Detection of miRNA expression in intact cells using activatable sensor oligonucleotides

Abstract

We describe a technology for the profiling of miRNA expression in intact cells. The technology is based on sensor oligonucleotides that are cleavable, completely complementary to a target miRNA, and dual-labeled with a fluorescent dye and a quencher. Upon entering the cell, the sensor oligonucleotide binds its specific miRNA target through complementary base-pairing. This triggers assembly of the endogenous RNA Induced Silencing Complex (RISC) around the miRNA-sensor duplex and cleavage of the sensor oligonucleotide, resulting in separation between the dye and quencher, and a fluorescence turn-on. In the presented feasibility studies, we focus on a specific miRNA (miR-10b) implicated in breast cancer metastasis. Using a human breast adenocarcinoma cell line, we illustrate the application of this technology for miRNA detection with nanomolar sensitivity in both a cell-free system and intact cells.