Self-assembling RNA Nanoparticle for Gene Expression Regulation in a Model System.

In the search for enzymatically processed RNA fragment, we found the novel three-way junction motif. The structure prediction suggested the arrangement of helixes at acute angle ~ 60o. This allows the design of trimeric RNA nanoparticle that can be functionalized with multiple regulatory fragments. Such RNA nano-object of equilateral triangular shape was applied for gene expression regulation studies in two independent cellular systems. Biochemical and functional studies confirmed predicted shape and structure of the nanoparticle. The regulatory siRNA fragments incorpo-rated into the nanoparticle were effectively released and triggered gene silencing. The regulatory effect was pro-longed when induced with structuralized RNA compared to unstructured siRNAs. In these studies, the enzymatic processing of the motif was utilized for function release from the nanoparticle, enabling simultaneous delivery of different regulatory functions. This methodology of sequence search, RNA structural prediction and applica-tion for rational design opens a new way for creating enzymatically processed RNA nanoparticles.