We have used two-dimensional 'H 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][565][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.The ability of nucleic acids to fold into a variety of different structures has been exploited in the development of techniques for the isolation of aptamers (1) which are DNA or RNA oligonucleotides that have been screened from a randomly generated population of sequences for their ability to bind a desired molecular target (2-4). The isolation process involves repeated cycles of selection for, and enrichment of, oligonucleotides with an affinity to a specific target, followed by amplification of these sequences using the PCR (5). Oligonucleotides with the selected characteristics-i.e., binding to a specific molecule-are finally identified through cloning and sequencing. The potential of these methods for the development of oligonucleotide-based therapeutics has recently been explored by their application in isolating oligonucleotide ligands that selectively inhibit the activity of a target protein. Selection of DNA aptamers that bind to thrombin and inhibit thrombin-catalyzed fibrin-clot formation in vitro (6) and more recently an RNA oligonucleotide that specifically inhibits cDNA synthesis by human immunodeficiency virus reverse transcriptase in vitro (7) have been reported.Here we report two-dimensional NMR studies of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (thrombin aptamer) that conforms to the consensus sequence d(GGtTGGN2_5GGtTGG), where an uppercase letter indicates an invariant base, a lowercase letter indicates a base bias at that position, and there are usually three central N nucleotides (6). The oligonucleotide folds into a unimolecular quadruplex containing two G-quartets (8) linked by two TT loops at one end and a TGT loop at the other end. The invariant thymines in the TT loops are potentially basepaired across the top of one G-quartet. This aptamer structure was determined by NMR techniques and illustrates that this molecule forms a specific folded structure. Knowledge of this structure should be useful in the further development of oligonucleotide-based therapeutics or as a starting point for small-molecule drug design...

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The DNA sequence d(G(4)T(4)G(4)) [Oxy-1.5] consists of 1.5 units of the repeat in telomeres of Oxytricha nova and has been shown by NMR and X-ray crystallographic analysis to form a dimeric quadruplex structure with four guanine-quartets. However, the structure reported in the X-ray study has a fundamentally different conformation and folding topology compared to the solution structure. In order to elucidate the possible role of different counterions in this discrepancy and to investigate the conformational effects and dynamics of ion binding to G-quadruplex DNA, we compare results from further experiments using a variety of counterions, namely K(+), Na(+)and NH(4)(+). A detailed structure determination of Oxy-1.5 in solution in the presence of K(+)shows the same folding topology as previously reported with the same molecule in the presence of Na(+). Both conformations are symmetric dimeric quadruplexes with T(4)loops which span the diagonal of the end quartets. The stack of quartets shows only small differences in the presence of K(+)versus Na(+)counterions, but the T(4)loops adopt notably distinguishable conformations. Dynamic NMR analysis of the spectra of Oxy-1.5 in mixed Na(+)/K(+)solution reveals that there are at least three K(+)binding sites. Additional experiments in the presence of NH(4)(+)reveal the same topology and loop conformation as in the K(+)form and allow the direct localization of three central ions in the stack of quartets and further show that there are no specific NH(4)(+)binding sites in the T(4)loop. The location of bound NH(4)(+)with respect to the expected coordination sites for Na(+)binding provides a rationale for the difference observed for the structure of the T(4)loop in the Na(+)form, with respect to that observed for the K(+)and NH(4)(+)forms.

NHP6A is a chromatin-associated protein fromSaccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three α-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13 C, 15 Nlabeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

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