Improved RNAi efficacy in insects using pcRNAs

Double-stranded RNA pesticides are an appealing alternative to conventional chemical pesticides, due to their potential to act in a species-specific manner. Their efficacy, however, can be severely limited by the action of nucleases that destroy the molecules before they reach their intracellular targets. We developed so-called paperclip dsRNAs (pcRNAs), with sealed ends, with the aim of protecting the dsRNA from exonuclease activity. In representative insects from a few different orders, including Coleoptera, Diptera, and Homoptera, we observed that pcRNAs were more resistant to gut nucleases than either linear dsRNAs or hairpin RNAs (hpRNAs). In feeding bioassays, we have observed that the pcRNAs can be equally, if not more potent than conventional linear insecticidal dsRNAs at knocking down transcripts and killing the pest insects. Curiously, the pcRNAs appear to enter target cells differently. In many insects, linear dsRNAs enter cells using clathrin-mediated endocytosis, but pcRNAs can bypass this mode of entry. In mosquito and lepidopteran cell lines, pcRNAs entered cells, even in the presence of chemical inhibitors of clathrin or following RNAi-induced knockdown of clathrin. By inhibiting various other cellular uptake pathways, we have recently gained insights into how the pcRNAs enter different insects’ cells. The ability of pcRNAs to enter cells differently suggests that they may serve as effective alternatives to linear dsRNA insecticides in those insects that are either naturally refractory or have become resistant to conventional dsRNA.