在活细胞中成像G-四重复核酸使用提奥夫拉T和光终身成像显微镜
Tigerlily Bradford1, Peter A Summers1, Aatikah Majid1
1Molecular Sciences Research Hub, Department of Chemistry, Imperial College London, London W12 0BZ, U.K.
Analytical chemistry
|December 11, 2024
概括
商用染料Thioflavin T (ThT) 和Thiazole Orange (TO) 现在可以可视化活细胞中的G-四重复 (G4) DNA结构. 这种光终身成像显微镜 (FLIM) 方法为研究这些重要的生物结构提供了一个更简单的替代方案.
科学领域:
- 细胞生物学 细胞生物学
- 生物物理学的生物物理.
- 分子生物学分子生物学
背景情况:
- 富含关氨酸的寡核酸在体内形成G-四重复 (G4) 结构.
- 这些非正规的DNA结构涉及至关重要的生物过程.
- 在活细胞中直接可视化G4s对于理解它们的功能至关重要.
研究的目的:
- 探索用于G4可视化的常见光染料的使用.
- 适应光终身成像显微镜 (FLIM) 用于使用可访问的试剂检测G4.
- 建立一种用于在活细胞中成像G4s的定量方法.
主要方法:
- 使用光终身成像显微镜 (FLIM).
- 研究了与不同DNA拓系结合的提奥夫拉T (ThT) 和酸 (TO) 的光寿命.
- 应用FLIM与ThT在不同条件下可视化活U2OS细胞中的G4s.
主要成果:
- ThT和TO的光寿命对DNA拓敏感,G4s表现出明显的,长时间的衰变时间.
- 在活的U2OS细胞中使用FLIM与ThT.成功地可视化了G4结构.
- 通过竞争性结合或核酶处理对G4水平的操纵证明了G4检测.
结论:
- 商用染料ThT和TO可以根据其光寿命作为G4结构的有效探针.
- 使用ThT的FLIM提供了一种可靠且易于使用的方法,用于活细胞中的定量G4成像.
- 这种方法简化了G4的可视化,促进了对其生物作用的进一步研究.
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