Interaction of DNA with actinocin amides containing cationoid centers in the amide groups

Complexes of DNA with actinocin derivatives containing ω-dialkylaminoalkyl groups in the1 and/or 9 positions of the chromophore were studied by spectrophotometric titration, circular dichroism, and viscometry. Induced circular dichroism (ICD) spectra of the DNA-ligand complexes were compared for the cases of the complexes of known structure established by other methods. It was shown that the presence of an isoelliptic point in the long-wavelength absorption band of the ICD spectra of the ligand under monomeric binding conditions could indicate intercalation of the actinocin chromophore into DNA. The separation of the cationoid center and the chromosphore upon elongation of the methylene chain increases the aggregability of the ligand pn the surface of the DNA double helix, which prevents the intercalation of the chromophore.     

Dependence of mode of DNA binding to benzo crown derivatives of actinocin on the size and distance from the chromophore of crown fragments

Complexes of DNA with benzocrown derivatives of actinocin were studied by viscometry and dynamic birefringence. Changes in the macromolecular structure of DNA caused by complex formation were determined. Models of DNA binding to the studied compounds were suggested on the basis of data obtained. The intercalation of actinocin chromophore of benzocrown derivatives of actinocin was shown to occur only when benzocrown groups were bound to the chromophore via glycine fragment. A change in the distance between the crown group and the chromophore prevents ligand intercalation. Increase in medium ionic strength results in appearance of new nonintercalational binding mode for crown-containing compounds with DNA caused by interaction of the crown groups with DNA.     

Dependence of Mode of DNA Binding to Benzocrown Derivatives of Actinocin on the Size and Distance from the Chromophore of Crown Fragments

Complexes of DNA with benzocrown derivatives of actinocin were studied by viscometry and dynamic birefringence. Changes in the macromolecular structure of DNA caused by complex formation were determined. Models of DNA binding to the studied compounds were suggested on the basis of data obtained. The intercalation of actinocin chromophore of benzocrown derivatives of actinocin was shown to occur only when benzocrown groups were bound to the chromophore via the glycine spacer. A change of the distance between the crown group and the chromophore prevented ligand intercalation. Increase of the ionic strength resulted in appearance of a new, nonintercalative binding mode for crown-containing compounds determined by interaction of the crown groups with DNA.     

Effect of a substituent origin in actinocin amides on their binding to DNA

The results of studies on the structure of complexes of DNA with compounds based on the actinocin chromophore, a center of binding of the antitumor antibiotic actinomycin D to DNA, were analyzed. In positions 1 and 9 of the chromophore of these compounds, pentapeptide lact ones of actinomycin D are replaced by groups of various origin. By using spectral, optical, and hydrodynamic methods a model of binding to DNA for each compound was constructed, and some regularities of complex formation depending on the structure of actinocin substituents and the amount of ligands in the complex were revealed.     

Interaction of DNA with benzocrown derivatives of actinocin

Complexes of DNA with actinocin derivatives containing benzocrown groups at the 1 and/or 9 positions of the chromophore were studied by spectrophotometric titration and circular dichroism. The actinocin chromophore and the crown fragments are the binding sites of the ligands with DNA. The mode of ligand-DNA binding is shown to depend on the size of the crown group, its distance to the actinocin chromophore, and the ionic strength of the medium. Selective toward Na+ ion benzocrown fragments combine with DNA phosphate groups. The simultaneous interaction of the actinocin chromophore with the DNA bases is possible only at optimal distance between both the binding sites of ligand molecule.     

Interaction of DNA with Benzocrown Derivatives of Actinocin

Complexes of DNA with actinocin derivatives containing benzocrown groups at positions 1 and/or 9 of the chromophore were studied by spectrophotometric titration and circular dichroism. The actinocin chromophore and the crown fragments are the DNA-binding sites of the ligands. The mode of ligand–DNA binding is shown to depend on the size of the crown group, its distance to the actinocin chromophore, and the ionic strength of the medium. Na⁺-selective benzocrown fragments combine with DNA phosphate groups. The simultaneous interaction of the actinocin chromophore with the DNA bases is possible only at an optimal distance between the two binding sites of ligand molecule.     

