Thrombin-Binding Aptamer (TBA) derivatives with one or two additional G-Tetrads: properties and potential antiproliferative activity

DNA and RNA aptamers are relatively short synthetic nucleic acids able to adopt distinctive three-dimensional structures and bind, with high affinity and specificity, a wide variety of molecular targets. Many aptamers, with significant biological activities, are characterized by G-rich sequences, thus adopting G-quadruplex structures as scaffolds. The Thrombin Binding Aptamer (TBA) represents one of the most investigated aptamers due to its structural and biological features. TBA is able to strongly bind thrombin showing a remarkable anticoagulant activity that has triggered its development and that of its derivatives, as promising anticoagulant drugs. Moreover, the TBA also possesses antiproliferative potential3. Unfortunately, the therapeutical development of the unmodified TBA as an anticoagulant agent halted, mainly due to some unfavourable aspects, such as the limited stability both from a thermodynamic point of view and in biological environments. On the other hand, the potential exploita0on of TBA as an anticancer molecule must also consider the anticoagulant activity, which, in this case, should be a side effect. To obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, and two further derivatives, in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics, have been prepared and studied in their proper0es. CD and PAGE results suggest that TBAG3 adopts a monomolecular parallel G-quadruplex conforma0on, while TBAG4 mainly folds into a very stable monomolecular an0parallel structure. Interes0ngly, the abasic site mimics in TBAG41S and TBAG4GS affect both molecularity and the topology of G4-structures. Furthermore, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of all TBA deriva0ves have been tested. Interestingly, none of the TBA analogs exhibits an an0coagulant ac0vity, while all of them show antiproliferative properties to the same extent. TBAG4 displays extraordinary nuclease stability, which preserves its structure up to 72 h, and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer drugs.