Portal de Periódicos da CAPES

Abasic site-based DNA aptamers for analytical applications

2010; Taylor & Francis; Volume: 22; Issue: 7-8 Linguagem: Inglês

10.1080/10610278.2010.484865

1061-0278

Seiichi Nishizawa, Yusuke Sato, Zhiai Xu, K. Morita, Minjie Li, Norio Teramae,

RNA Interference and Gene Delivery

Abstract 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 1.9 μM (at 20°C, pH 7.0, I = 0.11 M), 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 10 μM (at 5°C, pH 7.0, I = 0.11 M), 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 1–2 μM for adenosine. These results are discussed as a potential basis for the further development of an aptamer-based analytical assay. Keywords: aptameraptasensorabasic sitefluorescent signallingligandlabel free Acknowledgements This work was partially supported by Grants-in-Aid for Scientific Research (A) (No. 17205009), Scientific Research (B) (Nos 18350039 and 20350032), Exploratory Research (No. 21655023) and the G-COE Project from MEXT, Japan. Partial support from CREST, Japan Science and Technology Agency (JST) is also acknowledged. Notes 1 Present address: Division of Material Chemistry, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. 2 Present address: Chemical Engineering and Technology, College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, P.R. China. Additional informationNotes on contributorsKotaro Morita 1 1 Present address: Division of Material Chemistry, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. Minjie Li 2 2 Present address: Chemical Engineering and Technology, College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, P.R. China.