Flexible docking of DNA fragments and actinocin derivatives

We have applied molecular docking methods to systems containing nucleic acids as targets and biologically active substances as ligands. The complexes of DNA fragments and actinocin derivatives with different lengths of aminoalkyl side chains were obtained by molecular docking. It was observed that actinocin derivatives could form energetically favourable complexes with DNA both as intercalators and minor groove binders. It was shown that small changes in the binding energy (～1?kcal/mol) could result in complexes with substantially different structure. The complexes of actinocin derivatives and DNA fragments were stabilized by hydrogen bonding upon intercalation and minor groove binding. It was found that the change of solvent-accessible surface area upon binding of the actinocin derivative to DNA linear increased with the growth of methylene groups' number in ligand side chains. The solvation energy change upon binding of actinocin derivatives to DNA calculated by the WSAS method was favourable in the case of small uncharged ligands and unfavourable for positively charged ligands.     

Effect of synthetic carrier ampholytes on saturation of human serum transferrin.

We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds.     

Interaction of actinocin derivative with different poly(rC) structures

The interaction of actinocin derivative Act III with single- and double-stranded poly(rC) has been investigated by the methods of differential scanning microcalorimetry and UV-vis absorption spectroscopy. It was shown that, after the addition of the ligand, the temperature, enthalpy and entropy of poly (rC) melting decrease. The analysis of poly(rC)-ActIII absorption spectra indicated that the conformation of polynucleotide differs from that of free poly (rC) in the presence of ActHI at pH 4.46 and pH 6.0. Using the DALSMOD optimization program, the parameters of interaction of Act III with poly (rC) were calculated. It was found that the binding constant of ActHI with double-stranded poly (rC) is essentially higher than that with the single-stranded one upon monomeric binding. On the basis of these data, we conclude that the conformation changes of the matrix are the main cause of the decrease in melting temperature and enthalpy observed by calorimetry. Possible mechanisms of interaction of actinocin derivative with poly (rC) are discussed.     

Subclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5-6S and the other at 14-16S. The DNA-binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA-protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5-6-S and the 14-16-S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5-6-S subclass to viral control region DNA was optimal at 60-80 mM NaCl while specific DNA binding of the 14-16-S form was optimal at 150-200 mM NaCl; however, binding of the 14-16-S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5-6S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH 6-6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7-7.5. Binding of the 14-16-S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5-6-S form.     

DNA binding and anticancer activity of naphthalimides with 4-hydroxyl-alkylamine side chains at different lengths

A series of novel naphthalimide derivatives modified with various hydroxyl-alkylamines at 4-position have been synthesized. Their DNA binding properties were investigated by UV-Vis, fluoescence, and circular dichroism (CD) spectroscopies and thermal denaturation. The results showed that compounds 3a-e as the DNA intercalator exhibited middle binding affinities with Ct-DNA. The anticancer activities of 3a-e were preliminarily evaluated, compounds 3c and 3e exhibited potent anticancer activities against Bel-7402 cell line with IC(50) values of 5.57 and 9.17μM, respectively. More interestingly, enhancement of the fluorescence emission was found in the complexes of 3a-e with Ct-DNA, especially for 3c. This would make these compounds as potential DNA staining agents.     

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.     

Study of effect of water on the interaction of DNA and actinocin derivatives with various aminoalkyl chain length by infrared spectrophotometry and computer modeling

A comparative study of the effect of water on the interaction of DNA with actinocin derivatives having different numbers of methylene groups in side chains was performed by IR spectroscopy. It was found that, as relative humidity increases, water molecules simultaneously bind to hydrate-active sites of DNA and ligands. The absorption band at v = 1137 cm-1, caused by oscillations of the C-O and P-O groups of atoms in the DNA-ligand complex having two methylene groups, is due to the interactions between the cationic groups of the ligand and the sugar-phosphate backbone of DNA, which may be one of the reasons for the high stability of this complex. Using computer simulation of interaction of DNA fragments and actinocin derivatives in water environment, molecular models of the formation of their complexes for two ways of binding were constructed.     

