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

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:target between 0.25 and 2.00. The structure differentiates between target and mis-matched nucleic acid sequences, e.g., in the presence of a single base mismatch, the intensity is approximately a factor of two lower than a fully complementary target reflecting its lower association constant. 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 1µM. 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.