Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability

Small interfering RNAs (siRNAs) are potent therapeutic molecules, but despite recent progress, their administration in vivo remains challenging due to their low stability in the bloodstream. Thus, techniques for investigating the stability of siRNA are fundamental for the development of efficient siRNA delivery systems. We designed a simple FRET electrophoresis method to dynamically evaluate serum siRNA stability in parallel with its interaction with the serum components. Each strand of the siRNA was labeled with the fluorophore carboxyfluorescein (FAM) at the 5'-end and the quencher carboxytetramethylrhodamine (TAMRA) at the 3'-end. After incubation in serum, molecular stability was proportional to the FRET efficiency that could be quantified in-gel by ImageJ analysis. Compared to the usual gel-shift and other plate-based FRET assays, this method is more sensitive and allows investigation of the stability of serum siRNA and siRNA-based nanoparticles, as well as the extrapolation of degradation kinetics in parallel with interaction analysis.