Molecular beacons light the way: Imaging native mRNAs in living cells

Extract: In vivo techniques to investigate the pathways that mRNAs undergo from maturation to localization at specific regions within a cell have thus far been the exclusive domain of protein biology as our ability to probe for nucleic acids in vivo have been hindered. In vivo imaging of synthetic transcripts helped dissect such a fundamental cellular process. Yet, in order to fully grasp the diverse organization and behavior of mRNAs in living cells, it is important to visualize the distribution and dynamics of RNA molecules in their living context, much like green fluorescent protein and its variants that fluoresce in diverse colors, have allowed us to image the dynamics of proteins. In 1996, our laboratory described a novel approach for detecting nucleic acids in solution. We have proceeded to make significant adaptations to this technique to facilitate visualization of the distribution and trafficking of mRNAs in living cells. This technique utilizes hybridization probes, called "molecular beacons," that generate fluorescent signals only when hybridized to a complementary nucleic acid target sequence. Molecular beacons are single-stranded oligonucleotides that form a stem-and-loop structure, where the loop portion of the molecule is a probe sequence complementary to a target RNA sequence, and the stem is formed by the annealing of complementary arm sequences which envelope the probe sequence. A fluorophore and a quencher moiety are covalently linked to the end of each arm, respectively.