title: Detection and Live-Cell Imaging of a Micro-RNA Associated with the Cancer Neuroblastoma.
abstract: The ability of a molecular beacon to detect miR-132, a microRNA associated with the childhood cancer neuroblastoma, is reported in solution and within live cells. The stem-loop structure comprises a sequence complementary to miR-132, modified with a 6-FAM dye and dabcyl quencher on either end. In the absence of the target, self-binding occurs bringing the luminophore and quencher into close proximity, significantly decreasing the emission intensity. In the presence of miR-132, the signal is greatly enhanced, with a linear increase in intensity for mole ratios of beacon-to-target between 0.25 and 2.00. The structure differentiates between target and mismatched nucleic acid sequences, e.g., in the presence of a single-base mismatch, no increase in emission intensity beyond the background is observed. The stem-loop can be introduced into neuroblastoma cancer cells by electroporation, allowing miR-132 to be imaged within live cells. miR-132 appears to be localized within the nucleus of the cells, where its concentration is of the order of 1 μM. Significantly, transfection of the cells with a miR-132 mimic causes the emission intensity to more than double, demonstrating the sensitivity of the approach to changes in miR-132 concentration in live cells. This behavior opens up significant theranostic applications, such as the possibility of rapidly identifying retinoic acid resistant patients as well as providing a means to monitor therapeutic efficacy.
authors: National Centre for Sensor Research, School of Chemical Sciences, Dublin City University , Dublin 9, Ireland.
authors: Eoin
authors: E
authors: Brennan
authors: National Centre for Sensor Research, School of Chemical Sciences, Dublin City University , Dublin 9, Ireland.
authors: Roisin
authors: R
authors: Moriarty
authors: National Centre for Sensor Research, School of Chemical Sciences, Dublin City University , Dublin 9, Ireland.
authors: Tia E
authors: TE
authors: Keyes
authors: National Centre for Sensor Research, School of Chemical Sciences, Dublin City University , Dublin 9, Ireland.
authors: Robert J
authors: RJ
authors: Forster
mesh_terms: D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D009447:Neuroblastoma / Q000235:genetics*; D014162:Transfection; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; C547077:MIRN132 microRNA, human; D035683:MicroRNAs; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene