Chemical modifications of innate DNA/RNA aptamers facilitate the improvement of their function. Herein, we report our modular strategy to manipulate a thrombin-binding DNA aptamer (TBA) to improve its anticoagulation activity and binding affinity. A set of amino acid conjugates, termed amino acid–nucleic acid hybrids or ANHs, was synthesized and incorporated into a TBA loop sequences. We found that substitutions with hydrophobic amino acids in the loop region possessed significantly enhanced antithrombin activity, up to 3-fold higher than the native TBA. We investigated the correlations between thrombin-binding affinity and the features of our amino-acid conjugates using experimental techniques including circular dichroism spectroscopy, surface plasmon resonance assay, and molecular modeling. The present study demonstrates a systematic approach to aptamer design based on amino-acid characteristics, allowing the development of advanced aptamers.

G-quadruplex (G4) is the most well-known noncanonical conformation of DNA involved in diverse pharmacological and biological contexts. G4 ligands have been actively developed as molecular probes and tumor therapeutic reagent candidates. They have also been used to detect the presence of G4s and identify their biological roles. Currently used ligands are commonly aromatic, planar, and electron deficient for effective interaction with G4s. Recognizing that tropylium cations possess the aforementioned features of effective G4 ligands, we prepared tropylium derivatives to validate their binding affinity with G4s. Titration against various DNA sequences revealed gradual changes in the UV–vis spectra of the tropylium derivatives. A strong hypochromic effect, indicating intercalation or π–π stacking, was observed when c-kit DNA was present in a binding ratio of 2:1 (ligand:DNA). The energetically minimized binding model showed that the G4–ligand complexes were stabilized by electrostatic interactions. Encouraged by the present findings, the application of tropylium derivatives in cellular contexts is underway.

We report on the construction of Cu–histidine (His)–DNA hybrids as laccase-mimetic DNAzymes. Cu–His–DNAzymes provide new insights for the systematic construction of tailor-made active sites for biomimetics.