An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical method for cost-effective and sensitive detection of miRNA using a super 2-aminopurine (2-AP)-labeled hairpin probe (HP) and exonuclease I activity. Specifically, the fluorescence of 2-AP is strongly quenched when it is incorporated within DNA. In the presence of a target miRNA, HP attains an open conformation by hybridizing with the target miRNA to form a double-stranded structure with a protruding 3'-terminus. Next, the digestion of the protruding 3'-terminus is triggered by exonuclease I, during which 2-AP is released free in solution from the DNA, thereby increasing fluorescence. This method is highly sensitive, with a detection limit of 0.5 nM-10 times lower than a previously reported quencher-free fluorescence method. Furthermore, this method has potential applications in clinical diagnosis and biomedical research.