RNA Nanostructures

Kheiria Benkato, Benjamin O’Brien, My N. Bui, Daniel L. Jasinski, Peixuan Guo, Emil F. Khisamutdinov

Temperature gradient gel electrophoresis (TGGE) is a powerful tool used to analyze the thermal stabilities of nucleic acids. While TGGE is a decades-old technique, it has recently gained favor in the field of RNA nanotechnology, notably in assessing the thermal stabilities of RNA nanoparticles (NPs). With TGGE, an electrical current and a linear temperature gradient are applied simultaneously to NP-loaded polyacrylamide gel, separating the negatively charged NPs based on their thermal behavior (a more stable RNA complex will remain intact through higher temperature ranges). The linear temperature gradient can be set either perpendicular or parallel to the electrical current, as either will make the NPs undergo a transition from native to denatured conformations. Often, the melting transition is influenced by sequence variations, secondary/tertiary structures, concentrations, and external factors such as the presence of a denaturing agent (e.g., urea), the presence of monovalent or divalent metal ions, and the pH of the solvent. In this chapter, we describe the experimental setup and the analysis of the thermal stability of RNA NPs in native conditions using a modified version of a commercially available TGGE system.