Antisense-Induced Fas mRNA Degradation Produces Site-Specific Stable 3'-mRNA Fragment by Exonuclease Cleavage at the Complementary Sequence

Abstract

Antisense-mediated degradation of target mRNA is achieved by the enzymatic action of nuclease RNase H. The enzyme recognizes hybrid RNA-DNA duplexes and hydrolyzes the RNA strand. Here, we compared six different phosphorothioate oligonucleotides for their ability to induce target-specific mRNA degradation in cultured mouse AML12 cells. We targeted transcripts of the cell surface receptor Fas and analyzed the levels of mRNA by Northern blotting and ribonuclease protection assay (RPA). Four of the tested antisense oligonucleotides reduced the mRNA levels significantly. Cultures treated with one of the antisense molecules resulted in a shifted band on Northern blots. This band of lower molecular weight was not detected after 6 hours of transfection but appeared at 24 hours. By RPA, the product was shown to be a 3'-cleavage fragment of the full-length Fas mRNA. The RPA also mapped the stable fragment to start within the antisense complementary sequence.