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

• Published in: Nucleic acids research 1998-11, Vol.26 (21), p.4996-5003
• Main Authors: Gondeau, Claire, Maurizot, Jean Claude, Durand, Maurice
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - chemistry; Base Sequence; Circular Dichroism; DNA - chemistry; In Vitro Techniques; Ligands; Magnesium Chloride; Molecular Sequence Data; Netropsin - chemistry; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligodeoxyribonucleotides - chemistry; Potassium Chloride; Spectrophotometry, Ultraviolet; Thermodynamics
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/26.21.4996

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 triplex involves the oligonucleotide 5′-d(G4A4G4[T4]C4T4C4-[T4]G4T4G4) ([T4] 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([T4]G4T4G4), the presence of millimolar concentrations of MgCl2 generates an intramolecular triplex (via double hairpin formation). (ii) In contrast to the antiparallel triplex formed by the oligonucleotide 5′-d(G4T4G4[T4]G4A4G4[T4]C4T4C4), the parallel triplex melts in a biphasic manner (a triplex to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one. The enthalpy change associated with triplex 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 triplex remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A4/T4 of the duplex or triplex in an equimolar stoichiometry. Its association constant K is smaller for the parallel triplex (∼1 × 107 M−1) than for the antiparallel one (∼1 × 108 M−1). In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.