Ying Na, Ju Chuanjing, Sun Xiuwei, Li Letian, Chang Hongbiao, Song Guangping, Li Zhongyi, Wan Jiayu, Dai Enyong
Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
East China Sea Fisheries Research Institute, China Academy of Fishery Sciences, Shanghai, China.
PLoS One. 2017 Sep 25;12(9):e0185091. doi: 10.1371/journal.pone.0185091. eCollection 2017.
MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.
微小RNA（miRNA）构成了多种疾病的新型生物标志物。对miRNA表达进行准确和定量分析对于生物医学研究和临床诊疗至关重要。在本研究中，基于双链特异性核酸酶（DSN）和杂交链式反应（HCR）开发了一种用于灵敏且特异检测miRNA的双信号放大方法。设计并合成了一种报告探针（RP），其包含针对miR-21靶miRNA的识别序列（3'端用生物素修饰）以及针对HCR产物的捕获序列（5'端用Fam修饰）。HCR由引发探针（IP）的部分序列引发，IP的另一部分可与RP的捕获序列杂交，并由用生物素修饰的发夹探针（H1-bio和H2-bio）组装而成。miR-21触发固定在链霉亲和素（SA）包被磁珠（MB）上的RP的循环DSN切割。释放的Fam标记捕获序列随后与HCR产物杂交以产生可检测的双链DNA。然后将该聚合物滴加到侧流条上并观察到阳性结果。所提出的方法能够在100 fM至100 pM的动态范围内对miR-21进行定量序列特异性检测（检测限为2.1 fM，S/N = 3），具有出色的区分miRNA差异的能力。该方法在血清miRNA测定中显示出可接受的测试回收率。
