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Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.

Hammann, Christian and Martinez, E. and Moosbauer, J. and Hormes, R. and Tabler, M. (1999):
Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.
In: Antisense & nucleic acid drug development, pp. 25-31, 9, (1), ISSN 1087-2906,
[Article]

Abstract

The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.

Item Type: Article
Erschienen: 1999
Creators: Hammann, Christian and Martinez, E. and Moosbauer, J. and Hormes, R. and Tabler, M.
Title: Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.
Language: English
Abstract:

The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.

Journal or Publication Title: Antisense & nucleic acid drug development
Volume: 9
Number: 1
Divisions: 10 Department of Biology
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10 Department of Biology > Ribogenetics
Date Deposited: 29 Jul 2010 12:09
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