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