New toolkit to visualize RNAs in living cells

可视化活细胞中 RNA 的新工具包

基本信息

批准号：
8647672
负责人：
Karen Wu
金额：
$ 50.36万
依托单位：
LUCERNA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8647672
关键词：
Binding Biological Cell physiology Cells Cellular Structures Cellular biology Color Communities Complement Complex Culture Media Disease Dyes Elements Event Exhibits Fluorescence Fluorescence Microscopy Goals Green Fluorescent Proteins Human Genome Image Imaging Device Imaging Techniques Imaging technology Label Life Link Mammalian Cell Marketing Medical Messenger RNA MicroRNAs Modification Molecular Biology Movement Neurons Pathogenesis Pathway interactions Phase Plasmids Play Population Protein Binding Proteins RNA RNA Processing RNA Sequences Reagent Recording of previous events Research Research Personnel Rights Role Series Spinach - dietary Stimulus Structure Subcellular structure System Tandem Repeat Sequences Technology Universities Untranslated RNA Variant Vegetables aptamer base biological research commercialization design fascinate flexibility fluorophore improved insight interest meetings novel novel strategies protein expression public health relevance research study response

项目摘要

PROJECT SUMMARY Over the past 10 years it has become increasingly apparent that there is exceptional complexity in the RNA population in cells. This diverse RNA pool includes 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 is highly useful 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 "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 the Phase I portion of this project we markedly increased the brightness of Spinach in cells, and expanded the use of Spinach to novel noncoding RNAs. We also developed novel expression plasmids for expressing Spinach- tagged RNAs. In this Phase II proposal, we will develop novel concatamerization strategies to label RNAs with tandem repeats of Spinach, thereby increasing the sensitivity of this labeling approach. We also will tag a set of major noncoding RNAs that label cellular structures, in order to provide a suite of plasmids expressing pre-labeled RNAs that are ready to use for researchers. We also will expand the spectral imaging capacity of this approach by developing new fluorophores for Spinach, and new RNA-fluorophore complexes, which will provide the spectral versatility in biologic imaging that is needed by the research community. Based on the past history of successful commercialization of GFP expression systems, we expect that this expression system will have high commercial potential and will accelerate the pace of RNA research.

项目概要在过去的 10 年里，越来越明显的是，该领域存在着异常的复杂性。细胞中的 RNA 群体。这个多样化的 RNA 池包括 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 或蛋白质的改变与这些 RNA 结合的基因与多种医学疾病有关。的一个重大挑战分子生物学就是要确定这些令人着迷的新颖 RNA 种类的功能。为了了解这些 RNA 在细胞中的功能，对定位和成像进行成像非常有用在各种实验刺激下，活细胞中这些 RNA 的细胞内运动。我们有开发了一种新型基因编码系统，可以对细胞中的 RNA 进行荧光标记。这个系统利用称为 Spinach 的 RNA 序列元件，将其附加到感兴趣的 RNA 上，并且 “打开”其他非荧光染料的荧光。该染料基于以下结构 GFP 中的荧光团，使菠菜成为 GFP 的 RNA 模拟物。在本期第一阶段的部分项目中我们显着提高了菠菜在细胞中的亮度，并将菠菜的用途扩大到新型非编码RNA。我们还开发了用于表达菠菜的新型表达质粒标记的 RNA。在这个第二阶段提案中，我们将开发新颖的串联策略来标记 RNA 具有菠菜的串联重复序列，从而提高了这种标记方法的灵敏度。我们还将标记一组标记细胞结构的主要非编码 RNA，以便提供表达预标记 RNA 的质粒套件，可供研究人员使用。我们也会通过开发菠菜的新荧光团来扩展这种方法的光谱成像能力，以及新的 RNA-荧光团复合物，将为生物成像提供光谱多功能性这是研究界所需要的。基于过去的成功历史 GFP表达系统的商业化，我们预计该表达系统将具有高商业潜力并将加快RNA研究的步伐。