title: Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions.
abstract: In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Bella
authors.initials: B
authors.lastname: Katzman
authors.forename: Maria
authors.initials: M
authors.lastname: Vyazmensky
authors.forename: Olga
authors.initials: O
authors.lastname: Press
authors.forename: Micha
authors.initials: M
authors.lastname: Volokita
authors.forename: Stanislav
authors.initials: S
authors.lastname: Engel
mesh_terms: D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D016337:RNA, Catalytic; D011994:Recombinant Proteins; D019809:Streptavidin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
keywords: Class I RNA ligase; DNA-encoded compounds; Display selection; MDM2; Streptavidin