Phosphorothioate substitution can substantially alter RNA conformation

Phosphorothioate substitution-interference experiments, routinely used to s tereospecifically identify phosphoryl oxygen sites that participate in RNA- ligand binding and RNA-directed catalysis, rest in their interpretation on the untested assumption that substitution does not alter the conformation o f the modified molecule from its biologically active state. Using NMR spect roscopy, we have tested this assumption by determining the structural effec t of stereospecific phosphorothioate substitution at five positions in an R NA hairpin containing the binding site for bacteriophage MS2 capsid protein . At most sites, substitution has little or no effect, causing minor pertur bations in the phosphate backbone and increasing the stacking among nucleot ides in the hairpin loop. At one sire, however, phosphorothioate substituti on causes an unpaired adenine necessary for formation of the capsid protein -RNA complex to loop out of the RNA helix into the major groove. These resu lts indicate that phosphorothioate substitution can substantially alter the conformation of RNA at positions of irregular secondary structure, complic ating the use of substitution-interference experiments to study RNA structu re and function.