Synthetic antisense oligonucleotide probes the essentiality of metallothionein gene

The metallothionein II gene, whose structure is highly conserved throughout the animal kingdom, is composed of three exons and two introns. Synthetic antisense oligonucleotides (ODN), with sequence complementary to the messenger RNA coding for human metallothionein II, were prepared and tested for their ability to inhibit both constitutive- and cadmium-induced metallothionein protein synthesis in human Chang liver cells and hamster lung V79 cells in culture. The property of sense ODN was also examined as a measure of sequence specificity. The antisense inhibition of metallothionein protein synthesis rendered all cell lines more susceptible to the toxic effects of cadmium. However, the sense ODN had no effects on either metallothionein protein synthesis or sensitivity to cadmium. Phosphorothioate oligonucleotides were more potent in enhancing the toxicity of cadmium than phosphodiester oligonucleotides. The antisense oligonucleotides targeted to the splice donor region between exon 1 and intron 1 was significantly more effective as an inhibitor than those targeted to either the 5' end of the mRNA or within exon 3. Therefore, not all antisense oligonucleotide sequences are equally efficient as inhibitors of metallothionein synthesis. Therefore, the delineation of an obvious mechanism for predicting the most potent sequence is not apparent at this time. The results of this study are interpreted to indicate that (a) metallothionein is endowed with an essential gene, (b) modifications in oligonucleotide structures show specificity in inhibiting metallothionein synthesis, and (c) antisense inhibition of metallothionein gene expression may provide a useful tool in studying the action and perhaps the function(s) of metallothionein.