High-Affinity Six-Letter DNA XenoAptamer Generation by Genetic Alphabet Expansion

A method for generating high-affinity DNA aptamers (XenoAptamers) that bind to target proteins with a dissociation constant (K_D) in the tens of pM range was developed using a six-letter DNA containing two hydrophobic unnatural bases (UBs), Ds and Px/Pa', by genetic alphabet expansion. Introducing Ds increases the structural diversity of DNA, and Px/Pa' directly interacts with a hydrophobic region of target proteins. Each process in this method (six-letter ExSELEX) was optimized, resulting in the generation of high-affinity XenoAptamers with K_D values of 61 pM targeting interleukin-8 (IL8) and 1.7 pM targeting α-thrombin (thrombin). The sandwich-type ELISA using the anti-IL8 XenoAptamer-antibody combination exhibited higher sensitivity (limit of detection, LOD = 0.107 pg/mL) than that of an antibody-antibody pair (LOD = 1.227 pg/mL). The antithrombin XenoAptamer efficiently inhibits the thrombin-mediated cleavage of fibrinogen to fibrin. The six-letter ExSELEX method, with its increased DNA physicochemical and structural diversity, is expected to be innovative in creating DNA aptamers for diagnostic and therapeutic applications as an alternative to antibodies.