Introduction of a Double‐Headed Nucleotide Into G‐Quadruplex DNA: Position‐Dependent Stabilization and Structural Insights
- 1Department of Physics, Chemistry and Pharmacy University of Southern Denmark Odense M Denmark
- 2Laboratoire D'optique & Biosciences (LOB) Ecole Polytechnique CNRS Inserm Institute Polytechnique de Paris Palaiseau France
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, 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.
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BibTeX
@article{urup2026introduction,
title = {{Introduction of a Double‐Headed Nucleotide Into G‐Quadruplex DNA: Position‐Dependent Stabilization and Structural Insights}},
author = {Krista Urup and Peter Reinholdt and Kasper Munch Beck and Michael Petersen and Jean‐Louis Mergny and Poul Nielsen},
journal = {Chemistry (Weinheim an Der Bergstrasse, Germany)},
year = {2026},
month = aug,
doi = {10.1002/chem.71439},
url = {https://doi.org/10.1002/chem.71439},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Introduction of a Double‐Headed Nucleotide Into G‐Quadruplex DNA: Position‐Dependent Stabilization and Structural Insights
AU - Krista Urup
AU - Peter Reinholdt
AU - Kasper Munch Beck
AU - Michael Petersen
AU - Jean‐Louis Mergny
AU - Poul Nielsen
PY - 2026
DA - 2026/08/11
JO - Chemistry (Weinheim an Der Bergstrasse, Germany)
DO - 10.1002/chem.71439
UR - https://doi.org/10.1002/chem.71439
AB - 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.
SN - 0947-6539
SN - 1521-3765
ER -