SPECTROSCOPIC INVESTIGATION OF AN INTRAMOLECULAR DNA TRIPLEX CONTAINING BOTH G.G-C AND T.A-T TRIADS AND ITS COMPLEX WITH NETROPSIN

The triple helix formation by the oligonucleotide (5')d(G(4)T(4)G(4)- [T-4]-G(4)A(4)G(4)- [T-4]-C4T4C4) ([T-4] represents a stretch of 4 thy mine residues) has been investigated by UV absorption spectroscopy and circular dichroism. In a 10 mM sodium cacodylate, 0.2 mM disodium EDT A (pH 7) buffer, we show: the following significant results: i) in the absence of MgCl2, the oligonucleotide adopts a hairpin duplex structu re with the dangling tail (5')d(G(4)T(4)G(4)-[T-4]). This 5' extremity , which contains separated runs of four guanine residues, does not ass ume the expected tetraplex conformation observed when this sequence is free. ii) In the presence of MgCl2, the oligonucleotide folds back on itself twice to give a triple helix via a double hairpin formation, w ith [T-4] single-strand loops, iii) The addition of high concentration of KCl to the preformed tripler does not disrupt the structure. Never theless, if the oligonucleotide is allowed to fold back in the presenc e of K+, tripler formation is inhibited. Circular dichroism studies de monstrate that the oligonucleotide adopts a dimeric conformation, resu lting from the association of two hairpin duplexes, via the formation of an antiparallel G-quadruplex by the telomeric (5')d(G(4)T(4)G(4)-[T -4]) extremities, iv) Under the experimental conditions used in this r eport, the tripler melts in a monophasic manner, v) Netropsin, a DNA m inor groove ligand, binds to the central site A(4)/T-4 of the duplex a nd to that of the tripler in an equimolar stoichiometry. In contrast w ith previous studies concerning pyr.pur:pyr triplexes, thermal denatur ation experiments demonstrate that the netropsin binding stabilizes th e intramolecular tripler.