title: A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues.
abstract: The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
authors.forename: Andrey
authors.initials: A
authors.lastname: Arzumanov
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Stetsenko
authors.forename: Andrey D
authors.initials: AD
authors.lastname: Malakhov
authors.forename: Stefanie
authors.initials: S
authors.lastname: Reichelt
authors.forename: Mads D
authors.initials: MD
authors.lastname: Sørensen
authors.forename: B Ravindra
authors.initials: BR
authors.lastname: Babu
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D002117:Calcitriol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry; D019004:Galanin; D015696:Gene Products, tat / Q000037:antagonists & inhibitors*; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009687:Nuclear Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000494:pharmacology*; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects; D014862:Wasp Venoms; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D002117:Calcitriol; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors; D019004:Galanin; D015696:Gene Products, tat; D008156:Luciferases; D009687:Nuclear Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D014862:Wasp Venoms; D054322:tat Gene Products, Human Immunodeficiency Virus; C411462:1,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3); C496445:FGF4 protein, human; C071598:TARBP1 protein, human; D000099253:Trans-Activation Responsive RNA-Binding Protein; C477371:locked nucleic acid; C110068:transportan