title: Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.
abstract: The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression.
authors.affiliation: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, PO. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Peter A C
authors.initials: PA
authors.lastname: 't Hoen
authors.forename: Ruud
authors.initials: R
authors.lastname: Out
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.forename: F H D
authors.initials: FH
authors.lastname: van Batenburg
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Theo J C
authors.initials: TJ
authors.lastname: van Berkel
authors.forename: Erik A L
authors.initials: EA
authors.lastname: Biessen
authors.forename: Martin K
authors.initials: MK
authors.lastname: Bijsterbosch
mesh_terms: D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005982:Glutathione Transferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis; D021281:Quantitative Structure-Activity Relationship; D012333:RNA, Messenger / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012984:Software; D014158:Transcription, Genetic
chemical_list: D005982:Glutathione Transferase; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D016914:Ribonuclease H; C412602:glutathione S-transferase Mu 2