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Science 19 July 1991:
Vol. 253. no. 5017, pp. 314 - 317
DOI: 10.1126/science.1857967
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Articles

Science, Vol 253, Issue 5017, 314-317
Copyright © 1991 by American Association for the Advancement of Science

articles

Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes

WA Pieken, DB Olsen, F Benseler, H Aurup, and F Eckstein

Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen, Federal Republic of Germany.

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.


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