A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.