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 receptor mRNA were targeted with 2'-deoxyribonucleotide antisense oligonucleotides (ONs). The uptake of 1 microM concentration of these ONs into Chinese hamster ovary cells was facilitated by the use of cationic liposomes. The antisense sequences reduced binding of 125I-angiotensin II by 57-73%, while mismatch ONs and reverse sequence ONs produced little reduction in receptor binding. These reductions in AT1 receptor binding were accompanied by comparable decreases in AT1 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-type enzyme. When 2'-methoxyribonucleotide analogs of these same sequences were tested, AT1 receptor mRNA levels were unchanged even though small reductions in AngII binding were observed. Antisense effects seen with these 2'-methoxyribonucleotide sequences may have arisen through a translational arrest mechanism. Direct comparisons between 2'-deoxyribonucleotide analogs and their 2'-methoxyribonucleotide counterparts show that antisense effects are significantly larger when they are mediated through an RNase H-type mechanism. 2'-methoxyribonucleotide sequences were most effective when they were directed against the translation initiation codon.
Ho
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SP
CNS Department, DuPont Pharmaceuticals Research Laboratories, PO Box 80400 Experimental Station, Wilmington, DE 19880-0400, USA. siew.p.ho@dupontpharma.com
Bao
Y
Y
Lesher
T
T
Conklin
D
D
Sharp
D
D
D000803:Angiotensin I / Q000378:metabolism*; D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics*; D015152:Blotting, Northern; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology; D006224:Cricetinae; D005434:Flow Cytometry; D019793:Fluorescein; D005786:Gene Expression Regulation / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D014162:Transfection
D016375:Antisense Elements (Genetics); D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D000803:Angiotensin I; D016914:Ribonuclease H; D019793:Fluorescein