Anima Cell Metrology

University of Pennsylvania

Tel Aviv University

Anima Biotech Inc.

Zeev Smilansky

Barry S Cooperman

Yale E Goldman

Dongli Pan

Marcelo Ehrlich

Orna Elroy-Stein

Sima Barhoom

Yale E Goodman

Provided are methods for labeling transfer RNA comprising replacing the uracil component of a dihydrouridine of said transfer RNA with a fluorophore. The disclosed methods may comprise fluorescent labeling of natural tRNAs (i.e., tRNAs that have been synthesized in a cell, for example, in a bacterium, a yeast cell, or a vertebrate cell) at dihydrouridine (D) positions, or fluorescent labeling of synthetic tRNAs. In another aspect, the present invention provides methods for assessing protein synthesis in a translation system comprise providing a tRNA having a fluorophore substitution for the uracil component of a dihydrouridine in a D loop of the tRNA; introducing the labeled tRNA into the translation system; irradiating the translation system with electromagnetic radiation, thereby generating a fluorescence signal from the fluorophore; detecting the fluorescence signal; and, correlating the fluorescence signal to one or more characteristics of the protein synthesis in the translation system. The disclosed methods are useful in single molecule as well as in ensemble settings.

Fluorescent labeling of transfer RNA and study of protein synthesis

There are provided methods for detection and measurement of stress in a cell, the method including introducing a labeled tRNA into the cell and detecting a change in subcellular localization of the labeled tRNA in the cell, based on the signal emitted from the labeled tRNA. There are further provided methods and systems for the generation of a stress index of a living cell. There are further provided methods and systems for detection of stress in a living cell, comprising detection of changes in subcellular localization of labeled tRNA in a cell, wherein the detection is performed in real time.

Systems and methods for detection of cellular stress

The present invention relates to systems and methods for measuring the rate of translation of a target protein in cells, which are based on the detection of translation of one or more predetermined codon pairs during synthesis of the target protein. The detection is provided by a FRET signal emitted from labeled tRNA molecules which are juxtaposed during synthesis of the protein.

Systems and methods for measuring translation of target proteins in cells

A method and a device are disclosed for monitoring the synthesis of proteins by the ribosome, wherein the ribosome is bound to a first label, for example a donor fluorophore, and a tRNA and/or amino acid are is bound to a second label, for example an acceptor fluorophore, wherein the first and second labels together from a FRET pair. As the ribosome mechanism processes the mRNA and tRNA molecules and synthesizes a polypeptide chain, a light source illuminates the ribosome, exciting the donor fluorophores and thereby the acceptor fluorophores whenever these are in sufficient proximity to a donor. The resulting signals are detected and used as a key for database searching and identification of the protein being synthesized.

Protein synthesis monitoring (PSM)

A method and a device are disclosed for monitoring the synthesis of proteins by the ribosome in real time, in vivo as well as in in-vitro. The ribosome is engineered to carry a donor fluorophore, and tRNA and/or amino acids and/or some other part of the ribosome are either engineered to carry acceptor fluorophores or else their natural fluorescent properties are utilized as acceptors. As the ribosomes mechanism processed the mRNA and tRNA molecules and synthesizes a polypeptide chain, a light source illuminates the ribosome, exciting the donor fluorophores and thereby the acceptor fluorophores whenever these are in sufficient proximity to a donor. The resulting signals are detected and used as a key for real-time database searching and identification of the protein being synthesized. The resulting data can be tabulated and interpreted in different ways. FIG. () describes the properties of a FRET pair and the dependence of FRET on pair distance.

Growing community of inventors

D.N. Emek Sorek, Israel

Zeev Smilansky