New toolkit to visualize RNAs in living cells

项目摘要 在过去十年中，越来越明显的是， 细胞中的RNA群体。这种多样化的RNA库包括microRNA、Piwi相互作用RNA、末端RNA和末端RNA。 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结合的蛋白质，已经被证明与多种医学疾病有关。的一个重大挑战 分子生物学的目的是确定这些令人着迷的新型RNA物种的功能。为了 了解这些RNA如何在细胞中发挥作用，对定位进行成像是非常有用的， 在各种实验刺激下，这些RNA在活细胞中的细胞内运动。我们有 开发了一种新的遗传编码系统，用于荧光标记细胞中的RNA。该系统 利用被称为Spinach的RNA序列元件，其被附加到感兴趣的RNA上，和 “打开”了原本非荧光染料的荧光。这种染料是基于 这使得菠菜成为GFP的RNA模拟物。在第一阶段， 项目，我们显着增加了菠菜在细胞的亮度，并扩大了菠菜的使用， 新的非编码RNA。我们还开发了用于表达菠菜的新型表达质粒- 标签RNA。在第二阶段的建议中，我们将制定新的多媒体战略， RNA与菠菜的串联重复序列，从而增加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，以提供一个 一套表达预标记RNA的质粒，可供研究人员使用。我们也将 通过为菠菜开发新的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，这将为生物成像提供光谱多样性 这是研究界所需要的。根据过去成功的历史， 随着GFP表达系统的商业化，我们预计这种表达系统将具有很高的 商业潜力，并将加快RNA研究的步伐。

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.