Using phosphorothioate-substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex

Part of the binding affinity and specificity in RNA-protein complexes is of ten contributed by contacts between the protein and backbone phosphates tha t 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 sm all RNA hairpin. Using a short oligoribonucleotide which contains all the n ecessary sequence elements required for tight protein binding, a single pho sphorothioate linkage was introduced at 13 different positions. In each cas e, the lip and Sp 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 introd uction of a phosphorothioate only affects binding at sites where a protein- phosphate contact is observed in the crystal structure. This means that pho sphorothioate-containing oligoribonucleotides should also be useful for map ping phosphate contacts in RNA-protein complexes for which no crystal struc ture is available.