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（申请人提供）：随着基因和蛋白质编目技术的日趋成熟，细胞动力学研究正在成为生物学研究的下一个前沿领域。需要新技术来捕捉细胞动力学的复杂性。在此背景下，我们建议开发一种直接的方法来实时和亚细胞分辨率监测活细胞的蛋白质合成。该方法被称为 DiP（“Di-Peptide”），用荧光标记的 tRNA 转染细胞以形成 FRET 对。当细胞蛋白质合成机器处于活动状态时，此类 tRNA 将保留在活性核糖体的相邻位点（约 500 毫秒），产生 FRET 信号，在细胞生长和发育的正常条件下，其强度应与速率相关细胞内蛋白质的合成。 DiP 技术可以通过使用大量标记的 tRNA 来测量蛋白质合成的总体速率 (Overall DiP)，或者通过使用特定标记的 tRNA 来测量选定的感兴趣蛋白质的合成速率 (Specific DiP)。在这两种情况下，都具有高时间分辨率和亚细胞本地化很容易实现。这些能力在基础研究、临床试验和生物治疗药物的制造等领域具有许多有价值的应用。去年，我们成功地利用整体 DiP 在几种哺乳动物细胞系中产生 FRET 信号。我们在该项目第一阶段推进 DiP 开发的最重要任务是证明 1) 特定蛋白质的合成率可以通过 Specific DiP 进行监测； 2) 可校准 DiP 测定以提供定量测量； 3）此类测定可以在各种科学和制药应用中提供有用的信息。为了完成这些任务，我们将制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质的体内 FRET 强度和蛋白质表达之间的相关性。此外，还将开发方法、试剂、算法和软件来估计活性核糖体的浓度；将实施程序以区分活性核糖体产生的 FRET 信号与冷冻核糖体产生的 FRET 信号；将使用Overall DiP进行可行性研究，以测量一些与疾病相关的蛋白质的表达。在第二阶段，我们计划以第一阶段工作获得的知识为基础，进一步开发 DiP 的应用范围，并将 DiP 商业化，作为一种新的研究工具，广泛适用于研究蛋白质合成相关疾病和克隆选择以及治疗性蛋白质生产中的质量控制应用。 公共卫生相关性：困扰人类的许多（如果不是大多数）疾病都是由人类细胞内蛋白质生物合成异常引起的。此类疾病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变化发生时间和地点的能力。我们建议开发一种直接的方法来实时监测活细胞的蛋白质合成，并以亚细胞分辨率，该方法应该对蛋白质合成相关疾病的阐明和治疗具有广泛的适用性。

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