Enhancement of synergistic gene silencing by RNA interference using branched "3-in-1" trimer siRNA

Nanostructured RNA carrying three different siRNAs was assembled to silence three target genes (Axin, APC, and GSK-3β) in the Wnt/β-catenin signaling pathway. The trimer RNA nanostructure included equimolar concentrations of three oligonucleotide sequences. The three armed structures and the size of the trimer RNA were confirmed by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. In the presence of 10% human serum, the trimer RNA was able to resist degradation and maintained an intact structure for more than two hours. Protein expression analyses showed specific repression of the target proteins by siRNAs. As a result, the expression of luciferase in a β-catenin reporter vector was significantly increased by the trimer RNA compared with a pool of the three individual siRNAs. This high activity at a low concentration was considered to be due to the 3-in-1 format of the trimer and the long-term resistance to serum proteins by nanostructure formation. We demonstrated that a nanostructured "3-in-1" siRNA is effective in enhancing the effect of RNA interference.