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

ABSTRACT

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, G _G —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 G _G 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. G _G 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, G _G 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 G _G incorporation yielded exceptional stabilization (+34°C) but was accompanied by altered topological signatures. Together, the results presented herein establish G _G as a powerful but context‐dependent G‐quadruplex stabilizer and define design principles for its use in engineered G‐quadruplexes and aptamer development.