Oriented Tetrahedron-Mediated Protection of Catalytic DNA Molecular-Scale Detector against in Vivo Degradation for Intracellular miRNA Detection
Oriented Tetrahedron-Mediated Protection of Catalytic DNA Molecular-Scale Detector against in Vivo Degradation for Intracellular miRNA Detection
复制标题
催化 DNA 分子尺度检测器的定向四面体介导保护，防止细胞内 miRNA 检测的体内降解
DOI：
10.1021/acs.analchem.9b00860
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发表时间：
2019-09-17
影响因子：
7.4
通讯作者：
Wu, Zai-Sheng
中科院分区：
文献类型：
--
作者：
Li, Congcong;Xue, Chang;Wu, Zai-Sheng
我们报道了一种基于四面体的DNA酶探针（Tetra-ES）用于细胞内miRNA的检测。两个DNA四面体（Tetra）以独特的方向排列在酶（E）/底物（S）复合物的不同位置。Na+依赖性DNAzme被设计为最初锁定以抑制DNAzyme的活性。荧光成像和凝胶电泳分析表明，沉默的DNAzyme可以特异性地启动细胞内的目标miRNA。激活的DNAzyme重复切割底物，允许可控的信号转导和放大效应。DNA四面体的空间控制排列与锁定的DNAzyme的刺激响应行为的组合改善了细胞渗透性和期望的核酸酶抗性。Tetra-ES检测器表现出比非扩增分子信标对应物高至少10倍的检测灵敏度（LOD为16 pM），并且能够将miRNA靶标与相应的家族成员区分开。利用Tetra-ES检测器可靠地筛选目标细胞内的靶miRNA以及相同类型细胞系内的不同miRNA的表达水平。作为一种细胞内探针，Tetra-ES可以提供有价值的见解，开发一种基于均匀DNA纳米结构的可控信号转导策略，适用于检测miRNA和潜在的应用于癌症诊断，预后和治疗。
We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedra (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular target miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing a controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedra with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. The Tetra-ES detector exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the nonamplification molecular beacon counterpart and was capable of discriminating the miRNA target from the corresponding family members. The expression levels of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis, and therapeutics.