Engineering interlocked G-wire assembly by systematic modulation of G-triad•G Interface connections

The role of long G-tract position within heterogeneous G-tract sequences in the assembly of interlocked G-quadruplex (G4) architectures was systematically investigated. Assembly was dependent on four guanosines (Gs) in the long G-tract: two at the interface (one involved in the intramolecular G-triad, one that pairs with the neighboring G-triad yielding an intermolecular G-tetrad), with the remaining Gs contributing to the formation of stacked intramolecular G-tetrads. Our data demonstrated that apart from three heterogeneous G-tract sequences bearing both a 5'-terminal long G-tract and a 3'-terminal thymine-which specifically assemble into 5'-5' end-stacking interlocked G4 dimers-all other heterogeneous G-tract sequences, regardless of the presence of 3'-flanking non-G residues, readily formed interlocked G-wires via 5'-3' end-stacking. When the 5'-terminal G within the long G-tract paired with a neighboring G-triad to form an intramolecular G-tetrad, the resulting interlocked G-wires adopted 5-ring-interface stacking, exhibiting characteristic 330 nm emission (Assembly Pathway I). In contrast, when the 3'-terminal G within the long G-tract served this function, the assembled interlocked G-wires typically adopted 5/6-ring-interface stacking, generating a 385 nm emission (Assembly Pathway II). However, most of heterogeneous G-tract sequences harboring the long G-tract in the central position adopted either one preferential pathway-dependent on the ionic environment-or hybrid assembly pathways. Moreover, our study demonstrated that N-cyanoimidazole-driven end-ligation significantly enhanced the yield of interlocked G-wires. This work provides new perspectives and practical strategies for the rational assembly, precise regulation, and stability optimization of G4-based nanostructures.