Construction, stability, and activity of multivalent circular anticoagulant aptamers.
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 EC50 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.
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney NSW 2052, Australia.
Daniel A
DA
Di Giusto
Garry C
GC
King
D020816:Amino Acid Motifs; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology*; D000991:Antithrombins / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004270:DNA, Circular / Q000737:chemistry*; D019281:Dimerization; D005340:Fibrinogen / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013696:Temperature; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
D000925:Anticoagulants; D000991:Antithrombins; D017931:DNA Primers; D004270:DNA, Circular; D009841:Oligonucleotides; D010455:Peptides; D005340:Fibrinogen; D004247:DNA; D013917:Thrombin