New toolkit to visualize RNAs in living cells

描述(申请人提供)：在过去的10年里，越来越明显的是，细胞中的RNA群体具有特殊的复杂性，包括microRNAs、Piwi相互作用RNAs、末端相关RNAs和其他非编码RNAs。在许多情况下，这些RNA或与这些RNA结合的蛋白质的变化与广泛的医学疾病有关。分子生物学的一个主要挑战是确定这些迷人而新颖的RNA物种的功能。为了了解这些RNA如何在细胞中发挥作用，能够成像这些RNA在各种实验刺激下在活细胞中的定位和细胞内运动将是非常有价值的。我们已经开发了一种新的可遗传编码的系统来荧光标记细胞中的RNA。这个系统利用了一种名为菠菜的RNA序列元件，它被附加到感兴趣的RNA上，并“启动”了其他非荧光染料的荧光。这种染料是基于绿色荧光蛋白中荧光团的结构，使菠菜成为一种模仿绿色荧光蛋白的RNA。在这项提案中，我们将开发新的污染和稳定策略，使菠菜易于使用，并对单个RNA的成像高度敏感。第一阶段SBIR应用的结果将是RNA序列和原理证明数据，这将为商业、基于质粒的系统的开发奠定基础，以在细胞中表达荧光标记的RNA。基于过去成功商业化GFP表达系统的历史，我们预计该表达系统将具有很高的商业潜力，并有能力为更大的研究社区提供使能技术。 与公共卫生相关：人类基因组编码大量的mRNA和非编码RNA。虽然许多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一种新的基因编码的荧光RNA标记系统，轻松地将活细胞中的mRNAs和非编码RNA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。

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.!