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')GAAA(3') 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(DeltaG(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 Mg(2+) 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)(Mg(2+)) approximately 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 Cd(2+). These results may impact the interpretation of interference mapping experiments that use phosphorothioate substitutions to characterize RNAs in solution.
Horton
T E
TE
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
Maderia
M
M
DeRose
V J
VJ
D002104:Cadmium / Q000737:chemistry; D002151:Calorimetry; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D020871:RNA Stability; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry
D013873:Thionucleotides; D002104:Cadmium; D012313:RNA; D008274:Magnesium