Journal Article

Extending the cleavage rules for the hammerhead ribozyme: Mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17

János Ludwig, Martina Blaschke and Brian S. Sproat

in Nucleic Acids Research

Volume 26, issue 10, pages 2279-2285
Published in print May 1998 | ISSN: 0305-1048
Published online May 1998 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/26.10.2279
Extending the cleavage rules for the hammerhead ribozyme: Mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17

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In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17 to N16.2C16.1H17 (H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17 to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1 ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15.1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2′-O-alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2.

Journal Article.  4175 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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