Sensing miRNA: Signal amplifi cation by cognate RISC for intracellular detection of miRNA in live cells

Abstract

The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fl uorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fl uorophore free from its quencher. This results in fl uorescence “turn on.” Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecifi c cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.