Target-Mediated EV-SNA Clustering as an Amplifier for <i>In Situ</i> miRNA Imaging in Living Cells.

Abstract

To overcome the low efficiency of probe delivery and the comprised detection limit due to dispersion of fluorescent molecules, we develop a miRNA detection-amplification system, called the miRDAS, which can be rapidly internalized by cells and amplify miRNA signals <i>in situ</i> within living cells. The miRDAS consists of two types of extracellular vesicle spherical nucleic acid (EV-SNA), named EVsnater and EVsnafier, which are generated by hydrophobic coassembly of natural EVs with cholesterol-modified oligonucleotides and exhibit rapid cellular uptake dynamics. Upon specifically binding to its target miRNA, the EVsnater activates the fluorescence and then self-assembles with EVsnafier to form giant fluorescent clusters, resulting in a brighter and significantly amplified fluorescence signal. Additionally, the miRDAS exhibits excellent flexibility and orthogonality, thus enabling simultaneous detection of multiple miRNA targets. As a proof of concept, a miR-124 miRDAS was designed and employed for successfully monitoring miR-124 expression levels during P19 neuronal differentiation.