Hybridization arrest of cell-free translation of the malarial dihydrofolate reductase/thymidylate synthase mRNA by anti-sense oligodeoxyribonucleotides.
In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.
Sartorius
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Biocenter, Department Structural Biology, Basel, Switzerland.
Franklin
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RM
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010963:Plasmodium falciparum / Q000201:enzymology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D013940:Thymidylate Synthase / Q000096:biosynthesis / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase