The aim of the present study was to identify functional antisense
oligodeoxynucleotides (ODNs) against the rat glutathione
S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2.
These antisense ODNs would enable the study of the physiological
consequences of GSTM deficiency. Because it has been suggested
that the effectiveness of antisense ODNs is dependent on the
secondary mRNA structures of their target sites, we made mRNA
secondary structure predictions with two software packages,
Mfold and STAR. The two programs produced only marginally similar
structures, which can probably be attributed to differences
in the algorithms used. The effectiveness of a set of 18 antisense
ODNs was evaluated with a cell-free transcription/translation
assay, and their activity was correlated with the predicted
secondary RNA structures. Four phosphodiester ODNs specific
for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted
at both GSTM isoforms were found to be potent, sequence-specific,
and RNase H-dependent inhibitors of protein expression. The
IC50 value of the most potent ODN was approximately
100 nM. Antisense ODNs targeted against regions that were predicted
by STAR to be predominantly single stranded were more potent
than antisense ODNs against double-stranded regions. Such a
correlation was not found for the Mfold prediction. Our data
suggest that simulation of the local folding of RNA facilitates
the discovery of potent antisense sequences. In conclusion,
we selected several promising antisense sequences, which, when
synthesized as biologically stable oligonucleotides, can be
applied for study of the physiological impact of reduced GSTM
expression.