Supramolecular Aptamers on Graphene Oxide for Efficient Inhibition of Thrombin Activity

Abstract

Graphene oxide (GO), a two-dimensional material with a high aspect ratio and polar functional groups, can physically adsorb single-strand DNA through different types of interactions, such as hydrogen bonding and π-π stacking, making it an attractive nanocarrier for nucleic acids. In this work, we demonstrate a strategy to target exosites I and II of thrombin simultaneously by using programmed hybrid-aptamers for enhanced anticoagulation efficiency and stability. The targeting ligand is denoted as Supra-TBA 15/29 (supramolecular TBA 15/29 ), containing TBA 15 (a 15-base nucleotide, targeting exosite I of thrombin) and TBA 29 (a 29-base nucleotide, targeting exosite II of thrombin), and it is designed to allow consecutive hybridization of TBA 15 and TBA 29 to form a network of TBAs (i.e., supra-TBA 15/29 ). The programmed hybrid-aptamers (Supra-TBA 15/29 ) were self-assembled on GO to further boost anticoagulation activity by inhibiting thrombin activity, and thus suppress the thrombin-induced fibrin formation from fibrinogen. The Supra-TBA 15/29 -GO composite was formed mainly through multivalent interaction between poly(adenine) from Supra-TBA 15/29 and GO. We controlled the assembly of Supra-TBA 15/29 on GO by regulating the preparation temperature and the concentration ratio of Supra-TBA 15/29 to GO to optimize the distance between TBA 15 and TBA 29 units, aptamer density, and aptamer orientation on the GO surfaces. The dose-dependent thrombin clotting time (TCT) delay caused by Supra-TBA 15/29 -GO was >10 times longer than that of common anticoagulant drugs including heparin, argatroban, hirudin, and warfarin. Supra-TBA 15/29 -GO exhibits high biocompatibility, which has been proved by in vitro cytotoxicity and hemolysis assays. In addition, the thromboelastography of whole-blood coagulation and rat-tail bleeding assays indicate the anticoagulation ability of Supra-TBA 15/29 -GO is superior to the most widely used anticoagulant (heparin). Our highly biocompatible Supra-TBA 15/29 -GO with strong multivalent interaction with thrombin [dissociation constant ( K d ) = 1.9 × 10 −11 M] shows great potential as an effective direct thrombin inhibitor for the treatment of hemostatic disorders.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5′-3′ connecting linker on the aptamer stability and anticoagulant activity

“…In the past decades, both serendipity and targeted rational design have contributed to discover chemically modified TBA analogues endowed with markedly improved physico-chemical and biological properties. Among the approaches thus far explored to obtain modified TBA variants with promising pharmacokinetic profiles, either backbone modifications [7][8][9][10][11][12][13][14][15][16][17][18][19] or incorporation onto suitable nanoplatforms -including magnetic, [20][21][22] gold, [23][24][25][26][27] silica-based nanoparticles [28][29][30][31] and graphene [32,33] -have been successfully exploited.…”

Recent Advances in Micro/Nanomaterial-Based Aptamer Selection Strategies

“…However, many exhibit poor specificity and affinity, a short half-life, and susceptibility to nuclease present in the cell and blood, which limits their widespread applications in vivo. A recent study reported that nanocomposites and hybrid aptamer-immobilized GOx effectively inhibit thrombin’s coagulant activity in the blood [ 121 ]. This hybrid aptamer comprises two different aptamers (a 15-mer and a 29-mer) against thrombin that are linked with multiple poly (adenine) (A 20 ) segments.…”

Dimeric and Multimeric DNA Aptamers for Highly Effective Protein Recognition

“…In this context, TBA15 was incorporated onto very different nanoplatforms, including magnetic [ 126 , 127 , 128 ], gold [ 129 , 130 , 131 , 132 , 133 ], silica-based nanoparticles [ 134 , 135 , 136 , 137 ], and graphene [ 138 , 139 ].…”