title: Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection.
abstract: Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Dian-Ming
authors.initials: DM
authors.lastname: Zhou
authors.forename: Wen-Fang
authors.initials: WF
authors.lastname: Du
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xi
authors.forename: Jia
authors.initials: J
authors.lastname: Ge
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Jiang
mesh_terms: D045744:Cell Line, Tumor; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D051981:Uracil-DNA Glycosidase / Q000737:chemistry
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D004259:DNA-Directed DNA Polymerase; D051981:Uracil-DNA Glycosidase