Graphical abstract Two Ru(II) complexes [Ru(phen) 2 dicnq]2+ and [Ru(bpy) 2 dicnq]2+ have been synthesized and characterized. Binding properties of the two complexes with poly(U)·poly(A)*poly(U) triplex have been studied. Results indicate that the two stabilize the triplex by the stacking interaction, while they prefer to bind with the Watson-Crick base-paired duplex instead of the Hoogsteen base-paired strand of the triplex under the same conditions. Therefore, the two complexes stabilize the Watson-Crick base-paired duplex of the triplex almost without affecting the stability of third-strand. Abstract Using phen or bpy (phen = 1,10-phenanthroline, bpy = 2,2′-bipyridine) as ancillary ligands and dicnq (dicnq = 6,7-dicyanodipyrido[2,2- d :2′,3′- f ]quinoxaline) as the main ligand, two new Ru(II) polypyridyl complexes containing cyano groups, [Ru(phen) 2 dicnq]2+ (Ru 1) and [Ru(bpy) 2 dicnq]2+ (Ru 2) have been synthesized as binders for poly(U)·poly(A)*poly(U) triplex. The binding properties of Ru 1 and Ru 2 with the triplex has been studied by spectroscopic methods and viscosity measurements. Analysis of the electronic absorption spectra suggests that the binding of intercalating Ru 1 with the triplex is greater than that of Ru 2 , which is also supported by spectroscopic titrations and viscosity measurements. Thermal denaturation studies suggest that the two complexes stabilize the Watson-Crick base-paired duplex of the triplex structure almost without affecting the stability of the Hoogsteen base-paired third strand, suggesting that the electron-withdrawing effect of cyano group hardly increases the binding strengths of the third strand and the template duplex of the triplex under study here. Changes in the CD (CD = circular dichroism) spectra of the triplex indicate that the binding-induced CD perturbation of the triplex structure is more marked with Ru 1. The main results obtained here indicate that small differences in the ancillary ligands play important roles in the triplex binding when ruthenium(II) complexes contain the same intercalative ligands. [ABSTRACT FROM AUTHOR]