Bivalent ligands with long nanometer-scale flexible linkers.

High-affinity ligands recognizing biomolecules with high specificity are crucial for drug discovery and biomolecule detection. We describe here a simple approach to preparing aptamer-based ligands with enhanced binding affinity. In this approach, two aptamer ligands with suboptimal binding properties are covalently linked with a long flexible linker to create a bivalent ligand with significantly improved binding affinity. We first used a simple oligonucleotide-based model, which mimicked the interaction between bivalent ligands and their target molecules, to investigate the principles governing the affinity enhancement. These experiments showed that as long as the individual ligands had at least submicromolar binding affinities, they could be linked with a nanometer-scale flexible linker to produce bivalent ligands with improved binding affinity and specificity. Furthermore, comparison of the experimental data with the bivalent ligand properties predicted by a wormlike chain model showed that this model provided a good approximation of the binding properties of nanometer-scale flexible bivalent ligands. To verify the practicality of bivalent ligands with nanometer-scale flexible linkers, we constructed aptamer-based bivalent ligands for human alpha-thrombin. In agreement with the predictions derived from the model system, the binding affinities and the anticlotting activities of thrombin bivalent ligands were significantly improved compared to those of the individual ligands.