The long-term objective of this effort is to develop a generic gene shuffling-based technology to rapidly screen libraries of 10[10] proteins/peptides encoded by DNA libraries, for identifying biomolecules that can intercept both existing and emerging organophosphate-based chemical warfare nerve agents (CWNA). Enzymes identified in these screens should be capable of catalytically neutralizing the target agent under physiological conditions, thereby providing a basis for development of a new generation of therapeutic agents against CWNA. The major milestone is to integrate established components of enhanced molecular evolution techniques so as to provide a means of miniaturizing existing low-throughput assays, thereby dramatically increasing both sensitivity and throughput. Micro beads will be coated with multiple copies of recombinant human AChE (the CWNA physiological target). Genes of interest will be attached to the same beads. Gene libraries will be obtained by random mutagenesis of several genes that encode enzymes capable of hydrolyzing OPs (e.g., organophosphate hydrolases of both bacterial and mammalian origin, and a repertoire of AChEs, e.g. other vertebrate AChEs, insect AChEs). Beads coated with AChE and the corresponding genes will be compartmentalized in approximately 5fL emulsion droplets, and single genes transcribed and translated in individual droplets. The expressed biomolecules will be allowed to intercept the CWNA, preventing interaction with its target (viz. AChE), and genes that code for an effective interceptor will be isolated. Uniquely, this screen is for directly detoxifying the CWNA, not simply for binding it, thus allowing identification of biomolecules that prevent its action by degradation at the desired rate. Relevance: this technology is envisaged to provide rapid discovery of pretreatment and post challenge therapeutic drugs against existing and emerging CWNA threats and will shorten the time from emergence of a threat to identification of potential countermeasures to a few days or weeks. Once developed this technology can be extended to identification of interceptors for vesicants, pulmonary agents, metabolic/cellular poisons and biological warfare agents. .
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