Fluorescent tRNAs for Real-Time Monitoring of Protein Synthesis in Living Cells

用于实时监测活细胞中蛋白质合成的荧光 tRNA

• 批准号: 8001799
• 负责人: BARRY S. COOPERMAN
• 金额: $ 20万
• 依托单位: ANIMA CELL METROLOGY, INC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-05 至 2012-03-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8001799
• 关键词: Active Sites; Algorithms; Amino Acids; Area; Astrocytes; Basic Science; Biological Assay; Biological Response Modifier Therapy; Budgets; California; Cataloging; Catalogs; Cattle; Cell Line; Cells; Clinical Trials; Collaborations; Collagen Type I; Computer software; Cysteine-Specific tRNA; Detection; Development; Diagnosis; Disease; Dyes; Eukaryotic Cell; Feasibility Studies; Fluorescence Resonance Energy Transfer; Freezing; Gel; Gene Proteins; Glycine-Specific tRNA; Growth and Development function; Hela Cells; Human; Knowledge; Label; Laboratories; Libraries; Life; Liver; Liver Fibrosis; Lysine-Specific tRNA; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Mesenchymal Stem Cells; Messenger RNA; Methodology; Methods; Monitor; Multiple Myeloma; Mus; Peptides; Pharmacologic Substance; Phase; Phenylalanine-Specific tRNA; Preparation; Procedures; Production; Proline-Specific tRNA; Protein Biosynthesis; Proteins; Proteomics; Quality Control; Reagent; Research; Resolution; Rhodamine; Rhodamines; Ribosomes; Saccharomyces cerevisiae; Sampling; Signal Transduction; Techniques; Technology; Time; Tissues; Transfer RNA; Universities; Viral; Work; base; beta-Defensins; biological research; burden of illness; cell growth; clinical Diagnosis; combinatorial; commercialization; computerized data processing; cyanine dye 5; frontier; in vivo; interest; millisecond; new technology; outcome forecast; prevent; protein expression; public health relevance; response; synthetic protein; therapeutic protein; tool

DESCRIPTION (provided by applicant): As technologies of gene and protein cataloging are maturing, the study of cellular dynamics is becoming the next frontier in biological research. New technologies are required to capture the complexity of cellular dynamics. Against this background we propose the development of a straightforward method to monitor protein synthesis from living cells in real time and subcellular resolution. The method, termed DiP ("Di-Peptide"), transfects cells with tRNAs that are fluorescently-labeled to form FRET pairs. When the cellular protein synthetic machinery is active, such tRNAs will be held (for ~500 milliseconds) in adjacent sites of active ribosomes, generating FRET signals, the intensity of which should, under normal conditions of cell growth and development, correlate with the rate of protein synthesis in the cell. DiP technology can measure either overall rates of protein synthesis (Overall DiP), by employing bulk labeled tRNAs, or rates of synthesis of selected proteins of interest (Specific DiP), by using specific labeled tRNAs.In both cases, high temporal resolution and subcellular localization are easily achieved. Such capabilities have many valuable applications in areas ranging from basic research, to clinical trials, to the manufacture of biotherapeutics. Over the last year we have successfully employed Overall DiP to generate FRET signals in several mammalian cell lines. Our most important tasks in advancing DiP development in Phase 1 of this project are to demonstrate that 1) synthesis rates of specific proteins can be monitored by Specific DiP; 2) DiP assays can be calibrated to provide quantitative measurements; and 3) such assays can provide useful information in a variety of scientific and pharmaceutical applications. To accomplish these tasks we will prepare amino acid-specific fluorescently labeled tRNAs and use them to determine the correlation between in vivo FRET intensity and protein expression for two selected mammalian proteins. In addition, methodology, reagents, algorithms and software will be developed to estimate the concentration of active ribosomes; procedures will be implemented to differentiate FRET signals arising from active ribosomes from those arising from frozen ribosomes; and feasibility studies using Overall DiP will be conducted to measure the expression of a number of disease-related proteins. In Phase 2 we plan to develop further the spectrum of applications of DiP, building on the knowledge gained through phase 1 work and to commercialize DiP as a new research tool with wide applicability for the of study protein- synthesis related diseases, and for clone selection and quality control applications in production of therapeutic proteins. PUBLIC HEALTH RELEVANCE: Many if not most diseases afflicting humankind arise from abnormalities in the biosynthesis of proteins that are found within human cells. Advances in the diagnosis and treatment of such diseases depend in large measure on our ability to monitor changes in protein synthesis when and where they occur. We are proposing the development of a straightforward method to monitor protein synthesis from living cells in real time and at sub-cellular resolution that should have wide applicability for the elucidation and treatment of protein-synthesis related diseases.

