Formation of a catalytically active dimer by tRNAVal-driven short ribozymes

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.