HAMMERHEAD RIBOZYMES TARGETED TO THE FBN1 MESSENGER-RNA CAN DISCRIMINATE A SINGLE-BASE MISMATCH BETWEEN RIBOZYME AND TARGET

Hammerhead ribozymes are catalytic RNA molecules that can act in trans , with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin- 1 (FBN1) cause Marfan syndrome. The majority of mutations are single-b ase changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead riboz yme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to t he extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutan t protein and thus restore normal fibrillin function. This might be ac hieved by the mutation creating a ribozyme cleavage site that is not p resent in the normal allele, however this is likely to limit the numbe r of mutations that could be targeted. Alternatively, it might be poss ible to target the mutant allele via the ribozyme binding arms. To det ermine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme c leavage efficiency both in vitro and in vivo. The discrimination betwe en fully-matched and mismatched ribozyme varied with the length of hel ix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to desi gn hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucle otide, even if the mutation does not create a ribozyme cleavage site. (C) 1998 Academic Press.