Chapter 9 Tiny Molecular Beacons for in vivo mRNA Detection

The molecular beacon technology is an established approach for visualizing native mRNAs in living cells. These probes need to efficiently hybridize to accessible RNA regions in order to spatially and temporally resolve the dynamic steps of the RNA life cycle. A refined method using two computer algorithms, mfold and RNAstructure, is described for choosing shorter, more abundant target regions for molecular beacon binding. The probes are redesigned as small hairpins and are synthesized from 2¢-O-methyl RNA/LNA chimeric nucleic acids. These tiny molecular beacons are stable in the cellular environment and have a high affinity for binding to target RNAs. The user-friendly synthesis protocol and ability to couple to a variety of fluorophores make tiny molecular beacons the optimal technology to detect less abundant, highly structured RNAs, as well as small RNAs, such as microRNAs. As an example, tiny chimeric molecular beacons were designed to target regions of oskar mRNA, microinjected into living Drosophila melano-gaster oocytes and imaged via spinning disc confocal microscopy. Key words: Molecular beacons, 2¢-O-methyl RNA, LNA, Fluorescence live cell imaging, mRNA localization, mRNA transport Significant advances have been made over the last decade for visualizing and tracking of individual mRNAs within distinct mRNP complexes in subcellular space in real time (1, 2). Such approaches have provided a live glimpse of specific biological processes that have remained opaque thus far (3). Among them is the molecular beacon technology, which involves probes that fluoresce only upon hybridizing to specific complementary mRNA sequences. Introduced as an innovative and general approach, molecular beacons have been used in a variety of cell types, detecting mRNA at various levels of expression (4). Coupled with fast 3D imaging over time, molecular beacon technology has enabled highly time-resolved studies of RNA–protein interactions in viv