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Impact of Phosphorothioate Substitutions on the Thermodynamic Stability of an RNA GAAA Tetraloop:  An Unexpected Stabilization

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journal contribution
posted on 23.06.2000, 00:00 by Thomas E. Horton, Melissa Maderia, Victoria J. DeRose
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, Rp-A7, results in a stabilization corresponding to a Δ(ΔG37°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 tm's that are fit with a general electrostatic model to a dissociation constant of Kd(Mg2+) ∼ 2−3 mM (0.1 M NaCl). The Rp-A7 phosphorothioate-substituted hairpin showed an unusual decrease in tm 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.