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

• Published in: Biochemistry (Easton) Vol. 39; no. 28; pp. 8201 - 8207
• Main Authors: Horton, Thomas E, Maderia, Melissa, DeRose, Victoria J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.07.2000
• Subjects: Cadmium - chemistry; Calorimetry; Magnesium - chemistry; Nucleic Acid Conformation; Phosphorothioate; RNA - chemistry; RNA Stability; Thermodynamics; Thionucleotides - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi000141d

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 Δ(ΔG 37 ° 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+) ∼ 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.