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Visualizing the Movement of mRNAs in Living Cells

活细胞中 mRNA 运动的可视化

基本信息

批准号：
7222816
负责人：
SANJAY TYAGI
金额：
$ 28.48万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7222816
关键词：
Actins Amino Acid Sequence Binding Cell Line Cell Nucleus Cell fusion Cell membrane Cell surface Cells Chickens Circadian Rhythms Color Complementary DNA Cultured Cells Cytoplasm Cytoplasmic Protein DNA DNA Sequence Destinations Development Drosophila genus Elements Embryo Ensure Fiber Fibroblasts Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Transcription Green Fluorescent Proteins Image Individual Label Libraries Life Link Liposomes Location Mason-Pfizer monkey virus Mediating Membrane Messenger RNA Methods Molecular Molecular Conformation Monitor Movement Nuclear Nuclear Export Nucleic Acids Nucleotides Oligodeoxyribonucleotides Oligonucleotide Probes Oligonucleotides Oocytes Pathway interactions Peptide Sequence Determination Positioning Attribute Proteins RNA Rat-1 Reaction Rest Ribonuclease H Ribonucleoproteins Signal Transduction Site Specificity Structure Synthetic Genes Techniques Translations Travel Vesicle Work base fluorophore improved in vivo mRNA Expression member particle research study

项目摘要

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.

描述（由申请人提供）：分子信标将用于对活细胞中特定 mRNA 的合成、降解、运动和定位进行成像。这些探针是杂交时发出荧光的寡核苷酸。我们已经证明，由2'-O-甲基核糖核苷酸组成的分子信标在细胞内稳定，不会导致靶RNA的破坏，并且可用于可视化特定mRNA的分布。使用这些探针，我们将研究 mRNA 在细胞内自身运输和定位的机制。相同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。通过对果蝇卵母细胞中许多 mRNA 的定位和运输进行了深入研究，我们将寻求直接证据来证明含有 mRNA 的核糖核蛋白颗粒在细胞骨架纤维上移动到达目的地的观点。我们将借助分子信标探索这些核糖核蛋白颗粒的结构，其中荧光团通过光可裂解连接体与探针的其余部分连接。我们还将研究将不同 mRNA 发送到细胞中不同位点但利用相同运输机制的机制。为了观察培养细胞内单个 mRNA 分子的运动，我们将构建一个可诱导的人工基因，该基因编码包含 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 分子的运动，因为它们被转录、成熟、从细胞核输出，并被发送到细胞质的不同区域进行翻译。我们还提出了分子信标的新结构和配置，这将使我们能够控制它们在细胞内的分布，从而提高其信号的特异性。由于很难预测大 mRNA 中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有效方法将探针递送到细胞中。基于天然 SNARE 介导的囊泡融合的作用机制，该方法采用互补的单链寡脱氧核糖核苷酸，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，将有可能观察活细胞中特定 mRNA 的运动。