Construction, Stability, and Activity of Multivalent Circular Anticoagulant Aptamers

Abstract

Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra-or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification. In many cases, circular DNA aptamer half-lives are extended beyond 10 h in serum and plasma, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions are used as scaffold architectures around which two, three, or four aptameric motifs can be arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin display improved anticoagulant potency with EC 50 values 2-3-fold better than those of the canonical GS-522 thrombin DNA aptamer. Intrinsic duplex regions provide an opportunity to incorporate additional transcription factor binding motifs, whereas ancillary loops can be used to provide further functionality. These anticoagulant aptamers provide a starting point for merging the principles of DNA nanotechnology with aptameric functions.Since the first reports on nucleic acid aptamers more than a decade ago (1-3), this class of molecules has developed steadily toward general therapeutic (4 -6) and diagnostic (5, 7-9) applications. The relative ease of aptamer discovery has allowed for selection against a wide range of targets (10) by processes that have recently been automated (11). Simultaneous advances in signaling and detection strategies have improved the outlook for aptamer arrays in proteomic and biosensor applications. Whereas the high affinity and specificity of aptamers toward their molecular targets make them well suited for these tasks, the susceptibility of natural nucleic acids to nucleolytic degradation is a serious hurdle for uses in biological fluids. Strategies to improve the nuclease stability of aptamers, as for antisense nucleic acids, have included the use of chemical modifications such as phosphorothioate, locked nucleic acid, 2Ј-Omethyl-and 2Ј-fluoro-nucleotides (12), as well as mirror-image Spiegelmers (13). Of most relevance for potential therapeutic applications, some common nucleic acid stabilization strategies have encountered problems associated with toxic degradation products (14). In addition, modification performed post-selection can alter the subtle binding interactions of the selected natural aptamer (15, 16).Circularization of natural aptamers is an attractive alternative to chemical modification for improving aptamer stability. With the majority of nucleic acid degradation activity arising from plasma exonucleases (17), modification of exposed termini often achieves a sufficient improvement in stability for use in vivo (12), and circular constru...

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

“…It has been also shown that engineered aptamers like multivalent circular constructs exhibit increased stability against cleavage by exonucleases and improved (2 to 3-fold) anticoagulant activity in comparison with canonical single stranded aptamers [8]. Increased inhibitory activity against HIV-1 reverse transcriptase has been shown also for recently reported bimodular DNA aptamers [7].…”

“…Nowadays, increased interest in aptamer research is focused on improvement of these features for application in medical therapy, biotechnology and biosensor development [3,4]. A novel approach in aptamer engineering is based on dimerization [5][6][7] or multimerization [8], which possess two or more binding sites for the target ligand. Among DNA aptamers, those sensitive to thrombin are most studied.…”

Structure–activity relationships of a caged thrombin binding DNA aptamer: Insight gained from molecular dynamics simulation studies

“…Several studies have been reported to examine the biophysical and chemical properties of the G-quadruplex (Di Giusto and King, 2004;Peng and Damha, 2007;Sacca et al, 2005) and also showed the sensitive nature of the quadruplex upon chemical modification (Marathias et al, 1999;Sacca et al, 2005). The control of the inhibitory function of the aptamer is a challenging aspect.…”

Self-association of cyclic oligonucleotides through G:T:G:T minor groove tetrads

“…Firstly, cyclic oligonucleotides can be used to target singlestranded DNA 24,25 and RNA 26 with higher affinity and selectivity than their linear counterparts. Secondly, dumbbells can acts as aptamers 27,28 and transcription factor decoys. 29,30 Finally, small cyclic ribodinucleotides, such as c-di-AMP 31 and c-di-GMP, 32 are important second messenger signaling molecules that regulate many processes in bacteria.…”