Mini‐Hairpin DNA: Extraordinarily Stable Structure and Its Applications

Nucleic acids exhibit diverse biological functions through hierarchical structural organization, and higher-order architectures enable recognition beyond Watson-Crick base pairing. Here, we describe the discovery and characterization of an unusual, extraordinarily stable motif-mini-hairpin DNA-a compact hairpin structure composed of GCGNNAGC or GCGNAGC sequences (N = A, G, C, or T). About 40 years ago, we serendipitously found this unique structure from unusual mobility in denaturing gel electrophoresis. The tertiary structure of GCGAAGC was determined by NMR and contains two G-C pairs and one sheared G-A pair. Despite only three base pairs, the mini-hairpin structure remains nondenatured even in 7 M urea. This thermal stability cannot be accounted for by conventional thermodynamic or structural models and remains unexplained by current molecular dynamics predictions. In addition to thermal stability, this motif resists nuclease digestion. The motif has been identified not only in certain genomic contexts but also as a stabilizing element in engineered nucleic acids, where it enhances the performance of functional nucleic acid molecules. We hope that this review will accelerate further research and applications of mini-hairpin DNA.