Covalently linked dimers of a G-quadruplex-forming aptamer as HMGB1 inhibitors

We here studied a focused set of covalent dimers of the G-quadruplex(G4)-forming aptamer L12, a 26-mer previously selected as inhibitor of High Mobility Group Box 1 (HMGB1), protein involved in various inflammatory and autoimmune diseases as well as in cancer. Inspired by the ability of L12 to form dimeric parallel G4 structures, proved to be highly bioactive towards HMGB1, we investigated several covalent dimeric analogues of L12, whose design exploited linkers of different nature and length, maintaining or inverting the polarity of the two L12 strands in the sequence. Several biophysical techniques were used to analyze the conformational behaviour, molecularity and thermal/serum stability of these dimers, which formed G4 structures of parallel or hybrid topologies. Their ability to interact with the target HMGB1 protein and inhibit HMGB1-induced cellular migration was tested in comparison with L12 non-covalent dimer. The best candidate was L12d1 T3, containing a single thymidine as 3'-3' inversion of polarity motif in the junction between the two L12 strands. This oligonucleotide, forming a parallel G4 structure, showed strong affinity for the target protein (KDca. 40 nM), marked serum resistance (t1/2ca. 13 h), and excellent ability to hamper cell migration in A549 cells, with IC50 of 28 nM.