Numerical simulations of Raman spectra of guanine-cytosine Watson-Crick and protonated Hoogsteen base pairs

Large changes in the Raman spectra of calf thymus DNA are observed upon lowering the pH. In order to gain a better insight into these effects, several simulations of the Raman spectra of the guanine-cytosine (GC) Watson-Crick and Hoogsteen base pairs are performed. By comparing the Raman bands of GC base pairs in calf thymus DNA at high and low pH with the theoretical simulations of GC base pairs, it is found that the intensity changes in the theoretical bands located between 400 and 1000 cm(-1) are small compared to the experimental ones. The behavior of the cytosine band at 1257 cm(-1) upon lowering the pH is not reproduced in the GC theoretical spectra. The bands located above 1300 cm(-1) in the theoretical spectra display intensity changes that are similar to those found for GC base pairs in calf thymus DNA spectra.     

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).     

Comparative study of two cephalosporin antibiotics binding to calf thymus DNA by multispectroscopy,electrochemistry, and molecular docking

The over‐use of antibiotics has caused a number of problems such as contamination of antibiotic residues and virus resistance, and therefore has attracted global attention. In this study, spectroscopic techniques and molecular docking were employed to predict conformational changes and binding interaction between two cephalosporins (cefaclor and cefixime) and calf thymus DNA (ctDNA). Fluorescence and UV–vis spectra suggested that static quenching was predominant and cephalosporin bound to the groove region of ctDNA. Binding parameters calculated by the Stern–Volmer and Scatchard equations showed that cephalosporin bound to ctDNA with a binding affinity in the order of 10³ L mol^?1. Thermodynamic parameters further indicated that the reaction was a spontaneous process driven by enthalpy and entropy, and that the main binding force was an electrostatic force. The effects of iodide, denaturant, thermal denaturation and pH on a cephalosporin–Hoechst–DNA complex were also studied, and the results confirmed that cephalosporin bound to the groove area of DNA. Finally, these results were further confirmed by molecular docking and electrochemical studies.     

Resonance Raman spectroscopic studies of adriamycin and copper(II)-adriamycin and copper(II)-adriamycin-DNA complexes

Characteristic resonance Raman spectra are observed on ionization of the phenolic groups in adriamycin. On the basis of these results, vibrational assignments for the Raman bands of adriamycin are reported. Distinct Raman spectra are observed for Cu(II)-adriamycin complexes at pH approximately 5 and pH approximately 13. The data indicate that at lower pH a bis complex of Cu(II) is formed, which transforms to a polymeric Cu(II) chelate at higher pH. Upon interaction of the metal-drug complex with calf thymus DNA at pH approximately 5, a ternary complex is formed in which the Cu(II)-complexed adriamycin is intercalated into DNA.     

Form of DNA and the nature of interactions with proteins in chromatin.

Studies of native chromatins and of isolated nucleosomes (from calf thymus) show that the DNA is in the B form or modified B form. This was determined by Raman spectroscopy of chromatins, of nucleosomes (from calf thymus) and of DNA fibres and directly correlated with X-ray diffraction studies. The Raman spectra of three forms of DNA (A, B and C) have been characterized in fibres both by X-ray diffraction and Raman spectroscopy on the same sample. In particular, the Raman spectrum of the C form of DNA is characterized by a band of about 870 cm(-1). For the first time, chromatins of different origins with increasing content of non-histone proteins have been investigated by Raman spectroscopy. The site of interaction of the non-histone proteins appears to involve the N7 position of guanine while the histone core does not interact at this site. It is proposed that the mechanism of specific recognition in chromatin involves the large groove.     

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (?I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo‐denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of Ca2+ cations on low pH-induced DNA structural transitions

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.     

Thermal stability and DNA binding activity of a variant form of the Sso7d protein from the archeon Sulfolobus solfataricus truncated at leucine 54

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.     

