DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants K(que) = 7.25 x 10(4)M(-1), K(kae) = 3.60 x 10(4)M(-1), and K(del) = 1.66 x 10(4)M(-1). The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.     

A comparative study of DNA complexation with Mg(II) and Ca(II) in aqueous solution: major and minor grooves bindings

Although structural differences for the Mg-DNA and Ca-DNA complexes are provided in the solid state, such comparative study in aqueous solution has been less investigated. The aim of this study was to examine the bindings of Mg and Ca cations with calf thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (1.25 or 12.5 mM) and various concentrations of metal ions (2 microM-650 microM). Capillary electrophoresis, UV-visible, and Fourier transform infrared spectroscopic methods were used to determine the cation-binding modes, the binding constants, and DNA structural variations in aqueous solution. Direct Ca-PO(2) binding was evident by major spectral changes (shifting and splitting) of the backbone PO(2) asymmetric stretching at 1222 cm(-1) with K = 4.80 x 10(5) M(-1), whereas an indirect Mg-phosphate interaction occurred (due to the lack of shifting and splitting of the phosphate band at 1222 cm(-1)) with K = 5.6 x 10(4) M(-1). The metal-base bindings were directly for the Mg with K = 3.20 x 10(5) M(-1) and indirectly for the Ca cation with K = 3.0 x 10(4) M(-1). Both major and minor groove bindings were observed with no alteration of the B-DNA conformation.     

Thymol and carvacrol binding to DNA: model for drug-DNA interaction

Thymol and carvacrol can bind to major and minor grooves of B-DNA. The aim of this study was to examine the interaction of calf thymus DNA with thymol and carvacrol in aqueous solution and physiological pH with thymol/DNA and carvacrol/DNA (phosphate) molar ratios of 1/20, 1/10, 1/5, and 1/1. Fourier transform infrared and UV-visible difference spectroscopy were used to determine the thymol and carvacrol binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of thymol/DNA and carvacrol/DNA complexes in aqueous solution. Spectroscopic evidence showed that the thymol and carvacrol interaction occurred mainly through H-bonding of the thymol and carvacrol OH group to the guanine N7, cytosine N3, and backbone phosphate group with overall binding constant of K(thymol-DNA) = 2.43 x 10(3) M(-1), K(carvacrol-DNA) = 1.55 x 10(3) M(-1). In thymol and carvacrol-DNA complexes, DNA remains in the B-family structure.     

DNA interaction with saffron's secondary metabolites safranal, crocetin, and dimethylcrocetin

Saffron comes from the dried red stigmas of the Crocus sativus L. flower. Except for its use in cooking and in traditional medicine, it has numerous applications as an antitoxic, antioxidant, and anticancer agent due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). However, there has been no information on the interactions of these secondary metabolites with individual DNA at molecular level. This study was designed to examine the interaction of safranal, crocetin (CRT), and dimethylcrocetin (DMCRT) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) molar ratios from 1/48 to 1/2. FTIR and UV-visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants, and the effects of carotenoids and safranal complexation on the stability and conformation of DNA duplex. Both intercalative and external binding modes were observed, with overall binding constants K(safranal) = 1.24 x 10(3) M(-1), K(CRT) = 6.2 x 10(3) M(-1) and K(DMCRT) = 1.85 x 10(5) M(-1) A partial B- to A-DNA transition occurs at high carotenoids and safranal concentrations.     

Study of DNA interactions with steroidal and nonsteroidal estrogen-platinum (II)-based anticancer drugs

The interactions of the steroidal and nonsteroidal estrogen-platinum (Pt) (II)-based anticancer drugs 16beta-hydroxymethyl-16alpha-[8-(2-pyridin-2-yl-ethylamino)-3,6-dioxaoctyl]-1,3,5(10)-estratrien-3,17betadiol dichloroplatinum (II) (JPM-39), 4-[6-(2'-pyridylethylamino)-butyloxy)-phenyl]-7-methoxy-2,2-dimethyl-3-phenyl-chroman dichloroplatinum (II) (ATG-99), and 1-[(2-aminoethyl)amino]-9,10,10-tris(4-hydroxyphenyl)-9-decene dichloroplatinum (II) (GEB-28) with calf-thymus DNA in vitro using constant DNA concentration and various drug levels were studied. Fourier transform infrared (FTIR) and circular dichroism (CD) were studied with calf-thymus DNA in vitro using constant DNA concentration and various drug levels. FTIR, UV-visible, and CD spectroscopic methods were used to characterize the drug binding mode, the binding constant, and structural variations of DNA in aqueous solution. Spectroscopic evidence showed that the various Pt-based drugs bind indirectly to the major and minor grooves of DNA duplex with some degree of drug-phosphate interaction. The overall binding constants for JPM-39, GEB-28, and ATG-99 are K(JPM-39) = 4.2 (+/-0.75) x 10(3) M(-1), K(GEB-28) = 3.4 (+/-0.65) x 10(3) M(-1), and K(ATG-99) = 2.1 (+/-0.45) x 10(3) M(-1). DNA aggregation occurs at high drug concentration, while DNA remains in the B-family structure.     

DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.     

DNA interaction with antitumor polyamine analogues: a comparison with biogenic polyamines

Biogenic polyamines, putrescine, spermidine, and spermine, are ubiquitous cellular cations and exert multiple biological functions. Polyamine analogues mimic biogenic polyamines at macromolecular level but are unable to substitute for natural polyamines and maintain cell proliferation, indicating biomedical applications. The mechanistic differences in DNA binding mode between natural and synthetic polyamines have not been explored. The aim of this study was to examine the interaction of calf thymus DNA with three polyamine analogues, 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333), and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333), using FTIR, UV-visible, and CD spectroscopy. Polyamine analogues bind with guanine and backbone PO2 group as major targets in DNA, whereas biogenic polyamines bind to major and minor grooves as well as to phosphate groups. Weaker interaction with DNA was observed for analogues with respect to biogenic polyamines, with K(333) = 1.90 (+/-0.5) x 10(4) M(-1), K(BE-333) = 6.4 (+/-1.7) x 10(4) M(-1), K(BE-3333) = 4.7 (+/-1.4) x 10(4) M(-1) compared to K(Spm) = 2.3 (+/-1.1) x 10(5) M(-1), K(Spd) = 1.4 (+/-0.6) x 10(5) M(-1), and K(Put) = 1.02 (+/-0.5) x 10(5) M(-1). A partial B- to A-DNA transition was also provoked by analogues. These data suggest distinct differences in the binding of natural and synthetic polyamines with DNA.     

Thallium-DNA complexes in aqueous solution. Major or minor groove binding

Thallium (Tl) binds to the major and minor grooves of B-DNA in the solid state (Howerton et al., Biochemistry 40, 10023-10031, 2001). The aim of this study was to examine the binding of Tl(I) cation with calf-thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (12.5 mM) and various concentrations of metal ions (0.5 to 20 mM). UV-visible and FTIR spectroscopic methods were used to determine the cation binding site, the binding constant and DNA structural variations in aqueous solution. Direct Tl bindings to guanine and thymine were evident by major spectral changes of DNA bases with overall binding constant of K = 1.40 x 10(4) M(-1) and little perturbations of the backbone phosphate group. Both major and minor groove bindings were observed with no alteration of the B-DNA conformation. At low metal concentration (0.5 mM), the number of cations bound were 10 per 1000 nucleotides, while at higher cation concentration (10 mM), this increased to 30 cations per 1000 nucleotides.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Interaction of antioxidant flavonoids with tRNA: intercalation or external binding and comparison with flavonoid-DNA adducts

Antioxidants are essential to good health. Flavonoids are powerful antioxidants, and prevent DNA damage. The antioxidative protections are related to their binding modes to a DNA duplex and complexation with free radicals in vivo. Recently we reported the interaction of flavonoids with DNA in vitro (Kanakis et al., J. Biomol. Struct. Dyn. 22, 719-724, 2005), where polyphenol different binding modes were discussed. The aim of this study was to examine the interaction of transfer RNA with quercetin (que), kaempferol (kae), and delphinidin (del) in aqueous solution at physiological conditions and to make a comparison with the corresponding pigment-DNA adducts. Constant tRNA concentration (6.25 mM) and various drug/RNA(phosphate) molar ratios of 1/48 to 1/8 were used. FTIR and UV-visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants, and the effects of drug complexation on the stability and conformation of tRNA duplex. Both intercalative and external binding modes were observed. Structural analysis showed que, kae, and a del intercalate tRNA duplex with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants K (que) = 4.80 x 10(4) M(1), K (kae) = 4.65 x 10(4) M(1), and K (del) = 9.47 x 10(4) M(1). The stability of adduct formation is in the order of del > que > kae. A comparison with flavonoids-DNA adducts showed both intercalation and external bindings with the stability order K (que) = 7.25 x 10(4) M(1), K (kae) = 3.60 x 10(4) M(1), and K (del) = 1.66 x 10(4) M(1). Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial Bto A-DNA transition occurs at high drug concentration, while tRNA remains in the A-family structure.     

Interaction of tRNA with Safranal, Crocetin, and Dimethylcrocetin

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K(safranal) = 6.8 (+/- 0.34) x 10(3) M(-1), K(CRT) = 1.4 (+/- 0.31) x 10(4) M(-1), and K(DMCRT) = 3.4 (+/- 0.30) x 10(4) M(-1). Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.     

