PROBING THE EFFECTS OF CHEMICAL MODIFICATIONS ON THROMBIN BINDING APTAMER BIOLOGICAL ACTIVITY.

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin resulting in anticoagulant effect. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications to improve thermodynamic stability, resistance in biological environment and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modification, five TBA analogues have been designed and synthesized preserving its chair-like G-quadruplex structure, being crucial for its biological activity, and their structural and biological properties have been investigated by Circular Dichroism, Nuclear Magnetic Resonance, non-denaturing PAGE techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2′-deoxyguanosine (GBr) in G-syn glycosidic positions, while TBAL and TBAM contain also locked nucleic acid guanosine (GLNA) or 2’-Omethylguanosine (GOMe) in G-anti positions, respectively. In TBABL and TBABM two modifications have been introduced with the aim to obtain synergistic effects. In fact, both contain 8-bromo-2′-deoxyguanosine (GBr) in syn positions, while in positions adopting an anti-glycosidic conformation exhibit in turn locked nucleic acid guanosine (GLNA) and 2’-O-methylguanosine (GOMe). The most interesting results have been obtained for TBAB, which revealed an extraordinary thermal stability (Tm approximately 30◦C higher than that of TBA) and an anticoagulant activity higher than original aptamer. In the case of TBABM a promising cytotoxic activity against breast and prostate cancer cell lines were observed. These data indicate TBAB as the best TBA anticoagulant analogue here investigated, since some of the main limitations to therapeutic applications of this aptamer were overcome, and TBABM as a promising antiproliferative derivative.