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. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23-mer AP aptamer (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/5'-GTG TGC GTT GCC CTG GAC GCA GA-3'; X: the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 µM. Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and 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.

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 (10(6) 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 degrees 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 (DeltaC(p)), 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 (DeltaG(obs)) is dissected into its component terms.

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. From isothermal titration calorimetric experiments, we find that ligand incorporation into the AP sites is primarily enthalpy-driven. Examination of ionic strength dependency of ligand binding with DNA reveals that ligand-DNA complexation is a manifestation of both electrostatic and nonelectrostatic interactions and that the contribution from the nonelectrolyte effect is fundamental for the stabilization of the ligand-DNA complex. In comparison to riboflavin, reported previously as a T-selective ligand, lumiflavin binds to the DNA much more strongly and is a more promising ligand for efficient detection of T-related single nucleotide polymorphisms.

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 titration experiments reveal that, as compared to a parent ligand, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), capable of selectively binding C over T opposite an AP site in the duplex (K(d)/nM: C: 56, T: 100), APATMND shows a stronger binding affinity for T, while an affinity for C is reduced (K(d)/nM: C: 135, T: 37). An examination of salt dependence of binding constants reveals that a polyelectrolyte contribution (Delta G(pe)) is indeed increased for C- and T-bindings of APATMND, but a loss of non-polyelectrolyte contribution (Delta G(t)) is significant when binding to C. These binding properties of APATMND are discussed with a view towards further development of DNA-binding ligands suitable for gene detection.

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.

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.

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 = 0.11 M, at 20 degrees C). In this work, AMND is modified simply by introducing two methyl groups to the naphthyridine ring. The present ligand, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), shows a stronger binding affinity for cytosine, and a dissociation constant reaches 56 nM. ATMND is effectively applicable to the analysis of cytosine-related mutation of PCR products. These promising abilities of ATMND are presented based on the examination by melting temperature (T(m)) and fluorescence measurements.

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 7.0). From the analysis of salt dependence of binding constants, polyelectrolyte (DeltaG(pe)) and non-polyelectrolyte (DeltaG(t)) contributions are calculated as -1.7 kcal/mol and -6.9 kcal/mol, respectively, at 110 mM Na(+) concentration. The binding enthalpy determined by isothermal titration calorimetry (ITC) is -18.5 kcal/mol in 110 mM Na(+) at 20 degrees C. We discuss these results with a view towards further development of our ligand-based fluorescence assay for SNPs (single nucleotide polymorphisms) typing.

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.

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 7.0) containing 100 mM NaCl and 1.0 mM EDTA, Vitamin B2 is found to selectively bind to T (K11=1.8x10(6) M(-1) at 5 degrees C) over other nucleobases, and this is accompanied by significant quenching of its fluorescence. While the sensing functions depend on the flanking sequences to the AP site, Vitamin B2 is applicable to the detection of T/C (cytosine), T/G (guanine) and T/A (adenine) mutation sequences of the CYP2A6 gene, where the flanking nucleobases are guanines in both positions (-GXG-, X=AP site).

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 = 0.11 M), AMND is found to selectively recognize cytosine (C) base over other nucleotides, and the 1:1 association constant reaches 3.2x10(5) M(-1) when binding to C. The ligand-nucleotide interaction results in significant fluorescence quenching of AMND, which is highly selective to C. These sensing functions of AMND at the gap site are utilized for the development of ligand-based fluorescence assay for SNPs (single-nucleotide polymorphisms) typing.

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 = 2.9 x 10(5) M(-1)), and the fluorescence intensity at 424 nm is significantly quenched only when binding to T. It is highly likely that the selective interaction of CDPC with T is explained by a pseudo-base pairing, for which three point hydrogen bonds are formed along the Watson-Crick edge of the target T. These binding functions of CDPC at the AP site are presented to develop ligand-based fluorescence assay for SNPs (single-nucleotide polymorphisms) typing.

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, 7.0) containing 100mM NaCl and 1.0mM EDTA, AMND is found to strongly bind to C (K(d)=1.5x10(-6)M) in the target ODN while the binding affinity for G is relatively moderate (K(d)=50x10(-6)M). Significant fluorescence quenching of AMND is observed only when binding to C, making it possible to judge the C/G transversion with the naked eye.