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

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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 inflammatory, 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. • G4-forming anti-HMGB1 aptamers were analyzed as low concentrated samples after a typical annealing procedure. • Their functional properties and HMGB1-induced cell migration inhibitory activity were evaluated. • The annealed aptamers showed poor thermal and enzymatic stability and low affinity for HMGB1. • Upon annealing the aptamers lost the dimeric forms, exclusively forming monomeric G4 structures. • The conformational simplification obtained by annealing at low conc. does not lead to the most bioactive species of anti-HMGB1 aptamers.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.