Engineering RNA-activated fluorescence switches for RNA imaging and analyte sensi

描述（由申请人提供）：拟议研究的目的是扩展和开发一种用于成像活细胞中RNA和小分子的新颖且简单的方法。与可用于监测细胞中蛋白质的绿色荧光蛋白（GFP）不同，目前还没有类似的简单直接的方法来跟踪活细胞中RNA的运动。目前可用的方法有严重的局限性，妨碍了它们的广泛使用。我们设计了一种RNA受体和一种小分子配体，它们相互作用形成一种荧光复合物。RNA“打开”了原本无荧光的小分子的荧光。我们已经表征了这种RNA-荧光团复合物，优化了其序列以改善其荧光特性，并表明它可用于监测活细胞中的RNA。此外，我们已经表明，RNA适体可以融合到其他适体，以产生变构调节的荧光分析物传感器。为了将我们的系统发展成一种广泛使用的、简单的和灵敏的技术，从而允许RNA的细胞生物学的研究，本申请的具体目标是：（1）开发RNA-荧光团复合物的调色板;（2）优化RNA-荧光团复合物的荧光性质。为此，我们将使用亲和力成熟和其他技术来增加RNA-荧光团复合物的荧光;（3）使用RNA-荧光团复合物来可视化细胞中的mRNA和轴突中的mRNA运输。为此，我们描述了优化细胞中RNA-荧光团复合物的荧光并监测轴突转动过程中mRNA运输和mRNA降解的实验;（4）从RNA-荧光团复合物开发荧光传感器。我们提出了一个简单的和可推广的方法，用于产生荧光分析物传感器和使用它们在细胞中。总之，这四个目标的实验将产生一种多功能的RNA成像和荧光传感器技术，这种技术比目前可用的任何其他方法都更简单，更具体。

DESCRIPTION (provided by applicant): The objective of the proposed research is to expand and develop a novel and simple method for imaging RNA and small molecules in living cells. Unlike green fluorescent protein (GFP), which can be used to monitor proteins in cells, there are currently no analogous simple and straightforward approaches to track RNA movement in living cells. Currently available approaches have critical limitations that have precluded their widespread use. We have engineered both an RNA receptor and a small molecule ligand that interact to form a fluorescent complex. The RNA "switches on" the fluorescence of an otherwise nonfluorescent small molecule. We have characterized this RNA-fluorophore complex, optimized its sequence to improve its fluorescence properties, and shown that it can be used to monitor RNAs in living cells. Additionally, we have shown that the RNA aptamer can be fused to other aptamers to generate allosterically regulated fluorescent analyte sensors. In order to develop our system into a widely-used, simple and sensitive technique that would permit the study of the cell biology of RNA, the specific aims of this application are: (1) To develop a palette of RNA-fluorophore complexes; (2) To optimize the fluorescence properties of RNA-fluorophore complexes. In this aim, we will use affinity maturation and other techniques to increase the fluorescence of the RNA-fluorophore complexes; (3) To use RNA- fluorophore complexes to visualize mRNAs in cells and mRNA trafficking in axons. In this aim, we describe experiments to optimize the fluorescence of RNA-fluorophore complexes in cells and to monitor mRNA trafficking and mRNA degradation during axon turning; (4) To develop fluorescent sensors from RNA-fluorophore complexes. We present a simple and generalizable approach for generating fluorescent analyte sensors and using them in cells. Together, the experiments in these four aims will result in a versatile RNA imaging and fluorescent sensor technology that is simpler and more specific than any other methodology that is currently available.