Date of Award
Winter 12-19-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
As a result of its unique properties, RNA has been increasingly employed in emerging technologies with a wide range of environmental applications. For example, the higher rate of RNA degradation relative to DNA is leveraged in ecological monitoring to identify living organisms present at a specific sampling location in real time. Similarly, when using wastewater-based epidemiology (WBE) for surveillance of RNA viruses, the knowledge of RNA degradation rates is essential to relate measured RNA concentrations to the initial viral loads. Additionally, double-stranded (ds)RNA has been applied for agricultural pest control due to its ability to initiate a cellular process called RNA interference (RNAi). The successful deployment of ecological monitoring and WBE, and the assessment of ecological risk associated with the release of dsRNA biopesticides relies on an understanding of key fate processes governing RNA persistence in environmental systems (e.g., soils, surface waters). The first objective of this dissertation was to investigate whether RNA hydrolysis is catalyzed by metals under environmentally relevant conditions. I demonstrated that at concentrations ~10 µM, metal species possessing higher Lewis acidity (i.e., lower pKa; lead, copper) catalyzed the hydrolysis of single-stranded (ss)RNA at neutral pH. In contrast, metal species with weaker Lewis acidity (i.e., higher pKa; zinc, nickel) did not catalyze the hydrolysis of ssRNA. Unlike ssRNA, dsRNA was recalcitrant to metal-catalyzed hydrolysis regardless of metal Lewis acidity. I also found that more acidic solution pH and the presence of ligands (e.g., citrate) hindered lead- and copper-catalyzed ssRNA hydrolysis. These findings, coupled with the sub-micromolar concentration of metals in environmental systems, suggest that both ssRNA and dsRNA are unlikely to undergo metal-catalyzed hydrolysis in environmental aqueous systems. The second objective aimed to investigate how biotic degradation of RNA was impacted by dissolved organic matter (DOM) in environmental systems. In the environment, biotic or enzymatic degradation of RNA occurs rapidly on a timescale of days to weeks; thereby, controlling the environmental persistence of RNA. However, I hypothesized that DOM binds to RNA, suppressing its enzymatic degradation. I adapted a gel electrophoresis-based technique, previously used to assess RNA-protein binding, to examine RNA-DOM binding. I found that humic acids bound to RNA, whereas fulvic acids did not. Humic acids were also found to suppress RNA degradation to greater extents than fulvic acids at concentrations of 8–10 mgC/L, which represent the upper limit of DOM concentrations in surface waters. The suppression of enzymatic degradation of RNA was also observed in authentic soil extract and river water containing DOM. Thus, we determined that RNA is likely to undergo lowered enzymatic degradation in environmental systems with high DOM concentrations. The final objective evaluated the persistence of RNA in solutions under acidic conditions. My experimental findings revealed that dsRNA, likely owing to its duplex structure, did not exhibit degradation at acidic pH, invalidating prior assumptions regarding its chemical instability. In comparison, ssRNA exhibited orders-of-magnitude faster degradation. At pH 3–4, I found that the degradation rate constant of ssRNA increased proportionally with hydronium ion concentration. In contrast, at pH <3, I observed a greater than proportional increase of the degradation rate constant with hydronium concentration, suggesting an increased contribution of depurination to RNA degradation at elevated acidic conditions. Under conditions where both enzymatic and abiotic reactions occurred concurrently, I found that decreasing the solution pH to 3 served as a safeguard for both ssRNA and dsRNA against enzymatic degradation, without inducing extensive abiotic degradation. Overall, this dissertation (1) demonstrates that metal species are unlikely to be a significant catalyst for degradation of RNA released into the environment, (2) shows that dissolved organic matter can protect RNA from biological degradation, likely increasing its persistence in the environment, and (3) indicates that chemical stability of dsRNA relative to ssRNA is higher under acidic solution conditions. Thus, this dissertation contributes to an advanced understanding of processes impacting the persistence of RNA released into the environment, particularly in relation to its emerging applications.
Language
English (en)
Chair
Kimberly Parker