title: Label-free microRNA detection based on terbium and duplex-specific nuclease assisted target recycling.
abstract: In this paper, we describe a novel label-free fluorescence method for microRNA-21 (miR-21) detection based on terbium (Tb(3+)) and duplex-specific nuclease (DSN) assisted target recycling. Capture probes (Cps), containing a target-binding part and a signal-output part, are immobilized on magnetic beads (MBs). In the presence of the target miR-21, it hybridizes with the target-binding part of a Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzes the target-binding part of the Cp while liberating the intact target miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually, through magnetic separation, only the signal-output part of the Cp could remain in solution and function as a signalling flare to increase the fluorescence intensity of Tb(3+) dramatically. By employing the above strategy, this approach can gain an amplified fluorescent signal and detect as low as 8 fM miR-21 under the optimized conditions. Moreover, due to the high selectivity of DSN, the method shows little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human cell lysate samples of breast cancer patients.
authors.affiliation: The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, and College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China. 1601225385@qq.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wu
authors.forename: QiuXiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Mei
authors.initials: M
authors.lastname: Chen
authors.forename: Yaokun
authors.initials: Y
authors.lastname: Xia
authors.forename: Shuxian
authors.initials: S
authors.lastname: Cai
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.forename: Fang
authors.initials: F
authors.lastname: Wu
authors.forename: Jinghua
authors.initials: J
authors.lastname: Chen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004720:Endonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D013725:Terbium / Q000737:chemistry*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D013725:Terbium; D004720:Endonucleases