title: Sensing miRNA: Signal Amplification by Cognate RISC for Intracellular Detection of miRNA in Live Cells.
abstract: The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence "turn on." Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecific cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.
authors.affiliation: Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02129, USA. amol@nmr.mgh.harvard.edu.
authors.forename: Amol
authors.initials: A
authors.lastname: Kavishwar
authors.affiliation: Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.|Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: Zdravka
authors.initials: Z
authors.lastname: Medarova
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D014162:Transfection
chemical_list: D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D034743:RNA-Induced Silencing Complex
keywords: Fluorescence; Intracellular detection; Live cell s; RISC; Sensor; Turn on; miRNA