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 (N) moiety and a 3-hydroxypropylphosphate (p) either at the 5′- or 3′-end of the TBA sequence. Interestingly, N-TBA-p and p-TBA-N 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. N-TBA-p showed enhanced properties compared to both p-TBA-N 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 N-TBA-p than p-TBA-N and TBA.