Date of Award
Summer 8-15-2022
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Emerging agricultural biotechnology uses RNA interference (RNAi) to protect crops from pests. RNAi is a cellular mechanism in which double-stranded RNA (dsRNA) directs the degradation of the homologous messenger RNA (mRNA), leading to gene silencing and preventing the synthesis of essential proteins. As applied to agriculture, dsRNA biopesticides have been developed that can trigger RNAi inside of the pest tissues to cause reduced pest growth and/or increased pest mortality. When dsRNA biopesticides are released to receiving environments (e.g., agricultural soils and surface water), they pose potential ecological risks to non-target organisms. This dissertation aims to advance the understanding of the environmental fate of dsRNA to enable accurate ecological risk assessments of dsRNA biopesticides.The first objective of this dissertation was to establish a new method using quantitative reverse transcription polymerase chain reaction (RT-qPCR) to measure dsRNA in agricultural soils at environmentally relevant concentrations. Because dsRNA readily adsorbs to soil particles, a method was developed to efficiently transfer dsRNA from soil particles to solutions for RT-qPCR analysis. Furthermore, organic matter was removed using a validated cleanup protocol to prevent inhibition of RT-qPCR. Ultimately, the method was able to quantify dsRNA at orders of magnitude lower concentration than the estimated environmental concentration of dsRNA, indicating the adequate sensitivity of the method. Applying this new method, ≥75% of dsRNA was found to degrade in agricultural soils within 8 hours. The second objective was to characterize the chemical stability of dsRNA in aqueous solutions by examining the impacts of the duplex structure of dsRNA on alkaline hydrolysis. Although dsRNA shares the same primary structure as single-stranded RNA (ssRNA), dsRNA was found to undergo orders-of-magnitude slower alkaline hydrolysis than ssRNA. Furthermore, dsRNA remained intact for multiple months at neutral pH, challenging a prior assumption in dsRNA biopesticide risk assessment that dsRNA is chemically unstable. In systems enabling both enzymatic degradation and alkaline hydrolysis of dsRNA, increasing pH effectively attenuated enzymatic degradation without inducing alkaline hydrolysis that was observed for ssRNA. The third objective was to elucidate a novel abiotic reaction by which RNA, but not DNA, degraded upon adsorption to surfaces of goethite, a ubiquitous mineral in soils and sediments, under environmentally relevant physicochemical conditions. Upon adsorption to goethite, both ssRNA and dsRNA hydrolyzed on the timescale of hours. The reaction products were consistent with iron present in goethite acting as a Lewis acid to accelerate the hydroxide-catalyzed hydrolysis of random phosphodiester bonds comprising the RNA backbone. In contrast to well-established acid or base-catalyzed RNA hydrolysis in solution, mineral-catalyzed hydrolysis was fastest at circumneutral pH, which allowed for both sufficient RNA adsorption and hydroxide concentration. Contact of RNA with the mineral surface was necessary for hydrolysis to occur by demonstrating that RNA degradation was inhibited by compact RNA conformation at elevated ionic strength or competitive adsorption with orthophosphate and organic matter. In addition to goethite, hematite also catalyzed RNA hydrolysis, whereas aluminum-containing minerals (e.g., montmorillonite) did not. Overall, this work (1) contributes to the first method able to quantify dsRNA biopesticides at an environmentally relevant concentration, (2) demonstrates, for the first time, that key degradation pathways of dsRNA significantly differed from those of ssRNA, and (3) reports the first abiotic pathway to contribute to RNA degradation on environmentally relevant timescales. This study advances our understanding of the fate of dsRNA biopesticides in receiving environments and contributes to the ecological risk assessment of dsRNA biopesticides.
Language
English (en)
Chair
Kimberly Parker
Committee Members
Jeffrey Catalano, Daniel Giammar, Young-Shin Jun, Yinjie Tang,