Conformational Aspects of an Uncharged Phosphate Analogon Built In at the Branch-point of a DNA Four-way Junction

Electrostatic interactions appear to be important in the conformation and dynamics of DNA four-way junctions. Particularly, the Coulomb repulsion between two approaching phosphate groups at the site of strand exchange is commonly supposed to have a negative influence on the thermodynamic stability of the fully stacked conformation. We synthesized a unimolecular DNA four-way junction containing an uncharged methylene-acetal linkage, -O3′-CH 2-O5′-, instead of a regular phosphodiester linkage, -O3′-PO − 2-O5′-, between two specific residues at the branch-point to examine the effect of charge removal. Complete sequential 1H-NMR assignments of the non-exchangeable base protons and H1′/H2′/H2′′ sugar protons were obtained with the aid of NOESY and TOCSY experiments. On the basis of the NMR data it is concluded that the stacking arrangement at the branch-point of the modified oligonucleotide is similar to that of the previously studied parent four-way junction. Surprisingly, this is not the stacking arrangement in which the close phosphate-phos phate contact at the site of strand exchange would be absent. Some implications of this novel information regarding the role of the phosphate-phosphate repulsion in four-way junctions are discussed.