Fig. 2.

Invader assay. A) The assay involves two reactions. In the primary reaction, invasive and probe oligonucleotides with hairpin overhangs for enhancing stability are bound to the target miRNA. Hybridization results in the formation of a 5′ overlap-flap structure from the probe oligonucleotide, which serves as a substrate for the structure-specific 5′ nuclease, which causes release of the 5′ flap. In the secondary reaction, a FRET oligonucleotide labeled with a fluorophore (F) and a quencher (Q) is added to a secondary reaction template (SRT). The released 5′ flap from the primary reaction acts as an invasive probe in the secondary reaction, which leads to the formation of a second fluorophore-conjugated overlap-flap structure. Cleavage of this 5′ flap releases the fluorophore to generate a signal that can be quantified. B) An arrestor oligonucleotide complementary to the probe oligonucleotide is used in the secondary reaction that binds to the uncleaved probe to avoid its interference in the binding of the FRET probe to SRT, which reduces the background noise.