Department of Chemistry, University of Cambridge, Cambridge, UK.
Imperial College London, Chemistry Department, Molecular Science Research Hub, London, UK.
Nat Chem. 2020 Sep;12(9):832-837. doi: 10.1038/s41557-020-0506-4. Epub 2020 Jul 20.
Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
大量证据表明，DNA 形成 G-四链体（G4s）与基因表达改变有关。然而，需要开发能够在不干扰其折叠动力学的情况下在活细胞中探测 G4 的方法，以便更详细地了解它们的生物学作用。在此，我们报告了一种 G4 特异性荧光探针（SiR-PyPDS），它能够在活细胞中对单个 G4 结构进行单分子和实时检测。在使用低浓度 SiR-PyPDS（20 nM）的条件下进行活细胞单分子荧光成像，以提供有信息量的测量结果，代表活细胞中 G4 的群体，而不会全局干扰 G4 的形成和动力学。活细胞中单分子荧光成像和对未折叠 G4 的时变化学捕获表明，G4 在折叠和未折叠状态之间波动。我们还证明，活细胞中的 G4 形成是细胞周期依赖性的，并受到转录和复制的化学抑制的破坏。我们的观察结果为活细胞中动态 G4 形成提供了有力证据。
