Assessment of Intracellular Compartmentalization of RNA Nanostructures.

Nucleic acid nanoparticles (NANPs) are extensively investigated as diagnostic and therapeutic tools. These innovative particles can be composed of RNA, DNA, and/or modified nucleic acids. Due to the regulatory role of nucleic acids in the cellular system, NANPs have the ability to identify target molecules and regulate expression of genes in disease pathways. However, translation of NANPs in clinical settings is hindered due to inefficient intracellular delivery, chemical instability, and off-target immunostimulatory effects following immune recognition. The composition of nucleic acids forming NANPs has been demonstrated to influence immunorecognition, subcellular compartmentalization, and physicochemical properties of NANPs. This chapter first outlines the methods used to generate a panel of NANPs with a uniform shape, size, charge, sequence, and connectivity. This includes the procedures for replacing the RNA strands with DNA or chemical analogs in the designated NANPs. Second, this chapter will also describe experiments to assess the effect of the chemical modification on enzymatic and thermodynamic stability, delivery efficiency, and subcellular compartmentalization of NANPs.