Calculated frequency spectrum of Z-form poly(dG-dC).poly(dG-dC)

The calculated phonon spectrum of Z-form poly(dG-dC).poly(dG-dC) between 400 and 1600 cm-1 is reported. Comparison with the available data shows the very good agreement between theory and experiment. The eigenvector displacement is used to assign the characteristics of some of the important modes.     

Fluorescence-detected circular dichroism of ethidium bound to poly(dG-dC) and poly(dG-m5dC) under B- and Z-form conditions

The equilibrium binding of ethidium to poly(dG-dC) and poly(dG-m5dC) under conditions favoring B and Z forms was investigated with fluorescence-detected circular dichroism (FDCD) and optical titration methods. FDCD spectra indicate a similar geometry for the intercalated ethidium under both B- and Z-form conditions, even at low levels of bound ethidium. The magnitude of the 310-330-nm FDCD band as a function of the bound drug to base pair ratio (r) indicates ethidium binds to poly(dG-dC) in 4.4 M NaCl and to poly(dG-m5dC) in 25 mM MgCl2 by clustering. Under these conditions, circular dichroism spectra indicate the polymer is largely Z form. Thus, it appears ethidium clusters into regions it has induced into a right-handed form. For all conditions studied, the FDCD spectra provided no evidence for a left-handed binding site. Under B-form conditions, binding is random.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Comparison between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinum (II): immunological and spectroscopic studies

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

Alkaline titrations of poly(dG-dC).poly(dG-dC): microemulsion versus solution behavior

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid. In solution, the apparent pK(a) of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK(a) on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R(OH), defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R(OH) has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK(a) on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.     

Circular dichroism of polynucleotides: Interactions of NiCl2 with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) in a water-in-oil microemulsion

The thermal behavior of the synthetic, high molecular weight, double stranded polynucleotides poly(dA-dT).poly(dA-dT) [polyAT] and poly(dG-dC).poly(dG-dC) [polyGC] solubilized in the aqueous core of the quaternary water-in-oil cationic microemulsion CTAB|n-pentanol|n-hexane|water in the presence of increasing amounts of NiCl(2) at several constant ionic strength values (NaCl) has been studied by means of circular dichroism and electronic absorption spectroscopies. In the microemulsive medium, both polynucleotides show temperature-induced modifications that markedly vary with both Ni(II) concentration and ionic strength. An increase of temperature causes denaturation of the polyAT duplex at low nickel concentrations, while more complex CD spectral modifications are observed at higher nickel concentrations and ionic strengths. By contrast, thermal denaturation is never observed for polyGC. At low Ni(II) concentrations, the increase of temperature induces conformational transitions from B-DNA to Z-DNA form, or, more precisely, to left-handed helical structures. In some cases, at higher nickel concentrations, the CD spectra suggest the presence of Z'-type forms of the polynucleotide.     

Hoechst 33258--poly(dG-dC).poly(dG-dC) complexes of three types

It was found recently that Hoechst 33258, a dsDNA fluorescent dye used in cytological studies, is an efficient inhibitor of the interaction of TATA-box-binding protein with DNA, DNA topoisomerase I, and DNA helicases. In addition it proved to be a radioprotector. Biological activity of Hoechst 33258 may be associated with dsDNA complexes of not only monomeric, but also dimeric type. In this work, the Hoechst 33258 interaction with poly(dG-dC).poly(dG-dC) was studied using UV-vis and fluorescent spectroscopy, circular and flow-type linear dichroism. It was found that Hoechst 33258 formed with poly(dG-dC).poly(dG-dC) complexes of three types, namely, monomeric, dimeric, and, apparently, tetrameric, and their spectral properties were studied. Complexes of monomeric and dimeric types competed with distamycin A, a minor groove ligand, for binding to poly(dG-dC).poly(dG-dC). We proposed that Hoechst 33258 both monomers and dimers form complexes of the external type with poly(dG-dC).poly(dG-dC) from the side of the minor groove.     

B-Z transition in poly(dG-dC).poly(dG-dC) in the presence of formaldehyde amino derivatives

It was shown by circular dichroism that the B-Z transition of poly(dG-dC).poly(dG-dC) in high NaCl concentrations occurred more rapidly in the presence of formaldehyde and Tris. The product of formaldehyde and glycine interaction induces changes in the poly(dG-dC).poly(dG-dC) CD spectral characteristics of a 'B-like' conformation. It is supposed that the B-Z transition occurs without large-scale hydrogen bond breakage.     

