The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg under conditions that mimic the cytoplasm. We determined Mg binding constants for the top 15 metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics conditions, which we term "Eco80". We empirically determined that the mixture of metabolites in Eco80 approximated single-site binding behavior toward Mg in the biologically relevant free Mg range of ∼0.5 to 3 mM Mg, using a Mg-sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg between weak and strong Mg-metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function and enhances the biological relevance of mechanistic studies of RNA.