Probing the Effects of Chemical Modifications on Anticoagulant and Antiproliferative Activity of Thrombin Binding Aptamer

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin, resulting in anticoagulant effects. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications, particularly to improve thermodynamic stability, resistance in biological environment, and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modifications, five TBA analogues have been designed and synthesized considering that the chair-like G4 structure is crucial for biological activity. Their structural and biological properties have been investigated by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), native polyacrylamide gel electrophoresis (PAGE) techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2'-deoxyguanosine (<b>B</b>) in G-<i>syn</i> glycosidic positions, while TBAL and TBAM contain locked nucleic acid guanosine (<b>L</b>) or 2'-O-methylguanosine (<b>M</b>) in G-<i>anti</i> positions, respectively. Instead, both the two types of modifications have been introduced in TBABL and TBABM with the aim of obtaining synergistic effects. In fact, both derivatives include <b>B</b> in <i>syn</i> positions, exhibiting in turn <b>L</b> and <b>M</b> in the <i>anti</i> ones. The most appealing results have been obtained for TBABM, which revealed an interesting cytotoxic activity against breast and prostate cancer cell lines, while in the case of TBAB, extraordinary thermal stability (T_m approximately 30 °C higher than that of TBA) and an anticoagulant activity higher than original aptamer were observed, as expected. These data indicate TBAB as the best TBA anticoagulant analogue here investigated and TBABM as a promising antiproliferative derivative.