title: Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition.
abstract: We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.
authors.affiliation: Department of Chemistry, McGill University , 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada.
authors.forename: Adam
authors.initials: A
authors.lastname: Katolik
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , 7703 Floyd Curl Drive, San Antonio, Texas 78229, United States.
authors.forename: Nathaniel E
authors.initials: NE
authors.lastname: Clark
authors.affiliation: Department of Chemistry, McGill University , 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Tago
authors.affiliation: Departments of Biochemistry and Biomolecular Chemistry, University Of Wisconsin-Madison , 433 Babcock Drive, Madison, Wisconsin 53706, United States.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Montemayor
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , 7703 Floyd Curl Drive, San Antonio, Texas 78229, United States.|Department of Veterans Affairs, South Texas Veterans Health Care System , San Antonio, Texas 78229, United States.
authors.forename: P John
authors.initials: PJ
authors.lastname: Hart
authors.affiliation: Department of Chemistry, McGill University , 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada.
authors.forename: Masad J
authors.identifier: 0000-0002-4458-1623
authors.initials: MJ
authors.lastname: Damha
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D012313:RNA / Q000737:chemistry*; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D012313:RNA; D012316:RNA Nucleotidyltransferases