Introduction of a Double-Headed Nucleotide Into G-Quadruplex DNA: Position-Dependent Stabilization and Structural Insights.

G-quadruplexes are structurally diverse nucleic acid motifs with important biological properties as well as emerging applications in biotechnology and therapeutics. Here, we investigate whether a double-headed bis-guanine (G) nucleotide, GG-a substantial deviation from canonical nucleic acid architecture-can be accommodated within G-quadruplex structures and how its effects depend on sequence context and topology. Using a combination of circular dichroism (CD) spectroscopy, thermal denaturation analysis, UV thermal differential spectroscopy (TDS), fluorescence light-up assays, polyacrylamide gel electrophoresis (PAGE) analysis, and molecular dynamics (MD) simulations, we show that GG can substitute two consecutive guanosines in G-quadruplex-forming oligonucleotides (ONs) and directly participate in G-tetrad formation, but with strong sequence- and position-dependent consequences. GG incorporation is best tolerated in parallel G-quadruplexes and at G-tetrad steps with low native torsional twist, where local unwinding can be accommodated. In a tetramolecular TG4T system, GG positioned near the 3'-end of the G-stack preserved parallel G-quadruplex topology while providing significant thermal stabilization (+21°C). In the antiparallel thrombin-binding aptamer (TBA), a single GG incorporation yielded exceptional stabilization (+34°C) but was accompanied by altered topological signatures. Together, the results presented herein establish GG as a powerful but context-dependent G-quadruplex stabilizer and define design principles for its use in engineered G-quadruplexes and aptamer development.