Stereochemistry of Phosphorothioate Linkages Impacts the Structure and Binding Affinity of Aptamers and DNAzymes

The introduction of chemical modifications to oligonucleotides has been pivotal to their emergence as effective therapeutic agents. Understanding how these modifications influence the higher-order structure of oligonucleotides is one of the key considerations during the drug development process. Here, we use a G-quadruplex (thrombin-binding aptamer) and stem/loop structure (dehydroepiandrosterone sulfate binding aptamer) as model cases to show for the first time that the stereochemistry of phosphorothioate linkages can affect the stability and function of oligonucleotides that depend on their intramolecular conformation for activity (e.g., aptamers and DNAzymes). Differences up to 15 °C in melting temperature are observed between stereochemical variants of the same sequence. Moreover, using a hemin-DNAzyme assay and small molecule recognizing fluorogenic aptamer, we illustrate that stereochemistry can be used to tune the binding affinity and activity of the oligonucleotides examined in this study toward non-nucleic acid targets. Taken together, these discoveries highlight the potential influence of stereochemistry on the structure and function of other phosphorothioate-containing oligonucleotides, revealing important considerations for future drug design.