Expanded genetic alphabet increases structural and chemical diversity of six-letter DNA for high-affinity protein-targeting aptamers

Expanding the genetic alphabet through unnatural base pairs (UBPs) enables the creation of novel biopolymers with enhanced informational and functional properties. Hydrophobic UBPs, such as Ds-Px/Pa', exhibit high fidelity during PCR, facilitating the evolutionary engineering of UB-containing DNA aptamers (XenoAptamers) with exceptional target affinity and specificity. A series of XenoAptamers targeting dengue non-structural protein 1 (NS1), a key biomarker for dengue infection, can distinguish subtle amino acid differences among NS1 variants beyond serotypes. However, the molecular basis of this remarkable specificity and affinity remained unclear. Here, we determine cryo-EM structures of NS1-XenoAptamer complexes. Each XenoAptamer adopts a unique stable tertiary structure that precisely complements NS1's surface, whose remarkable rigidity is key in achieving high affinity and specificity to its targets. The hydrophobic Ds base introduces these unique stable structural motifs through diverse stacking interactions. Meanwhile, the propynyl group of Pa' inserts deeply into a hydrophobic pocket of NS1. These findings reveal that UBs expand DNA's structural and physicochemical diversity, demonstrating their potential to create new nucleic acid modalities and opening promising avenues for diagnostics and therapeutics.