Quantifying Mg ²⁺ dependence on conformational equilibrium in the two-state 7SK RNA stem-loop 3

titration experiments, expanding the utility of chemical probing for detecting noncanonical base pairs. Our findings demonstrate how modest differences in cation affinity between conformational states can modulate RNA population ensembles. More broadly, this work establishes a generalizable thermodynamic framework to quantitatively dissect ion-dependent conformational equilibria in structurally heterogeneous RNAs and advances structural understanding of the essential SL3 regulatory element in the 7SK RNP.