“Lock and Key”: Binding Behavior of Malachite Green and Functional Nucleic Acids and Their Multidimensional Applications

Small molecules often suffer from poor druggability, and their binding conformations with nucleic acids play a critical role in determining biological function and application potential. The interaction between small molecules and functional nucleic acids (FNAs) offers key insights into nucleic acid conformational mechanisms and structural adaptability. Among them, malachite green (MG), a triphenylmethane dye, has emerged as a model molecule due to its unique photophysical properties, especially its fluorescence activation upon conformational restriction. Importantly, MG has also drawn significant attention due to its known toxicity and widespread use in aquaculture and industry, raising concerns in areas such as food safety and environmental health. Therefore, studying MG's interaction with FNAs advances molecular design and bioengineering while supporting safer, more effective detection and detoxification strategies. Using MG as a representative case, this review systematically discusses its binding mechanisms with five typical FNA structures—RNA aptamers, DNA aptamers, G‐quadruplexes, double‐stranded DNA, and thymine‐Hg(II)‐thymine motifs. Herein, eight major application areas of MG‐FNA complexes, including MG biosensing, target biosensing, bioimaging, synthetic biology, structural monitoring, biocatalysis, masking application, and physiological regulation, is further summarized. Finally, the future directions for enhancing MG‐FNA‐based molecular systems are highlighted, offering guidance for advancing nucleic acid nanotechnology.