AbstractWe have made an accidental discovery of an unusual, single-stranded, guanine-rich DNA molecule that is capable of adopting a folded structure in 7 M urea (7MU) known to denature nucleic acid structures. The folding of this molecule requires Na⁺ and Mg²⁺ and the folded structure remains stable when subjected to denaturing (7MU) polyacrylamide gel electrophoresis. Results from sequence mutagenesis, DNA methylation, and circular dichroism spectroscopy studies suggest that this molecule adopts an intramolecular guanine-quadruplex structure with 5 layers of guanine tetrads. Our finding indicates that DNA has the ability to create extremely stable structural folds despite its limited chemical repertoire, making it possible to develop DNA-based systems for unconventional applications.