SBIR-STTR Award

The goal of this Phase I proposal is the development of a simple, cost-effective, and high-throughput method for direct and quantitative RNA detection. In molecular medicine, such a method would greatly facilitate the analysis of RNA viruses and the measurement of specific gene expression, currently pressing problems in the field. Despite this need, few techniques have emerged that are truly direct and quantitative over a broad range of concentration differences, while being readily adaptable to any genetic system. This proposal describes the development of a novel enzymatic assay for direct and quantitative RNA analysis in a microtiter plate-based format. This assay relies on structure-specific cleavage of oligonucleotides hybridized to the target RNA and can be used to detect any sequence. A combination of protein engineering and solid-phase capture approaches will be used to attain a sensitivity of detection of subattomole quantities of RNA without intervening conversion to DNA. The expected outcome of Phase I is an assay for quantitative monitoring of gene expression in 1 mg or less of tissue that will be adaptable to any RNA sequence. This system will be adaptable to a variety of low-cost readout platforms, including chemiluminescent, fluorescent and colorimetric detection

The goal of this Phase II Proposal is the development of a simple, cost- effective, and high-throughput method for detecting RNA directly and quantitatively. The advent of genome-wide sequencing projects and expression array analysis has led to an unprecedented demand for an accurate method to measure specific gene expression that can accommodate large numbers of samples. This Phase II Proposal describes the further development of an enzymatic assay for direct and quantitative analysis of RNA in a microtiter plate-based format. The assay relies on structure-specific cleavage of oligonucleotides hybridized to the target RNA strand, and can be adapted to any sequence. During the Phase I Project, we successfully increased the sensitivity of the detection to about 10/5 copies, through the use of solid phase capture and ELISA detection of the cleavage products, allowing direct detection of common mRNAs from the cell. In this Phase II Proposal, we propose to use novel chimeric enzymes and reaction modifications to drive the sensitivity down to levels suitable for detection of rare mRNAs, at least a 10-fold, and ideally a 100- fold improvement. The assay will be able to quantitate mRNAs from tissue culture cells without prior RNA purification in about 4 hours. The assay will also be configured to use a fluorescence resonance energy transfer readout, eliminating post-reaction sample processing, reducing variation, and making the assay more suitable for high-throughput screening. PROPOSED COMMERCIAL APPLICATION: This project could result in a low-cost, high-throughput, microtiter plate- based method for the direct detection of RNA for monitoring gene expression of a wide variety of genes. This versatile technology could capture a significant portion of the $100 million research market for monitoring cytokine expression, and see broad application in expression screening for drug development. In addition, the enhanced detection may improve capability for mRNA quantitation