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

The effect of various monovalent, divalent and oligovalent cations on the r eaction of tripler formation by GT and AG motif tripler-forming oligonucleo tides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr gen es, 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 MgCl2 the triple helices were progressively destabilized by addin g increasing amounts of NaCl, from 20 to 140 mM, to the solution. We also o bserved 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 eq uilibria involving the G-rich tripler-forming oligonucleotides. Potassium w as found to destabilize tripler DNA even when the triple helices are prefor med in the absence of K+. However, footprinting experiments also showed tha t the inhibitory effect of K+ on tripler DNA is partially compensated for b y millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2 +, which upon coordinating to N7 of guanine are expected to enhance hydroge n-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich tripler-forming oligonucleotide s. Tripler 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 tripler DNA is rationalized in terms of competition among the different valence cations to bind to tripler DNA, and differential cat ion stabilization of unusual quadruplex structures formed by the tripler-fo rming oligonucleotides.