title: Ultrasensitive and rapid detection of miRNA with three-way junction structure-based trigger-assisted exponential enzymatic amplification.
abstract: Aberrant expression of micro RNA (miRNA) is associated with development of cancers and diseases, so miRNA has become a tissue-based biomarker for cancer prognosis and diagnosis. Herein, a novel trigger-assisted exponential enzymatic amplification (T-EXPEA) method for ultrasensitive miRNA detection based on three-way junction (3-WJ) structure driven has been reported, which can be used in potential applications in cancer prognosis and diagnoses. In this assay, target miRNA can unfold hairpin probe and start the reaction, and thus specifically form stable 3-WJ structure with helper. Then it can produce triggers under the synergetic polymerase and restriction endonucleases amplification. The produced triggers could be used to unfold molecular beacon (MB) and initiate T-EXPEA process. In the EXPEA part, the exponential triggers were generated to initiate new T-EXPEA and high enhancement fluorescence amplification efficiency was obtained. The feature of our strategy lies in the T-EXPEA combining with 3-WJ structure has been utilized for fluorescence miRNA detection. It is worth noting that the sequence of the triggers in T-EXPEA part is the same to that of triggers generated from the 3-WJ part. In addition, the design of restriction enzyme cutting sites using the same restriction enzyme (Nt.BbvCI) in hairpin probe and MB respectively, improved reaction efficiency cost-efficiently. This method can quantitatively detect sequence-specific miRNA in a dynamic range from 10 aM to 10 pM with a detection limit as low as 7.8 aM.
authors.affiliation: Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Resources and Environment, University of Jinan, Jinan 250022, PR China.
authors.forename: Su
authors.initials: S
authors.lastname: Liu
authors.affiliation: College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Yu
authors.affiliation: College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Wang
authors.affiliation: College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China.
authors.forename: Yuna
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China; College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China. Electronic address: chm_huangjd@ujn.edu.cn.
authors.forename: Jiadong
authors.initials: J
authors.lastname: Huang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D005453:Fluorescence; D056945:Hep G2 Cells; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D035683:MicroRNAs; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes
keywords: EXPAR; Isothermal synergetic enzyme amplification; Three-way junction structure; miRNA detection