Probing the Site-Selective Binding of an Antiretroviral Drug,Stavudine to Calf Thymus Dna

The interaction of an anti-HIV drug, stavudine (STV) with calf thymus deoxyribonucleic acid (DNA) was investigated employing acridine orange (AO) as a fluorescence probe. Spectroscopic investigations revealed the intercalative mode of binding of STV to DNA. The analysis of fluorescence data indicated the presence of static quenching mechanism between STV and DNA. Thermodynamic parameters indicated the presence of van der Waals forces in addition to intercalative mode of binding. CD data revealed the partial B→A conformational transition of DNA upon intercalative mode of binding with STV.     

Poly(ADP-ribose) polymerase-1 dimerizes at a 5' recessed DNA end in vitro: a fluorescence study

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.     

The binding of an avian myeloblastosis virus basic 12,000 dalton protein to nucleic acids.

The binding of a basic 12,000 dalton protein (p12) from avian myeloblastosis virus to viral RNA and heterologous DNA has been investigated. The binding stoichiometries and constants were determined by an extrinsic fluorescence assay. In both cases each bound p12 molecule occupies four nucleotides and the apparent binding constant is approximately 1 x 10(6) M-1. Binding is non-cooperative and there is no apparent difference in the interaction of p12 with viral RNA or heterologous single-strand DNA. The relative binding constant at various ionic strengths was assayed by the nitrocellulose filter procedure. Analysis of the data revealed that each bound p12 molecule forms three ion pairs with the nucleic acid.     

Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Synthesis, and DNA-binding and DNA-photocleavage properties of multiply charged porphyrins

Four new cationic porphyrins, compounds 1-4, with five to seven positive charges, were synthesized, characterized, and investigated for their binding properties towards calf-thymus DNA (CT-DNA). UV/VIS and fluorescence-titration data indicated strong binding, the apparent binding constants (K(app); (1.3-10)x10(-6) M) increasing with increasing number of charges, as determined by competitive fluorescence titration using ethidium bromide (EB) as molecular probe. These results were qualitatively confirmed by the observed photocleavage efficiency of the porphyrins towards plasmid pBR322 DNA.     

Synthesis, DNA binding, and cleavage studies of novel PNA binding cyclen complexes

A novel coumarin‐appended PNA binding cyclen derivative ligand, C1 , and its copper(II) complex, C2 , have been synthesized and characterized. The interaction of these compounds with DNA was systematically investigated by absorption, fluorescence, and viscometric titration, and DNA‐melting and gel‐electrophoresis experiments. DNA Melting and viscometric titration experiments indicate that the binding mode of C1 is a groove binding, and C2 is a multiple binding mode that involves groove binding and electrostatic binding. From the absorption‐titration data, we can state that the primary interaction between CT DNA and the two compounds may be H‐bonds between nucleobases. Fluorescence studies indicate that the binding ability of C1 to d(A)₉ is as twice or thrice as that of other oligodeoxynucleotides. Agarose gel‐electrophoresis experiments demonstrate that C2 is an excellent chemical nuclease, which can cleave plasmid DNA completely within 24 h.     

Measurement of the absolute temporal coupling between DNA binding and base flipping

The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 相似文献   

Evidence for nonintercalative complexes formed from the reversible binding of benzo[a]pyrene metabolites to closed-circular, single-stranded M13mp19 DNA

The fluorescence excitation spectrum of complexes formed from the reversible binding of the proximate carcinogen, trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene (BP78D) to closed-circular, single-stranded, viral M13mp19 DNA (SS M13 DNA) exhibits a red-shift of 5 nm compared to the spectrum of BP78D measured without DNA or with native, calf thymus DNA. In SS M13 DNA which is 0.10 mM in PO4-, the fluorescence intensity of BP78D is 2.3 times smaller than the intensity measured without DNA; however, the fluorescence lifetime (42.7 nsec) of BP78D with SS M13 DNA is 1.7-1.8 times larger than the lifetimes of BP78D measured without DNA or with calf thymus DNA. These results are consistent with the conclusion that, in addition to binding sites which cause fluorescence quenching, SS M13 DNA contains sites which permit formation of BP78D inclusion complexes that have weaker interactions with nucleotide bases than those occurring in intercalated complexes. The association constant (1.45 +/- 0.01 x 10(5) M-1) for the binding of BP78D to SS M13 DNA is more than 9.0 times larger than that for binding to calf thymus DNA. It is 7.1 times larger than that for the binding of the less genotoxic metabolite, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (BP45D) to SS M13 DNA. UV Photoelectron data and results from ab initio molecular orbital calculations suggest that a difference in polarizability contributes to the greater SS M13 DNA binding of BP78D compared to that of BP45D.     

