他汀，端粒和金属salphens）取代探针从端粒区域的能力。这将为小分子与活细胞中的G-四链体的结合动力学提供独特的见解，并最终可以作为一种技术来筛选靶向端粒中的G-四链体的非荧光分子作为潜在的抗癌药物。接下来，我们将使用我们的探针在细胞内研究DNA解旋酶对G-四链体生物学的相互作用和功能。体外研究表明，包括ATRX在内的一些组蛋白伴侣具有结合G-四链体的能力。然而，目前尚不清楚该因子是否在体内和细胞周期中识别并作用于G-四链体。在这个项目中，我们将首次尝试直接可视化ATRX在G4维护中的作用。