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

描述（由申请人提供）：非编码RNA、mRNA和毒性RNA是对神经元功能有重要影响的RNA种类的例子。然而，由于缺乏直接的技术来在活细胞中真实的时间成像RNA定位，我们发现这些不同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，其能够使用Spinach在活细胞中遗传编码荧光RNA，Spinach是一种98-nt长的RNA适体，其结合并激活类似于GFP发色团的条件荧光分子的荧光。然而，该技术不能应用于成像低丰度RNA，如mRNA。此外，由于这种方法能够以单一颜色成像，因此它不能应用于以不同颜色同时成像不同RNA。最后，由于大多数RNA通过选择性结合特定蛋白质发挥作用，因此主要需要对活细胞中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，本项目的目标功能方面是：（1）开发 用于多重RNA成像的红色荧光蛋白的RNA模拟物。在这里，我们将开发一种新的方法来同时成像不同的RNA在活细胞中使用新的高度明亮的黄色，橙子和红色荧光RNA成像标签。(2)开发含有明亮且光稳定的RNA-荧光团复合物的串联重复序列的盒，用于成像低丰度RNA。我们将开发一种方法来成像低丰度的mRNA，因为他们在轴突和生长锥使用多个串联重复的一个新的，高度光稳定的RNA荧光团复合物，南瓜交通。(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的直接RNA-蛋白质相互作用。这种方法使用了一种新的和简单的策略，允许在传统的荧光显微镜FRET成像。我们将使用这种方法来鉴定直接结合与脆性X相关震颤和共济失调综合征相关的毒性RNA的蛋白质。总之，该提案中的实验旨在从根本上实现技术，这将大大提高我们研究神经元中不同RNA种类功能的能力。

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.