Abstract
Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA. We have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications. The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA.
Highlights
The application of dsRNA for the targeted, sequence specific inhibition of specific genes via RNA interference (RNAi) is emerging as an important tool for the development of novel RNAi-based sustainable insect and fungal management strategies.[1,2,3] There are a wide range of future potential applications of dsRNA based biocontrols for agricultural insect pests as well as for prevention of diseases in beneficial insects
Large quantities of dsRNA can be readily synthesised in either bacterial systems, by in vitro transcription, in cell-free systems or in planta in order to block the expression of essential genes, resulting in insect mortality
In this study we focused on the production of dsRNA using both in vitro transcription and transcription in E. coli by growing E. coli HT115 cells transformed with plasmids to express dsRNA
Summary
Variety of robust analytical techniques to fully characterise and accurately quantify dsRNA prior to RNAi applications. Large scale synthesis of dsRNA for RNAi applications, including the production of RNA-based insecticides and fungicides has created a demand for biochemical and biophysical techniques to analyse the long dsRNA produced. Such analytical approaches are aimed at the characterisation (including analysis of the structural homogeneity of the dsRNA) prior to downstream applications. In this study we have used ion pair reverse phase chromatography (IP RP HPLC) under both non-denaturing and denaturing conditions in conjunction with high resolution AFM imaging to characterise long dsRNA and their associated dsRNA multimers or higher order assemblies generated both in vitro and in vivo in E. coli for use as RNA-based insecticides
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