Abasic site-based DNA aptamers for analytical applications

We here describe a class of duplex DNA aptamers that possess an abasic site as an active cavity for binding events. A structurally optimised 23-meric duplex (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/3'-AGA CGC AGG TCT CGT TGC GTG TG-5', X=abasic site; a propyl linker, T=receptor base) binds to riboflavin with a dissociation constant of 1.9M (at 20 degrees C, pH 7.0, I=0.11M), and it exhibits a high selectivity for riboflavin over flavin mononucleotide and flavin adenine dinucleotide. A fluorescent signalling aptamer is also developed by the incorporation of fluorescent 2-aminopurine (2-AP) into the duplex to flank the abasic site. This 2-AP-modified abasic site-containing duplex DNA (5'-TCTGC GTCCT PXT TAACG CACAC-3'/3'-AGACG CAGGA TCA ATTGC GTGTG-5', P=2-AP, X=abasic site; a propyl linker, C=receptor base) is capable of selectively binding to the bronchodilator theophylline with a dissociation constant of 10M (at 5 degrees C, pH 7.0, I=0.11M), and it is applicable to monitoring serum theophylline concentrations. In addition, we describe a design strategy for label-free aptamer-based fluorescence-signalling systems based on an abasic site-binding fluorescent ligand. A DNA aptamer against adenosine is examined as a model system, and an abasic site is designed to be incorporated into the aptamer system, so that the adenosine binding causes either a release or binding of abasic site-binding ligands with a clear fluorescent signalling. The designed system is highly selective and sensitive with a detection limit of 1-2M for adenosine. These results are discussed as a potential basis for the further development of an aptamer-based analytical assay.