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Miscellaneous

Hammerhead ribozymes for target validation

Pages 235-247 | Published online: 25 Feb 2005

Bibliography

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  • ••Discriminates between rRNAs that differ atonly a single position within the cleavage site. Suppression of the mutant oncogene had the desired effect on the neoplastic phenotype showing the importance of this protein in transformation.
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  • •Discrimination between rRNA targets that differ by only a single nucleotide is possible even when the difference lies outside the cleavage site.
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  • ••Determines the tolerance for mismatchesbetween the hammerhead and its substrate and shows that the ability of the hammerhead to discriminate decreases if it forms more than about 12 base pairs with the substrate.
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  • ••Uses ribonuclease H and antisenseoligonucleotides to compare the accessibility of different GUC sites. This does not correlate with predictions of substrate secondary structure. Two - four mismatches are needed in the stems to prevent cleavage. Ribozymes seem to work better in cells than in vitro.
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  • •Using different promoters and flanking sequences, hammerheads can be localised within the nucleus or transported into the cytoplasm. Greater activity in the cytoplasm suggests better accessibility of the RNA target.
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  • ••Injection of anti-K-ras hammerheadadenoviral vectors into mice can give complete regression of some pre-existing tumours.
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  • •A heat shock inducible promoter was used to switch on hammerhead expression at a specific stage of development in transgenic fruit fly embryos.
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  • ••Modification of the ribozyme increases itshalf-life 14,000 times in serum. Injection of liposome-bound hammerhead into tumours suppresses growth and sheds light on the function of PKC-a.
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  • ••In vivo validation of a target for restenosisusing a synthetic hammerhead. The crippled control ribozyme with a single change in the catalytic core was inactive.
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  • •Modified hammerheads survive up to 7 days in the synovium and are taken up into cells. This targets a protease involved in tissue damage caused by arthritis.
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  • •In vivo validation of a target for restenosis using a synthetic hammerhead.
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