Module Assembly Strategy for Single‐Cell Nucleic Acid Imaging at the Sub‐Molecule Level

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 m6 A methylation. Remarkably, we highlight the key strategy, "Module Assembly", for high-performance molecular recognition and demonstrate the required improvements in future research.