Strict Interactions of Fifth Letters, Hydrophobic Unnatural Bases, in XenoAptamers with Target Proteins

XenoAptamers are DNA fragments containing additional letters (unnatural bases, UBs) that bind specifically to their target proteins with high affinities (sub-nanomolar K_D values). One of the UBs is the highly hydrophobic 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which significantly increases XenoAptamers' affinities to targets. Originally, Ds was developed as a third base pair with a complementary UB, 2-nitro-4-propynylpyrrole (Px), for replication, and thus it can be used for aptamer generation by an evolutional engineering method involving PCR amplification. However, it is unclear whether the Ds base is the best component as the hydrophobic fifth-letter ligand for interactions with target proteins. To optimize the ligand structure of the fifth letter, we prepared 13 Ds variants and examined the affinities of XenoAptamers containing these variants to target proteins. The results obtained using four XenoAptamers prepared by the replacement of Ds bases with variants indicated that subtle changes in the chemical structure of Ds significantly affect the XenoAptamer affinities. Among the variants, placing either 4-(2-thienyl)pyrrolo[2,3-b]pyridine (Ys) or 4-(2-thienyl)benzimidazole (Bs) at specific Ds positions in each original XenoAptamer greatly improved their affinities to targets. The Ys and Bs bases are variants derived by replacing only one nitrogen with a carbon in the Ds base. These results demonstrate the strict intramolecular interactions, which are not simple hydrophobic contacts between UBs and targets, thus providing a method to mature XenoAptamers' affinities to targets.