When annealing is detrimental: The case of HMGB1-targeting G-quadruplex aptamers

In this work, we present the case of the G-quadruplex(G4)-forming aptamers we recently identified for the recognition of HMGB1, protein involved in inflammation, autoimmune diseases and cancer. These aptamers were previously analyzed, without annealing them, after proper dilution of the stock solution in a pseudo-physiological buffer mimicking the extracellular environment where the protein exerts its pathological activity, and showed high thermal stability and nuclease resistance, good protein affinity and remarkable in vitro activity. These features were more marked for the aptamers forming dimeric, parallel G4 structures in solution. Herein, we fully characterized the same anti-HMGB1 aptamers after a standard annealing procedure performed on diluted samples. Notably, upon a thermal unfolding/folding cycle, these aptamers, and particularly the best ones in the not-annealed form, showed significant conformational switches compared to the same systems analyzed without annealing, forming exclusively monomeric G4 structures, featured by poor thermal and enzymatic stabilities, along with lower protein affinities. These results prove that, for these aptamers, analyzed in the chosen conditions, annealing at low concentration does not produce a beneficial effect in terms of favouring the most bioactive species.