Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

(A) Divalent metal-ion midpoint ratios of P4–P6 variants folded in either Mg²⁺ or in Mn²⁺ in a background of 2 M NaCl (OS: outer-sphere metal-ion ligand; IS: inner-sphere metal-ion ligand). (B) Calculated ΔΔG values (relative to unmodified P4–P6) associated with Mg²⁺-induced (left) and Mn²⁺-induced (right) folding of P4–P6 phosphorothioate variants with respect to unmodified P4–P6. The values were calculated according to a thermodynamic framework in which two site-bound metal ions, derived from solution or from the ion atmosphere, bind within the metal-ion core (Model 1, Supplemental Material). (C) Calculated ΔΔΔG values associated with Mg²⁺- vs. Mn²⁺-induced folding of phosphorothioate-subsituted P4–P6 variants in a background of 2 M NaCl.