title: (A,G)-oligonucleotides form extraordinary stable triple helices with a critical R.Y sequence of the murine c-Ki-ras promoter and inhibit transcription in transfected NIH 3T3 cells.
abstract: The promoter of the murine c-Ki-ras proto-oncogene contains a critical homopurine-homopyrimidine sequence which is recognized by a protein factor and is a potential site for triplex-forming oligonucleotides (TFOs). The TFOs designed to bind this critical c-Ki-ras target have either an AG or a GT sequence motif. Of the two types, the first is found to form triplexes with extraordinarily high stability. For instance, both d(AGGGAGGGAGGAAGGGAGGG) (20AG) and d(GGGAGGGAGGGAAGGAGGGAGGGAGGGAGC) (30AG) are able to bind the c-Ki-ras target at 65 degrees C and to resist a polyacrylamide gel temperature of 55 degrees C. By contrast, the triplex formed by d(TGGGTGGGTGGTTGGGTGGG) (20GT) is largely dissociated at a gel temperature of 55 degrees C. The affinity constants of the TFOs at 37 degrees C, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl2 (standard buffer) were determined through band-shift experiments and found to be respectively 1.0 x 10(6), 4.0 x 10(6), and 2.5 x 10(7) M-1 for 20GT, 30AG, and 20AG. The AG-triplexes exhibit in standard buffer monophasic melting profiles (Tm approximately 75 degrees C) and circular dichoroism spectra showing the typical negative ellipticity at 212 nm, which is a hallmark for triplex DNA. The rate at which the TFOs bind to the c-Ki-ras target at 37 degrees C was examined under pseudo-first-order conditions. When the TFOs are in excess over the target and in the micromolar concentration range, the kinetics of triplex formation are slow, characterized by association half-lives of about 1 h. The ability of the TFOs to act as artificial transcription repressors was examined in a cellular system employing transient transfection experiments. Cultured NIH 3T3 fibroblast cells were cotransfected with a DNA mixture composed by a TFO and plasmid pKRS-413 containing the chloramphenicol acetyltransferase (CAT) gene driven by the c-Ki-ras promoter. It was found that the CAT activity is specifically inhibited by the TFOs in a dose-dependent manner. As expected, stronger CAT repression is obtained with 20AG, the oligonucleotide which forms the more stable triplex. These data suggest that (A,G)-oligonucleotides may provide a valuable means for the selective repression of the c-Ki-ras gene expression.
authors.affiliation: Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Alunni-Fabbroni
authors.forename: D
authors.initials: D
authors.lastname: Pirulli
authors.forename: G
authors.initials: G
authors.lastname: Manzini
authors.forename: L E
authors.initials: LE
authors.lastname: Xodo
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D011905:Genes, ras; D007700:Kinetics; D051379:Mice; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D016283:Proto-Oncogene Proteins p21(ras)