title: An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA.
abstract: 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.
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. macb2012@csu.edu.cn.
authors.forename: Changbei
authors.initials: C
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. 15675888046@163.com.
authors.forename: Haisheng
authors.initials: H
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. Kefeng@csu.edu.cn.
authors.forename: Kefeng
authors.initials: K
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. macblab@163.com.
authors.forename: Mingjian
authors.initials: M
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. zg_zly@126.com.
authors.forename: Liyang
authors.initials: L
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha 410013, China. junwang@csu.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
mesh_terms: D004247:DNA; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D057230:Limit of Detection; D035683:MicroRNAs; D013050:Spectrometry, Fluorescence
chemical_list: D035683:MicroRNAs; D004247:DNA; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I
keywords: 2-aminopurine; exonuclease I; hairpin probe; microRNA; quencher-free