EAGER: Environmental fate of double stranded RNA-based bionanocomposites

Unrine, Jason<br/>1712323 <br/><br/>Recent advances in RNA interference technology have enable the introduction of double stranded RNA (dsRNA)-based pesticides, which are currently being commercialized by the agrochemical industry. This technology hypothetically enables the targeting of pest species with only a very specific genetic sequence using silencing of genes required for their survival, thereby potentially eliminating environmental health and safety concerns associated with chemical pesticides. dsRNA has poor stability in the environment and is not readily absorbed by many pest species. Using nanoparticles to deliver dsRNA can help overcome problems with stability and facilitate absorption by pests. However, limited information on the persistence and long-term fate of these materials in soil is available and there are little established techniques for detecting and characterizing these materials in the environment. This project will develop techniques to track these materials in soil and characterize their degradation. The tools developed and insights gained by this project will enable the development of more benign nanocomposite pesticides. This will aid in increasing global food security while helping to avoid unwanted environmental impacts resulting from pesticide application. <br/><br/>The objective of this project will be to establish a set of methods to track the fate and transformations of dsRNA nanocomposites in soil and in soil organisms, to do preliminary testing of their stability in soil solutions, and to assess possible bio-uptake non-target effects in soil organisms. It will focus on biopolymer polyplex dsRNA nanocomposites and inorganic (calcium phosphate) core with a biopolymer/dsRNA coating. The main hypothesis that will be tested is that binding of dsRNA to a nanocomposite makes it more persistent in soil and in non-target organisms, increasing the likelihood of adverse effects. The PI will synthesize diethylaminoethyl dextran (DEAE), chitosan, poly-L-lysine and poly-L-histidine coated calcium phosphate particles loaded with dsRNA complimentary to the green fluorescent protein gene. This gene is present within a genetically modified strain of the nematode Caenorhabditis elegans (PD4251) which the PI will use as a model organism. Because the gene is only expressed within cells in the body wall, silencing of this gene ensures that the dsRNA has been taken up and internalized within the worm. Nanocomposites will then be aged in soil solutions of varying composition for varying lengths of time to determine how transformations in soil affect their bioactivity. The PI will track the fate of the nanoparticles in soil solution and in organisms by isotopically labeling calcium phosphate with the stable 44Ca isotope and fluorescently labelling the dsRNA. The florescence label will be tracked using asymmetrical field flow fractionation (AF4) coupled to fluorescence spectroscopy. The isotopically labelled core will be detected using AF4 coupled to ICP-MS. Bio-uptake of the materials will be assessed using laser confocal microscopy. The PI will systematically characterize how soil chemistry and aging time affect aggregation, disassembly, dsRNA degradation, bio-uptake, specific bioactivity and non-target effects.