New toolkit to visualize RNAs in living cells

可视化活细胞中 RNA 的新工具包

• 批准号: 8315429
• 负责人: Karen Wu
• 金额: $ 31.54万
• 依托单位: LUCERNA, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315429
• 关键词: Binding; Cell physiology; Cells; Cellular biology; Communities; DNA Polymerase II; DNA Polymerase III; Data; Development; Disease; Dyes; Elements; Eukaryotic Cell; Event; Fluorescence; Fluorescence Microscopy; Human Genome; Image; Imagery; Imaging technology; Individual; Life; Link; Medical; Messenger RNA; MicroRNAs; Molecular Biology; Monitor; Movement; Pathogenesis; Phase; Plasmids; Population; Protein Binding; RNA; RNA Processing; RNA Sequences; RNA Stability; Recording of previous events; Research; Research Personnel; Rights; Site; Small Business Innovation Research Grant; Spinach - dietary; Staging; Stimulus; Structure; System; Techniques; Technology; Universities; Untranslated RNA; Vegetables; base; cell type; commercialization; fascinate; fluorophore; insight; interest; novel; research study; small molecule; trafficking

DESCRIPTION (provided by applicant): Over the past 10 years it has become increasingly apparent that there is exceptional complexity in the RNA population in cells, including microRNAs, Piwi-interacting RNAs, termini-associated RNAs and other noncoding RNAs. In many cases, alterations in these RNAs, or proteins that bind to these RNAs, have been linked to a wide range of medical disorders. A major challenge of molecular biology is to determine the function of these fascinating and novel RNA species. In order to understand how these RNAs function in cells, it will be exceptionally valuable to be able to image the localization and intracellular movements of these RNAs in living cells under a variety of experimental stimuli. We have developed a novel genetically encodable system to fluorescently tag RNAs in cells. This system utilizes an RNA sequence element, termed Spinach, which is appended to an RNA of interest, and which "switches on" the fluorescence of an otherwise non-florescent dye. This dye is based on the structure of the fluorophore in GFP, making Spinach an RNA mimic of GFP. In this proposal, we will develop novel contamerization and stabilization strategies to make Spinach simple to use and highly sensitive for imaging of individual RNAs. The results of this phase I SBIR application will be RNA sequences and proof- of-principle data which will set the stage for the development of a commercial, plasmid based system for expressing fluorescently tagged RNAs in cells. Based on the past history of successful commercialization of GFP expression systems, we expect that this expression system will have high commercial potential, and the ability to provide an enabling technology to the larger research community. PUBLIC HEALTH RELEVANCE: The human genome encodes a large number of mRNAs and noncoding RNAs. Although many RNAs are linked to disease processes, cellular mechanisms by which these RNAs function in cells are not fully clear. This project will develop a toolkit whic will enable researchers to easily image both mRNAs an noncoding RNAs in living cells using a novel genetically encoded fluorescent RNA tagging system, thereby providing insights into the function of these RNAs in cell biology and disease pathogenesis.!

描述（由申请人提供）：在过去的 10 年里，越来越明显的是，细胞中的 RNA 群体存在异常的复杂性，包括 microRNA、Piwi 相互作用 RNA、末端相关 RNA 和其他非编码 RNA。在许多情况下，这些 RNA 或与这些 RNA 结合的蛋白质的改变与多种医学疾病有关。分子生物学的一个主要挑战是确定这些令人着迷的新颖 RNA 物种的功能。为了了解这些 RNA 在细胞中的功能，能够对这些 RNA 在各种实验刺激下在活细胞中的定位和细胞内运动进行成像将非常有价值。我们开发了一种新型遗传编码系统，可以对细胞中的 RNA 进行荧光标记。该系统利用称为“菠菜”的 RNA 序列元件，将其附加到感兴趣的 RNA 上，并“打开”其他非荧光染料的荧光。这种染料基于 GFP 中荧光团的结构，使菠菜成为 GFP 的 RNA 模拟物。在本提案中，我们将开发新颖的异构化和稳定化策略，使 Spinach 易于使用且对单个 RNA 成像高度敏感。这一阶段 SBIR 应用的结果将是 RNA 序列和原理验证数据，这将为开发基于质粒的商业系统奠定基础，用于在细胞中表达荧光标记的 RNA。基于 GFP 表达系统过去成功商业化的历史，我们预计该表达系统将具有很高的商业潜力，并且能够为更大的研究界提供支持技术。 公共卫生相关性：人类基因组编码大量 mRNA 和非编码 RNA。尽管许多 RNA 与疾病过程有关，但这些 RNA 在细胞中发挥作用的细胞机制尚不完全清楚。该项目将开发一个工具包，使研究人员能够使用新型基因编码荧光 RNA 标记系统轻松对活细胞中的 mRNA 和非编码 RNA 进行成像，从而深入了解这些 RNA 在细胞生物学和疾病发病机制中的功能。