TY - JOUR
T1 - Repression of human thymidylate synthase mRNA translation by antisense 2'-O-methyl oligoribonucleotides
AU - Schmitz, John C.
AU - Agrawal, Sudhir
AU - Chu, Edward
PY - 1998
Y1 - 1998
N2 - Previous studies have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end product TS in a negative autoregulatory manner. Disruption of this process results in increased synthesis of TS and may be associated with the development of cellular drug resistance to TS-directed anticancer agents. As one strategy to inhibit TS expression, we have designed antisense RNA oligoribonucleotides (ORNs) that directly target the 5'-upstream binding site (nt 80-109) of TS mRNA, a critical cis-acting regulatory element. ORNs were analyzed for their ability to specifically inhibit translation of human TS mRNA in an in vitro rabbit reticulocyte lysate translation system. Native 2'-hydroxyl(OH) ORNs inhibited TS mRNA translation in a dose-dependent manner but did not repress translation of control mRNAs, including p53 or Escherichia coli TS. A control sense 2'-OH ORN was unable to repress translation of either human TS mRNA, or control mRNAs. Modified antisense ORNs with 2'-O-methyl phosphodiester or 2'-O-methyl phosphorothioate backbones (or both) repressed human TS mRNA translation in a dose-dependent manner, and they were both more effective than the respective 2'-OH ORN. However, nonspecific effects on mRNA translation were observed with the 2'-O-methyl phosphorothioate ORN. In vitro translation experiments revealed that in the presence of antisense ORNs, the target TS mRNA remained intact. These findings demonstrate that antisense ORNs targeted at the 5'-upstream cis-acting element represent effective inhibitors of TS mRNA translation.
AB - Previous studies have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end product TS in a negative autoregulatory manner. Disruption of this process results in increased synthesis of TS and may be associated with the development of cellular drug resistance to TS-directed anticancer agents. As one strategy to inhibit TS expression, we have designed antisense RNA oligoribonucleotides (ORNs) that directly target the 5'-upstream binding site (nt 80-109) of TS mRNA, a critical cis-acting regulatory element. ORNs were analyzed for their ability to specifically inhibit translation of human TS mRNA in an in vitro rabbit reticulocyte lysate translation system. Native 2'-hydroxyl(OH) ORNs inhibited TS mRNA translation in a dose-dependent manner but did not repress translation of control mRNAs, including p53 or Escherichia coli TS. A control sense 2'-OH ORN was unable to repress translation of either human TS mRNA, or control mRNAs. Modified antisense ORNs with 2'-O-methyl phosphodiester or 2'-O-methyl phosphorothioate backbones (or both) repressed human TS mRNA translation in a dose-dependent manner, and they were both more effective than the respective 2'-OH ORN. However, nonspecific effects on mRNA translation were observed with the 2'-O-methyl phosphorothioate ORN. In vitro translation experiments revealed that in the presence of antisense ORNs, the target TS mRNA remained intact. These findings demonstrate that antisense ORNs targeted at the 5'-upstream cis-acting element represent effective inhibitors of TS mRNA translation.
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U2 - 10.1089/oli.1.1998.8.371
DO - 10.1089/oli.1.1998.8.371
M3 - Article
C2 - 9826264
AN - SCOPUS:0031794562
SN - 1087-2906
VL - 8
SP - 371
EP - 378
JO - Antisense and Nucleic Acid Drug Development
JF - Antisense and Nucleic Acid Drug Development
IS - 5
ER -