Paperclip RNAs bypass clathrin mediated endocytosis in insect and fungal cells

Insects and fungi are among the most destructive pests and pathogens affecting agriculture and public health. Although chemical pesticides have long been the primary means of control, their broad‑spectrum activity and widespread use have accelerated resistance in many systems. RNA interference (RNAi) has therefore emerged as a promising species‑specific alternative. RNAi is initiated by double‑stranded RNA (dsRNA), which triggers sequence‑directed degradation of target transcripts. However, RNAi efficiency varies markedly across species, influenced by nuclease activity, dsRNA length, and the cellular uptake route. We previously showed that long dsRNA uptake in Aedes aegypti depends on clathrin‑mediated endocytosis (CME), whereas paperclip RNA (pcRNA), a compact dsRNA structure developed in our laboratory, bypasses CME and enters cells through a distinct pathway. Here, we compared the uptake and RNAi performance of two dsRNA formats, long dsRNA (210–230 bp) and pcRNA (21–23 bp), in three representative species: Aedes aegypti (mosquito), Spodoptera frugiperda (moth), and Sclerotinia sclerotiorum (fungus). To assess cellular uptake of these dsRNAs, we used two CME inhibitors, chlorpromazine (CPZ) and bafilomycin A1 (BafA1), along with fluorescein‑labeled dsRNAs. CME inhibition prevented long dsRNA from entering the cytoplasm and abolished its ability to induce RNAi. In contrast, pcRNAs were readily internalized and produced significant gene knockdowns under both inhibitor treatments. Microscopy further revealed that all three cell types, pretreated with either CPZ or BafA1, prevented long dsRNA from entering the cytoplasm. pcRNA, consistently reached the cytoplasm and showed superior uptake across all species. Together, these findings demonstrate that pcRNA uses an uptake mechanism distinct from long dsRNA, enabling it to circumvent CME-dependent barriers. This unique internalization route likely reflects its compact size and structural configuration and highlights pcRNA as a promising platform for improving RNAi-based pest and pathogen control.