title: Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells.
abstract: We have previously demonstrated, in vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methylphosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherichia coli and human RNases H in vitro. Intact human leukaemia cells 'biochemically microinjected' with antisense effectors demonstrated efficient suppression of target mRNA expression. It was noted that the chimeric methylphosphonodiester/phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity.
authors.affiliation: Department of Biochemistry, University of Liverpool, UK.
authors.forename: R V
authors.initials: RV
authors.lastname: Giles
authors.forename: C J
authors.initials: CJ
authors.lastname: Ruddell
authors.forename: D G
authors.initials: DG
authors.lastname: Spiller
authors.forename: J A
authors.initials: JA
authors.lastname: Green
authors.forename: D M
authors.initials: DM
authors.lastname: Tidd
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects; D016373:DNA, Antisense / Q000138:chemical synthesis / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D004952:Esters / Q000138:chemical synthesis; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D017354:Point Mutation; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D012333:RNA, Messenger / Q000037:antagonists & inhibitors*; D016914:Ribonuclease H / Q000378:metabolism*; D013301:Streptolysins / Q000494:pharmacology; D013873:Thionucleotides / Q000138:chemical synthesis; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D016373:DNA, Antisense; D004952:Esters; D009943:Organophosphorus Compounds; D011089:Polydeoxyribonucleotides; D012333:RNA, Messenger; D013301:Streptolysins; D013873:Thionucleotides; D016159:Tumor Suppressor Protein p53; C025964:streptolysin O; D016914:Ribonuclease H