Competitive assay for theophylline based on an abasic site-containing DNA duplex aptamer and a fluorescent ligand.

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin.

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes.

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.

2-Aminopurine-modified abasic-site-containing duplex DNA for highly selective detection of theophylline.

2-Aminopurine-modified abasic-site-containing duplex [DNA 5'-TCTGC GTCCT PXT TAACG CACAC-3'/3'-AGACG CAGGA TCA ATTGC GTGTG-5'; P = 2-aminopurine, X = abasic site (Spacer-C3), C = receptor base] is capable of selectively binding to the bronchodilator theophylline with a dissociation constant of 10 microM (5 degrees C, pH 7.0, I = 0.11 M) and is applicable to monitoring serum theophylline concentrations.

Small-molecule binding at an abasic site of DNA: strong binding of lumiflavin for improved recognition of thymine-related single nucleotide polymorphisms.

The binding behavior of lumiflavin, a biologically vital ligand, with DNA duplexes containing an abasic (AP) site and various target nucleobases opposite the AP site is studied. Lumiflavin binds selectively to thymine (T) opposite the AP site in a DNA duplex over other nucleobases. Using 1H NMR spectroscopy and fluorescence measurements, we show that ligand-DNA complexation takes place by hydrogen-bond formation between the ligand and the target nucleobases and by stacking interactions between the ligand and the nucleobases flanking the AP site.

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 detection of cytosine/guanine transversion based on a hydrogen bond forming ligand.

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate potential use of a hydrogen bond forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), for the fluorescence detection of the cytosine (C)/guanine (G) mutation sequence of the cancer repression gene p53. Our method is based on construction of the AP site in ODN duplexes, which allows small synthetic ligands to bind to target nucleobases accompanied by fluorescence signaling: an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. In 10mM sodium cacodylate buffer solutions (pH, 7.

Effect of an alkyl amino group on the binding of 1,8-naphthyridines to AP site-containing DNA duplexes.

A 1,8-naphthyridine derivative having a positively charged side-chain, N-(3-aminopropyl)-5,6,7-trimethyl-1,8-naphthyridine-2-amine (APATMND), is synthesized, and its binding to AP site-containing DNA duplexes (5'- GCA GCT CCC GXG GTC TCC TCG-3'/ 5'-CGA GGA GAC CNC GGG AGC TGC-3', X = AP site; dSpacer, N = C, T) is examined in solutions buffered to pH 7.0 (I = 0.11 M, at 20 degrees C).

Fluorescence and electrochemical detection of pyrimidine/purine transversion by a ferrocenyl aminonaphthyridine derivative.

A novel hydrogen bond-forming ligand for pyrimidine/purine transversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.

Strong binding of naphthyridine derivatives to cytosine in an AP site-containing DNA duplex and their application to fluorescence detection of single nucleotide polymorphisms.

Here we report on a significant enhancement of the binding affinity of naphthyridine-based fluorescence ligands that can selectively bind to cytosine opposite an AP site in a DNA duplex (5'-TCC AGX GCA AC-3'/3'-AGG TCC CGT TG -5', X = AP site, C = target). We have previously reported that 2-amino-7-methyl-1,8-naphthyridine (AMND) binds to cytosine with a dissociation constant of 370 nM (pH 7.0, I = 0.

Electrochemical and fluorescence detection of cytosine-related SNPs using a ferrocenyl naphthyridine derivative.

A novel hydrogen bond-forming ligand for cytosine-related single nucleotide polymorphism, which contains both a fluorescent naphthyridine moiety and a ferrocene group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase within an abasic site-containing DNA duplex was confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1: 1 complex with the target nucleobase opposite an AP site, and the selectivity was in the order of cytosine > thymine > adenine, guanine, reflecting the stability of hydrogen bond formation.

Use of vitamin B2 for fluorescence detection of thymidine-related single-nucleotide polymorphisms.

In combination with abasic site (AP site)-containing DNAs, potential use of a biotic fluorescence compound, Vitamin B2 (riboflavin), is demonstrated for the fluorescence detection of the thymine (T)-related single-nucleotide polymorphisms. Our method is based on construction of the AP site in DNA duplexes, which allows small ligands to bind to target nucleotides accompanied by fluorescence signaling: an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleotides through stacking and hydrogen-bonding interactions. In 10 mM sodium cacodylate buffer solutions (pH 7.

Thermodynamic characterization of the binding of naphthyridines to the AP site-containing DNA duplexes.

We here report on the thermodynamics of the hydrogen bond-mediated binding of 2-amino-7-methyl-1,8-naphthyridine (AMND) to a cytosine base opposite an abasic site (AP site) in a 21-meric DNA duplex (5'-GCA GCT CCC GXG GTC TCC TCG-3'/3'-CGT CGA GGG CCC CAG AGG AGC-5', X= AP site, C = target). The examination by fluorescence titration experiments shows a 1:1 binding constant of 2.7x10(6) M(-1) at 20 degrees C in solutions containing 110 mM Na(+) (pH 7.

Fluorescence detection of thymidine-related single-nucleotide polymorphisms by 3, 5-diaminopyrazine derivatives.

We report on a highly selective fluorescence ligand for thymine (T) base opposite an abasic site (AP site) in DNA duplexes. From the examination of the binding behaviors in solutions buffered to pH 7.0, we find that 6-chloro-3,5-diamino-2-pyrazinecarbonitrile (CDPC) can selectively recognize T with high affinity (Ka = 2.

Hydrogen bond-mediated binding of ligands to a nucleobase at a gap site in a DNA duplex and its use for fluorescence detection of single-nucleotide polymorphisms.

Here we report on the strong and selective binding of a hydrogen bond-forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), to a nucleobase at a gap site in DNA duplexes. In solutions buffered to pH 7.0 (at 20 degrees C, I = 0.

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.

Strong and selective binding of amiloride to thymine base opposite AP sites in DNA duplexes: simultaneous binding to DNA phosphate backbone.

Amiloride (N-amidino-3,5-diamino-6-chloro-pyrazinecarboxamide hydrochloride) has two sets of hydrogen-bond forming sites suitable for target nucleotides and the phosphodiester DNA backbone by which a thymine base opposite an abasic site in DNA duplexes can be recognized with high selectivity and affinity, and it is applicable to the fluorescence detection of thymidine-related SNPs (single-nucleotide polymorphisms) of PCR amplification products.