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

Abstract

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine±polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl 2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K + . However, footprinting experiments also showed that the inhibitory effect of K + on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn 2+ and Ni 2+ , which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.Keywords: bcr gene; bivalent cations; Ki-ras gene; monovalent cations; purine±purine±pyrimidine triplex.Tracts of polypurine±polypyrimidine [poly(R´Y)] sequences often occur in regulation regions of DNA [1]. 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. Among these, the use of triplex-forming ODNs as artificial transcription repressors has drawn the attention of many researchers [12,13]. This strategy is based on the notion that the interaction of triplex-forming ODNs with critical sequences within promoter regions should interfere with transcription, with the consequence of inhibiting the expression of the target gene, without affecting the overall activity of the cell. This original strategy, called the antigene...

Potential in vivo roles of nucleic acid triple-helices

“…High concentrations of multivalent cations promote triplex stability by compensating for the unfavorable electrostatic repulsion of the three negatively charged oligonucleotide backbones. 158,182,183 It is well established that metal cations are naturally present in DNA in vivo. 184 However, the stabilizing effect of any multivalent cation is limited by its competition with available monovalent counterfeits.…”

pH and Cation Effects on the Properties of Parallel Pyrimidine Motif DNA Triplexes

“…The resulting sample versus buffer scans collected for each sample were averaged, and the buffer versus buffer baseline was subtracted and then normalized by the strand concentration and the sample volume to obtain the excess heat capacity, ∆C P , versus temperature, T, profiles. The transition temperature was determined by the peak height maximum of the baseline-corrected ∆C P vs T profiles, and the calorimetric enthalpy was calculated from the integration area (11,17,21,33,34). SPR Measurements.…”

Evaluation of effects of bivalent cations on the formation of purine-rich triple-helix DNA by ESI-FT-MS

“…Since self-association of G-rich oligonucleotides could decrease triplex formation, inhibitory effects of monovalent cations (especially K ϩ ) on purine-rich triplex formation might relate to the promotion of such competing self-structures [6,9]. In this work, Ba 2ϩ ions have been found to enhance the formation of homodimers, so it might inhibit triplex formation like K ϩ does.…”

“…Besides binding phosphate, divalent cations have been found to interact with various sites on nucleic acid, including N and O of bases [26,27]. When an electrophile interacts with N7, the formation of reverse-Hoogsteen hydrogen bonds would be enhanced, thus improving the stability of the purinerich triplex [6,10,28]. Although Mg 2ϩ may coordinate with N7 and O6 of guanine (Figure 1), it is primarily bound to the phosphate group [27] and slightly assists the formation of triplexes.…”

“…For the purine motif, the triplex will be inhibited in the presence of monovalent cations, such as sodium and potassium, which serve to coordinate and stabilize the G-rich oligonucleotide dimmers and tetramers [1,4]. The stabilization of the bivalent cation to triplex is complicated-i.e., it is not a simple electrostatic phenomenon-and has been investigated using circular dichroism, UV absorption spectroscopy, footprinting techniques, electrophoresis mobility shift assay, and laser densitometry [5][6][7][8][9][10][11].…”