The ability to generate RNA molecules that can catalyze complex organic transformations not only facilitates the reconstruction and plausibility of possible prebiotic reaction pathways but is also crucial for elucidating the potential of the application of RNA catalysts in organic syntheses. Iterative RNA selection previously identified a ribozyme that catalyzes the Michael addition of a cysteine thiol to an alpha,beta-unsaturated amide. This reaction is chemically similar to the rate limiting step of the thymidylate synthase reaction, which is the corresponding reaction of a cysteine thiol to the double-bond of the uracil nucleobase. Here we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its characterization and the investigation of the background reaction. We also describe the further characterization of the ribozyme with respect to substrate specificity. We show that the thiol group of the cysteine nucleophile is essential for the reaction to proceed. When substituted for a thiomethyl group, no reaction takes place.