Easy chemical modifications to explore the ‘Janus face’ of TBA: anticoagulant vs antiproliferative properties.

RNA and DNA aptamers can be defined as short synthetic ribo- and deoxyribonucleic acids able to bind with high affinity and specificity a broad range of molecular targets as small molecules, proteins and other nucleic acids (1). Under certain conditions and in aqueous solution, aptamers are able to fold in stable three-dimensional structures conferring on them the ability to bind their cognate ligands. The thrombin binding aptamer (TBA) is a 15-base long oligodeoxynucleotide (5'-GGTTGGTGTGGTTGG-3') endowed with interesting anticoagulant properties. According to both X-ray and NMR spectroscopy investigations, TBA adopts a monomolecular antiparallel G-quadruplex structure, characterized by two stacked G-tetrads and three edge loops (two TT loops and one TGT loop, Figure) (2,3). Several studies have shown that G-tetrads are mostly responsible for the thermal stability of the aptamer, while loops are involved in the interaction with its target protein, namely thrombin, which is a serine protease playing a key role in the blood coagulation pathway (4). After its discovery, TBA has been subjected to a plethora of chemical modifications aimed at improving thermal stability, enhancing nucleases resistance and increasing anticoagulant activity (5). Besides the anticoagulant activity, just as other G-rich oligonucleotides, TBA has also shown antiproliferative properties (6). In this frame, the simultaneous anticoagulant activity of TBA represents a drawback in exploiting this additional biological property. In an effort to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones, we have prepared some TBA derivatives exhibiting appropriate site-specific replacement of the residues in the loops with a dibenzyl linker or commercially available thymine analogues, such as 2'-deoxyuridine (U), 5-bromo-2'-deoxyuridine (B) and 5-hydroxymethyl-2'-deoxyuridine (H). All the new quadruplex-forming TBA based sequences were studied for their structural (CD, CD melting, NMR) and biological (PT and MTT assays) properties in comparison with the parent aptamer. The whole of data open up the possibility to modulate the TBA properties by using simple tiny modifications concerning specific positions.