Rapid and highly sensitive hairpin structure-mediated colorimetric detection of miRNA

Abstract Herein, we report a novel hairpin structure–mediated diagnostic method for the simple and rapid colorimetric detection of miRNA through the sensing of pyrophosphate. When the hairpin structure of the template DNA (h-Probe) was hybridized with the primer, the DNA primer extension mediated by nPfu special enzyme was blocked. However, this h-Probe was extended using nPfu special enzyme, upon the structural change of the template DNA, from a hairpin to a linear structure, in the presence of the target miRNA. The miRNA-hybridized template DNA sequence was cleaved by a duplex-specific nuclease (DSN), which cleaved the DNA from the RNA–DNA hybrid, thereby allowing the target miRNA to be recycled. Primer extension using nPfu special enzyme produced pyrophosphate when nucleotide triphosphate was incorporated into the DNA; this pyrophosphate was sensed in terms of a color change, from pink to colorless, when using pp Probe, a probe developed previously by our group. This novel system for the colorimetric detection of target miRNA operated with high sensitivity (LOD = 132 aM) and selectivity, with the whole detection process requiring only 30 min. Furthermore, this system could also detect miRNA fluorimetrically with similar sensitivity (LOD = 105 aM), highlighting the dual-sensing properties of pp Probe. This unique, extremely simple, and rapid system for the detection of miRNA through a highly sensitive color change would presumably be useful in applications requiring point-of-care detection.