Spectrometric and voltammetric investigation of interaction of neutral red with calf thymus DNA: pH effect

The interaction of neutral red (NR) with calf thymus DNA (CT DNA) was investigated by spectrometric (UV-vis, circular dichroism and fluorescence) and voltammetric techniques. It was shown that the interaction of NR with DNA depended on the values of R (R is defined as the ratio of the concentration of NR to that of CT DNA) and pH of the solution. NR intercalated into CT DNA base pairs at lower R value (R < 2.4) and following by NR aggregating along the helical surface of DNA at higher R value (R > 2.4) in pH 6.0 solution. Interestingly, we found that at lower R value, NR intercalated into CT DNA with its long axis perpendicular or parallel to the dyad axis of DNA in the solution of pH 6.0. While in pH 7.0 solution, NR bound with CT DNA through intercalation and electrostatic interactions. The electrochemical inactive complexes, NR-2CT DNA, 3NR-CT DNA, and NR-CT DNA were formed when NR interacted with nucleic acids in pH 6.0 and 7.2 solutions, respectively. The corresponding intrinsic binding constants for these complexes were obtained by UV-vis and fluorescence spectrometric methods, respectively. The CD spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to left-handed Z-DNA due to the aggregating of NR along the surface of DNA in pH 6.0 solution, whereas a conversion from B-DNA to C-DNA was induced due to the interaction of DNA with NR in pH 7.2 solution. Finally, two binding modes of NR with CT DNA in aqueous with different values of pH were shown in the scheme.     

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.     

Synthesis and evaluation of oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids as novel gene expression control compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T(m) values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.     

Comparative circular dichroism and fluorescence studies of oligodeoxyribonucleotide and oligodeoxyribonucleoside methylphosphonate pyrimidine strands in duplex and triplex formation

An analogue of the homopyrimidine oligodeoxyribonucleotide d(CT)8 has been synthesized. This analogue, d(CT)8 contains nonionic methylphosphonate internucleoside linkages. The pH-dependent conformational transitions of d(CT)8 have been studied and its ability to form duplexes and triplexes with the normal homopurine oligonucleotide d(AG)8 has also been investigated as a function of pH. Circular dichroism spectroscopy and ethidium bromide fluorescence enhancement have been used to monitor pH-dependent conformational transitions driven by the protonation of cytosine residues, and the different behavior of d(CT)8 and d(CT)8 has been compared. It was possible to form self-associated complexes by using either d(CT)8 or d(CT)8, and both compounds combined with d(AG)8 to form duplex or triplex DNA. At neutral pH, the CD spectrum of d(AG)8.d(CT)8 duplex was quite different from the CD spectrum of d(AG)8.d(CT)8 duplex, reflecting most likely a difference in conformation. The duplex to triplex transition characteristic of this DNA sequence occurred at a lower pH when d(CT)8 was substituted for d(CT)8; however, at pH 4.2, triplex containing d(CT)8 was similar in conformation to triplex containing d(CT)8. Several of these observations can be related to the alterations in electrostatic and steric interactions that occur when the negatively charged phosphodiester backbone of d(CT)8 is replaced with a nonionic methylphosphonate backbone.     

Conformation and reactivity of DNA in the complex with proteins. III. Helix-coil transition and conformational studies of model complexes of DNA''s with poly-L-histidine.

Differences in the interaction of poly-L-histidine with DNA of various base composition have been demonstrated using melting and CD measurements. The two types of complexes formed with DNA at pH values below the pK of 5.9 and in the region of pH 6.5 are very different in their CD spectral properties. The binding effects with highly protonated poly-L-histidine are AT-dependent as reflected by large negative CD spectra indicating the formation of psi-DNA as a condensed state of the double helix. GC-rich DNA may, however, also form psi-DNA structures with poly-L-histidine under certain conditions. At pH 6.5 complex formation with the weakly protonated polypeptide is GC-dependent. From the results it is concluded that protonated poly-L-histidine interacts more specifically at AT base pairs, prabably along the small groove while the weakly protonated poly-L-histidine tends to interact preferentially with GC regions which seems to occur rather in the large groove.     

Optical studies of the interaction of 33258 Hoechst with DNA,chromatin, and metaphase chromosomes

The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.     

Synthesis and Evaluation of Oligonucleotide-Conjugated Pyrrole Polyamide-2′-Deoxyguanosine Hybrids as Novel Gene Expression Control Compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2′-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T_m values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.