Molecular beacons will be used to image the synthesis, degradation, movement and localization of specific mRNAs in living cells. These probes are oligonucleotides that become fluorescent upon hybridization. We have shown that molecular beacons composed of 2'-O-methylribonucleotides are stable within cells, do not cause the destruction of target RNAs, and can be used to visualize the distribution of specific mRNAs. Using these probes, we will study the mechanisms that mRNAs use for their own transport and localization within cells. The same probes will be used to study cyclical changes in gene expression that are associated with cellular circadian rhythms. Working with fruitfly oocytes, in which the localization and transport of many mRNAs has been studied intensively, we will seek direct proof of the view that ribonucleoprotein particles containing mRNAs travel on cytoskeletal fibers to reach their destinations. We will explore the structure of these ribonucleoprotein particles with the aid of molecular beacons in which the fluorophore is linked to the rest of the probe via a photocleavable linker. We will also study mechanisms that send different mRNAs to different sites in the cell, yet utilize the same transport machinery. In order to observe the movement of single mRNA molecules within cultured cells, we will construct an inducible artificial gene that encodes an mRNA containing 256 tandem molecular beacon targets. With this construct, we will be able to follow the movement of individual mRNA molecules as they are transcribed, undergo maturation, are exported from the nucleus, and are sent to different regions of the cytoplasm for translation. We are also proposing new structures and configurations for molecular beacons that will enable us to control their distribution within the cell, which improves the specificity of their signals. Since it is difficult to predict the location of probe-accessible sequences in large mRNAs, we will develop a new experimental approach for finding those sites. Finally, we will develop a new and efficient method for delivering probes into cells. Based on the mechanism of action of natural SNARE-mediated vesicle fusion, this method employs complementary single-stranded oligodeoxyribonucleotides that are anchored to the surfaces of cells and vesicles to facilitate their fusion. Through the development of these synergistic techniques, it will be possible to observe the movement of specific mRNAs in living cells.
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