title: Protein-dependent ribozymes report molecular interactions in real time.
abstract: Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.
authors.affiliation: Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
authors.forename: Jörg S
authors.initials: JS
authors.lastname: Hartig
authors.forename: S Hani
authors.initials: SH
authors.lastname: Najafi-Shoushtari
authors.forename: Imke
authors.initials: I
authors.lastname: Grüne
authors.forename: Amy
authors.initials: A
authors.lastname: Yan
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
authors.forename: Michael
authors.initials: M
authors.lastname: Famulok
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D015694:Gene Products, rev / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004277:DNA, Single-Stranded; D015694:Gene Products, rev; D011506:Proteins; D016337:RNA, Catalytic