Factors affecting stability of RNA – temperature, length, concentration, pH, and buffering species

RNA is prone to both chemical degradation and/or physical instability. Some of the factors affecting stability of RNA in solution are its length, 3’ poly A tail and 5’ cap integrity, excipients, buffering species, pH of the solution, nucleases, and divalent cations. In this work, we showed the effect of temperature, messenger RNA (mRNA) length, buffering species, pH of the solution, and the concentration of mRNA on its chemical and physical stability. Our thermodynamic analysis of a 4000 nucleotide-long mRNA measured an activation energy of 31.5 kcal/mol normalized per phosphodiester backbone. We found mRNA length to be negatively correlated to its stability. Buffering species and pH of the solution affected mRNA integrity along with affecting the onset temperature of melting obtained by Differential Scanning Calorimetry (DSC) thermograms. It was also found that increasing the concentration of mRNA in solution increased its stability.