Consequences of mRNA Secondary Structure on Stability against both Hydrolysis and Aggregation: The Role of Electrostatic, π–π Stacking, and Thermal Effects

The seemingly unrelated massive aggregation of free mRNA under certain solution conditions and the well-known autohydrolysis of mRNA are actually both closely linked through its secondary and possibly tertiary structure (s/t). This hypothesis posits that s/t partially stabilizes mRNA against both autohydrolysis and massive aggregation. Destabilization of s/t via denaturant guanidine-HCl (Gd), or temperature, has profound effects on both aggregation rates and final degree of autohydrolysis. These denaturant effects occurred for a variety of mRNA, ranging from 700 to 3000 nucleotides but showed very different quantitative behavior among themselves, suggesting some of the methods presented here might help characterize mRNA stability and robustness. Light scattering monitoring during dialysis of mRNA against Gd revealed an "aggregation window", over 0.5-3 M Gd, whereas dialyzing against a nondenaturing electrolyte (NaCl) showed semireversible monotonic increase of aggregation up to 4 M. Massive aggregation of mRNA in solutions with monovalent ions and in denaturing solutions has not been previously reported. A phenomenological model involving intermolecular electrostatic repulsion and attractions due chiefly to π-π stacking helps interpret the various phenomena.