| Aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] confers resistance to several clinically important aminoglycoside antibiotics. One strategy to preserve the effectiveness of existing aminoglycosides involves using antisense oligodeoxynucleotides (ODNs) to inhibit expression of AAC(6')-Ib. The purpose of this research is to identify antisense ODN target sites within aac(6')-Ib mRNA. The structure of the mRNA was characterized using a combination of mapping with specific ribonucleases and computer generated secondary structure models. Several single stranded regions were predicted, but only a few were available for hybridization with ODNs. This information was used to design antisense 20-mer and 30-mer ODNs complementary to each accessible site. In vitro RNase H cleavage assays, ODN-RNA binding experiments, and in vitro transcription/translation were conducted to determine the effect of antisense ODNs on the expression of aac(6 ')-Ib. The results indicate that antisense ODNs interfered with the expression of aac(6 ')-Ib in vitro through RNase H-dependent degradation of the mRNA and physically preventing translation. |
