Kotaro Morita

Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization

Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (106 M−1) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na+ (pH 7.0, at 20°C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (ΔCp), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (−161 cal/mol K) and ATMND (−217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (ΔGobs) is dissected into its component terms.

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 1.9 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.

Conversion of thioureas to fluorescent isothiouronium-based photoinduced electron transfer sensors for oxoanion sensing

A convenient conversion is described of thiourea-based receptors to fluorescent isothiouronium-based photoinduced electron transfer (PET) sensors for oxoanion sensing. Naphthalene- or anthracene-functionalized mono-isothiouroniums are synthesized from the corresponding thioureas, in which the fluorophore is connected to the sulfur atom of the thiourea moiety by a methylene or an ethylene spacer. Even though all of the isothiouroniums with a methylene spacer readily decompose in MeOH upon excitation of the fluorophore moiety, the isothiouronium with an ethylene spacer shows good stability under identical conditions. The naphthyl isothiouronium with an ethylene spacer shows a significant fluorescence enhancement upon formation of a 1 ∶ 1 complex with oxoanions in MeOH, and the selectivity follows the order of hydrogen phosphate > acetate ≫ dihydrogen phosphate ≫ chloride. The results in the present work indicate that various types of fluorescent PET sensors might be readily obtainable from non-fluorescent thiourea-based receptors by the introduction of appropriate fluorophores at the sulfur atom of thiourea-binding sites.

Electrochemical SNPs detection using an abasic site-containing DNA on a gold electrode

An abasic site-containing DNA combined with lumiflavin allows amperometric determination of single nucleotide polymorphism through hydrogen bond-mediated nucleobase recognition in water by using abasic sites as a molecular recognition field.