Fluorescent reporters for real-time single-cell Pol III transcription measurement

用于实时单细胞 Pol III 转录测量的荧光报告基因

基本信息

批准号：
9254931
负责人：
Karen Wu
金额：
$ 17.62万
依托单位：
LUCERNA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9254931
关键词：
Area Biological Assay CRISPR/Cas technology Cell Line Cells Communities Complex DNA Polymerase II DNA Polymerase III DNA-Directed RNA Polymerase Detection Disease Drug Targeting Dyes Elements Engineering FRAP1 gene Feasibility Studies Flow Cytometry Fluorescence Gene Expression Genetic Transcription Goals Green Fluorescent Proteins Growth Guava Fruit Heterogeneity Homeostasis Image In Vitro Individual Label Link Luciferases Malignant Neoplasms Measurement Measures Messenger RNA Methods Monitor Northern Blotting Nutrient Obesity Pathogenesis Pathway interactions Phase Photobleaching Plasmids Population Process Proteins RNA RNA Sequences RNA Splicing Reporter Reporter Genes Reporting Research Ribosomal RNA Scientist Serum Signal Transduction Signaling Molecule Small Business Innovation Research Grant Spinach - dietary Stimulus System Tail Technology Testing Time Transcript Transcriptional Regulation Transfer RNA Translating Translations Universities Untranslated RNA Variant base cell growth drug discovery extracellular genetic regulatory protein high throughput screening inhibitor/antagonist interest live cell imaging novel promoter rapid growth red fluorescent protein response small molecule small molecule inhibitor tool

项目摘要

SUMMARY RNA polymerase III (Pol III) is increasingly recognized as an important drug target and a rapidly developing area of academic research. Pol III is responsible for generating RNAs that are critical for cell growth, such as tRNAs, certain rRNAs, and various noncoding RNAs involved in processes such as mRNA splicing. These RNAs are required for cell growth, proliferation, and differentiation and disrupting the pathways that control Pol III is now thought to be valuable for targeting cancer and obesity. Despite the importance of Pol III, little is known about the pathways that regulate Pol III transcription. This is largely due to the absence of simple and robust reporters for Pol III activity. This is in contrast to reporter assays for Pol II promoters for mRNA transcription—these reporters comprise Pol II promoters controlling reporter genes such as green fluorescent protein, and have been the major driver for drug discovery for mRNA transcription inhibitors. However, Pol III generates RNAs that cannot be translated. As a result, nearly all studies of Pol III rely on Northern blotting, which is slow and not suited to high-throughput screening efforts. At Lucerna, we have developed a photostable RNA mimic of red fluorescent protein (termed Corn) and demonstrated that this RNA can serve as a robust fluorescent reporter for Pol III promoters. By simply creating a Pol III promoter that controls the synthesis of the Corn RNA, fluorescence can be readily detected without the need to synthesize a protein. We have demonstrated this concept using the 5S subtype of a Pol III promoter. Interestingly, we found marked cell-to-cell variation in Pol III transcription after cells are exposed to serum and other growth-promoting signals. Therefore, the goal of this Phase I SBIR project is to develop this simple and genetically encodable Corn reporters for live cell imaging and measurement of all three subtypes of Pol III transcription. Further, we will develop this reporter system so that it can be used in flow cytometry analysis, allowing Pol III transcription to be monitored in hundreds of thousands of cells over time. For phase II, we will adapt our Pol III reporters into marketable toolkits, validate the Pol III reporters in disease-relevant cell lines, and use the Cas9/CRISPR technology to image endogenous Pol III transcription activity. Together, the plasmids and kits generated in this project will result in the first Pol III reporters, and will therefore constitute an important commercial product that will have wide utility in the academic RNA research community and will enable discovery of small molecule inhibitors of this previously hard-to-assay class of drug targets.

概括 RNA 聚合酶 III (Pol III) 越来越被认为是重要的药物靶点和快速发展的药物靶点。 Pol III 负责生成对细胞生长至关重要的 RNA，例如 tRNA、某些 rRNA 和参与 mRNA 剪接等过程的各种非编码 RNA。 RNA 是细胞生长、增殖和分化所必需的，并且会破坏控制 Pol 的途径尽管 Pol III 很重要，但目前认为 III 对于治疗癌症和肥胖具有重要价值。人们对调节 Pol III 转录的途径知之甚少，这主要是由于缺乏简单和有效的途径。这与 Pol II mRNA 启动子的报告分析相反。转录——这些制剂包含控制报告基因（例如绿色荧光）的 Pol II 启动子然而，Pol III 一直是 mRNA 转录抑制剂药物发现的主要推动力。产生无法翻译的 RNA 因此，几乎所有 Pol III 的研究都依赖于 Northern blotting。这很慢并且不适合高通量筛选工作。在 Lucerna，我们开发了一种方法。红色荧光蛋白（称为玉米）的光稳定 RNA 模拟物，并证明该 RNA 可以充当通过简单地创建一个控制 Pol III 启动子的强大荧光报告基因。玉米RNA的合成，无需合成蛋白质即可轻松检测荧光。已经使用 Pol III 启动子的 5S 亚型证明了这一概念。细胞暴露于血清和其他生长促进信号后，Pol III 转录的细胞间差异。因此，第一期SBIR项目的目标是开发这种简单且可基因编码的玉米此外，我们将开发该报告系统，使其可用于流式细胞术分析，使 Pol III 转录能够随着时间的推移，我们将在数十万个细胞中进行监测。对于第二阶段，我们将调整我们的 Pol III 生产。适销对路的工具包，在疾病相关细胞系中验证 Pol III 生产者，并使用 Cas9/CRISPR 技术对内源性 Pol III 转录活性进行成像，其中生成的质粒和试剂盒一起。项目将产生第一批 Pol III 生产商，因此将构成一个重要的商业产品将在学术 RNA 研究界具有广泛的实用性，并将促进小分子的发现这类以前难以测定的药物靶点的抑制剂。