Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes.
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.
Pieken
W A
WA
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Olsen
D B
DB
Benseler
F
F
Aurup
H
H
Eckstein
F
F
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
D002712:Chlorides; D017895:Manganese Compounds; D016337:RNA, Catalytic; D012265:Ribonucleotides; D015636:Magnesium Chloride; D008345:Manganese; D012260:Ribonucleases; C025340:manganese chloride