Bradford Tigerlily, Summers Peter A, Majid Aatikah, Sherin Petr S, Lam Jeff Yui Long, Aggarwal Savyasanchi, Vannier Jean-Baptiste, Vilar Ramon, Kuimova Marina K
Molecular Sciences Research Hub, Department of Chemistry, Imperial College London, London W12 0BZ, U.K.
Telomere Replication and Stability Group, MRC London Institute of Medical Sciences, London W12 0NN, U.K.
Anal Chem. 2024 Dec 24;96(51):20223-20229. doi: 10.1021/acs.analchem.4c04207. Epub 2024 Dec 11.
Visualization of guanine-rich oligonucleotides that fold into G-quadruplex (G4) helical structures is of great interest in cell biology. There is a large body of evidence that suggests that these noncanonical structures form and play important biological roles. A promising recent development highlighted fluorescence lifetime imaging microscopy (FLIM) as a robust technique for the direct and quantitative imaging of G4s in live cells. However, this method requires specialized, bespoke synthetic dyes that are not widely available. Herein, we demonstrate that the fluorescence lifetime of commercially available environmentally sensitive dyes Thioflavin T () and Thiazole Orange () is strongly dependent on the type of DNA topology they bind to, with G4s showing long and distinctive decay times that should allow G4 detection in the biological environment. We applied this observation to visualize G4s in live U2OS cells using FLIM of , upon alteration in G4 levels due to competitive binding or nuclease treatment of cells.
可视化折叠成G-四链体（G4）螺旋结构的富含鸟嘌呤的寡核苷酸在细胞生物学中备受关注。有大量证据表明，这些非经典结构会形成并发挥重要的生物学作用。最近一项有前景的进展突出了荧光寿命成像显微镜（FLIM）作为一种在活细胞中直接定量成像G4的强大技术。然而，这种方法需要专门定制的合成染料，而这些染料并未广泛可得。在此，我们证明市售的环境敏感染料硫黄素T（Thioflavin T）和噻唑橙（Thiazole Orange）的荧光寿命强烈依赖于它们所结合的DNA拓扑结构类型，G4显示出长且独特的衰减时间，这应能在生物环境中检测G4。我们利用Thioflavin T的FLIM，在细胞因竞争性结合或核酸酶处理导致G4水平改变时，将这一观察结果应用于可视化活U2OS细胞中的G4。
