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G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4–protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4–protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4–protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations. Affinity enrichment methods and G-quadruplex (G4) ligand-mediated photo-crosslinking methods can be used to screen new G4-binding proteins (G4BPs) with high throughput and accuracy. Many in vitro methods based on classic nucleic acid–protein interaction assays have been developed to validate G4BPs, but methods for investigating G4–protein interactions within the cellular milieu are still under demand. The development of more highly sensitive, selective, and high-throughput biochemical methods will greatly facilitate the development of G4BP biology. [ABSTRACT FROM AUTHOR]

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]

Single‐cell imaging has unique advantages of maintaining the in situ physiological state, morphology, and microenvironment, becoming a powerful tool to unravel the nature of intracellular nucleic acids. The analysis of nucleic acids unprecedentedly demands the sub‐molecule details at segment or subunit, secondary structure and monomer levels, instead of just probing the sequence and the abundance of nucleic acids. Detection of nucleic acids at the sub‐molecule level requires higher specificity and higher sensitivity, which becomes a new challenge in nucleic acid analysis. Herein, we summarize the recent progress in the design and the application of single‐cell nucleic acid imaging methods at the sub‐molecule level, including the visualization of RNA splicing variants, RNA G‐quadruplexes in an individual gene, single nucleotide variation of mitochondrial DNA, and RNA m6A methylation. Remarkably, we highlight the key strategy, "Module Assembly", for high‐performance molecular recognition and demonstrate the required improvements in future research. [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]

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]