Ribogreen Fluorescent Assay Kinetics to Measure Ribonucleic Acid Loading into Lipid Nanoparticle Carriers

New generations of vaccines have been developed by encapsulating messenger ribonucleic acid (mRNA) in lipid nanoparticle (LNP) carriers. In addition to the physicochemical properties of LNPs, the encapsulation efficiency (EE) of mRNA in LNPs is a key factor to screen vaccine assembly assays. Fluorescent dyes with amplified signals upon binding with mRNA are at the core of developing assays to quantify EE. However, disregarding the temporal effects during the assay impacts the accuracy of the assay. Here, the kinetics of temporal decay in fluorescence intensity of dye-RNA complex-in Ribogreen assay-are reported and shown how this dynamic process can be impeded in the presence of a nonionic surfactant. Further, the impact of this dynamic process on the calculated EE is studied. The corrections needed to accurately assay dynamic mRNA loading processes are presented. Ribogreen fluorescence assay is widely used to quantify the amount of messenger ribonucleic acid (mRNA) encapsulated in lipid nanoparticles (LNPs). However, the kinetics of fluorophore binding with mRNA in the presence of lipid components and surfactants are complex. Here, the time-dependency of the Ribogreen assay on surfactant and lipid additions is discussed. image