Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.