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

描述(申请人提供)：非编码RNA、信使核糖核酸和有毒核糖核酸是对神经元功能有重要影响的核糖核酸种类的例子。然而，我们发现这些不同的RNA的作用和功能的能力受到了限制，因为缺乏直接的技术来实时成像活细胞中的RNA定位。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个核苷酸长的RNA适体，它结合并激活一个类似于GFP发色团的条件荧光分子的荧光。然而，这项技术不能用于成像低丰度的RNA，如mRNAs。此外，由于该方法能够在单一颜色下成像，因此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的功能是通过选择性地结合特定的蛋白质来实现的，因此对成像活细胞中RNA-蛋白质相互作用的空间和时间动态的技术是非常必要的。作为我们总体目标的一部分，本项目目标的功能方面是：(1)开发 核糖核酸模拟红色荧光蛋白，用于多重核糖核酸成像。在这里，我们将开发一种新的方法，使用新的高亮度黄色、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)开发含有明亮的、光稳定的RNA-荧光团络合物的串联重复序列的盒，用于低丰度RNA的成像。我们将开发一种方法来成像低丰度的mRNAs，因为它们在轴突和生长锥体中流动，使用一种新的、高度光稳定的RNA-荧光团复合体的多个串联重复序列，Squash。(3)开发一种简化的方法来成像活细胞中的RNA-蛋白质相互作用。我们将开发第一个基于FRET的方法来成像神经元中直接的RNA-蛋白质相互作用。这种方法使用了一种新颖而简单的策略，允许在传统荧光显微镜上进行FRET成像。我们将使用这种方法来识别直接结合与脆性X相关震颤和共济失调综合征相关的有毒RNA的蛋白质。总之，这项提案中的实验旨在从根本上使技术成为可能，这些技术将大大提高我们研究神经元中不同RNA物种的功能的能力。

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.