Designed Alteration of Binding Affinity in Structure-Switching Aptamers through the Use of Dangling Nucleotides

The ability to change binding affinity in a controlled fashion is a key step in the rational design of biomolecules in general and functional nucleic acids in particular. Here, we use dangling nucleotides to alter the binding affinity of structure-switching aptamers. Dangling nucleotides can stabilize or destabilize a nucleic acid structure with a known ΔG°₃₇. When the dangling nucleotide stabilizes the structure, less free energy from ligand binding is needed to fold the molecule and hence the ligand is observed to bind tighter than in the absence of the unpaired nucleotide. For a destabilizing dangling nucleotide, the opposite occurs, and the observed binding is weaker. We demonstrate this concept using both the cocaine-binding aptamer and the ATP-binding aptamer systems. We find that for both aptamers there is a direct, but different, relationship between the predicted stabilization and the change in the observed binding free energy.