Abstract

Pest management strategies utilizing RNA interference (RNAi) led to significant advancements in pesticide development and have generated considerable optimism in the field of plant protection. However, the broad application of RNAi-based approaches in complex ecological settings is hindered by significant challenges, including low environmental stability and high susceptibility to nuclease degradation. In our study, we found that d -ribose can prevent RNase A from degrading dsRNA. Inspired by this finding, we incorporated chemically modified nucleotides, 2’- O -methyl nucleoside triphosphates (2’-OMe-NTPs), to synthesize dsRNA. The results indicated that the stability of 2’-OMe-dsRNA was significantly enhanced under different conditions such as elevated temperatures, ultraviolet radiation, RNase A treatment, and the homogenate of the cotton bollworms midgut. RNAi efficiency experiments on cotton aphids showed that using 2’-OMe-dsRNA to synthesize two target genes, 2’-OMe-dsHem and 2’-OMe-dsCYP6DA2, significantly increased gene silencing efficiency of cotton aphids by more than 20%, correspondingly, the survival rates decreased by 16.7% and 18.9%, respectively. Further analysis of molecular mechanisms indicates that 2’-OMe-dsRNA induces higher levels of apoptosis and gene silencing by activating the tumor necrosis factor (TNF) pathway and upregulating Argonaute ( AGO2 ) gene expression. These results indicate that incorporating modified nucleotides, specifically 2’-OMe-NTPs, during the synthesis of dsRNA can enhance the stability of RNAi biopesticides under challenging conditions, thereby broadening their range of applications.