Visualizing the Movement of mRNAs in Living Cells

描述(由申请人提供)： 分子信标将被用来成像活细胞中特定mRNAs的合成、降解、移动和定位。这些探针是寡核苷酸，杂交后会变成荧光。我们已经证明了由2‘-O-甲基核糖核苷酸组成的分子信标在细胞内是稳定的，不会导致靶RNA的破坏，并且可以用来可视化特定RNA的分布。使用这些探针，我们将研究mRNAs在细胞内自身运输和定位的机制。同样的探针将用于研究与细胞昼夜节律相关的基因表达的周期性变化。在对许多mRNAs的定位和运输进行了深入研究的果蝇卵母细胞中，我们将寻求含有mRNAs的核糖核蛋白颗粒通过细胞骨架纤维到达目的地的直接证据。我们将在分子信标的帮助下探索这些核糖核蛋白颗粒的结构，分子信标中的荧光团通过可光裂解的连接物连接到探针的其余部分。我们还将研究将不同的mRNAs发送到细胞内不同位置，但使用相同的运输机制的机制。为了观察单个mRNA分子在培养细胞内的运动，我们将构建一个可诱导的人工基因，编码包含256个串联分子信标靶标的mRNA。有了这个结构，我们将能够跟踪单个mRNA分子在转录、成熟、从细胞核输出并被送到细胞质的不同区域进行翻译时的运动。我们还提出了分子信标的新结构和配置，使我们能够控制它们在细胞内的分布，从而提高它们信号的特异性。由于在大的mRNA中很难预测探针可访问序列的位置，我们将开发一种新的实验方法来寻找这些位点。最后，我们将开发一种新的高效的方法将探针输送到细胞中。这种方法基于天然圈套介导的囊泡融合的作用机制，使用互补的单链寡核苷酸锚定在细胞和囊泡的表面以促进它们的融合。通过这些协同技术的发展，将有可能观察特定的mRNAs在活细胞中的运动。

DESCRIPTION (provided by applicant): 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.