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J. Biol. Chem., Vol. 279, Issue 31, 32063-32070, July 30, 2004

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Engineered RNase P Ribozymes Increase Their Cleavage Activities and Efficacies in Inhibiting Viral Gene Expression in Cells by Enhancing the Rate of Cleavage and Binding of the Target mRNA^*

Hua Zou, Jarone Lee, Ahmed F. Kilani, Kihoon Kim, Phong Trang, Joseph Kim, and Fenyong Liu¶

From the Program in Infectious Diseases and Immunity, Program in Comparative, Biochemistry, School of Public Health, University of California, Berkeley, California 94720

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G⁹⁵ U⁹⁵) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A²⁰⁰ C²⁰⁰) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.

Received for publication, March 19, 2004 , and in revised form, May 26, 2004.

^* The research has been supported in part by grants from the March of Dimes Birth Defects Foundation and the American Heart Association and by the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Block predoctoral grant (UC-Berkeley Graduate Division).

Recipient of a predoctoral fellowship from the American Heart Association (Western States Affiliate).

^¶ A Scholar of Leukemia and Lymphoma Society and an Established Investigator of American Heart Association. To whom correspondence should be addressed: School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720. Tel.: 510-643-2436; Fax: 510-643-9955; E-mail: liu_fy{at}uclink4.berkeley.edu.

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