Research Project
6589708
DESCRIPTION: (Applicant's Description) We propose to develop a technology to simultaneously detect and quantify large numbers individual proteins and post-translationally modified proteins. This technology will be based on a spatially-arrayed aptamer microchip and sensitive fluorescence-based optical detection techniques. The system will provide a powerful new tool for biomedical research and diagnosis of diseases and cancers. Currently, the levels of cellular proteins are inferred from cellular mRNA levels by using DNA oligonucleotide microchips. These microchips allow the detection of mRNA levels by base-pairing interactions to homologous DNA oligomers located at different sites in the microchip array. However, this indirect assay of protein levels suffers from two problems: First, mRNA levels may not accurately reflect the level of encoded protein because translation and degradation rates vary from protein to protein and are regulated in response to cell physiology. Second, post-translational modifications proteins often alter critical protein activities. This technology we propose to develop is comprised of three parts: 1) Aptamers with high-affinity and high-specifity for individual proteins. These aptamers will be designed to have ligand-dependent changes in fluorescence properties. 2) A microchip array with each array site occupied by aptamers against a different target molecule. 3) An evanescent field fluorescence detector which detects target molecules binding to aptamers at the array sites. Our proposal includes plans for: 1) selection of aptamers that can discriminate between different post-translational modifications of the same protein; 2) development of fluorescence-based techniques for detecting oligonucleotide:protein interactions; 3) design and construction of an optimized fluorescence detector; 4) scaling up methods to allow large numbers of independent aptamer selections against different proteins; 5) construction of a prototype protein chip.
