Assessing G‑Quadruplex Stability and Nonspecific Interactions in Living Cells Using 19F NMR.

G-quadruplex (G4) structures play key physiological roles and serve as functional building blocks in DNA/RNA-based aptamer biosensors. Their function critically depends on their structural stability and nonspecific interactions in cells. However, the behavior of exogenously delivered G4s in living cells remains poorly understood. Here, using 19F NMR, we monitored three representative G4 sequences, HT, hVEGFP, and TBA, in intact Xenopus laevis oocytes, HeLa cells, and derived lysates. We found that G4 structural stability, interactions, and NMR signal detectability are cell-type-dependent. In oocytes, all three G4s maintain detectable folded signals, with TBA showing slight time-dependent degradation. In HeLa cells, folded signals are absent for all three G4s due to intracellular interactions. HT and TBA undergo degradation in the cytoplasm rather than in the nucleus, whereas hVEGFP uniquely remains protected from degradation owing to both its higher thermal stability and protective interactions. These findings highlight the non-negligible effect of the cellular environment and the necessity of studying G4s in their native cellular context. Furthermore, we demonstrated that cell lysates can serve as a practical platform for reflecting G4 stability in intact cells. Collectively, this work deepens our understanding of G4 behavior in cells and provides a basis for improvement of G4-based agents.