Formation of 8-hydroxydeoxyguanosine in DNA treated with fecapentaene- 12 and -14

Fecapentaene-12 and -14, direct-acting mutagens in human feces, were found to hydroxylate the C-8 position of guanine residues in DNA in vitro. Fecapentaene-12 or -14 was incubated with 0.5 mg of calf thymus DNA in 1 ml of reaction mixture at pH 7.4 for 2 h at 37°C in the dark, and then 8-hydroxydeoxyguanosine (8-OH-dG) was analyzed. In these conditions 8-OH-dG was formed dose-dependently at levels of 1.1–4.6 residues/10⁴ dG with concentrations of 0.5–3.0 mM of fecapentaene-12. Similar results were obtained with fecapentaene-14. The amount of 8-OH-dG in untreated DNA was 0.2–0.3 residue/10⁴ dG.     

The effect of HCl on the solution structure of calf thymus DNA: A comparative study of DNA denaturation by proton and metal cations using fourier transform IR difference spectroscopy

The interaction of HCl with calf thymus DNA was investigated in aqueous solution at pH 7-2 with H⁺/DNA(P)(P:phosphate) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform (FTIR) difference spectroscopy. Correlations between spectral changes, proton binding mode, DNA denaturation, and conformational variations are established. A comparison was also made between their spectra of denaturated DNA, in the presence of proton and Cu ions with similar cation concentrations. The FTIR difference spectroscopic results have shown that at low proton concentrations of r = 1/80 and 1/40 (pH 7–5), no major spectral changes occur for DNA, and the presence of H⁺ results in an increased base-stacking interaction and helical stability. At higher proton concentrations of r > 1/40, the proton binding to the cytosine and adenine bases begins with major destabilization of the helical duplex. As base protonation progresses, a B to C conformational conversion occurs with major DNA spectral changes. Protonation of guanine bases occurs at a high cation concentration r > 1/2 (pH < 3) with a major increase in the intensity of several DNA in-plane vibrations. Copper ion complexation with DNA exhibits marked similarities with proton at high cation concentrations (r > 1/10), whereas at low metal ion concentrations, copper–PO₂ and copper–guanine N-7 bindings are predominant. No major DNA conformational transition was observed on copper ion complexation. © 1995 John Wiley & Sons, Inc.     

Electrochemically induced oxidative damage to double-stranded calf thymus DNA adsorbed on gold electrodes

Electrochemically induced oxidative damage to DNA was studied with double-stranded calf thymus DNA immobilized directly on a gold electrode surface. Pre-polarization of the DNA-modified electrodes at +0.5 V versus Ag/AgCl reference electrode, in a free from DNA blank buffer solution, pH 7.4, allowed for subsequent detection of direct electrochemical oxidation of adsorbed on gold DNA, in the potential range from +0.7 to +0.8 V. The redox potential of the process corresponded to the potentials of the oxidation of guanine bases in DNA. It is shown that with increasing potential scan rate, v, the mechanism of electrochemical oxidation of DNA changes from the irreversible 4e^– oxidative damage of DNA at low v to reversible 1e^– oxidation at high v, keeping the electrochemical activity of the adsorbed DNA layer virtually the same.     

Melting and premelting phenomenon in DNA by laser Raman scattering.

Raman spectra of DNA from calf thymus DNA have been taken over a wide range of temperatures (25°–95°) in both D₂O and H₂O. A study of the temperature dependence of the Raman spectra shows that the temperature profiles of the intensities and frequencies of the various bands fall into four different categories: (1) base bands that show a reversible increase in intensity prior to the melting region, i.e., a definite premelting phenomenon; (2) base bands that show little or no temperature dependence; (3) deoxyribose-phosphate backbone vibrations that show no temperature dependence up to the melting region, at which point large decreases in intensity occur; and (4) slow frequency changes in certain in-plane vibrations of guanine and adenine due to deuteration of the C-8 hydrogen of these purines in D₂O. Certain Raman bands arising from each of the four bases, adenine, thymine, guanine, and cytosine have been found to undergo a gradual increase in intensity prior to the melting region at which point large, abrupt increases in intensity occur. The carbonyl stretching band of thymine, involved in the interbase hydrogen bonding actually undergoes both a gradual shift to a lower frequency as well as an increase in intensity. These changes provide evidence that some change in the geometry of the bases relative to each other begins to occur around 50°C, well below the melting region of 70°–85°C. From the spectra taken at various temperatures, the DNA appears to remain in the B conformation until the melting point is reached, at which time the DNA progresses into a disordered random-coil form. No A-form conformation is found either in the premelting or the melting region.     

