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Molecules 2017, 22(10), 1770;

Construction of a Bivalent Thrombin Binding Aptamer and Its Antidote with Improved Properties

Western Australian Centre for Thrombosis and Haemostasis, Discovery Way, Murdoch University, Perth, WA 6150, Australia
Perth Blood Institute, Hollywood Private Hospital, Monash Avenue, Perth, WA 6009, Australia
Centre for Comparative Genomics, Discovery Way, Murdoch University, Perth, WA 6150, Australia
Perron Institute for Neurological and Translational Science, Perth, WA 6009, Australia
Authors to whom correspondence should be addressed.
Received: 27 September 2017 / Revised: 18 October 2017 / Accepted: 19 October 2017 / Published: 19 October 2017
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Aptamers are short synthetic DNA or RNA oligonucleotides that adopt secondary and tertiary conformations based on Watson–Crick base-pairing interactions and can be used to target a range of different molecules. Two aptamers, HD1 and HD22, that bind to exosites I and II of the human thrombin molecule, respectively, have been extensively studied due to their anticoagulant potentials. However, a fundamental issue preventing the clinical translation of many aptamers is degradation by nucleases and reduced pharmacokinetic properties requiring higher dosing regimens more often. In this study, we have chemically modified the design of previously described thrombin binding aptamers targeting exosites I, HD1, and exosite II, HD22. The individual aptamers were first modified with an inverted deoxythymidine nucleotide, and then constructed bivalent aptamers by connecting the HD1 and HD22 aptamers either through a triethylene glycol (TEG) linkage or four consecutive deoxythymidines together with an inverted deoxythymidine nucleotide at the 3′-end. The anticoagulation potential, the reversal of coagulation with different antidote sequences, and the nuclease stability of the aptamers were then investigated. The results showed that a bivalent aptamer RNV220 containing an inverted deoxythymidine and a TEG linkage chemistry significantly enhanced the anticoagulation properties in blood plasma and nuclease stability compared to the existing aptamer designs. Furthermore, a bivalent antidote sequence RNV220AD efficiently reversed the anticoagulation effect of RNV220 in blood plasma. Based on our results, we believe that RNV220 could be developed as a potential anticoagulant therapeutic molecule. View Full-Text
Keywords: modified nucleotide; aptamer; thrombin binding aptamer; triethylene glycol linkage modified nucleotide; aptamer; thrombin binding aptamer; triethylene glycol linkage

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Hughes, Q.W.; Le, B.T.; Gilmore, G.; Baker, R.I.; Veedu, R.N. Construction of a Bivalent Thrombin Binding Aptamer and Its Antidote with Improved Properties. Molecules 2017, 22, 1770.

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