Post-SELEX optimisation of split aptamers via a supramolecular anchoring strategy

Split aptamers (SPAs) display unique functions to bind one another in the presence of a specific small-molecule or protein target, thus recognised as compelling tools in diagnostics and therapy. However, only a limited number of SPAs have been proven successful so far due to the largely compromised affinity, specificity and stability arising from cleaving a long parent aptamer. To this end, we propose a supramolecular anchoring approach to improve SPA function after the Systematic Evolution of Ligands by Exponential Enrichment process. With the macrocyclic host cucurbituril and its guest molecule tethered to separate strands of an SPA for the fluorescence dye Thioflavin T (TT), the anchoring module was constructed by host-guest complexation once TT was bound to the SPA and brought the discrete aptamer units into proximity. With the supramolecular anchor to facilitate the aptamer-target complex formation and stabilise the assembled structure via dynamic crosslinking, SPA function for bio-imaging and sensing purposes was largely improved as witnessed by the increased fluorescence readout of TT in the aptamer-bound form, even when the nucleic acid sequences were shortened below the duplex-formation level. The obtained fluorescence response tracked well with the anchoring strength (none, weak or strong), thereby validating the supramolecular entity as a sophisticated and versatile strategy to rehash the otherwise incompetent SPAs.