Analogs of naturally occurring substances obtained by chemical modifications are powerful tools to study intra- and intermolecular interactions. We have used the phosphorothioate technique to analyze RNA-protein interactions, here the interactions of transfer RNAs (tRNAs) with the three ribosomal binding sites. We describe preparation and purification of thioated tRNAs, formation of functional complexes of programmed ribosomes with tRNAs, and the evaluation of the observed phosphorothioate footprints on the tRNAs.