Engineering RNA-activated fluorescence switches for imaging RNA and RNA biology

工程化 RNA 激活荧光开关，用于 RNA 成像和 RNA 生物学

基本信息

批准号：
8773962
负责人：
SAMIE R JAFFREY
金额：
$ 37.08万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8773962
关键词：
Axon Binding Biology Cell Nucleus Cell physiology Cells Cessation of life Code Color Communities Complex Cytoplasmic Granules Disease Disease Pathway Engineering Exposure to FXTAS Fluorescence Fluorescence Resonance Energy Transfer Functional RNA Gene Expression Generations Genetic Genome Goals Green Fluorescent Proteins Growth Cones Image Imaging technology Lasers Lead Life Link Messenger RNA Methods Neurons Neurosciences Nuclear Structure Oranges Pathogenesis Pathway interactions Photobleaching Proteins RNA RNA Sequences RNA Splicing RNA-Protein Interaction Regulation Regulatory Pathway Role Signal Transduction Spinach - dietary Squash Tandem Repeat Sequences Techniques Technology Time Translating Translations Trinucleotide Repeats Untranslated RNA Variant Work aptamer axon growth axon guidance base chromophore design directed evolution fluorescence microscope fluorophore insight instrument mRNA tagging new technology novel novel strategies public health relevance red fluorescent protein research study trafficking

项目摘要

DESCRIPTION (provided by applicant): Noncoding RNA, mRNA, and toxic RNAs are examples of RNA species that have important influences on neuronal function. However, our ability to uncover the roles and functions of these diverse RNAs has been limited by the lack of straightforward technologies to image RNA localization in real time in living cells. Recently, we described RNA mimics of green fluorescent protein (GFP), which enable the genetic encoding of fluorescent RNA in living cells using Spinach, a 98-nt long RNA aptamer that binds and activates the fluorescence of a conditionally fluorescent molecule that resembles the chromophore of GFP. However, this technology cannot be applied for imaging low abundance RNAs, such as mRNAs. Furthermore, since this approach enables imaging in a single color, it cannot be applied for simultaneous imaging of different RNAs in different colors. Lastly, because most RNAs function by selectively binding specific proteins, there is a major need for technologies to image the spatial and temporal dynamics of RNA-protein interactions in living cells. As part of our overall goal functional aspects of goals of this project are: (1) To develop RNA mimics of red fluorescent protein for multiplexed RNA imaging. Here we will develop a novel approach to simultaneously image different RNAs in living cells using new highly bright yellow, orange and red fluorescent RNA imaging tags. (2) To develop cassettes containing tandem repeats of bright and photostable RNA-fluorophore complexes for imaging low abundance RNAs. We will develop an approach to image low abundance mRNAs as they traffic in axons and growth cones using multiple tandem repeats of a new, highly photostable RNA-fluorophore complex, Squash. (3) To develop a simplified approach to image RNA-protein interactions in living cells. We will develop the first FRET-based approach to image direct RNA-protein interactions in neurons. This approach uses a novel and straightforward strategy that allows FRET imaging on conventional fluorescence microscopes. We will use this approach to identify proteins that directly bind toxic RNAs linked to fragile X-associated tremor and ataxia syndrome. Together, the experiments in this proposal are designed to lead to fundamentally enabling technologies that will considerably advance our ability to study the functions of diverse RNA species in neurons.

描述（由申请人提供）：非编码 RNA、mRNA 和毒性 RNA 是对神经元功能具有重要影响的 RNA 种类的例子。然而，由于缺乏在活细胞中实时成像 RNA 定位的直接技术，我们揭示这些不同 RNA 的作用和功能的能力受到限制。最近，我们描述了绿色荧光蛋白 (GFP) 的 RNA 模拟物，它能够使用菠菜在活细胞中对荧光 RNA 进行遗传编码，菠菜是一种 98 nt 长的 RNA 适体，可结合并激活类似于发色团的条件荧光分子的荧光绿色荧光蛋白。然而，该技术不能应用于低丰度 RNA 的成像，例如 mRNA。此外，由于这种方法能够以单色成像，因此它不能应用于不同颜色的不同RNA的同时成像。最后，由于大多数 RNA 通过选择性结合特定蛋白质发挥作用，因此迫切需要对活细胞中 RNA-蛋白质相互作用的空间和时间动态进行成像的技术。作为我们总体目标的一部分，该项目目标的功能方面是：(1) 开发红色荧光蛋白的 RNA 模拟物，用于多重 RNA 成像。在这里，我们将开发一种新方法，使用新的高亮黄色、橙色和红色荧光 RNA 成像标签同时对活细胞中的不同 RNA 进行成像。 (2) 开发包含明亮且光稳定的 RNA-荧光团复合物串联重复序列的盒，用于对低丰度 RNA 进行成像。我们将开发一种方法，使用新型、高度光稳定的 RNA-荧光团复合物 Squash 的多个串联重复，对低丰度 mRNA 在轴突和生长锥中运输时进行成像。 (3) 开发一种简化的方法来对活细胞中的 RNA-蛋白质相互作用进行成像。我们将开发第一个基于 FRET 的方法来对神经元中的直接 RNA-蛋白质相互作用进行成像。这种方法采用了一种新颖且简单的策略，可以在传统荧光显微镜上进行 FRET 成像。我们将使用这种方法来鉴定直接结合有毒 RNA 的蛋白质，这些有毒 RNA 与脆性 X 相关震颤和共济失调综合征有关。总之，本提案中的实验旨在从根本上引导技术的发展，从而大大提高我们研究神经元中不同 RNA 种类功能的能力。