Developing optical probes for cellular imaging of G-quadruplexes.

G-四链(G4)DNA是一种非规范结构，形成于基因组的鸟嘌呤富集区。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G4S。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G4S进行成像。由于我们的探针随着时间的推移具有高度的稳定性，而且探针的发光很明亮，即使在衍射有限的分辨率下，我们也应该能够检测到染色体尖端。一旦我们能够看到端粒区域的G4S(这将是第一次用小分子而不是抗体完成这一过程)，我们将探索许多细胞过程。我们将首先研究增加大量成熟的G-四链结合体(例如，吡哚他汀、端粒酮和金属盐)取代端粒区域探针的能力。这将为了解活细胞中小分子与G-四链的结合动力学提供独特的见解，并最终可能被用作一种筛选以端粒G-四链为靶点的非荧光分子作为潜在抗癌药物的技术。接下来，我们将使用我们的探针来研究纤维素酶、DNA解旋酶在G-四链生物学中的相互作用和功能。体外研究表明，包括ATRX在内的一些组蛋白伴侣蛋白具有结合G-四联体的能力。然而，目前还不清楚该因子是否在体内和细胞周期中识别并作用于G-四联体。在这个项目中，我们将首次尝试将ATRX在G4维护中的作用直接形象化。

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.