School of Biological Sciences and Technology, University of Jinan, Jinan 250022, P.R. China.
Analyst. 2019 Aug 16;144(17):5245-5253. doi: 10.1039/c9an00953a.
We have formulated a rapid and high-efficiency fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced exponential rolling circular amplification (RCA) strategy for the ultrasensitive detection of miR-21. In this strategy, there are several aspects that are worthwhile. First, the functionalized THMS, comprising a typical triplex structure (T-A·T), specific recognition sequence for nicking endonuclease, complementary sequence for miR-21, and RCA product-annealing sequence, was concurrently used to perform signal transduction with one fluorophore and one quencher. As compared to the traditional double-helix molecular switches or molecular beacons, one of the biggest differentiating factors is that the properties of THMSs are independent of any specific binding sequence that they may contain. As far as we know, this is the first time that an ingeniously designed THMS not only contains the primer for exponential RCA, but also functions as the tracer for fluorescence assay. In the presence of miR-21, targets can induce conformation changes in THMS with the release of the trapped DNA segment (P), which, in turn, can activate the first run of the RCA process. Meanwhile, the RCA reaction is also initiated by the formation of a similar primer (SP) as the trapped DNA through a continuous "extension-nicking" reaction. Secondly, the resultant first-generation RCA product consists of numerous tandem repeated regions that can attach to countless THMSs, resulting in the release of the trapped DNA segment (P) for initiating the second run of the RCA reaction. Significantly, a large amount of THMSs were continuously consumed to yield a remarkably strong fluorescent signal. In addition, this biosensor was demonstrated to exhibit improved sensitivity owing to the high efficiency and rapid amplification kinetics of the exponential RCA and high selectivity toward miR-21 with a limit of detection as low as 1.1 aM. Hence, the target-mediated THMS-conformation-change-initiated exponential RCA strategy presents an optimal detection performance toward analytes for potential applications in related fundamental research and clinical diagnosis.
我们基于靶标激活的三链体分子开关（THMS）构象变化引发的指数滚环扩增（RCA）策略，构建了一种快速高效的荧光生物传感平台，用于超灵敏检测 miR-21。在该策略中，有几个方面值得注意。首先，功能化的 THMS 由典型的三链体结构（T-A·T）、切口内切酶的特异性识别序列、miR-21 的互补序列和 RCA 产物杂交序列组成，同时使用一个荧光团和一个猝灭剂进行信号转导。与传统的双链分子开关或分子信标相比，THMS 的最大区别之一是其性质不依赖于它们可能包含的任何特定结合序列。据我们所知，这是第一次设计巧妙的 THMS 不仅包含指数 RCA 的引物，而且还作为荧光测定的示踪剂。在 miR-21 的存在下，靶标可以诱导 THMS 的构象变化，释放被捕获的 DNA 片段（P），这反过来又可以激活 RCA 过程的第一轮。同时，通过连续的“延伸-切口”反应，形成与被捕获的 DNA 相似的引物（SP）也可以启动 RCA 反应。其次，所得的第一代 RCA 产物包含许多串联重复区域，可以附着无数的 THMS，从而释放被捕获的 DNA 片段（P），以启动 RCA 反应的第二轮。重要的是，大量的 THMS 被连续消耗，产生了非常强的荧光信号。此外，由于指数 RCA 的高效率和快速扩增动力学以及对 miR-21 的高选择性，该生物传感器表现出了更好的灵敏度，检测限低至 1.1 aM。因此，靶介导的 THMS 构象变化引发的指数 RCA 策略为分析物的检测提供了最佳性能，有望在相关基础研究和临床诊断中得到应用。
