Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.
Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2024;2713:453-462. doi: 10.1007/978-1-0716-3437-0_30.
In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.
除了典型的 B-DNA 构象外，DNA 还可以折叠成不同的二级结构。其中包括 G-四链体结构（G4s）。G4 结构非常稳定，可以在 DNA 和 RNA 中的特定富含鸟嘌呤的区域折叠。不同的计算机模拟、体外和细胞内实验表明，迄今为止，在所有测试的生物体中都形成了 G4 结构。在高等真核生物中，有超过 700,000 个预测的 G4，到目前为止，人们假设并非所有的 G4 都会同时形成。它们的形成受到蛋白质的动态调节，具有细胞类型特异性，甚至在细胞周期或不同的外源或内源刺激（例如感染或发育阶段）期间发生变化时，G4 水平也会发生变化。已经表明 G4 在癌细胞中积累，它们有助于基因表达变化和肿瘤的突变负担。目前正在讨论针对 G4 结构进行特异性靶向以影响致癌基因表达作为癌症治疗方法。在肿瘤微环境中，不仅肿瘤细胞会受到 G4 稳定的靶向作用，而且巨噬细胞等免疫细胞也会受到靶向作用。尽管在多种生物体和不同类型的细胞中都检测到了 G4，但它们在免疫细胞中的作用知之甚少。在这里，我们提供了一种详细的方案，通过显微镜成像来检测脊椎动物和无脊椎动物巨噬细胞核中 G4 的形成。
