title: Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes.
abstract: The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.
authors.affiliation: Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
authors.forename: W A
authors.initials: WA
authors.lastname: Pieken
authors.forename: D B
authors.initials: DB
authors.lastname: Olsen
authors.forename: F
authors.initials: F
authors.lastname: Benseler
authors.forename: H
authors.initials: H
authors.lastname: Aurup
authors.forename: F
authors.initials: F
authors.lastname: Eckstein
mesh_terms: D001483:Base Sequence; D002712:Chlorides; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012265:Ribonucleotides; D013379:Substrate Specificity
chemical_list: D002712:Chlorides; D017895:Manganese Compounds; D016337:RNA, Catalytic; D012265:Ribonucleotides; D015636:Magnesium Chloride; D008345:Manganese; D012260:Ribonucleases; C025340:manganese chloride