A Kinetic Study of Triple‐Helix Formation at a Critical R · Y Sequence of the Murine c‐Ki‐ras Promoter by (A,G)‐ and (G,T) Oligonucleotides

Abstract

(20AG), d(TGGGTGGGTGGTTGGGTGGG) (20GT) and a 29-bp polypurine-polypyrimidine sequence located in the c-Ki-ms promoter (D) was studied by electrophoretic experiments in 50 mM Tridacetate, pH 7.4, 50 mM NaC1, 5 mM MgC1,. Rates of triplex formation were determined at three different temperatures (20°C, 37 "C and 45 "C), under pseudo-first order conditions obtained by using the triplex-forming oligonucleotide (TFO) 500-fold in excess over the target duplex ( 5 nM). Measurements at TFO/target ratios of 20 and 100 were also carried out. At 37°C the pseudo first-order constants, k,,,,,, were 18.9X10-' s-' for 20AG and 13.0X10-5 s ' for 20GT, yielding association half-lives of 1 h and 1.5 h, respectively.Second-order association constants were found to be in the order of 10' M-' s-': these are slightly lower if compared with those measured for triplex formation by polypyrimidine (C,T) oligonucleotides Mol. Bid. 62,. Dissociation rate constants, k I, were indirectly obtained from equilibrium constants (&) and found to be, at 37"C, 6.7X10-7 s I and 5.4X10-' s-I for 20AG and 20GT, respectively. From the rate constants obtained at 20"C, 37°C and 45°C we estimated activation energies of triplex formation between D plus 20AG and D plus 20GT of respectively 1 3 4 2 2 9 and 88?21 kJ/mol. Moreover, the activation energies for the reaction of triplex dissociation were 385 -C 50 kJ/mol for 20AG and 330 -C-42 kJ/mol for 20GT. Decreasing the TFO/target ratio from 500 to 100 or 20, we observed a concomitant decrease of the association rate, in keeping with the finding that triplex formation occurs through a bimolecular process. We found that the effect of salt on triplex formation is rather complex, as, the addition of 2 mM spermidine boosted the binding rate of 20GT, but slightly reduced that of 20AG; the increase of NaCl from SO mM to 100 mM or 150 mM decreased the rate of triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex-forming oligonucleotides specific for the c-Ki-rus promoter are discussed.Keywords: c-Ki-rus promoter; R . R . Y triple helices; kinetics; activation energy.Targeting synthetic triplex-forming oligonucleotides (TFO) against critical promoter sequences is the basis of a new strategy aiming at the selective inhibition of gene expression (Cooney et al

Specificity of DNA triple helix formation analyzed by a FRET assay

“…A quantitative analysis results in bimolecular rate constants of association of 5.6 × 10 4 min -1 M -1 for TFO1 and DS1 and 8.1 × 10 4 min -1 M -1 for TFO2 and DS2. These rate constants are in the range of results obtained with polypyrimidine TFOs and slightly higher than values previously determined for a polypurine TFO using a heterophasic assay [26]. This result shows that the 10-fold increase in the stability of triplex 2 is in part due to a faster rate of formation of this complex.…”

New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex

“…The large conformational changes, mandatory for the formation of the RH DNA triplex with non-isomorphic G*GC and T*AT triplets, are expected to warrant much higher activation energy compared to that required for the formation of isomorphic DNA triplex. This gains support from the kinetic study which suggests that such triplex formation, in fact, needs higher activation energy of 88 ± 21 kJ m −1 compared to nearly zero value required for the formation of isomorphic T*AT and C + *GC containing triplexes (41). …”

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.…”

“…In previous studies we have characterized the binding properties of AG and GT motif triplex‐forming ODNs targeted against critical poly(R·Y) sites located in the murine Ki ‐ras [5–8] and human bcr [22] genes. The sequences of these naturally occurring targets are shown in Fig.…”