Methods for construction and characterization of simple or special multifunctional RNA nanoparticles based on the 3WJ of phi29 DNA packaging motor.

The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNA nanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications have been extensively explored. Now it is time to offer different levels of RNA construction methods for both the beginners and the experienced researchers or enterprisers. The first and second parts of this article will provide instructions on basic and simple methods for the assembly and characterization of RNA nanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA packaging motor. The third part of this article will focus on specific methods for the construction of more sophisticated multivalent RNA nanoparticles for therapeutic applications. In these parts, some simple protocols are provided to facilitate the initiation of the RNA nanoparticle construction in labs new to the field of RNA nanotechnology. This article is intended to serve as a general reference aimed at both apprentices and senior scientists for their future design, construction and characterization of RNA nanoparticles based on the pRNA-3WJ of phi29 DNA packaging motor.