Circular dichroism and UV melting studies on formation of an intramolecular triplex containing parallel T*A:T and G*G:C triplets:: netropsin complexation with the triplex

We have used circular dichroism and UV absorption spectroscopy to characterize the formation and melting behaviour of an intramolecular DNA triple helix containing parallel T*A:T and G*G:C triplets. Our approach to induce and to stabilize a parallel tripler involves the oligonucleotide 5'-d(G(4)A(4)G(4)[T-4]C4T4C4- [T-4]G(4)T(4)G(4)) ([T-4] represents a stretch of four thymine residues). In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we have shown the following significant results. (i) While in the absence of MgCl2 this oligonucleotide adopts an intramolecular hairpin duplex structure prolonged by the single strand extremity 5'-d([T-4]G(4)T(4)G(4)), the presence of millimolar concentrations of MgC(l)2 generates an intramolecular tripler (via double hairpin formation), (ii) In contrast to the antiparallel tripler formed by the oligonucleotide 5'-d(G(4)T(4)G(4)[T-4]G(4)A(4)G(4)[T-4]C4T4C4), the parallel tripler melts in a biphasic manner (a tripler to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one, The enthalpy change associated with tripler formation (-37 kcal/mol) is approximately half that of duplex formation (-81 kcal/mol), (iii) The parallel triple helix is disrupted by increasing the concentration of KCl (> 10 mM), whereas, under the same conditions, the antiparallel tripler remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A(4)/T-4 of the duplex or tripler in an equimolar stoichiometry, Its association constant K is smaller for the parallel tripler (similar to 1 x 10(7) M-1) than for the antiparallel one (similar to 1 x 10(8) M-1), In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.