Te. Horton et al., Impact of phosphorothioate substitutions on the thermodynamic stability ofan RNA GAAA tetraloop: An unexpected stabilization, BIOCHEM, 39(28), 2000, pp. 8201-8207
This study analyzes the impact of phosphorothioate substitutions on the the
rmodynamic stability of a 12-nt RNA hairpin containing a 5'GAAA3' tetraloop
. The thermodynamic consequences of stereospecific phosphorothioate substit
utions 5' to each adenosine in the loop region are measured using optical m
elting and calorimetry experiments. Surprisingly, a single stereospecific p
hosphorothioate substitution 5' to the second adenosine of the tetraloop, R
-p-A7, results in a stabilization corresponding to a Delta(Delta G(37 degre
es C)) Of approximately -2.9 kcal mol(-1) (0.1 M NaCl) when compared with t
hat of an unmodified sample. Five other phosphorothioate-substituted sample
s did not show significant thermodynamic differences in comparison with the
unsubstituted samples. Addition of Mg2+ to all of the hairpins studied res
ults 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+. Th
ese results may impact the interpretation of interference mapping experimen
ts that use phosphorothioate substitutions to characterize RNAs in solution
.