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G‐quadruplex (G4) structures exist in the single‐stranded DNA of chromatin and regulate genome function. However, the native chromatin G4 landscape in living cells has yet to be fully characterized. Herein, a genetic‐encoded live‐cell G4 identifier probe (LiveG4ID) is constructed and its cellular localization, biocompatibility, and G4‐binding specificity is evaluated. By coupling LiveG4ID with cleavage under targets and tagmentation (CUT&Tag), LiveG4ID‐seq, a method for mapping native chromatin G4 landscape in living cells with high accuracy is established. Compared to the conventional G4 CUT&Tag method, LiveG4ID‐seq can identify more chromatin G4 signals and have a higher ratio of true positive signals. Using LiveG4ID‐seq, the dynamic landscape of chromatin G4 structures during the cell cycle is profiled. It is discovered that chromatin G4 structures are prevalent in the promoter regions of cell cycle‐specific genes, even in the early M phase when the chromatin is condensed. These data demonstrate the capacity of LiveG4ID‐seq to profile a more accurate G4 landscape in living cells and promote future studies on chromatin G4 structures. [ABSTRACT FROM AUTHOR]

G‐quadruplexes (G4s), non‐canonical nucleic acid secondary structure, regulate many biological functions and are considered potential molecular targets for cancer therapeutics. However, due to the lack of analytical methods, the regulating mechanism of monogenic G4s is still unclear. Here, we developed a Module Assembled Multifunctional Probes Assay (MAMPA) for visualizing endogenous G4s in individual genes in single cells. Two modular probes separately recognize G4 structures and the adjacent RNA sequences, and the module assembly enables imaging of G4s in an individual RNA with high specificity. Through imaging G4s in several individual genes, we found that G4s were steadily occupied by G4 Binding Proteins (G4BPs) in various mRNAs in every cell line and defined "Occupied G4 Ratio". We demonstrated MAMPA was suitable for most experimental situations and found that Occupied G4 Ratios had the potential to become a new parameter for the study of G4s in living cells. [ABSTRACT FROM AUTHOR]