使用可激活的传感器寡核苷酸检测完整细胞中的miRNA表达。
Detection of miRNA expression in intact cells using activatable sensor oligonucleotides.
作者信息
Yoo Byunghee, Kavishwar Amol, Ghosh Subrata K, Barteneva Natalie, Yigit Mehmet V, Moore Anna, Medarova Zdravka
机构信息
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, 200 Longwood Avenue, D-239, Boston, MA 02115, USA.
出版信息
Chem Biol. 2014 Feb 20;21(2):199-204. doi: 10.1016/j.chembiol.2013.12.007. Epub 2014 Jan 16.
We describe a technology for the profiling of miRNA expression in intact cells. The technology is based on sensor oligonucleotides that are cleavable, completely complementary to a target miRNA, and dual-labeled with a fluorescent dye and a quencher. Upon entering the cell, the sensor oligonucleotide binds its specific miRNA target through complementary base-pairing. This triggers assembly of the endogenous RNA Induced Silencing Complex (RISC) around the miRNA-sensor duplex and cleavage of the sensor oligonucleotide, resulting in separation between the dye and quencher, and a fluorescence turn-on. In the presented feasibility studies, we focus on a specific miRNA (miR-10b) implicated in breast cancer metastasis. Using a human breast adenocarcinoma cell line, we illustrate the application of this technology for miRNA detection with nanomolar sensitivity in both a cell-free system and intact cells.
我们描述了一种用于完整细胞中微小RNA（miRNA）表达谱分析的技术。该技术基于可切割的传感器寡核苷酸，其与目标miRNA完全互补，并由荧光染料和淬灭剂进行双标记。进入细胞后，传感器寡核苷酸通过互补碱基配对与特定的miRNA靶标结合。这会触发内源性RNA诱导沉默复合体（RISC）围绕miRNA-传感器双链体组装，并导致传感器寡核苷酸的切割，从而使染料和淬灭剂分离，荧光开启。在本可行性研究中，我们聚焦于一种与乳腺癌转移相关的特定miRNA（miR-10b）。使用人乳腺腺癌细胞系，我们展示了该技术在无细胞系统和完整细胞中以纳摩尔灵敏度检测miRNA的应用。
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