Recent advance in single-molecule counting for in vitro detection and in vivo imaging of RNA methylation and demethylation

RNA methylation and demethylation function as “molecular switches” to regulate the metabolism and function of RNA through a dynamic equilibrium. Accurate and sensitive detection of RNA methylation and demethylation is significant for molecular diagnostics and clinical therapeutics. Single-molecule fluorescence detection represents a cutting-edge bioanalytical technology characterized by its operational simplicity, minimal sample consumption, rapid analysis, and high sensitivity. By simply counting individual fluorescent events, single-molecule counting allows precise quantification of specific target molecules. In recent years, numerous single-molecule counting-based methods have been introduced for highly sensitive sensing of RNA methylation and demethylation, but a comprehensive review remains lacking. Herein, we aim to bridge this gap by providing an overview of the advance in single-molecule counting for in vitro detection and in vivo imaging of RNA methylation and demethylation, and discuss their fundamental principles, characteristics, and biomedical applications. Furthermore, we emphasize the existing challenges and prospective future trends in this field. • We review the advance in single-molecule counting for methylation and demethylation assays. • We summarize their applications in in vitro and in vivo biosensing. • We discuss the challenges and prospects in this field.