Search

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]

Imaging agents, molecular recognition, nucleic acids, single-cell imaging, sub-molecule level Frontispiece: Module Assembly Strategy for Single-Cell Nucleic Acid Imaging at the Sub-Molecule Level Here, the key strategy, "Module Assembly", for single-cell imaging of nucleic acids at the sub-molecule level is reviewed. [Extracted from the article]