A method for generating high-affinity DNA aptamers (XenoAptamers) that bind to target proteins with a dissociation constant (KD) 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 KD 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.
Matsunaga et al. (Sun,) studied this question.