Abstract Number: PB0159
Meeting: ISTH 2020 Congress
Background: Basic DNA aptamer containing G-quadruplex and interacting with thrombin exosite I consists of 15 nucleotides (5′-GGTTGGTGTGGTTGG-3) (15TBA, Thrombin Binding Aptamer). 15TBA inhibits exosite I binding with fibrinogen and platelet thrombin receptors and suppressed thrombin-induced fibrin formation and platelet aggregation.
Aims: We compared properties of G-quadruplex related aptamers (GQRAs), containing in addition to 15TBA structure hinge region (4 nucleotides) and duplex region with different number of complementary nucleotides. Earlier we have shown that aptamer RE31 (31 nucleotides) with 6 nucleotide pairs in the duplex region is more stable and has higher antithrombin activity than 15TBA [Dobrovolsky et al, 2011; Spiridonova et al, 2015].
Methods: GQRAs with 2-8 nucleotide pairs in the duplex region (RE23-RE35 series) and RE31 analogues with nucleotide insertions in the hinge region were synthesized. Stability of aptamers was assessed by measuring melting temperature at which 50% of G-quadruplexes are unfolded. Antithrombin activity of aptamers was evaluated by prolongation of thrombin, prothrombin and activated partial thromboplastin clotting times. RE31 effects on fibrinolysis were tested in in vitro model and its antithrombotic effects in in vivo FeCl3 thrombosis model in rats.
Results: Increasing the number of nucleotide pairs in the duplex region of GQRAs enhanced their stability and antithrombin activity. Stability curves reached saturation at 6 and clotting time curves at 5 nucleotide pairs (RE31 and RE29 aptamers respectively). Thus according to combined results of both tests RE31 aptamer has an optimal length of the duplex region. Nucleotide insertions in RE31 hinge region decreased its antithrombin activity. RE31 potentiate fibrinolysis in in vitro model by inhibiting activation of thrombin activated fibrinolysis inhibitor and prevents thrombosis in in vivo model.
(1) Hinge and duplex regions increase stability and antithrombin activity of GQRAs.
(2) Minimal optimal structure was achieved in RE31 aptamer containing 6 nucleotide pairs in the duplex region.
To cite this abstract in AMA style:Mazurov A, Spiridonova V, Novikova T, Sizov V, Shashkovskaya V, Zharikova E, Titaeva E, Dobrovolsky A. G-Quadruplex Related Aptamers to Thrombin Exosite I with Additional Duplex Region. Relationships between their Structure, Stability and Antihtrombin Activity [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/g-quadruplex-related-aptamers-to-thrombin-exosite-i-with-additional-duplex-region-relationships-between-their-structure-stability-and-antihtrombin-activity/. Accessed December 3, 2021.
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