T. Kozu et al., Ribonuclease H attack of leukaemic fused transcripts AML1-MTG8 (ETO) by DNA/RNA chimeric hammerhead ribozymes, GENES CELLS, 5(8), 2000, pp. 637-647
Background: Catalytic anti-sense oligonucleotides might be useful tools for
controlling specific gene expression. However, to obtain effective oligonu
cleotides of the desired function in vivo is still a difficult task.
Results: To evaluate time usefulness of synthesized DNA/RNA hammerhead ribo
zymes targeting AML1-MTG8 (ETO) leukaemic fusion transcripts in vivo, we an
alysed their effects on cell growth and the mechanism of action using isola
ted cell nuclei. These ribozymes inhibited the growth of leukaemic cell lin
es expressing the AML1-MTG8 and degraded AML1-MTG8 mRNA in isolated nuclei
of these cells. However, the reactions gave rise to additional cleavage pro
ducts. Systematic cleavage analyses using an anti-sense oligonucleotide arr
ay revealed that the cleavage was induced by endogenous RNase H at specific
sites, in accordance with their calculated melting temperature (T-m) value
s. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme h
ad a ribozyme catalytic activity. In addition, the ribozymes and anti-sense
oligonucleotides suppressed the AML1-MTG8 protein in the leukaemic cells.
Conclusions: The DNA/RNA ribozymes inhibited cell growth primarily via anti
-sense effects, the main role of which was the activation of RNase H-digest
ion by their DNA arms. In addition, the isolated nuclei provided a useful a
ssay system for modelling in vivo conditions for the quantitative evaluatio
n of anti-sense/ribozyme activity.