Nwokeoji Alison Obinna, Nwokeoji Eleojo Ahuva, Chou Tachung, Togola Abou
Chemical and Biological Engineering, University of Sheffield, Sheffield, S1 3JD, UK.
School of Biosciences, University of Sheffield, Sheffield, S10 2TN, UK.
Bioresour Bioprocess. 2022 Oct 6;9(1):107. doi: 10.1186/s40643-022-00596-2.
RNA 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.
RNA干扰（RNAi）是真核细胞进化出的用于调控基因表达的最保守途径之一。RNAi利用不可翻译的双链RNA（dsRNA）分子来隔离或降解基因的mRNA分子。在RNAi中，递送至细胞的经过特殊设计的外源dsRNA可使靶基因沉默，这一现象已在许多功能研究中得到利用，并在生物农药应用中得到探索。对安全且可持续的作物害虫管理方案的探索推动了抵消无机农药对生物多样性影响的需求。用dsRNA作物喷雾剂替代无机农药的前景越来越受到欢迎，因其具有高靶向特异性和低环境影响。然而，要使dsRNA进入农药市场，必须以具有成本效益且可持续的方式进行生产。在本文中，我们开发了一种高产表达培养基，与使用1 mM异丙基-β-D-硫代半乳糖苷（IPTG）的现有表达培养基相比，其dsRNA产量提高了15倍。我们还优化了一种低成本的纯化方法，可产生高质量的纯化dsRNA。所开发的方法避免了商业试剂盒或商业核酸酶中常见的用于消除污染性DNA或单链RNA（ssRNA）的有害化学试剂的需求。我们还证明了该生产平台具有可扩展性，从259 mg湿大肠杆菌细胞沉淀中可产生6.29 mg dsRNA。研究结果还为微生物来源的dsRNA库中的异质dsRNA种类提供了结构见解。最后，我们还表明，未经预先配制的纯化“裸”dsRNA在田间条件下可诱导昆虫毒性。本研究提供了一种新颖、完整、低成本的dsRNA平台制备方法，具有在工业dsRNA生产中应用的潜力。
