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

描述（申请人提供）：随着基因和蛋白质编目技术的日益成熟，细胞动力学研究正在成为生物学研究的下一个前沿。需要新技术来捕捉细胞动力学的复杂性。在此背景下，我们提出了一种直接的方法来监测蛋白质合成从活细胞实时和亚细胞分辨率的发展。这种方法被称为DiP（“双肽”），用荧光标记的trna转染细胞，形成FRET对。当细胞蛋白质合成机制处于活跃状态时，这些trna将在活性核糖体的邻近位点被保留（约500毫秒），产生FRET信号，在细胞生长发育的正常条件下，FRET信号的强度应该与细胞中蛋白质合成的速度相关。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，高时间分辨率和亚细胞定位很容易实现。这种能力在基础研究、临床试验和生物治疗药物生产等领域有许多有价值的应用。在过去的一年里，我们已经成功地利用总体DiP在几种哺乳动物细胞系中产生FRET信号。在本项目第一阶段，我们推进DiP开发的最重要任务是证明：1)特异性DiP可以监测特定蛋白质的合成速率；2) DiP测定可校准以提供定量测量；3)这种检测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、算法和软件来估计活性核糖体的浓度；将实施程序，区分活跃核糖体产生的FRET信号与冷冻核糖体产生的FRET信号；以及使用Overall DiP进行可行性研究，以测量一些疾病相关蛋白的表达。在第二阶段，我们计划在第一阶段工作中获得的知识的基础上，进一步开发DiP的应用范围，并将DiP作为一种新的研究工具商业化，该工具广泛适用于研究蛋白质合成相关疾病，以及克隆选择和治疗蛋白生产中的质量控制应用。

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.