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

Abstract

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.

Figure 1.

(A) Schematic illustration of the ligand binding to nucleotides opposite an AP site in a DNA duplex. For the detection of SNPs, an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site towards a target nucleotide, by which a hydrophobic binding pocket is provided for aromatic ligands to bind to target nucleotide. (B) Structures of the series of 2-amino-1,8-naphthyridines examined in this work.

Figure 2.

Thermal denaturation profiles of a 11-meric AP site-containing DNA duplex [5′ -TCC AGX GCA AC-3′/3′-AGG TCC CGT TG-5′, X = AP site (dSpacer), C = target cytosine]. (a) DNA alone, and in the presence of (b) AND, (c) AMND, (d) ADMND and (e) ATMND. [DNA duplex] = 30 µM, [ligand] = 580 µM, in 100 mM NaCl, 1.0 mM EDTA and 10 mM sodium cacodylate (pH 7.0). Absorbance of DNA was measured at 260 nm as a function of temperature, which ranged from 2°C to 92°C with a heating rate of 1.0°C/min. Optical path length = 1 mm.

Figure 3.

Fluorescence reponses of ATMND (500 nM) to 21-meric AP site-containing DNA duplex [5′-GCA GCT CCC GXG GTC TCC TCG-3′/3′-CGT CGA GGG CCC CAG AGG AGC-5′, X = AP site (dSpacer), C = target cytosine], measured in solutions buffered to pH 7.0 (10 mM sodium cacodylate) containing 100 mM NaCl and 1.0 mM EDTA. Excitation wavelength, 350 nm; temperature, 20°C. Inset: nonlinear regression analysis of the changes in the fluorescence intensity ratio at 403 nm based on a 1:1 binding isotherm model. F and F₀ denote the fluorescence intensities of ATMND in the presence and absence of DNA duplexes, respectively.

Figure 4.

Fluorescence titration curves for the binding of (A) AND (5.0 µM), (B) AMND (1.0 µM), (C) ADMND (1.0 µM) and (D) ATMND (0.5 µM) to 21-meric AP site-containing DNA duplexes [5′-GCA GCT CCC GXG GTC TCC TCG-3′/3′-CGT CGA GGG CNC CAG AGG AGC-5′, X = AP site (dSpacer), N = G, C, A or T]. Sample solutions were buffered to pH 7.0 with 10 mM sodium cacodylate, containing 100 mM NaCl and 1.0 mM EDTA. Excitation wavelength, 350 nm; temperature, 20°C. Analysis: AND, 392 nm; AMND, 400 nm; ADMND, 400 nm; ATMND, 403 nm.

Figure 5.

ITC data obtained at 20°C for the addition of ATMND aliquots (each 15 µl of 175 µM) into the solution containing DNA duplex [1.43 ml of 20 µM, 5′-GCA GCT CCC GXG GTC TCC TCG-3′/3′-CGT CGA GGG CCC CAG AGG AGC-5′, X = AP site (dSpacer), C = target cytosine]. Sample solutions were buffered to pH 7.0 with 10 mM sodium cacodylate, containing 100 mM NaCl and 1.0 mM EDTA. The data were best fitted to a model that assumes a single set of identical binding sites, giving the binding enthalpy (ΔH_obs) of −12.8 kcal/mol with a binding stoichiometry (n) of 1.1.

Figure 6.

Temperature dependence of the binding enthalpy for 2-amino-1,8-naphthyridine-cytosine interactions: (a) AND and (b) ATMND. Errors are the standard deviations obtained from at least three independent measurements. The linear least squares fit to the data yielded the heat capacity change, ΔC_p, of –161 cal/mol K for AND (r = 0.9906), and –217 cal/mol K for ATMND (r = 0.9999), respectively. See Table 2 for further details.

Figure 7.

Proposed binding modes of 2-amino-1,8-naphthyridines with cytosine or thymine opposite the AP site in DNA duplexes.

Figure 8.

Fluorescence detection of single-base mutation of PCR products present in (A) sense strand and (B) antisense strand of K-ras gene (107-mer, codon 12). After PCR, the products were analyzed in solutions buffered to pH 7.0 with 100 mM sodium cacodylate containing 1.6 mM EDTA, 50 nM ATMND and 5.0 μM AP site-containing probe DNA. Excitation 350 nm; detection 403 nm. Temperature 5°C.