Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, Fujian Engineering Research Center for Drug and Diagnoses-Treat of Photodynamic Therapy, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry , Fuzhou University , Fuzhou 350002 , People's Republic of China.
Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences , Wenzhou Medical University , Wenzhou 325035 , People's Republic of China.
Anal Chem. 2019 Sep 17;91(18):11529-11536. doi: 10.1021/acs.analchem.9b00860. Epub 2019 Aug 26.
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.
我们报告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。设计了一种 Na 依赖性 DNAzme 以最初锁定来抑制 DNA 酶的活性。荧光成像和凝胶电泳分析表明，沉默的 DNA 酶可以被细胞内目标 miRNA 特异性启动。激活的 DNA 酶反复切割底物，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 DNA 酶的刺激响应行为的结合提高了细胞通透性和理想的核酸酶抗性。Tetra-ES 探测器的检测灵敏度至少比非扩增分子信标对照物高 10 倍（LOD 为 16 pM），并且能够从相应的家族成员中区分 miRNA 靶标。利用 Tetra-ES 探测器可以可靠地筛选感兴趣的细胞内靶标 miRNA 以及同一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 可能为开发基于同质 DNA 纳米结构的可控信号转导策略提供有价值的见解，该策略适用于 miRNA 的检测，并有可能应用于癌症诊断、预后和治疗。
