The three-dimensional structure of a short DNA fragment, d(GCGAAGC) exhibiting an extraordinarily stable hairpin structure was determined by nuclear magnetic resonance spectroscopy. Two possible models were obtained by molecular modelling using distance and torsion constraints. Only one of these two models is the correct structure, which can clearly explain all the 1H chemical shifts. d(GCGAAGC) is folded back on itself between A4 and A5, and all the sugars in the fragment adopt the C2'-endo conformation. This compact molecule is stabilized by regular extensive base-stacking interaction within each B-form helical strand of G1C2G3A4 and AAG6CA, and by two G-C and one G3-A5 base pairs. The molecule is hard to differentiate into stem and loop regions, so that we classify it as a turn (hairpin-turn) structure exerted by a single-stranded DNA. This highly stacked structure shows high thermostability and strong resistance against nucleases contained in E.coli extracts and in human serum.

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d(GCGAAAGC) and d(GCGAAGC) fragments form extraordinarily stable DNA minihairpins containing only two G-C base pairs and a GAAA or GAA loop, respectively, with a Tm of 76 degrees C. These sequences are frequently found in some important regions such as replication origins and promoter regions for transcription. We examined all 64 possible DNA fragments, d(GCNNNGC), in which the triloop region of the d(GCGAAGC) minihairpin was randomized and found that only four fragments, d(GCGNAGC) (N = A, G, C, or T), formed extraordinarily stable minihairpins as shown by their gel mobility and resistance to a single-stranded DNA-specific exonuclease. Structural and thermodynamic analyses suggest that the extraordinary stability is caused by a unique structural property of the trinucleotide sequences corresponding to the GNA loop.