Ligand Selectivity by Inserting GCGC‐Tetrads into G‐Quadruplex Structures

G‐Quadruplexes (G4s) assembled from tandem G‐rich repeat sequences exhibit significant biological functions and applications, which may well depend on their structural features, such as the planar arrangement of G‐tetrads and flexibility of loop regions. It has been found that cytosine‐intercalated G‐repeat sequences also assemble to be quadruplex structures, involving the formation of nonplanar GCGC‐tetrads. Herein, to investigate the effect of GCGC‐tetrads on structural properties of G4s, some previously studied quadruplexes with or without GCGC‐tetrads were selected, and were used to interact with various developed G4 ligands. Our data show that stacked G‐tetrads in quadruplexes are important for the π–π stacking interactions, thus promoting the combination with end‐stacking ligands, such as porphyrins or planar small molecules. This is confirmed by the observation that the quadruplex formed by d(GGGCT4GGGC) with two internal G‐tetrads and two external GCGC‐tetrads can bind to planar ligands in the presence of specific G4‐stabilizing cations, including K+ and Pb2+, and can realize the sensitive detection of Pb2+. However, the quadruplex composed of two external G‐tetrads and two internal GCGC‐tetrads formed by d(GCGGT3GCGG) facilitates the binding of nonplanar ligands, such as triphenylmethane (TPM) dyes, owing to the structural flexibility induced by internal GCGC‐tetrads. This work provides new insights into the interaction between DNA quadruplexes and specific ligands, which is beneficial to the development of quadruplex‐based biosensors and the design of anticancer drugs. GCGC‐tetrad‐contained quadruplexes: One quadruplex with two internal GCGC‐tetrads formed by d(GCGGT3GCGG) and another quadruplex structure containing two stacked G‐tetrads assembled by d(GGGCT4GGGC) have been shown to preferentially interact with nonplanar and planar G‐quadruplex ligands, respectively.