Electrochemical determination of interaction parameters for DNA and mitoxantrone in an irreversible redox process

Mitoxantrone (MXT), an anti-tumor antibiotic, shows irreversible electrochemical behavior at a waxed graphite electrode in a 0.05 M Tris-HCl buffer (pH 7.4) solution. The interaction between MXT and calf thymus DNA (ctDNA) in solution has been studied using cyclic voltammetry. An electrochemical equation suitable for examining the binding of irreversibly electroactive molecules to DNA is established. Determination of diffusion coefficients of both free and binding MXT (D(f), D(b)), the binding constant (K) and binding site size (s base pairs per molecule, bp) of MXT with DNA was performed on the basis of the equation. A nonlinear fit analysis of the experimental data yielded: D(f)=3.76 x 10(-5) cm(2)s(-1), D(b)=2.73 x 10(-7) cm(2)s(-1), K=8.7 x 10(9) cm(3)mol(-1), s=2.8 bp. The results demonstrate that MXT binds tightly to ctDNA and covers three base pairs. The anthraquinone of MXT, which is a planar heterocyclic ring, intercalates between the DNA's base pairs. The two aminoethylamino side-chains of the drug fit to the major groove reinforce the combination of MXT and DNA. The results show that MXT is a DNA intercalator with a high binding constant compared to those of other anthraquinones.     

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p: reinterpretation of recent single molecule experiments

Brewer et al. (Biophys. J. 85 (2003) 2519-2524) have studied the compaction of dsDNA in a double flow cell by observing the extension of stained DNA tethered in buffer solutions with or without Abf2p. They use a Langmuir adsorption model in which one Abf2p molecule adsorbs on one site on the DNA, and the binding constant, K, is given as the ratio of the experimental rates of adsorption and desorption. This paper presents an improved interpretation. Instead of Langmuir adsorption we use the more appropriate McGhee-von Hippel (J. Mol. Biol. 86 (1974) 469-489) theory for the adsorption of large ligands to a one-dimensional lattice. We assume that each adsorbed molecule shortens the effective contour length of DNA by the foot print of Abf2p of 27 base pairs. When Abf2p adsorbs to DNA stretched in the flowing buffer solution, we account for a tension effect that decreases the adsorption rate and the binding constant by a factor of 2 to 4. The data suggest that the accessibility to Abf2p decreases significantly with increasing compaction of DNA, resulting in a lower adsorption rate and a lower binding constant. The kinetics reported by Brewer et al. (Biophys. J. 85 (2003) 2519-2524) lead to a binding constant K=3.6 x 10(6) M(-1) at the beginning, and to K=5 x 10(5) M(-1) near the end of a compaction run, more than an order of magnitude lower than the value K=2.57 x 10(7) M(-1) calculated by Brewer et al. (Biophys. J. 85 (2003) 2519-2524).     

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.     

Interaction of glycyrrhizin and glycyrrhetinic acid with DNA

Glycyrrhizin (GL), a molecule of glycyrrhetinic acid (GA), is an aqueous extract from licorice root. These compounds are well known for their anti-inflammatory, hepatocarcinogenesis, antiviral, and interferon-inducing activities. This study is the first attempt to investigate the binding of GL and GA with DNA. The effect of ligand complexation on DNA aggregation and condensation was investigated in aqueous solution at physiological conditions, using constant DNA concentration (6.25?mM) and various ligands/polynucleotide (phosphate) ratios of 1/240, 1/120, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2, and 1/1. Fourier transform infrared and ultraviolet (UV)-visible spectroscopic methods were used to determine the ligand binding modes, the binding constants, and the stability of ligand-DNA complexes in aqueous solution. Spectroscopic evidence showed that GL and GA bind DNA via major and minor grooves as well as the backbone phosphate group with overall binding constants of K(GL-DNA)=5.7×10(3) M(-1), K(GA-DNA)=5.1×10(3) M(-1). The affinity of ligand-DNA binding is in the order of GL>GA. DNA remained in the B-family structure, whereas biopolymer aggregation occurred at high triterpenoid concentrations.     

Effects of some flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

The effects of six flavonoids viz., apigenin, genistein, morin, naringin, pelargonidin and quercetin on the susceptibility of low-density lipoprotein (LDL) to oxidative modification were investigated. Flavonoids were added to plasma and incubated for 3 hr at 37 degrees C, and the LDL fraction was separated by ultracentrifugation. Oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS), after cupric sulfate solution was added. Quercetin and morin significantly (P<0.01 by ANOVA) prolonged the lag time before initiation of oxidation reaction in dose-dependent manner. They also suppressed the formation of lipid peroxides and TBARS more markedly than other flavonoids. The ability to prolong lag time and suppression of lipid peroxides and TBARS formation was in the following order: quercetin >morin >pelargonidin >genistein >naringin >apigenin. LDL exposed to flavonoids reduced oxidizability. These findings suggest that flavonoids may have a role in ameliorating atherosclerosis.