A technique is being developed for the visualization of specific mRNA sequences in living cells. This technique utilizes molecular beacons, which are oligonucleotide probes that become fluorescent upon hybridization. Use of molecular beacons allows the simultaneous mapping of the intracellular distribution of multiple mRNAs within living cells and imaging of gene expression on a cell-by-cell basis. Using a model system that employs syncytial embryos of Drosophila melanogaster, we will show that the dynamic changes that occur in the localization of different mRNAs can be observed in real-time in living cells. The distribution of multiple mRNAs will be mapped within the same cell by introducing a mixture of several different molecular beacons, where each molecular beacon is specific for a different mRNA target, and each molecular beacon is labeled with a differently colored fluorophore. The distribution of a group of mRNAs that localize at postsynaptic dendritic sites in response to the synaptic stimuli will be mapped by microinjecting molecular beacons into the perinuclear space of neurons maintained in primary cultures. The intracellular transport of mRNAs within neurons in the form of ribonucleoprotein particles will be monitored using confocal laser scanning microscopy, in order to shed light on the mechanism of mRNA localization. Techniques will be developed for an effective delivery of molecular beacons into cells in suspension and gene expression will be monitored in individual cells using fluorescence-activated cell sorting. The ability to detect mRNAs in living cells with molecular beacons will allow us to explore the molecular complexities of mRNA localization and transport within neurons and other cells with a sharper focus than has been possible before.
|Bratu, Diana P (2006) Molecular beacons: Fluorescent probes for detection of endogenous mRNAs in living cells. Methods Mol Biol 319:1-14|
|Marras, Salvatore A E; Kramer, Fred Russell; Tyagi, Sanjay (2003) Genotyping SNPs with molecular beacons. Methods Mol Biol 212:111-28|
|Bratu, Diana P; Cha, Byeong-Jik; Mhlanga, Musa M et al. (2003) Visualizing the distribution and transport of mRNAs in living cells. Proc Natl Acad Sci U S A 100:13308-13|
|Marras, Salvatore A E; Kramer, Fred Russell; Tyagi, Sanjay (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30:e122|
|Mhlanga, M M; Malmberg, L (2001) Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25:463-71|
|Tyagi, S; Marras, S A; Kramer, F R (2000) Wavelength-shifting molecular beacons. Nat Biotechnol 18:1191-6|