EAGER: Environmental Fate of Double Stranded RNA-Based Bionanocomposites.

Background Recent advances in RNA interference technology have enable the introduction of double stranded RNA (dsRNA)-based pesticides, which are currently being commercialized by companies such as Monsanto. This technology hypothetically enables the targeting of pest species with only a very specific genetic sequence using silencing of genes required for its survival, thereby eliminating environmental health and safety concerns associated with chemical pesticides. However, this hypothesis is essentially untested. Further, dsRNA has poor stability in the environment and is not readily taken up by many pest species. This has led to the development of nanocomposite-based delivery of the dsRNA, for which U.S. and Chinese patents have been recently awarded. Nanocomposite delivery vehicles protect the dsRNA from degradation in the environment and in the gut of pest species, enhance cell penetration, and enhance escape of the dsRNA from endosomes. The structure and composition of these nanocomposites has been inspired by drug delivery devices designed for gene therapy in humans, which in turn have been inspired by viruses. Nanocomposites make the dsRNA far more efficacious in certain pest species and potentially persistent in the environment. At present, no studies have been conducted to assess the environmental fate and effects of these bionanocomposites. In fact, this would be the first study we are aware of that would investigate the environmental fate of active bionanomaterials. Objectives and Hypotheses The objective of this project will be to establish a set of methods to track the fate and transformations of these bionanomaterials in soil and in soil organisms, to do preliminary testing of their stability in soil solutions, and to assess possible biouptake 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. Approach We will synthesize diethylaminoethyl dextran (DEAE) /dsRNA polyplex particles and DEAE 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 we 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. We 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. Biouptake of the materials will be assessed using laser confocal microscopy. We will systematically characterize how soil chemistry and aging time affect aggregation, disassembly, dsRNA degradation, biouptake, specific bioactivity and non-target effects. Appropriateness for EAGER program The hybrid bionanomaterials under investigation are different than the passive nanomaterials that have been studied thus far. That is because they are designed to be absorbed by specific organisms, go to a certain subcellular compartment, sense that condition, and release their dsRNA cargo. Therefore, they are active nanostructures. Because this is the first study we are aware of to track the environmental fate of an active bionanomaterial, the research is exploratory in nature and is of high risk. The project would need to develop new tools to track these materials. However, it is potentially transformative because it would initiate a paradigm shift in environmental nanosciences away from studying passive nanomaterials such as TiO2, CeO2 and Ag, and move toward more complex active nanostructures, which are actually simple forms of synthetic biology with behavior similar to viruses (although they are not designed to reproduce). The forward-thinking and exploratory nature of this research make it difficult to fund through other funding mechanisms.