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Encyclopedia of Physical Science and Technology EN014F-661 July 28, 2001 20:35

Ribozymes 259

A. Generalized hammerhead ribozyme

Cleavage site

I I III
III
5’NNNNNUHNNNNN3’
5’NNNNNUHNNNNN3’
| | | | | | | | | | |
| | | | | | | | | | |
3’NNNNNA NNNNN5’
3’NNNNNA NNNNN5’
A C C U U G G A A
A
A A N N
G G A AG G
--P
Y Y--P
N--N
N--N
N--N II II
N--N
N--N
N--N
N N N N
N N N N

FIGURE 2 Generalized hammerhead ribozyme. The ribozyme catalytic core is flanked by stems I, II and III. Cleavage
is directed after the H (A, C or U). The N’s represent any nucleotide, Y = pyrimidine, and P = purine. Other bases are
A = adenosine, C = cytosine, G = guanosine, and U = uracil.

Ribozyme mediated target validation can also be used phenotype as a gene knockout. Localized retroviral deliv-
to identify specific member(s) of a protein family involved ery of the same ribozyme later in development allowed
in a specific phenotype. Carefully designed ribozymes can dissection of the neuregulin biochemical pathway.
selectively knockdown expression of each protein in a
gene family. F. Ribozyme Mediated RNA Repair
A novel therapeutic application of ribozymes exploits the
E. Transgenic Animals Expressing Ribozymes
trans-splicing activity of the Tetrahymena ribozyme. This
Therapeutics and target validation studies will certainly ribozyme has been used to repair defective mRNAs by
be tested in animals. Ribozymes have been used in trans- trans-splicing onto these RNAs a functional sequence.
genic mice to create disease models such as diabetes by These ribozymes are designed to bind and cleave the target
selectively downregulating the hexokinase mRNA in pan- RNAs 5 of the undesired mutation. Since the ribozyme in

creatic islets. In this case, the ribozyme expression was this case is an intron, it is engineered to carry with it the
under the control of the insulin promoter, and was there- correct RNA sequence as the 3 -exon. Following cleavage

fore only expressed in the pancreatic beta cells. Retroviral of the mutant target RNA, the ribozyme catalyzes ligation
delivery of ribozymes targeted against neuregulin-1 in a of wild-type sequence onto the cleaved transcript. This
chick blastoderm resulted in the same embryonic lethal was successfully demonstrated with the correction of a