New toolkit to visualize RNAs in living cells

描述(由申请人提供)：在过去的10年里，细胞中的RNA群体具有特殊的复杂性，这一点变得越来越明显。这个多样化的RNA池包括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，在各种实验刺激下对这些RNA在活细胞中的定位和细胞内运动进行成像是非常有用的。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。该系统利用一种名为菠菜的RNA序列元件，将其附加到感兴趣的RNA上，并“启动”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。在这个项目的第一阶段，我们显著提高了菠菜在细胞中的亮度，并将菠菜的用途扩大到新的非编码RNA。我们还开发了用于表达菠菜标记的RNA的新型表达载体。在这个第二阶段的提案中，我们将开发新的串连策略来标记带有菠菜串联重复序列的RNA，从而提高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，以便提供一套表达预标记RNA的质粒，供研究人员使用。我们还将通过开发新的菠菜荧光团和新的RNA-荧光团络合物来扩展这种方法的光谱成像能力，这将在生物成像中提供研究界所需的光谱多功能性。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，并将加快RNA研究的步伐。

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. 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.