Analysis of RNA Interference (RNAi) Biopesticides: Double-Stranded RNA (dsRNA) Extraction from Agricultural Soils and Quantification by RT-qPCR

Abstract

Double-stranded RNA (dsRNA) molecules are used as a novel class of biopesticides. To enable assessments of the ecological risk associated with their release to receiving environments, we developed an approach to quantify dsRNA in agricultural soils using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). To allow quantification of dsRNA adsorbed to particles, we also developed a protocol to transfer dsRNA from particles to the extraction buffer by changing particle surface charge and adding constituents to compete with dsRNA for adsorption sites. Our approach could quantify dsRNA amounts as low as 0.003 ng dsRNA /g soil . This approach is the first available field-applicable approach able to quantify dsRNA biopesticides down to environmentally relevant concentrations. We applied this approach to investigate dsRNA dissipation (including dilution, degradation, and adsorption) in two agricultural soils. When we applied a low amount of dsRNA (1 ng dsRNA /g soil ) to the soils, we observed that a greater fraction of dsRNA was adsorbed to and extractable from soil particles in a silty clay loam soil than in a fine sandy loam soil. In both soils, dsRNA dissipated on the timescale of hours. Overall, these results demonstrate that our approach can be applied to assess the environmental fate of dsRNA biopesticides at concentrations relevant to their release to soils.

Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA

“…The resultant liquid was stored at −20 °C until use. For experiments using RNase from soil, we mixed a slurry prepared with 50 g of fine sandy loam soil (characterized previously 40 ) and 50 mL of sterile water in an uncovered 250 mL Erlenmeyer flask using a stir bar (1000 rpm) at 24 °C for 2 days. We then centrifuged the slurry at 21,100g for 1 min and collected the supernatant.…”

RNA-based pesticides aim to get around resistance problems

“…Kimberly Parker, assistant professor of environment and chemical engineering at Washington University, has had to come up with an entirely new technique to detect particles of dsRNA in the environment because they are much larger than the molecules in traditional pesticides (7). "Our big concerns are actually understanding what happens to them in the environment and how they undergo different degradation processes," she says.…”

dsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology

“…SIGS using naked dsRNA has been shown to be an effective control method against both insect pests and fungal pathogens [31,52,108]. Despite these advances, topically applied dsRNA molecules are vulnerable to degradation.…”

“…A number of studies in both fungi and insects have demonstrated the effectiveness of this technology [31,45,49,50]. Unlike many chemical pesticides, environmental persistence of dsRNAs appears to be limited, which, from an environmental protection perspective, is an attractive feature [51,52]. Studies have shown a half-life of less than 24 h for dsRNA within the soil, due primarily to bacterial degradation [51].…”