Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers

The expansion of the genetic alphabet through unnatural base pairs (UBPs) enables novel biotechnologies to create biopolymers with enhanced informational and functional properties. Hydrophobic UBPs, such as Ds − Px and Ds − Pa′, exhibit high fidelity during PCR amplification to enable the generation of UB-containing DNA aptamers (XenoAptamers) with exceptional affinity and specificity for target proteins. We developed XenoAptamers containing Ds and Px/Pa′ that bind to each serotype or variant of dengue non-structural protein 1 (DENV-NS1), a key biomarker of dengue infection, with picomolar level dissociation constants. To elucidate the mechanisms of their high affinity and specificity, we solved cryo-electron microscopy (cryo-EM) structures of oligomeric NS1 − XenoAptamer complexes. Each XenoAptamer adopts a unique tertiary structure tailored to the target protein’s surficial cavity, with Ds enhancing the structural diversity by an aromatic core formation, while Pa′ fits precisely into NS1’s hydrophobic pocket, expanding and strengthening binding interactions. Rigidity in the XenoAptamers’ tertiary structures by e.g. enhancing dipole-dipole interactions between nucleobases further improved their binding affinity. These findings elucidate the molecular mechanisms underlying XenoAptamers’ specificity and highlight the potential of genetic alphabet expansion for developing antibody alternatives. This approach opens new avenues for therapeutic and diagnostic applications.