Spectroscopic and calorimetric studies on the triplex formation with oligonucleotide-ligand conjugates

Several triplex-forming 9-mer oligonucleotide (TFO) conjugates with a methyl- or methoxy-substituted 5-phenyl-6H-indolo[3,2-b]quinoline (PIQ) attached at the 5'-terminus or 3'-terminus or at an internal C5 thymine position were synthesized and tested for their ability to form and stabilize a triple helix with a double-helical DNA target employing UV melting experiments, fluorescence titrations, and isothermal titration calorimetry (ITC). A considerable thermal stabilization by up to 14 degrees C at pH 6.0 was observed for the 5'- and 3'-conjugates with little influence on the type of substituent but also for a conjugate with the ligand tethered by a short linker to the interior of the 9-mer TFO. A detailed thermodynamic characterization of the unmodified TFO and its 5'-conjugate with a methyl-substituted ligand by ITC experiments yielded a DeltaDeltaG degrees of -1.8 kcal mol(-1) at pH 6.0 for the TFO-attached PIQ-triplex interaction and also revealed a favorable entropic contribution as the major determinant for the free energy of PIQ binding in the conjugate. The pH dependence of triplex thermal stability highlights the importance of ring protonation of the triplex-bound ligand for its effective interaction and triplex stabilization near physiological conditions.