title: Target-triggered three-way junction structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine for highly specific, one-step, and rapid microRNA detection at attomolar level.
abstract: MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3'-end, the 5'-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3' end modification (2'-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay.
authors: Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University , Xianning West Road, Xi'an, Shaanxi 710049, P. R. China.
authors: Qing
authors: Q
authors: Zhang
authors: Feng
authors: F
authors: Chen
authors: Feng
authors: F
authors: Xu
authors: Yongxi
authors: Y
authors: Zhao
authors: Chunhai
authors: C
authors: Fan
mesh_terms: D045744:Cell Line, Tumor; D004256:DNA Polymerase I / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D035683:MicroRNAs; D004256:DNA Polymerase I; D003850:Deoxyribonuclease I