Adenine and guanine 8CH exchange in nucleic acids: resolution and measurement by Raman optical multichannel analysis

Deuterium exchange of 8C protons of adenine and guanine in nucleic acids is conveniently monitored by laser Raman spectrophotometry, and the average exchange rate so determined [kA + kG] can be exploited as a dynamic probe of the secondary structure of DNA or RNA [J. M. Benevides and G. J. Thomas, Jr. (1985) Biopolymers 24, 667-682]. The present work describes a rapid Raman procedure, based upon optical multichannel analysis, which permits discrimination of the different 8CH exchange rates, kA of adenine and kG of guanine, in a single experimental protocol. For this procedure, simultaneous measurements are made of the intensity decay or frequency shift in separately resolved Raman bands of adenine and guanine, each of which is sensitive only to 8C deuteration of its respective purine. Resolution of the rates kA and kG is demonstrated for the mononucleotide mixtures, 5'-rAMP + 5'-rGMP and 5'-dAMP + 5'-dGMP, for the polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), for calf thymus DNA, and for the 17 base-pair operator OR3. We show that the different exchange rates of adenine and guanine, in nucleotide mixtures and in DNA, may also be calculated independently from intensity decay of the composite 1481-cm-1 band, comprising overlapped adenine and guanine components, over a time domain that encompasses two distinct regimes: (1) a relatively more rapid exchange of guanine, and (2) a concurrent slower exchange of adenine. Both methods developed here yield consistent results. We find, first, that exchange of guanine is approximately twofold more rapid than that of adenine when both purines are present in the same structure and solvent environment, presumably a consequence of the greater basicity of the 7N site of guanine. Second, we find that adenine suffers greater retardation of exchange than guanine when both purines are incorporated into a "classical" B-DNA secondary structure, such as that of calf thymus DNA. This finding suggests different microenvironments at the 7N-8C loci of adenine and guanine in aqueous B-DNA. We also confirm that adenine residues of B-form poly(dA-dT).poly(dA-dT) exchange much more slowly than those of other B-DNA sequences, implying a secondary structure for the alternating-AT sequence with unusual stereochemistry in the major groove. The greater resistance of adenine than guanine to 8CH exchange in the B-DNA secondary structure is more evident in high molecular weight calf thymus DNA and in the alternating AT and GC copolymer duplexes than in the smaller 17 base-pair operator OR3.(ABSTRACT TRUNCATED AT 400 WORDS)     

抗癌药物ADM与DNA相互作用的紫外共振拉曼光谱的研究

用紫外共振拉曼光谱研究了ＡＤＭ与小牛胸腺ＤＮＡ相互作用,分析表明：ＡＤＭ插入ＤＮＡ的ＧＣ－ＣＧ位置;ＡＤＭ与ＤＮＡ之间的主要相互作用是蒽环π电子与碱基Ｇ和Ｃ的π电子形成的π－π电子相互作用,并通过碱基Ｇ、Ｃ的ＮＨ＿２的氨的孤对Ｐ电子与ＡＤＭ的π电子形成的π－Ｐ电子相互作用以及ＡＤＭ和ＤＮＡ碱基Ｇ、Ｃ和ＰＯ＿２之间形成的氢键使相互作用加强;ＡＤＭ插入ＤＮＡ使其构象产生一定变化,但未破坏ＤＮＡ碱基对间的氢键。     

Differential scanning calorimetry of chromatin at different levels of condensation

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.     

