RNA interference (RNAi) is a biological process in which double-stranded RNA (dsRNA) directs the degradation of homologous messenger RNA (mRNA), preventing the synthesis of essential proteins. In agriculture, several RNAi-based products have been developed using dsRNA as active agents (i.e., dsRNA biopesticides) to protect crops from pests. The ongoing development of dsRNA biopesticides raises the importance of developing a fundamental understanding of the chemical stability of dsRNA molecules, which defines their ultimate potential to persist in biological and environmental systems in the absence of enzymes or microorganisms. In this study, we examined, for the first time, the specific physicochemical conditions that lead to dsRNA hydrolysis. We hypothesized that base-catalyzed dsRNA hydrolysis, which involves an intermolecular attack by the 2’-hydroxyl group, would be impeded relative to ssRNA due to the decreased the freedom of the 2’-hydroxyl group resulting from the double-helix secondary structure of dsRNA. We first determined that the hydrolysis of dsRNA occurred orders of magnitude more slowly than ssRNA at alkaline pH using gel electrophoresis with quantitative image analysis. As a complementary approach, we used reverse transcription quantitative polymerase chain reaction (RT-qPCR) which amplifies individual strands of the dsRNA to ensure that the strands of dsRNA were more persistent than that of ssRNA. Lastly, we evaluated the persistence of both dsRNA and ssRNA at neutral pH and found that both molecules were completely stable for at least 74 days. Overall, this finding suggests that dsRNA biopesticides in solution are unlikely to be degraded in the absence of catalyzing agents like enzymes or metals due to both inherently slow alkaline hydrolysis and rigid duplex structure. Our results specifically challenge prior assumptions that ssRNA hydrolysis rates can be used to predict hydrolysis rates of dsRNA biopesticides and better define the environmental fate of these biopesticides.

Duplex structure of double-stranded RNA increases chemical stability of RNA interference biopesticides

ACS Fall 2021

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