Tiny Molecular Beacons: LNA/2′-<i>O</i>-methyl RNA Chimeric Probes for Imaging Dynamic mRNA Processes in Living Cells

Abstract

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2′-<i>O</i>-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2′-<i>O</i>-methyl RNA chimera oligonucleotides “tiny molecular beacons”. We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons and the flexibility to couple them to a large variety of fluorophores and quenchers render them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for <i>oskar</i> mRNA and microinjected them into living <i>Drosophila melanogaster</i> oocytes. We then imaged the live oocytes <i>via</i> spinning disk confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons and are able to access previously inaccessible target regions