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

设计用于 RNA 成像和分析物传感的 RNA 激活荧光开关

• 批准号: 8394525
• 负责人: SAMIE R JAFFREY
• 金额: $ 6万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8394525
• 关键词: Affinity; Axon; Binding; Biological Assay; Cell physiology; Cells; Cellular biology; Complex; Cytoplasmic Granules; Dendrites; Detection; Development; Disease; Engineering; Fluorescence; Fluorescent Dyes; Foundations; Generations; Green Fluorescent Proteins; Health; Image; Life; Ligands; Messenger RNA; Methodology; MicroRNAs; Molecular; Molecular Biology; Monitor; Movement; Neurons; Nobel Prize; Physiology; Preclinical Drug Evaluation; Property; Protein Binding; Proteins; RNA; RNA Processing; Research; Role; Signal Transduction; Small RNA; Stress; Subcellular structure; System; Techniques; Technology; Untranslated RNA; analog; aptamer; axon guidance; base; chemical synthesis; enhanced green fluorescent protein; fluorophore; imaging modality; improved; in vitro Assay; in vivo; mRNA Transcript Degradation; novel; novel strategies; promoter; public health relevance; receptor; research study; sensor; small molecule; trafficking

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.

描述（由申请人提供）：拟议研究的目标是扩展和开发一种新的简单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，它可以用来监测细胞中的蛋白质，目前还没有类似的简单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，妨碍了它们的广泛使用。我们设计了RNA受体和小分子配体，它们相互作用形成荧光复合物。RNA“开启”了一种非荧光小分子的荧光。我们对这种rna -荧光团复合物进行了表征，优化了其序列以提高其荧光特性，并表明它可以用于监测活细胞中的rna。此外，我们已经证明RNA适体可以与其他适体融合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为一种广泛使用的、简单而敏感的技术，从而可以研究RNA的细胞生物学，本应用的具体目标是：(1)开发RNA-荧光团复合物的调色板；(2)优化rna -荧光团配合物的荧光性质。为此，我们将使用亲和成熟和其他技术来增加rna -荧光团复合物的荧光；(3)利用RNA-荧光团复合物可视化细胞内mRNA和轴突内mRNA的运输。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)利用rna -荧光团复合物制备荧光传感器。我们提出了一种简单而通用的方法来产生荧光分析物传感器并在细胞中使用它们。总之，这四个目标的实验将产生一种通用的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。公共卫生相关性：RNA越来越被认为是细胞功能的关键调节剂；然而，目前还没有简单直接的方法来跟踪活细胞中的RNA运动。所提出的实验将产生一种功能强大的RNA成像和荧光传感器技术，该技术比目前可用的任何其他方法都更简单，更具体。这将提供一个分子工具包，大大提高我们研究RNA在健康和疾病中的作用的能力。