STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF POTENT ANTITHROMBOTIC OLIGONUCLEOTIDES POSSESSING BOTH QUADRUPLEX AND DUPLEX MOTIFS

We report the results of a selection for single-stranded DNA oligonucl eotide ligands to the serine protease thrombin using recently develope d methods, This selection yielded a family of DNA sequences that confo rm to a consensus structure comprised of a unimolecular quadruplex mot if and complementary flanking sequences capable of forming an addition al Watson-Crick duplex motif. This novel quadruplex/duplex structure w as not reported in a previous selection for DNA molecules which bind t o thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/du plex molecules tested bound to thrombin with higher affinity than quad ruplex structures lacking the duplex structure. However, binding affin ities did not always correlate with inhibitory potency since some mole cules with high affinity were not potent inhibitors in vitro. H-1 NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimole cular structures. Constraining these molecules to the unimolecular qua druplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity, All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex mot if. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more tha n 40-fold over the unbridged quadruplex. In addition, we have found th at both the length and sequence of the duplex motif are important for inhibition.