title: Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level.
abstract: Sensitive and specific analysis of microRNAs (miRNAs) in a single tube without the need of thermal cycler instrument would greatly facilitate the investigation of miRNA-associated regulatory circuits and diseases. Homogeneous isothermal amplification assays are attractive in conducting single tube assays that can minimize contamination-prone steps and simplifies assay procedures. However, the relative low amplification efficiency and high detection background remain as bottlenecks restricting their more versatile applications. In this work, we have developed a novel isothermal exponential enzymatic amplification (IEEA) strategy for miRNAs analysis. By rational triple cascade amplification cycles of target recycling, nicking-replication reaction, and DNAzyme catalysis, the strategy exhibited high signal amplification efficiency (10(4)-10(9) folds of amplification in 1h) with very low detection background and excellent specificity. As a result, the miR-27a target model was rapidly determined with a limit of detection down to 0.79 aM (S/N=3), corresponding to 94 copies of the miRNA molecule in a 200 μL sample solution. The levels of miR-27a in atherosclerotic sprague-dawley rats were accurately quantified. The strategy is anticipated to have an important impact on the development of simple and rapid molecular diagnostic applications for any short oligonucleotides.
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Xueqing
authors: X
authors: Zhou
authors: College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
authors: Yongqian
authors: Y
authors: Liang
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Yuzhi
authors: Y
authors: Xu
authors: College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
authors: Xiulian
authors: X
authors: Lin
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Jun
authors: J
authors: Chen
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Yingjun
authors: Y
authors: Ma
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Li
authors: L
authors: Zhang
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
authors: Danping
authors: D
authors: Chen
authors: College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
authors: Fenyun
authors: F
authors: Song
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China. Electronic address: daizong@mail.sysu.edu.cn.
authors: Zong
authors: Z
authors: Dai
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China; SYSU-CMU Shunde International Joint Research Institute, Shunde, Guangdong 528300, PR China. Electronic address: ceszxy@mail.sysu.edu.cn.
authors: Xiaoyong
authors: X
authors: Zou
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods; D057230:Limit of Detection; D008297:Male; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017207:Rats, Sprague-Dawley
chemical_list: C000594049:MIRN27 microRNA, rat; D035683:MicroRNAs
keywords: Atherosclerotic sprague-dawley rats; Homogeneous isothermal amplification assays; Isothermal exponential enzymatic amplification (IEEA) strategy; MicroRNAs analysis