Design of RNAs That Inhibit the Activated c-Ha-ras Gene in Mammalian Cells MAKOTO KOIZUMI AND EIKO OHTSUKA Faculty of Pharmaceutical Sciences Hokkaido University Sapporo, 060, Japan To determine the structural bases in self-cleaving RNA domains, we synthesized oligoribonucleotides and their analogs to create a series of “hammerhead ribozymes.” We found several essential bases for the enzymatic activity of the catalytic domain and possible target sequences in the substrates.l On the basis of the foregoing information, ribozymes that cleave mRNA from mutated c-Ha-ras genes, which contain a single mutation at codon 12 (Y = G, normal c-Ha-ras mRNA; Y = U, activated c-Ha-ras mRNA), were designed (FIG.1). Experiments in vitro show that the ribozymes specifically cleaved the mutated mRNA.* We used NIH-3T3 cells to investigate the ability of ribozymes to cleave c-Ha-ras mRNA. Plasmids containing various ribozyme genes were constructed and expressed under the control of the long terminal repeats of Rous sarcoma virus. The ribozymes inhibited the formation of foci in transfected NIH-3T3 cells by approximately 50%. 5‘ 3‘

\ I

COG G-C COG G-C A * U C-Ha-faS mRNA

UACGAC

UGGUGUAG-

\ Riboryme

FIGURE 1. Secondary structure of the complex between a designed ribozyme and c-Hams mRNA. The consensus sequences are boxed and the cleavage site is indicated by an arrow.

3’

5‘

REFERENCES

1. KOIZUMI,M., S. IWAI8~E. OHTSUKA. 1988. FEBS Lett. 2 2 8 228-230. 2. KOIZUMI,M., Y. HAYASE,S. IWAI, H. KAMIYA, H. INOUE& E. OHTSUKA. 1989. Nucl. Acids Res. 17: 7059-7071. 276