Ultrasensitive Quantification of microRNA Copy Number in Individual Extracellular Vesicles Using DNA Tetrahedron-Based Single-Molecule Imaging

The ultrasensitive detection of microRNAs (miRNAs) in extracellular vesicles (EVs) can accurately reflect the progress and metastasis of miRNA-mediated intercellular communication, providing an unprecedented opportunity for liquid biopsy. However, due to the low abundance and high heterogeneity of miRNAs in EVs, the ultrasensitive quantification and establishment of a distribution model for miRNA within native EVs remain challenging. Here, we have developed a DNA tetrahedron-based single-molecule fluorescence imaging strategy to overcome this challenge. The internalization efficiency of the probe was as high as 70% without disrupting the native structure of EVs, and combined with single-molecule fluorescence imaging, we achieved in situ imaging analysis of single-copy miRNA in individual EVs without amplification for the first time. A new distribution model for miRNAs has been revealed by statistical analysis of the copy number of miRNAs in EVs across multiple cell lines, characterized by low occupancy and a heterogeneous distribution. More importantly, we found that drug resistance cancer cells promote an increase in the number of drug resistance-related miRNAs within EVs without a corresponding increase in the number of EVs secreted, providing new insights into the EV miRNA sorting mechanisms. We anticipate that this technology will rapidly advance miRNA-mediated intercellular communication based on EVs.