tRNAVal-heterodimeric maxizymes with high potential as geneinactivating agents: simultaneous cleavage at two sites in HIV-1 Tat mRNA in cultured cells.
It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNAVal-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNAVal-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNAVal-driven maxizymes tested to date have been more effective than tRNAVal-driven "standard" hammerhead ribozymes, the tRNAVal-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.
National Institute for Advanced Interdisciplinary Research, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Tsukuba Science City 305-8566, Japan.
T
T
Kuwabara
M
M
Warashina
A
A
Nakayama
J
J
Ohkawa
K
K
Taira
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012366:RNA, Transfer, Val / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
D016337:RNA, Catalytic; D012366:RNA, Transfer, Val