Application of RNA Chemical Modifications to Modulate RNAi in Insects

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either have no current means of control, or have developed resistance against traditional chemical pesticides. Therefore, there is currently significant demand to develop new types of insecticide, capable of overcoming issues with resistance, or lack of target species selectivity. Long dsRNA based biocontrols are emerging as a novel alternative for sustainable insect management strategies. Long dsRNA based biocontrols are capable of inducing RNA interference (RNAi) in insects resulting in knockdown of a target mRNA and therefore reduced levels of its protein product. Targeting of the mRNA for a protein essential to the growth and survival of the insect results in mortality of the target insect. Therefore long dsRNA based biocontrols are emerging as novel species-specific insecticides. This approach has successfully been demonstrated for a wide range of target mRNAs and target species. A major barrier to the technology, is degradation of the dsRNA by nucleases in the environment and within the insect before it can induce RNAi. Therapeutic siRNAs and DNA antisense oligonucleotides are ineffective in whole organism mammalian systems unless they contain chemically modified nucleotides which prevent their degradation in the bloodstream by extracellular nucleases, as well as improving delivery and transport of the siRNA or DNA oligo. It was hypothesised that such chemical modifications could improve the nuclease resistance of dsRNA based biocontrols, increasing their efficacy in insects. In this study, I have optimised the synthesis and purification of long dsRNA containing a range of different chemical modifications including phosphorothioate, 2’-fluoro and 5-hydroxymethyl. The effects of chemical modifications of the long dsRNA were studied both in vitro and in vivo. The results showed for the first time that dsRNA containing phosphorothioate modifications demonstrated increased resistance to stink bug salival nucleases. In addition, both phosphorothioate and 2’-fluoro modified dsRNAs demonstrated increased RNAi efficacy in Drosophila cell cultures. Furthermore, the effects of the chemical modification of long dsRNA on RNAi efficacy were also studied in live insects in both southern green stink bug using injection assays, and in western corn rootworm using feeding assays. The results showed that the chemically modified long dsRNA resulted in successful RNAi in live insects as measured by insect mortality. However, no increase in RNAi efficacy was observed compared to unmodified dsRNA. To our knowledge this is the first time that RNAi has successfully been triggered by chemically modified dsRNA in insect cells or live insects. These results provide further insight into the effects of chemical modifications of dsRNA based biocontrols. It is anticipated that these results will provide important information for developing new alternative dsRNA based biocontrols with improved nuclease resistance and RNAi efficacy.