title: Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.
abstract: MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics.
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan 250012, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
authors.forename: Yongshun
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan 250012, PR China. Electronic address: wangl-sdu@sdu.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
mesh_terms: D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation*; D008168:Lung / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000295:instrumentation*
chemical_list: D035683:MicroRNAs; D004247:DNA
keywords: DNA tetrahedron substrate; Selectivity; Sensitivity; Toehold-mediated SDR; miRNA detection