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Ribozymes 261

has been used to create RNA-cleaving Rzs with smaller ually improving. These molecules must retain their cat-
catalytic domains, DNA-cleaving ribozymes, and new cat- alytic potential, reach an accessible site on the substrate,
alytic motifs. Even RNA-cleaving DNAzymes have been and effectively impact on the steady-state levels of target
generated through in vitro evolution. These “evolved” molecules to be useful as either surrogate genetic tools or
enzymes exemplify the power of in vitro evolution and therapeutic agents. Great progress has been made in all of
will no doubt find many applications. these areas and should allow extensive use of the highly
specific reagents for down-regulating expression of target
RNAs.
VI. CONCLUSIONS

The transformation of ribozyme sequences from naturally SEE ALSO THE FOLLOWING ARTICLES
occurring, cis-cleaving molecules to target-specific, trans-
cleaving reagents has stimulated a great deal of interest
BIOMATERIALS,SYNTHESIS,FABRICATION, AND APPLI-
in their potential applications. Ribozymes targeting viral
CATIONS • CELL DEATH (APOPTOSIS) • GENE EXPRES-
genes are now in clinical evaluation; ribozymes targeting
SION,REGULATION OF • IMMUNOLOGY-AUTOIMMUNITY
cellular genes are moving into transgenic animals; and
• MAMMALIAN CELL CULTURE • TRANSLATION OF RNA
the use of ribozymes is expanding into RNA evolution,
TO PROTEIN
mRNA repair, and gene discovery.
For ribozymes to become generally useful surrogate
genetic tools and realistic therapeutic agents, several ob-
stacles need first to be overcome. These obstacles are the BIBLIOGRAPHY
efficient delivery to a high percentage of the cell popula-
tion, efficient expression of the ribozyme from a vector Castanotto, D., Rossi, J. J., and Deshler, J. O. (1992). “Biological and
Functional Aspects of Catalytic RNAs,” Crit. Rev. Eukaryot. Gene
or intracellular ribozyme concentration, colocalization of
Expr. 2, 331.
the ribozyme with the target, specificity of ribozyme for Cech, T. R. (1990). “Self-Splicing of Group I Introns,” Annu. Rev.
the desired mRNA, and an enhancement of ribozyme- Biochem. 59, 543.
mediated substrate turnover. Despite these reservations, Doherty, E. A., and Doudna, J. A. (2000). “Ribozyme Structures and
results with ribozymes so far look promising, particularly Mechanisms,” Annu. Rev. Biochem. 69, 597.
Fedor, M. J. (2000). “Structure and Function of the Hairpin Ribozyme,”
in the HIV-1 studies. As our knowledge of RNA struc-
J. Mol. Biol. 297, 269.
ture, secondary and tertiary, increases, we will be able to Hauswirth, W. W., and Lewin, A. S. (2000). “Ribozyme Uses in Retinal
target RNAs more rationally, which may help with the Gene Therapy,” Progr. Retin. Eye Res. 19, 689.
problems of specificity. At the same time, the understand- James, H. A. (2000). “Therapeutic Potential of Ribozymes in Haemato-
ing of the physical localization of RNA in cells and its logical Disorders,” Expert Opin. Investig. Drugs 9, 1009.
Kurz, J. C., and Fierke, C. A. (2000). “Ribonuclease P: A Ribonucleo-
tracking as it moves from the nucleus to cytoplasm will
protein Enzyme,” Curr. Opin. Chem. Biol. 4, 553.
also help in ensuring colocalization of the ribozyme and Rossi, J. J. (1999). “Ribozymes, Genomics and Therapeutics,” Chem.
target. Modifications of the ribozymes, for example, the Biol. 6, R33.

2 -ribose with various agents such as methyl, allyl, flu- Rossi, J. J. (2000). “Ribozyme Therapy for HIV Infection,” Adv. Drug.
oro, and amino groups, increases the stability to nucle- Deliv. Rev. 44, 71.
Szostak, J. W. (1997). “In Vitro Selection and Directed Evolution,” Har-
ases quite dramatically. Similarly, chimeric DNA–RNA
vey Lect. 93, 95.
ribozymes increase the stability. The efficiency of deliv- Watanabe, T., and Sullenger, B. A. (2000). “RNA Repair: A Novel Ap-
ery to cells with viral vectors or liposomes is also contin- proach to Gene Therapy,” Adv. Drug Deliv. Rev. 44, 109.