A novel sustainable platform for scaled manufacturing of double‑stranded RNA biopesticides

Open Access Journal; Published online: 06 Oct 2022RNA interference (RNAi) represents one of the most conserved pathways evolved by eukaryotic cells for regulating gene expression. RNAi utilises non-translatable double-stranded RNA (dsRNA) molecules to sequester or degrade mRNA molecules gene. In RNAi, specifically designed exogenous dsRNA delivered to the cell can silence a target gene, a phenomenon that has been exploited in many functional studies and explored in biopesticide applications. The search for safe and sustainable crop pest management options drives the need to offset the effect of inorganic pesticides on biodiversity. The prospect of replacing inorganic pesticides with dsRNA crop spray is gaining popularity, enhanced by its high-target specificity and low environmental impact. However, for dsRNA to reach the pesticide market, it must be produced cost-effectively and sustainably. In this paper, we develop a high-yield expression media that generates up to 15-fold dsRNA yield compared to existing expression media utilising 1 mM IPTG. We also optimise a low-cost purification method that generates high-quality and purified dsRNA. The developed method circumvents the need for hazardous chemical reagents often found in commercial kits or commercial nucleases to eliminate contaminating DNA or single-stranded RNA (ssRNA) species. We also demonstrate that the production platform is scalable, generating 6.29 mg dsRNA from 259 mg wet E. coli cell pellet. The results also provide structural insights into the heterogeneous dsRNA species within the microbial-derived dsRNA pool. Finally, we also show that the purified ‘naked’ dsRNA, without prior formulation, can induce insect toxicity under field conditions. This study provides a novel, complete, low-cost process dsRNA platform with potential for application in industrial dsRNA production

Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry

Accurate quantification of nucleic acids using hypochromicity measurements in conjunction with UV spectrophotometry

UV absorbance spectrophotometry is widely used for the quantification of nucleic acids. For accurate quantification it is important to determine the hypochromocity of the oligonucleotide or complex nucleic acid structure. The use of thermal denaturation studies in conjunction with UV spectrophotometry to determine hypochromicity requires prolonged, elevated temperatures, which may cause partial hydrolysis of RNA. In addition, dsRNA is difficult to denature even at elevated temperature and the extinction coefficients of nucleic acids are also affected by temperature, which makes it difficult to accurately determine the nucleic acid concentration. To overcome these caveats, we have utilised the chemical denaturant dimethyl sulfoxide which, in conjunction with a short thermal denaturation prevents renaturation of the duplex nucleic acids (dsDNA/RNA). Using this approach, we have measured the absorbance of both the unstructured and structured nucleic acids to accurately measure their hypochromicity and determine their extinction coefficients. For a range of different dsRNA we have for the first time determined values of 46.18-47.29 µg/ml/A260 for the quantification of dsRNA using UV spectrophotometry. Moreover, this approach enables the accurate determination of the relative proportion of duplex nucleic acids in mixed ds/ss nucleic acid solutions, demonstrating significant advantages over current methods