title: Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis.
abstract: The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5' terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.
authors.affiliation: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China.
authors.forename: Xueqing
authors.initials: X
authors.lastname: Zhou
authors.affiliation: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China.
authors.forename: Yingjun
authors.initials: Y
authors.lastname: Ma
authors.affiliation: College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China.
authors.forename: Xiulian
authors.initials: X
authors.lastname: Lin
authors.affiliation: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China daizong@mail.sysu.edu.cn.
authors.forename: Zong
authors.initials: Z
authors.lastname: Dai
authors.affiliation: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China SYSU-CMU Shunde International Joint Research Institute, Shunde, Guangdong 528300, China.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Zou
mesh_terms: D001710:Biotin / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic; D002583:Uterine Cervical Neoplasms / Q000235:genetics
chemical_list: D035683:MicroRNAs; C487931:mirnlet7 microRNA, human; D001710:Biotin; D004259:DNA-Directed DNA Polymerase