Detection of Extracellular Vesicle RNA Using Molecular Beacons

Summary Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.
Graphical Abstract
Highlights • RBC and RBC-EVs contain unequal amount of RNA • Efficient detection of miRNA in vitro by MBs using nano-flow cytometry • CPPs effectively deliver cargo to cells and extracellular vesicles • MB-CPP can be used to detect cell-specific EV miRNAs
Biochemistry Methods; Biomolecules; Cell Biology; Functional Aspects of Cell Biology