Impact of phosphorothioate substitutions on the thermodynamic stability of an RNA GAAA tetraloop: An unexpected stabilization

This study analyzes the impact of phosphorothioate substitutions on the thermodynamic stability of a 12-nt RNA hairpin containing a 5'GAAA3' tetraloop. The thermodynamic consequences of stereospecific phosphorothioate substitutions 5' to each adenosine in the loop region are measured using optical melting and calorimetry experiments. Surprisingly, a single stereospecific phosphorothioate substitution 5' to the second adenosine of the tetraloop, R-p-A7, results in a stabilization corresponding to a Delta(Delta G(37 degrees C)) Of approximately -2.9 kcal mol(-1) (0.1 M NaCl) when compared with that of an unmodified sample. Five other phosphorothioate-substituted samples did not show significant thermodynamic differences in comparison with the unsubstituted samples. Addition of Mg2+ to all of the hairpins studied results in increased t(m)'s that are fit with a general electrostatic model to a dissociation constant of K-d(Mg2+) similar to 2-3 mM (0.1 M NaCl), The R-p-A7 phosphorothioate-substituted hairpin showed an unusual decrease in t(m) and apparent increase in enthalpy of unfolding upon addition of Cd2+. These results may impact the interpretation of interference mapping experiments that use phosphorothioate substitutions to characterize RNAs in solution.