Guanine‐Rich Oligonucleotides Targeted to a Critical R · Y Site Located in the Ki‐ras Promoter

Abstract

The promoter of the murine JS-rus proto-oncogene contains a (C+G)-rich homopurine . homopyrimidine (R . Y) sequence that is essential for transcription activity. We have designed two G-rich oligonucleotides, d(TGGGTGGGTGGTTGGGTGGG) (20GT) and d(AGGGAGGGAGGAAGGGAGGG) (20AG), that have the potential to bind the critical Ki-rus sequence via tripIex-helix formation. Band-shift experiments have shown that 20GT binds the Ki-rus R . Y duplex with a AG value of -40 -t 5 kJ/mol, while 20AG appeared to have a lower affinity under the experimental conditions adopted: 50 mM Tris/HCI, pH 7.4, 50 inM NaC1, 5 mM MgCl,, 25°C. In the absence of Mg2+, 20GT did not bind to the Ki-rus R . Y target, while 20AG exhibited the same affinity observed in the magnesium-containing buffer. To gain insight into the solution properties of 20GT and 20AG, we have performed several experiments including polyacrylamide gel electrophoresis (PAGE), hydroxyapatite chromatography, ultraviolet absorption melting and circular dichroism (CD). We found that 20AG rapidly self-associates into presumably a duplex, even at low concentration (< 1 pM), while 20GT forms aggregates slowly, a process favoured by high oligonucleotide concentrations (> 25 pM). The critical Ki-rus sequence was inserted in Bluescript KS+, downstream from the T7 promoter, to investigate to what extent 20AG and 20GT, which are directed against the R . Y target, are able to inhibit T7 RNA polymerase transcription, under nearphysiological conditions. Transcription experiments conducted in vitro at pH 7.4 have shown that oligonucleotide 20GT produced a remarkable repression of T7 RNA polymerase activity in the concentration range (10-25 pM), whereas 20AG had little effect on transcription. In conclusion, the results of this work together with other data reported in the literature

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

“…Our results are in agreement with previously published results on purine-motif triplexes (Xodo 1995;Alunni-Fabbroni et al 1996;Arimondo et al 1998;Raghavan et al 2005). …”

(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

“…In a previous work, we found that at room temperature 20AG exhibited a weaker affinity than 20GT for the c-Ki- ras target because of its strong self-association property (Alunni-Fabbroni et al, 1996). In this study, we show that the capacity of the (A,G) oligonucleotides 20AG and 30AG to bind the c-Ki-ras target is surprisingly enhanced in the temperature range of 37-65°C.…”

“…Through band-shift analyses carried out at 25°C, these authors have shown that the affinity for the human c-Ki-ras promoter of 22-mer TFOs was approximately (0.2-0.5) × 10 6 M -1 . Moreover, other K a values determined under different experimental conditions (different T and MgCl 2 concentrations) have been reported in the literature for G-rich triplex-forming oligonucleotides, varying from 10 6 to 10 8 M -1 (Durland et al, 1991;McShan et al, 1992;Vasquez et al, 1995;Noonberg et al, 1995;Olivas & Maher, 1995a,b;Xodo, 1995;Alunni-Fabbroni et al, 1996).…”

Effect of cations on purine·purine·pyrimidine triple helix formation in mixed‐valence salt solutions

“…In this DNA motif, each purine contains a bidentate acceptor–donor hydrogen‐bond system that can be recognized by a third guanine‐rich oligonucleotide (ODN), through the formation of G·G·C and A·A·T or T·A·T base triplets. The resulting triple‐stranded complex is thermodynamically stable under physiological or near‐physiological conditions [2–11]. As triplex‐forming ODNs bind to poly(R·Y) targets in a highly sequence‐specific manner, they represent an interesting class of DNA ligands that allow a number of applications in biotechnology and pharmacology.…”