New toolkit to visualize RNAs in living cells

可视化活细胞中 RNA 的新工具包

• 批准号: 8878350
• 负责人: Karen Wu
• 金额: $ 50.75万
• 依托单位: LUCERNA, INC.
• 依托单位国家: 美国
• 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/8878350
• 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; Health; 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; imaging platform; imaging system; improved; insight; interest; meetings; novel; novel strategies; protein expression; research study; response; spectrograph; transcriptome sequencing

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.

描述（由申请人提供）：在过去的10年中，越来越明显的是，细胞中的RNA群体具有异常的复杂性。这种多样化的RNA库包括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或与这些RNA结合的蛋白质的改变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种类的功能。为了了解这些RNA在细胞中的功能，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遗传编码系统来荧光标记细胞中的RNA。该系统利用了一种RNA序列元件，称为Spinach，其被附加到感兴趣的RNA上，并“打开”原本非荧光染料的荧光。这种染料基于GFP中荧光团的结构，使Spinach成为GFP的RNA模拟物。在该项目的第一阶段，我们显著增加了Spinach在细胞中的亮度，并将Spinach的用途扩展到新型非编码RNA。我们还开发了用于表达Spinach标记的RNA的新型表达质粒。在这个第二阶段的建议，我们将开发新的concatamerization策略，以标记RNA与菠菜的串联重复序列，从而增加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，以提供一套表达预标记RNA的质粒，供研究人员使用。我们还将通过为菠菜开发新的荧光团和新的RNA荧光团复合物来扩大这种方法的光谱成像能力，这将为研究界提供所需的生物成像光谱多功能性。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，并将加快RNA研究的步伐。