Department of Chemistry , Liaocheng University , Liaocheng , 252059 , Shandong , China.
Dongchangfu county education bureau , Liaocheng , 252059 , Shandong , China.
Bioconjug Chem. 2018 Apr 18;29(4):1399-1405. doi: 10.1021/acs.bioconjchem.8b00098. Epub 2018 Mar 20.
Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative approach for simple, sensitive, and selective miRNA quantification.
已经开发出了各种用于检测 miRNA 的荧光传感系统，但它们大多包含酶促扩增反应和标记程序。工具酶的严格反应条件和标记的高成本限制了它们的潜在应用，特别是在复杂的生物基质中。在这里，我们解决了这些难题，并报告了一种基于无酶非线性杂交链式反应（HCR）介导的多个 G-四链体的无标记荧光 DNA 树突状聚合物的策略，用于简单、灵敏和选择性地检测具有低背景信号的 miRNA。在该策略中，巧妙地在两条双链 DNA 的两端设计了分裂的 G-四链体（3:1）序列，将其用作非线性 HCR 组装的构建块，从而获得低背景信号。发夹开关探针（HSP）被用作识别和转导元件。在感测到靶 miRNA 后，在两条单链 DNA 辅助物的帮助下，两个块（块-A 和块-B）的非线性 HCR 组装被启动，导致具有多个 G-四链体掺入的 DNA 树突状聚合物的链分支生长。由于锌（II）原卟啉 IX（ZnPPIX）选择性地插入多个 G-四链体中，因此获得了荧光 DNA 树突状聚合物，导致荧光强度呈指数增加。受益于非线性 HCR 和低背景信号的出色性能，该策略具有简化的反应操作过程以及高灵敏度的特点。此外，所提出的荧光传感策略还表现出较好的选择性，并且无需修饰 DNA 块即可实现。重要的是，该策略还在乳腺癌细胞中进行了 miRNA 定量测试，具有较高的置信度。因此，这种基于非线性 HCR 介导的多个 G-四链体的无标记荧光 DNA 树突状聚合物的策略将成为一种用于简单、灵敏和选择性 miRNA 定量的替代方法。
