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

描述(申请人提供)：拟议研究的目标是扩展和开发一种新的和简单的方法来成像活细胞中的RNA和小分子。与绿色荧光蛋白(GFP)不同，绿色荧光蛋白可用于监测细胞中的蛋白质，目前还没有类似的简单和直接的方法来跟踪活细胞中的RNA运动。目前可用的方法有严重的局限性，使其无法广泛使用。我们已经设计了一个RNA受体和一个小分子配体，它们相互作用形成荧光复合体。这种核糖核酸“开启”了原本无荧光的小分子的荧光。我们已经对这种RNA-荧光团络合物进行了表征，优化了它的序列以改善其荧光性质，并表明它可以用于监测活细胞中的RNA。此外，我们还证明了RNA适配子可以与其他适配子融合，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为一种广泛使用的、简单和灵敏的技术，从而能够对RNA的细胞生物学进行研究，这一应用的具体目的是：(1)开发一系列RNA-荧光团络合物；(2)优化RNA-荧光团络合物的荧光性质。为此，我们将使用亲和成熟和其他技术来增强RNA-荧光团复合体的荧光；(3)使用RNA-荧光团复合体来可视化细胞中的mRNAs和轴突中的mRNAs。在这一目标中，我们描述了优化细胞中RNA-荧光团复合体的荧光并监测轴突转动过程中的mRNA运输和mRNA降解的实验；(4)从RNA-荧光团复合体开发荧光传感器。我们提出了一种简单和可推广的方法来产生荧光分析物传感器并在细胞中使用它们。这四个目标的实验加在一起，将产生一种多功能的RNA成像和荧光传感器技术，比目前可用的任何其他方法都更简单和更具体。与公共卫生相关：RNA被越来越多地认为是细胞功能的关键调节因素；然而，目前还没有简单和直接的方法来跟踪活细胞中的RNA运动。拟议的实验将产生一种非常强大的RNA成像和荧光传感器技术，它比目前可用的任何其他方法都更简单和更具体。这将提供一个分子工具箱，大大增强我们研究RNA在健康和疾病中的作用的能力。

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. PUBLIC HEALTH RELEVANCE: RNA is increasingly recognized as being a critical regulator of cellular function; however, there are currently no simple and straightforward approaches to track RNA movement in living cells. The proposed experiments will result in a highly powerful RNA imaging and fluorescent sensor technology that is simpler and more specific than any other methodology that is currently available. This will provide a molecular toolkit that will substantially enhance our ability to study the role of RNA in health and disease.