G-Quadruplex-Forming Aptamers as Selective Inhibitors of HMGB1 Protein: a Journey from Design to Their Functional Validation

High Mobility Group Box 1 (HMGB1) is a multifunctional nuclear protein which, extracellularly released, acts as a pro-inflammatory alarmin implicated in inflammatory, autoimmune and cancer-related pathologies. This review provides a detailed account on G-quadruplex (G4)-forming aptamers targeting HMGB1 and inhibiting HMGB1-induced cell migration. A SELEX-based approach allowed identifying a set of G4-forming anti-HMGB1 aptamers, with L12 as the most promising one forming monomeric hybrid along with dimeric parallel G4 structures. The latter forms displayed higher affinity and activity towards HMGB1 but, upon annealing, were irreversibly converted into less active monomeric G4 structures. Covalently linked L12 dimers were designed to lock the most bioactive species, with L12d1T3 as the best candidate forming unimolecular parallel G4 structures featured by reversible folding/unfolding, marked affinity for HMGB1 and high serum resistance. These findings highlight the role of G4 structuring in anti-HMGB1 activity, showcasing covalently linked dimeric aptamers as tools for HMGB1-targeted therapy and diagnostics.