title: Fluorescent RNA labeling using self-alkylating ribozymes.
abstract: The ability to fluorescently label specific RNA sequences is of significant utility for both in vitro and live cell applications. Currently, most RNA labeling methods utilize RNA-nucleic acid or RNA-protein molecular recognition. However, in the search for improved RNA labeling methods, harnessing the small-molecule recognition capabilities of RNA is rapidly emerging as a promising alternative. Along these lines, we propose a novel strategy in which a ribozyme acts to promote self-alkylation with a fluorophore, providing a robust, covalent linkage between the RNA and the fluorophore. Here we describe the selection and characterization of ribozymes that promote self-labeling with fluorescein iodoacetamide (FIA). Kinetic studies reveal a second-order rate constant that is on par with those of other reactions used for biomolecular labeling. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with RNA.
authors.affiliation: Department of Chemistry and the Center for Cell and Genome Science and ‡Department of Biology and the Center for Cell and Genome Science, University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: Ashwani K
authors.initials: AK
authors.lastname: Sharma
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Plant
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Rangel
authors.forename: Kirsten N
authors.initials: KN
authors.lastname: Meek
authors.forename: April J
authors.initials: AJ
authors.lastname: Anamisis
authors.forename: Julie
authors.initials: J
authors.lastname: Hollien
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Heemstra
mesh_terms: D000478:Alkylation; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D012313:RNA