The effect of Cr(III) upon the thermal denaturation of DNA

Thermal denaturation studies of Cr-DNA solutions at pH 6.0 were carried out by monitoring the uv absorbance at 260 nm. The melting curves of solutions of calf thymus and Escherichia coli DNA with added Cr(ClO₄)₃ were broadened and shifted to higher temperatures. As the ratio of Cr: DNA increased, the melting temperature increased until it reached a maximum at Cr: DNA ratios of 0.7 (E. coli) and 0.9 (calf thymus). At higher concentrations of Cr³⁺ the melting temperature decreased and then leveled off, but it never fell as low as that of the pure DNA.     

Laser Raman identification of an interaction site on DNA for arginine containing histones in chromatin.

Comparisons of the Raman spectra of DNA, chromatin, and complexes of DNA with poly-L-arginine and N-α-acetylarginine have been made. Both in native chromatin and in complexes of DNA with the arginine derivatives there is a marked decreased in the Raman intensity of the 1490±2 cm^?1 band due to guanine. Considerable evidence is presented to show that a decrease in the intensity of the 1490 cm^?1 Raman band of quanine in DNA is strong indication of a hydrogen bond being attached to the N-7 position of quanine. A specific model is presented for the interaction of the arginine residues with the guanine residues in the major groove of DNA. The Raman frequency of the histone Amide 1 band indicates that these protein molecules have a high α-helical content while the phosphate diester stretch frequency of the DNA shows the DNA to be in the B-family.     

Histone-like protein in the prokaryote Thermoplasma acidophilum.

The DNA of the prokaryote Thermoplasma acidophilum is associated with a histone-like protein that has the following properties: it has a high content (23%) of basic amino acids, is positively charged at neutral pH, is soluble in acid, and can stabilize DNA against thermal denaturation. In polyacrylamide gel electrophoresis, in the presence of either sodium dodecylsulfate or urea, it migrates at the same rate as histone IV (F2a1) of calf thymus. The amino acid composition, however, it unusually rich in the amides of acidic amino acids (16-20%), and it does not appear to be closely homologous to any of the classes of eukaryotic histones. Escherichia coli DNA, on the other hand, was associated with no detectable acid-soluble proteins, and the nucleoprotein thermally denatured at a lower temperature than pure DNA.     

Dynamic light-scattering studies of internal motions in DNA. III. Evidence for titratable joints associated with bound polycations

Dynamic light-scattering techniques are employed to study the internal Brownian motions of a commercial calf thymus DNA, clean and contaminated ?29 DNAs, and a clean ?29 DNA with bound spermidine as a function of pH. The Rouse-Zimm model parameters of both calf thymus and contaminated ?29 DNAs differ substantially from those of clean ?29 DNA in the neutral-pH region. However, this difference is largely removed by adding 0.01M EDTA (which has no effect on clean ?29 DNA) to the calf thymus DNA sample. These findings imply the existence in that preparation of polycation contaminants, presumably basic proteins, that can substantially alter the local mechanical properties of the DNA near their binding sites. The internal motion parameters k_BT/f and b of both calf thymus and contaminated ?29 DNAs are found to exhibit pronounced characteristic variations between pH 8.5 and 10.5, over which range there is essentially no detectable titration to a resolution of about 1% of the base pairs. These variations, which are not observed for clean ?29 DNA, are qualitatively similar to those previously reported for a ?29 DNA with 21 single-strand breaks per chain. This indicates the formation of titratable joints associated with bound polycation contaminants. These basic ligands presumably facilitate local denaturation by stabilizing the titration of one or more protons on base-ring nitrogens near their binding sites. Spermidine binding up to 85–87% of neutralization of the total DNA charge has only a relatively minor effect on the internal motion parameters at neutral pH in 0.01M NaCl. However on raising the pH to 10.2, the internal motion parameter k_BT/f undergoes a marked decrease similar to that observed for both calf thymus and contaminated ?29 DNAs and also ?29 DNA with single-strand breaks. This indicates that spermidine, too, is capable of inducing titratable joints. Evidence is presented that the titratable joints associated with bound polycations on the calf thymus DNA may serve primarily as torsion joints, as was found previously for the titratable joints associated with single-strand breaks.     

Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.