Part of the binding affinity and specificity in RNA−protein complexes is often contributed by contacts between the protein and backbone phosphates that are held in position by the RNA structure. This study focuses on the well-characterized interaction between a dimer of the MS2 coat protein and a small RNA hairpin. Using a short oligoribonucleotide which contains all the necessary sequence elements required for tight protein binding, a single phosphorothioate linkage was introduced at 13 different positions. In each case, the R_P and S_P stereoisomers were separated and their affinities to the MS2 coat protein were determined. Comparison of these biochemical data with the crystal structure of the protein−hairpin complex indicates that introduction of a phosphorothioate only affects binding at sites where a protein−phosphate contact is observed in the crystal structure. This means that phosphorothioate-containing oligoribonucleotides should also be useful for mapping phosphate contacts in RNA−protein complexes for which no crystal structure is available.