描述（由申请人提供）：随着基因和蛋白质分类技术的成熟，细胞动力学的研究正成为生物学研究的下一个领域。需要新技术来捕获细胞动力学的复杂性。在这种背景下，我们提出了一种直接的方法，以实时监测活细胞和亚细胞分辨率的蛋白质合成。该方法称为浸入（“二肽”），用荧光标记的TRNA转染细胞形成fret对。当细胞蛋白质合成机器活跃时，将在活性核糖体的相邻部位（约500毫秒）保持这种TRNA（约500毫秒），产生FRET信号，在细胞生长和发育的正常条件下，应与细胞中蛋白质合成的速率相关。通过使用特定的标记的TRNA。在两种情况下，浸入技术可以通过采用大量标记的TRNA（特定的DIP）合成速率，或通过使用特定标记的TRNA。这种功能在从基础研究到临床试验到生物治疗药的生产方面都有许多有价值的应用。在过去的一年中，我们已经成功地使用了总体倾斜来在几个哺乳动物细胞系中产生FRET信号。我们在该项目的第1阶段推进DIP开发方面的最重要任务是证明1）可以通过特定的DIP监测特定蛋白质的合成速率； 2）可以校准DIP分析以提供定量测量； 3）此类测定可以在各种科学和药物应用中提供有用的信息。为了完成这些任务，我们将准备氨基酸特异性荧光标记的TRNA，并使用它们来确定两种选定的哺乳动物蛋白的体内FRET强度与蛋白质表达之间的相关性。此外，将开发方法，试剂，算法和软件以估计活性核糖体的浓度。将实施程序，以区分由活性核糖体与冷冻核糖体产生的核心体引起的特征信号；并将使用总体DIP进行可行性研究，以测量许多与疾病相关的蛋白质的表达。在第2阶段，我们计划进一步开发DIP的应用，基于通过第一阶段工作获得的知识的基础，并将DIP作为一种新的研究工具将其用于研究蛋白质合成相关疾病的新研究工具，以及在生产治疗蛋白质中的克隆选择和质量控制应用。 公共卫生相关性：许多疾病（即使不是大多数疾病）遭受人类的疾病是由于人类细胞中发现的蛋白质的生物合成异常引起的。这种疾病的诊断和治疗的进展在很大程度上取决于我们监测蛋白质合成的变化的能力。我们提出了一种直接的方法，以实时和亚细胞分辨率监测活细胞的蛋白质合成，该方法应该广泛适用于阐明和治疗蛋白质合成相关疾病。

项目成果

Tb(3+)-tRNA for LRET studies of protein synthesis.

Tb(3)-tRNA 用于蛋白质合成的 LRET 研究。

• DOI: 10.1021/bc400062d
• 发表时间: 2013
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: Alonso,Dulce;Liu,Wei;Rosenblum,Gabriel;Mani,Tomoyasu;Goldman,YaleE;Cooperman,BarryS
• 通讯作者: Cooperman,BarryS

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

• DOI: 10.1093/nar/gkr601
• 发表时间: 2011-10
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Barhoom S;Kaur J;Cooperman BS;Smorodinsky NI;Smilansky Z;Ehrlich M;Elroy-Stein O
• 通讯作者: Elroy-Stein O