ABSTRACT

The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Fo¨ rster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fltRNA Ile UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Nucleic Acids Research, 2013, Vol. 41, No. 18 e177 doi:10.1093/nar/gkt686

Sima Barhoom1, Ian Farrell2, Ben Shai1, Dvir Dahary2, Barry S. Cooperman3, Zeev Smilansky2, Orna Elroy-Stein1,* and Marcelo Ehrlich1,

1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel,

2Anima Cell Metrology, Inc., Bernardsville, NJ 07924-2270, USA and

3Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA

Received May 29, 2013; Accepted July 2, 2013

The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

DiCodon Diagnostics, LLC is a biotechnology company developing a diagnostic platform technology based on a novel methodology called Diagnostic DiCodon Monitoring of Protein Synthesis (Di-Di). The technology is patented by and licensed from the University of Pennsylvania and based upon research by Dr. Barry Cooperman.

Di-Di presents a new approach to disease diagnosis. It is based on distinguishing diseased from normal cells on the basis of differences in dicodon usage during translation, and visualizing such differences through the use of specific fl-tRNA FRET pairs that bind to specific dicodons within a ribosome actively catalyzing protein synthesis. As many as 1035 discrete fl-tRNA pairs can be monitored, different sets of which can serve as biomarkers for specific diseases.

Our goal is to provide point of care results which will lead to earlier detection of diseases and better patient outcomes by getting patient treatment faster. We are seeking grant, joint development and commercialization opportunities for this technology.

The company is in the early stages of developing a microfluidic device coupled with very sensitive optics for rapid, point of care diagnoses of specific infections. We are currently working on developing grant opportunities for this development. DiCodon Diagnostics is interested in partnering its diagnostic development programs while also planning to provide DiCoMPS FRET monitoring as a central lab.

The patent is based upon United States Patent Application, “Fluorescent Labeling of Transfer RNA and Study of Protein Synthesis”, number 0140329234. The patent application was filed on November 6, 2014 by The Trustees of the University of Pennsylvania.

The following research papers would be of interest for more information on this technology:

Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

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

Kinetics of initiating polypeptide elongation in an IRES-dependent system.

Earle Hager has accepted the role of CEO of this company.