Thermodynamically Stable RNA three-way junctions as platform for constructing multi-functional nanoparticles for delivery of therapeutics

RNA nanoparticles can potentially be used to treat cancers and viral infection but their instability has hindered their therapeutic applications. The lack of covalent linkage or cross-linking in the nanoparticles causes dissociation in vivo. Here we show the assembly of thermodynamically stable three-way junction (3WJ) of the motor pRNA of bacteriophage phi29 from 3–6 pieces of RNA oligomers without the use of metal salts to form stable multifunctional nanoparticles. Each RNA oligomer contains either a receptor-binding ligand, aptamer, siRNA or ribozyme functional module. When mixed together, they self-assemble into tri-stars nanoparticles with a 3WJ core. The nanoparticles are resistant to 8M urea denaturation, stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting the 3WJ core can be used as a platform for building a variety of multifunctional nanoparticles. Of the 25 different 3WJ motifs evaluated, only one other motif in biological RNA shares similar characteristics as the phi29 3WJ.