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Detection and Live-Cell Imaging of a Micro-RNA Associated with the Cancer Neuroblastoma

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journal contribution
posted on 01.09.2016, 00:00 by Eoin Brennan, Roisin Moriarty, Tia E. Keyes, Robert J. Forster
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.

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