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.

现在有大量证据支持DNA G-四链体（G4s）的形成与基因表达的改变有关。但是，需要使我们能够在不干扰其折叠动力学的情况下在活细胞中探测G4的方法才能更详细地了解其生物学作用。在此，我们报告了一种G4特异性荧光探针（SiR-PyPDS），该探针能够对活细胞中的单个G4结构进行单分子和实时检测。G4s的活细胞单分子荧光成像是在使用低浓度SiR-PyPDS（20 nM）的条件下进行的，以提供代表活细胞中G4种群的信息性测量，而不会全局干扰G4的形成和动力学。活细胞中未折叠的G4的单分子荧光成像和时间依赖性化学捕获显示，G4在折叠状态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，并受到化学抑制转录和复制的破坏。我们的观察结果提供了有力的证据支持活细胞中动态G4的形成。