Nucleic Acids Research Advance Access published online on May 2, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn242
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Molecular Biology |
Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation
1Department of Biochemical Science and Technology, P.le Kolbe 4, 33100 Udine, Italy, 2Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand and 3Nucleic Acid Center, Institute of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark
*To whom correspondence should be addressed. Tel: +39 0432 494395; Fax: +39 0432 494301; Email: lxodo{at}makek.dstb.uniud.it
Received November 23, 2007. Revised April 14, 2008. Accepted April 15, 2008.
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.