We have used two-dimensional 1H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355, 564-566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.

Intramolecular DNA triplexes that contain eight base triplets formed from the folding of a single DNA strand tolerate a single X.G.C mismatch in the third strand at acidic pH. The structure and relative stability of all four triplets that are possible involving a G.C Watson-Crick base pair were determined with one- and two-dimensional proton nuclear magnetic resonance techniques. Triplexes containing A.G.C, G.G.C, or T.G.C triplets were less stable than the corresponding parent molecule containing a C.G.C triplet. However, all mismatched bases formed specific hydrogen bonds in the major groove of the double helix. The relative effect of these mismatches on the stability of the triplex differs from the effect assayed (under different conditions) by two-dimensional gel electrophoresis and DNA cleavage with oligonucleotide EDTA.Fe(II).