Detection of miRNA expression in intact cells using activatable sensor oligonucleotides.
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.
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA.
Byunghee
B
Yoo
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA.
Amol
A
Kavishwar
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA.
Subrata K
SK
Ghosh
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, 200 Longwood Avenue, D-239, Boston, MA 02115, USA.
Natalie
N
Barteneva
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA.
Mehmet V
MV
Yigit
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA. Electronic address: amoore@helix.mgh.harvard.edu.
Anna
A
Moore
MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA. Electronic address: zmedarova@partners.org.
Zdravka
Z
Medarova
D020029:Base Pairing; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D002474:Cell-Free System; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism
D005456:Fluorescent Dyes; C547183:MIRN10 microRNA, human; D035683:MicroRNAs; D009841:Oligonucleotides; D012260:Ribonucleases