A terminal functionalization strategy reveals unusual binding abilities of anti-thrombin anticoagulant aptamers

Despite their unquestionable properties, oligonucleotide ap-tamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to over-come these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moi-eties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 50 and 30 ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallo-graphic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/ DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel abil-ity is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge -transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.

Automated summary

This article discusses the advantages and disadvantages of oligonucleotide aptamers and proposes strategies for improvement. Crystallographic analysis of an engineered oligonucleotide in complex with thrombin reveals that terminal modifications enhance the interaction with thrombin, thereby expanding its potential.