title: Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.
abstract: Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.
authors.affiliation: Clatterbridge Cancer Research Trust, J. K. Douglas Research Laboratories, Clatterbridge Hospital, Bebington, Wirral CH63 4JY, UK. bryonyl@ccrt.co.uk
authors.forename: B H
authors.initials: BH
authors.lastname: Lloyd
authors.forename: R V
authors.initials: RV
authors.lastname: Giles
authors.forename: D G
authors.initials: DG
authors.lastname: Spiller
authors.forename: J
authors.initials: J
authors.lastname: Grzybowski
authors.forename: D M
authors.initials: DM
authors.lastname: Tidd
authors.forename: D R
authors.initials: DR
authors.lastname: Sibson
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D001667:Binding, Competitive; D002463:Cell Membrane Permeability / Q000187:drug effects; D016373:DNA, Antisense / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012680:Sensitivity and Specificity; D013301:Streptolysins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D020298:U937 Cells
chemical_list: D001426:Bacterial Proteins; D016373:DNA, Antisense; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D012333:RNA, Messenger; D013301:Streptolysins; D014409:Tumor Necrosis Factor-alpha; C025964:streptolysin O; D016914:Ribonuclease H; D013755:Tetradecanoylphorbol Acetate