Agricultural RNA interference (RNAi) technology involves the application of double-stranded RNA (dsRNA) biopesticides to control crop pests. The subsequent release of dsRNA biopesticides to agricultural soils and surface waters poses ecological risks. To evaluate the potential risks caused by the release of dsRNA, the environmental fate of dsRNA biopesticides needs to be assessed. The fate of released dsRNA in aquatic environments is influenced by hydrolysis, both biotic and abiotic. While biotic hydrolysis is recognized to be a major degradation pathway, the assessment of abiotic hydrolysis may provide the absolute limit of environmental persistence of released dsRNA. Although dsRNA can undergo abiotic hydrolysis catalyzed by acidic or alkaline solutions, the rate of hydrolysis is slow at circumneutral pH. An abiotic pathway that can cause sufficiently rapid dsRNA degradation to account for degradation on the requisite timescale has yet to be identified. As a result, we investigate the effect of metal ions on dsRNA hydrolysis at conditions relevant to aquatic environments. We find that the metal to phosphodiester bond molar ratio is the key governing factor influencing the extent of metal-catalyzed hydrolysis. Specifically, dsRNA at 0.01 ng/µL degraded up to 90% in 25 hours when incubated with lead at nominal 1 µM at pH 7 and 23 °C. Further, we found that the ability of metal ions (lead, copper, zinc, and nickel) to catalyze RNA hydrolysis correlates with lower pK<sub>a</sub> values (stronger Lewis acidity) of the aqueous metal-hydroxide complexes. The metal ion hydrolyzes RNA through a transesterification reaction which involves an intramolecular nucleophilic attack on the phosphorus center of the phosphodiester bond by the deprotonated 2’-OH group, leading to the production of 2’ and 3’ nucleoside monophosphates as end products. Overall, our results suggest that metal-catalyzed dsRNA hydrolysis can be a significant degradation pathway for released dsRNA at environmentally relevant conditions.

Environmental fate of dsRNA biopesticides: Metal-catalyzed RNA hydrolysis in aquatic environments

ACS Fall 2021

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