When the Position of Pendant Groups Makes the Difference in G-Quadruplex Behavior: The Case of Bis-Conjugated Thrombin-Binding Aptamers

In the search for effective and low-toxicity anticoagulant agents, the G-quadruplex-forming thrombin-binding aptamer (TBA) with sequence 5'-GGTTGGTGTGGTTGG-3', able to selectively recognize the fibrinogen-binding exosite I of the thrombin enzyme, emerged as a promising therapeutic and surgical tool. In this frame, we recently synthesized and evaluated a library of TBA analogues carrying a naphthalene diimide (<b>N</b>) moiety and a 3-hydroxypropylphosphate (<b>p</b>) either at the 5'- or 3'-end of the TBA sequence. Interestingly, <b>N-TBA-p</b> and <b>p-TBA-N</b> analogues, having the same pendant groups at 5'- or 3'-end but in reversed position, showed very different behavior in terms of thermal stability, nuclease resistance in serum, and anticoagulant activity. <b>N-TBA-p</b> showed enhanced properties compared to both <b>p-TBA-N</b> and the parent TBA and thus emerged as a very promising candidate for future in vivo studies. Here, by in-depth molecular dynamics-based analyses, we disclosed the structural features determining the higher thermal stability and nuclease resistance as well as the higher anticoagulant activity due to thrombin recognition, experimentally observed for <b>N-TBA-p</b> than <b>p-TBA-N</b> and TBA.