title: Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation.
abstract: Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA-TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA-TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA-TFOs were found to abrogate the formation of a DNA-protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA-TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome.
authors.affiliation: Department of Biochemical Science and Technology, P.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Manikandan
authors.initials: M
authors.lastname: Paramasivam
authors.forename: Susanna
authors.initials: S
authors.lastname: Cogoi
authors.forename: Vyacheslav V
authors.initials: VV
authors.lastname: Filichev
authors.forename: Niels
authors.initials: N
authors.lastname: Bomholt
authors.forename: Erik B
authors.initials: EB
authors.lastname: Pedersen
authors.forename: Luigi E
authors.initials: LE
authors.lastname: Xodo
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D005786:Gene Expression Regulation; D005990:Glycerol / Q000031:analogs & derivatives / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D009841:Oligonucleotides / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D011721:Pyrenes / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection; D018631:ras Proteins / Q000235:genetics
chemical_list: C507245:(R)-1-O-(4-(1-pyrenylethynyl)phenylmethyl)glycerol; D004268:DNA-Binding Proteins; C117307:KRAS protein, human; C000715470:MAS1 protein, human; D009841:Oligonucleotides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011721:Pyrenes; C051802:triplex DNA; D006147:Guanine; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D005990:Glycerol; D011188:Potassium