Ultrasensitive Quantification of microRNA Copy Number in Individual Extracellular Vesicles Using DNA...

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.