Regulation of the angiotensin type-1 receptor by antisense oligonucleotides occurs through an RNase H-type mechanism

Multiple, diverse sites in the coding region of the angiotensin type-1 rece ptor mRNA were targeted with 2'-deoxyribonucleotide antisense oligonucleoti des (ONs). The uptake of 1 mu M concentration of these ONs into Chinese ham ster ovary cells was facilitated by the use of cationic liposomes. The anti sense sequences reduced binding of I-125-angiotensin II by 57-73%, while mi smatch ONs and reverse sequence ONs produced little reduction in receptor b inding. These reductions in AT(1) receptor binding were accompanied by comp arable decreases in AT(1) receptor mRNA levels. Furthermore, mRNA cleavage fragments corresponding in size to 3'-cleavage fragments were observed with two of the antisense ONs, consistent with the involvement of an RNase H-ty pe enzyme. When 2'-methoxyribonucleotide analogs of these same sequences we re tested, AT(1) receptor mRNA levels were unchanged even though small redu ctions in AngII binding were observed. Antisense effects seen with these 2' -methoxyribonucleotide sequences may have arisen through a translational ar rest mechanism. Direct comparisons between 2'-deoxyribonucleotide analogs a nd their 2'-methoxyribonucleotide counterparts show that antisense effects are significantly larger when they are mediated through an RNase H-type mec hanism. 2'-methoxyribonucleotide sequences were most effective when they we re directed against the translation initiation codon. (C) 1999 DuPont Pharm aceuticals Company. Published by Elsevier Science B.V.