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

Citation
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
Citations number
46
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
28
Year of publication
2000
Pages
8201 - 8207
Database
ISI
SICI code
0006-2960(20000718)39:28<8201:IOPSOT>2.0.ZU;2-R
Abstract
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 .