Abstract
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.
Highlights
In 1990, two reports described the isolation of short single-stranded oligonucleotide sequences that can bind to a target molecule with high affinity and specificity [1,2]
The process described was referred to as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which uses oligonucleotide sequence libraries (~1015 –1018 members) to eventually isolate sequences called aptamers that bind to a selected target molecule with high affinity and specificity [3,4]
The high affinity of the selected aptamer is due to its ability to adopt unique secondary and tertiary structure dictated by Watson–Crick base-pairing interactions
Summary
In 1990, two reports described the isolation of short single-stranded oligonucleotide sequences that can bind to a target molecule with high affinity and specificity [1,2]. The process described was referred to as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which uses oligonucleotide sequence libraries (~1015 –1018 members) to eventually isolate sequences called aptamers that bind to a selected target molecule with high affinity and specificity [3,4]. One of the most well studied aptamers is HD1, an aptamer targeting the exosite I moiety of thrombin (an important haemostatic protein) that was first described in 1992 [10]. The discovery of HD1 was soon followed by the development of a second thrombin targeting aptamer in 1997, HD22, which targets exosite
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