We designed an octahedron shaped RNA structure. Each side of the structure consists of a 27 base pair long double stranded region and a 4 nucleotide single stranded loop at the edges. In five edges a staple strand was nicked and two nucleotides removed in order to create the 2 nt 3' overhangs recognized by the Dicer enzyme. The idea was that the octahedron should be able to contain a high molecular weight drug as well as being degraded by Dicer into siRNA. Polyacrylamide gel electrophoresis (PAGE) confirms that a complex is formed when the staple strands are added to the scaffold. Furthermore Förster resonance energy transfer (FRET) and small angle x-ray scattering (SAXS) together confirm that the structure does indeed form as predicted.
In Vitro Dicer experiments show that a structure designed with 3' overhangs has way higher affinity for the Dicer enzyme than a structure with no overhangs, leading us to conclude that the structure interacts with Dicer as intended. Furthermore the dual-luciferase assay confirms that the structure will indeed cause statistically significant gene knockdown in human cancer cells expressing renilla luciferase.