Direct Observation of Specific Messenger RNA in a Single Living Cell under a Fluorescence Microscope

Abstract

We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

“…12,27 In those experiments the researchers used the Bodipy493-Cy5 FRET pair to construct their binary probes obtaining similar results to ours. In our design we decided to use Alexa488 due to its high fluorescence quantum yield (0.94).…”

“…11 These conditions are generally fulfilled when there is no light absorption by the acceptor at the donor excitation wavelength, when the distance between the fluorophores is optimal, and when there is a good overlap between the donor fluorescence spectrum and the acceptor absorption spectrum. 27 One of the advantages of BPs is their high specificity since two distinct probes must bind the target to produce the characteristic detection signal. BPs avoid false positive signals due to non-specific opening since the fluorescent detection signal occurs only when the probes are brought together, and this occurs only when both probes are specifically hybridized with the target.…”

“…BPs avoid false positive signals due to non-specific opening since the fluorescent detection signal occurs only when the probes are brought together, and this occurs only when both probes are specifically hybridized with the target. 28 Some potential applications of BPs include in vitro monitoring of RNA transcription, 12 cellular imaging, 27 and identification of gene translocation. 11 Nevertheless, there is still much room for improvement in the design of BPs.…”

Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes

“…A few recent reports have described the use of beacon-type or linear FRET probes for microscopic imaging of mRNAs in eukaryotic cells (19)(20)(21)(22)(23)(24). A comparison of the present results with those prior ones offers some useful insights.…”

“…However, a small number of molecular approaches are under development (3,4,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Prominent among these approaches are quenched probe strategies, including molecular beacon (MB) probes (19,(22)(23)(24) and quenched autoligating (QUAL) probes (25)(26)(27)(28).…”