LARGE PARTICLE SORTING FOR THE SELECTION OF OPTIMAL APTAMER BINDERS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. While current droplet sorters can be used to sort the particles 20-70 microns in diameter, the size of the particles limit the sort speed to about a thousand per second. As aptamer libraries could contain >109 beads, sorting speed of larger particles will be at a premium. The technologies developed in this grant (Project 2) will result in the development of a high speed sorter (with initial target rates of 100,000 per second) that would greatly assist in sorting one bead one compound aptamer libraries (or library of libraries). This would greatly improve the ability of investigators to select aptamer and thioaptamer high affinity ligands to critical protein targets. The NFCR will develop sorting technologies as described in project 2. The sorting technologies will be integrated into a functional sorter, which will be initially tested at the NFCR to sort aptamer libraries provided by Dr. Gorenstein. Once instrument performance has been determined to be sufficient, a second generation prototype will be assembled in Dr. Gorenstein's laboratory for use by his lab. Dr. Gorenstein will provide aptamer libraries and protein targets that interest his programs for selection and sorting at the NFCR. Our goal in this project is to utilize our bead-based technology for selecting thioaptamers utilizing multi-color flow cytometry targeting the differentially expressed proteins within the host proteomes in two major projects funded by NIH. In DARPA and more recently an NIAID-funded U01 partnership grant focused on biodefense, Dr. Gorenstein (PI) is developing a new thioaptamer proteomics chip technology to investigate protein expression associated with pathogen-induced inflammatory responses. He will utilize the NFCR facility to select thioaptamers targeting specific cytokines and chemokines and other identified proteins to dissect immune responses of selected viruses of concern in bioterrorism by analyzing the dynamic changes in protein expression. Changes in protein expression are being determined in human cell models as well as rodent models of immune responses to arena viruses such as Lassa and Pichinde (hemorrhagic fever viruses. These results will aid in the design of drugs to inhibit the identified protein interactions underlying immune response processes and so ameliorate cytopathological immune responses resulting in shock or to enhance "innate immunity" to help mount effective immune response. Elucidating these protein expression changes will allow early diagnosis and enhanced prognosis of viral disease, and subsequent development of effective pharmacological and immunological interventions. UTMB has a BSL-4 facility for handling these select agents such as Lassa and is constructing one of two National Biocontainment Laboratories and this project is in collaboration with a number of noted virologists at UTMB such as Drs. C. J. Peters, Alan Barrett, Judy Aronson and Norbert Herzog. Dr. Gorenstein will also use the NFCR facility for selecting thioaptamers (again those involved in the immune response) to develop a thioaptamer proteomics chip as a proteomics biomarker/biosignature diagnostic tool to identify and quantify the differential expression of key proteins involved in RSV-infections related to asthma in an NHLBI-funded Proteomics Center at UTMB (one of ten in the country). The working hypothesis is that by utilizing the massive proteome HTS shown in the figure, a limited range of proteins in serum and lung lavage samples can be targeted. Accomplishing this will provide this program the opportunity to construct a biosignature "chip" which will allow identification and quantification of the various important proteins expressed during disease progression. In both projects, the long-term objectives are to develop a real-time thioaptamer chip-based identification and readout of the levels of proteins and protein+protein complexes to allow these researchers to correlate changes in protein expression to predict biomarkers/biosignatures for progressive disease and therapeutic responses. Dr. Gorenstein's lab will serve as a beta testing facility for new high speed large particle sorters and will communicate with the NFCR to optimize instrument performance.