Inhibition of c-Ha-ras gene expression by hammerhead ribozymes containing a stable C(UUCG)G hairpin loop.
Catalytic RNAs recognize specific sequences of RNA and cleave at a specific site. In this study, we designed hammerhead ribozymes with a thermodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage activities of these ribozymes were examined using a synthetic pentadecamer with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GGU-->GUU). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. When this ribozyme-encoding gene and the activated c-Ha-ras gene were cotransfected into NIH3T3 cells, morphologically normal cells were obtained. We also determined that the expression of the c-Ha-ras gene was inhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the regulation of specific gene expression in vivo.
Koizumi
M
M
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Kamiya
H
H
Ohtsuka
E
E
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D005786:Gene Expression Regulation; D011905:Genes, ras; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D009841:Oligonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger