Qin Xinshu, Wang Xingyu, Xu Ke, Zhang Yi, Tian Hongye, Li Yinglei, Qi Bangran, Yang Xingbin
College of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang'an Avenue, Xi'an 710062, Shaanxi, China.
Department of Joint Surgery, Hong Hui Hospital, Xi'an Jiaotong University, Xi'an 710054, Shaanxi, China.
Mol Ther Nucleic Acids. 2022 Dec 27;31:241-255. doi: 10.1016/j.omtn.2022.12.015. eCollection 2023 Mar 14.
Here, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miRNAs differing only one nucleotide), wide dynamic range (seven orders of magnitude) and ability to accurately detect plant miRNAs (immune to hindrance of 2'-O-methyl moiety) enable SPLICER comparable with the commercially available TaqMan and miRCURY assays. SYBR green I, rather than expensive hydrolysis or locked nucleic acid probes indispensable to TaqMan and miRCURY assays, is adequate for SPLICER. The method was efficient (<1 h), economical ($7 per sample), and robust (able to detect xeno-miRNAs in mammalian bodies), making it a powerful tool for molecular diagnosis and corresponding therapy.
在此，我们建立了一种利用SplintR连接酶介导的、由核糖核酸酶H增强的互补配对探针连接法（SPLICER）来定量检测微小RNA（miRNA）的方法。该策略有两个步骤：（1）将两个与目标miRNA特异性杂交的DNA探针进行连接；（2）对连接后的探针进行定量聚合酶链反应（qPCR）扩增。探针的miRNA结合区域呈茎环结构，这种基序在高连接温度（65°C）下能显著减少非特异性连接。探针的末端设计成互补的，以形成配对探针，便于识别低浓度的目标miRNA。事实证明，核糖核酸酶H能够稳定由探针和目标miRNA形成的异源双链体，从而提高灵敏度（检测限 = 60个拷贝）。高特异性（区分仅相差一个核苷酸的同源miRNA）、宽动态范围（七个数量级）以及准确检测植物miRNA的能力（不受2'-O-甲基部分的阻碍）使得SPLICER与市售的TaqMan和miRCURY检测方法相当。SYBR绿I，而非TaqMan和miRCURY检测方法所必需的昂贵水解探针或锁核酸探针，对于SPLICER来说就足够了。该方法高效（<1小时）、经济（每个样品7美元）且稳健（能够检测哺乳动物体内的外源miRNA），使其成为分子诊断及相应治疗的有力工具。
