Bioimaging of miRNA biogenesis using a color-tunable molecular beacon

Molecular imaging is a novel technology used to study cellular and molecular mechanisms. MicroRNAs (miRNAs or miRs) play important roles in clinical diseases. Bioimaging of miRNA is a powerful tool used to study a variety of biological phenomena. Current bioimaging of miRNA biogenesis uses a reporter gene and a mono-fluorophore-based molecular beacon (MB) to visualize miRNA-regulating cellular developments. However, the miRNA reporter gene is unable to determine the miRNA function-regulating signal-off imaging activity of cellular death. In addition, a miR MB has limited accuracy in detecting miRNA expression when fluorescence intensity in cells is weak. To overcome the disadvantages of both miRNA imaging systems, our group developed dual-fluorophore-based color-tunable miR MBs (ColoR MBs). These MBs consist of a partial double-stranded DNA oligonucleotide containing a target miRNA binding site with a fluorophore (Cy3)/black hole quencher 1 (BHQ1) at one end as a reporter probe and another fluorophore (Cy5.5) without the quencher at the other end as a reference probe. The reference probe of ColoR MB is always turned on regardless of miRNA expression. In the absence of miRNA of interest, the fluorescence activity of the reporter probe from ColoR MB was quenched due to close proximity between the fluorophore and quencher. Therefore, cells expressing little or no miRNA of interest were only visualized by the reference probe, resulting in red color. When the target miRNA was expressed in cells, it bound to the miRNA binding site of the ColoR MB. The quencher then separated from the MB, activating the fluorescence signals of the reporter probe. Cells were visualized as a yellow color due to merging of green from Cy3 and red from Cy5.5. The reference probe of ColoR MB successfully differentiated the low fluorescence activity of the reporter probe resulting from weak expression of target miRNA or low transfection of the ColoR MB in cells. The color-tunable specificity of the ColoR MB for sensing color changes based on miRNA expression and miRNA-regulating cellular development overcame limitations of the miRNA reporter gene, which shows signal-off imaging activity in the presence of target miRNA.