Interactions of diastereomeric tripeptides of lysyl-5-fluorotryptophyllysine with DNA. 1. Optical and 19F NMR studies of native DNA complexes

Lysyl-5-fluoro-L-tryptophyllysine and lysyl-5-fluoro-D-tryptophyllysine were synthesized, and their interactions with double-stranded DNA were investigated as a model for protein-nucleic acid interactions. The binding to DNA was studied by monitoring various 19F NMR parameters, the fluorescence, and the optical absorbance in thermal denaturation. The 19F resonance of the L-Trp peptide shifts upfield in the presence of DNA, and that of the D-Trp peptide shifts downfield with DNA present. The influence of ionic strength on the binding of each peptide to DNA and the fluorescence quenching titration of each with DNA indicate that electrostatic bonding (approximately 2 per peptide-DNA complex) dominates the binding in each case and accounts for the similar binding constants determined from the fluorescence quenching, i.e., 7.7 X 10(4) M-1 for the L-Trp complex and 6.2 X 10(-1) for the D-Trp complex. The 19F NMR chemical shift, line width, 19F[1H] nuclear Overhauser effect, and spin-lattice relaxation time (T1) changes all indicate that the aromatic moiety of the L-Trp complex, but not that of the D-Trp complex, is stacked between the bases of DNA. The relative increases in DNA melting temperature caused by binding of the tripeptide diastereomers are also consistent with stacking in the case of the L-Trp peptide. The magnitude of the changes and the susceptibility of the 19F NMR chemical shift to altering the solvent isotope (H2O vs. D2O) suggest that the L-Trp ring is not intercalated in the classical sense but is partially inserted between the bases of one strand of the double helix.     

HU binding to DNA: evidence for multiple complex formation and DNA bending

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.     

Study on the interaction between cannabinol and DNA using acridine orange as a fluorescence probe

The interaction between cannabinol (CBN) and herring‐sperm deoxyribonucleic acid was investigated by using acridine orange as a fluorescence probe in this work. UV‐Vis spectroscopy, fluorescence spectroscopy, and DNA melting techniques were used. The fluorescence of DNA acridine orange was quenched by CBN. The results indicated that CBN can bind to DNA. The binding constant for the CBN and herring‐sperm deoxyribonucleic acid was obtained at 3 temperatures, respectively. Results of molecular docking corroborated the experimental results obtained from spectroscopic investigations. The influence of ionic strength on the fluorescence properties was also investigated. The thermodynamic results indicated that hydrophobic interaction played a major role in the binding between CBN and DNA.     

The DNA binding activity of p53 displays reaction-diffusion kinetics

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.     

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double-stranded DNA, thus behaving like a DNA-melting protein.     

New phthalimide‐appended Schiff bases: Studies of DNA binding,molecular docking and antioxidant activities

Herein, we investigated new phthalimide‐based Schiff base molecules as promising DNA‐binding and free radical scavenging agents. Physicochemical properties of these molecules were demonstrated on the basis of elemental analysis, ultraviolet–visible (UV–Vis), infra‐red (IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. All spectral data are agreed well with the proposed Schiff base framework. The DNA‐binding potential of synthesized compounds were investigated by means of UV–visible, fluorescence, iodide quenching, circular dichroism, viscosity and thermal denaturation studies. The intrinsic binding constants (K _b) were calculated from absorption studies were found to be 1.1 × 10⁴ and 1.0 × 10⁴ M^?1 for compounds 2a and 2b suggesting that compound 2a binding abilities with DNA were stronger than the compound 2b. Our studies showed that the presented compounds interact with DNA through groove binding. Molecular docking studies were carried out to predict the binding between Ct‐DNA and test compounds. Interestingly, in silico predictions were corroborated with in vitro DNA‐binding conclusions. Furthermore, the title compounds displayed remarkable antioxidant activity compared with reference standard.     

Interaction of quinacrine mustard with mononucleotides and polynucleotides

1. The interaction between quinacrine mustard and mononucleotides and polynucleotides was investigated by fluorimetry and absorbance spectrophotometry. 2. The fluorescence spectrum of quinacrine mustard is independent of the ionic strength and pH. The dependence of the quinacrine mustard fluorescence intensity on ionic strength, pH and anions is described. 3. The fluorescence intensity of quinacrine mustard was enhanced with the mononucleotide adenylic acid and polynucleotides such as poly(rA), poly(rU) and poly(rA,rU). 4. Quenching of the fluorescence intensity of quinacrine mustard occurred with the mononucleotide guanylic acid and with poly(rG) and poly(rC,rG). 5. The mononucleotide cytidylic acid or poly(rC) showed no effect on the fluorescence intensity of quinacrine mustard. 6. The interaction between the dye and native DNA species was also dependent on the presence of base-specific binding sites in the DNA. The higher the (G+C) content was in the native DNA tested the higher was the quenching effect on the fluorescence intensity of quinacrine mustard. 7. No interaction was found between the dye and methylated DNA. The binding between quinacrine mustard and apurinic DNA was confirmed to be in the phosphate groups of the purines.     

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity

BACKGROUND: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS: The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE: Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.     

Studies on the interaction of isoxazolcurcumin with calf thymus DNA

The interaction of isoxazolcurcumin (IOC), a synthetic derivative of curcumin, with calf thymus-DNA (ct-DNA) has been investigated by UV-Vis, fluorescence, circular dichroism spectroscopies, viscosity measurements and docking studies. From these analyses, the binding constant, number of binding sites and mode of binding of IOC to ct-DNA has been determined. The binding constant of IOC to DNA calculated from both UV-Vis and CD spectra was found to be in the 10(4)M(-1) range. Analyses of fluorescence spectra, viscosity measurements and molecular modeling of IOC-DNA interactions indicate that IOC is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide displacement studies revealed that IOC did not have any effect on ethidium bromide bound DNA which is indicative of groove binding. To elucidate the preferred region of binding of IOC to DNA, docking studies have been performed and changes in accessible surface area (DeltaASA) of nucleobases determined due to IOC-DNA complexation.