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.
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