New toolkit to visualize RNAs in living cells

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群体具有异常的复杂性，包括microRNAs、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的改变，或与这些rna结合的蛋白质，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的新RNA物种的功能。为了了解这些rna在细胞中的功能，能够在各种实验刺激下对活细胞中这些rna的定位和细胞内运动进行成像将是非常有价值的。我们已经开发了一种新的基因编码系统来荧光标记细胞中的rna。该系统利用一种称为“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。这种染料基于绿色荧光蛋白中的荧光团结构，使菠菜成为绿色荧光蛋白的RNA模拟物。在本提案中，我们将开发新的接触和稳定策略，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序列和原理验证数据，这将为在细胞中表达荧光标记RNA的商业化、基于质粒的系统的开发奠定基础。基于过去GFP表达系统成功商业化的历史，我们预计这种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一种使能技术。

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.!