SBIR-STTR Award

Reporter genes are essential for accurate and sensitive gene activity measurements. Linked to specific DNA response elements they provide a means to screen compounds that alter specific signaling pathways. Efficient reporter genes encode enzymes that act on pro-fluorescent and pro-chemi-luminescent substrates and give highly amplified signals. Certain reporter gene / substrate combinations allow detection of gene expression changes over a range of 5-7 orders of magnitude. However, the number of suitable reporter genes is small and limits simultaneous screening of multiple compounds and signaling pathways. Fluorescence quenched probes are highly suitable reagents for genomic, biochemical, and immunoassays. Because the fluorescent signal is generated only by uncoupling of the fluorophore and quencher, the probes' have nearly undetectable background signals. The signal is typically generated in one of three ways: a change in conformation of a fluorescence-quenched probe (e.g. DNA hybridization), cleavage of the probe (e.g. proteolysis), or hydrolysis of a substrate-fluorophore conjugate (e.g. galactose removal from fluorescein-digalactoside). Recently, we conceived and have initiated development of a novel mechanism for eliciting fluorescent signals, by direct enzymatic reduction of azo bonds. Our azoreductase cuts azo bonds of quencher molecules leading to loss of quenching and thus to generation of fluorescence. Under this grant we propose to apply azoreductase to develop a powerful and versatile new reporter technology. A number of assays can be envisioned employing the azo reduction mechanism, including reporter gene assays to study cell signaling pathways and enzyme linked immunological detection of proteins such as western analysis. The principle objectives of Phase I of this grant are to optimize the azoreductase gene for functional expression in mammalian cells to evaluate its potential as a reporter gene. The versatility of the enzyme's azo reduction capabilities on model fluorescence-quenched substrates and in vitro applications of this novel technology will be explored. Technology to be developed in the proposed research should find broad applications in the general study of cell and molecular biology as well as to high throughput screening of drug candidates

Abstract Reporter genes are essential for accurate and sensitive gene activity measurements. Linked to DNA response elements they provide a means to screen compounds that alter specific signaling pathways. Efficient reporter genes encode enzymes that act on pro- fluorescent and pro-chemiluminescent substrates to give highly amplified signals. Certain enzyme / substrate combinations allow detection of gene expression changes over a range of 5-7 orders of magnitude. Few suitable reporter genes limit simultaneous high throughput screening of multiple compounds and signaling pathways. Fluorescence quenched probes are highly suitable for genomic, biochemical, and immunoassays. Because the fluorescent signal is generated only by uncoupling of the fluorophore and quencher, the probes' have nearly undetectable background signals. The signal is typically generated in one of three ways: a change in conformation of a fluorescence-quenched probe (e.g. DNA linearization via hybridization), cleavage of the probe (e.g. proteolysis of peptide substrate), or hydrolysis of a substrate-fluorophore conjugate (e.g. galactose removal from fluorescein-digalactoside). Recently, we conceived and have initiated development of a novel mechanism for eliciting fluorescent signals by direct enzymatic reduction of azo bonds. Our azoreductase cuts azo bonds of our Black Hole QuencherTM dark quencher molecules to anilines leading to loss of quenching and thus to generation of fluorescence. Under this grant we propose to apply azoreductase to develop a powerful and versatile new reporter technology. A number of assays can be envisioned employing the azoreduction mechanism, including reporter gene assays to study cell signaling pathways and immunological detection of proteins such as western analysis and ELISA. Principle objectives of this Phase II grant are to assemble a functional, simple, yet versatile azoreductase-based reporter gene system for expression in mammalian cells. New enzyme variants and pro-fluorescent substrates we will be combined with existing reporter technology for extended versatility and multiplexing. Azoreductase will also be explored for further in vitro applications to facilitate multiplex detection Technology to be developed under the proposed research should find broad applications in the general study of cell and molecular biology as well as to high throughput screening of drug candidates. Reporter genes are essential to accurately measure specifc gene activity . In combination with fluorescent molecules they provide for high quality replacements for older radioactivity based methods. Technology to be developed under the proposed research at Biosearch Technologies should find broad applications in the general study of cell and molecular biology as well as in high throughput screening of drug candidates.