title: A ribozyme with michaelase activity: synthesis of the substrate precursors.
abstract: 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.
authors.affiliation: Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Eisenführ
authors.forename: Paramjit S
authors.initials: PS
authors.lastname: Arora
authors.forename: Gerhard
authors.initials: G
authors.lastname: Sengle
authors.forename: Leo R
authors.initials: LR
authors.lastname: Takaoka
authors.forename: James S
authors.initials: JS
authors.lastname: Nowick
authors.forename: Michael
authors.initials: M
authors.lastname: Famulok
mesh_terms: D000577:Amides / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives; D003545:Cysteine / Q000737:chemistry; D015723:Gene Library; D006151:Guanosine / Q000031:analogs & derivatives; D007700:Kinetics; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000378:metabolism*; D010759:Phosphorus Isotopes; D010777:Photochemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013698:Templates, Genetic; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism
chemical_list: D000577:Amides; D009841:Oligonucleotides; D010759:Phosphorus Isotopes; D016337:RNA, Catalytic; D013438:Sulfhydryl Compounds; D006151:Guanosine; C032239:fumaramide; D001710:Biotin; D013940:Thymidylate Synthase; D003545:Cysteine