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.     

Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC).

The solution properties of the B and Z forms of poly(dG-dC).poly(dG-dC) have been measured by static and dynamic laser light scattering. The radius of gyration, persistence length, translational and segmental diffusion coefficients, and the Rouse-Zimm parameters have been evaluated. The persistence length of the Z form determined at 3 M NaCl is about 200 nm compared to 84 and 61 nm respectively for the B forms of poly(dG-dC).poly(dG-dC), and calf thymus DNA, both determined at 0.1 M NaCl. The data on persistence length, diffusion coefficients and the Rouse-Zimm parameters indicate a large increase in the chain stiffness of Z DNA compared to the B form. These results are opposite to the ionic strength effects on random sequence native DNAs, for which the flexibility increases with ionic strength and levels off at about 1 M NaCl.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

Effect of basic oligopeptides on the B-Z transition of poly(dG-dC). poly(dG-dC) in water-methanol solutions

The effect of basic oligopeptides (Lys-Ala-Ala)n (n = 1-5, 10) and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-dC).poly(dG-dC) in water-methanol solutions was investigated using CD and uv spectroscopy. In the absence of peptides, the concentration of methanol at the midpoint of the B-Z transition is 64% at 25 degrees C. The transition is temperature dependent and the B conformation is preferred at higher temperatures. All peptides tested shift the midpoint of the B-Z transition to lower concentrations of methanol. For shorter peptides this effect increases with an increasing number of monomeric units, showing the importance of the number of positive charges in the peptide molecule. Al conditions of low methanol content, the trimer and tetramer of the (Lys-Leu-Ala)n series have a greater effect on the B-Z transition than the corresponding oligomers of the (Lys-Ala-Ala)n series. This indicates an important influence of the presence of hydrophobic groups in the peptide side chains on the binding. In the presence of peptides, the B-Z transition is also temperature dependent and the B conformation is preferred at higher temperatures. The addition of peptides results in an increase of the transition midpoint and of the transition width. These parameters were used for the calculation of the transition enthalpy delta HB-Z in 65% methanol, which is -1.15 +/- 0.25 kcal/base pair. Since the van't Hoff enthalpy delta HVH calculated from the temperature dependence of the B-Z transition in the absence of peptides is -130 kcal/mol, the length of the cooperative unit is about 110 base pairs. The results suggest that the mechanism of Z-DNA induction is similar but not identical with that involved in the action of metal cations in aqueous solution.     

Conformations of poly(dG-dC).poly(dG-dC) modified by the O-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) has been modified by reaction with 4-acetoxyaminoquinoline 1-oxide (Ac-4 HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide. The circular dichroism (CD) spectra of the modified and unmodified polymers have been compared under various experimental conditions. The CD spectra were recorded in 1 mM phosphate, 50% (v/v) ethanol, 3.8 M LiCl and 95% (v/v) ethanol, conditions in which poly(dG-dC).poly(dG-dC) adopts the B-, Z-, C- and A-form respectively. In 1 mM phosphate buffer, poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO seems not to contain regions in the Z-form. Z-form induction could be progressively obtained by the addition of ethanol as follows: in the buffer with about 30% ethanol the modified polymer started to adopt the Z structure, while 40% of ethanol in the buffer was necessary for the unmodified polymer. In the 50% ethanol-1 mM phosphate buffer mixture (v/v), poly(dG-dC).poly(dG-dC) was entirely in the Z-form while poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO remained partially in the B-form. Enzymatic digestions with the nuclease S1 which is specific of the single-stranded DNA were carried out in order to support the modified poly(dG-dC).poly(dG-dC) CD study conclusions. The role played by the two major adducts on the conformational characteristics of modified polymer is discussed.     

Comparison of the conformation of poly(dI-dC) with poly(dI-dbr5C) and the B and Z forms of poly(dG-dC). One- and two-dimensional NMR studies

One- and two-dimensional nuclear Overhauser effects (2D NOE) have been used to compare the conformational properties of 60-80 base pair long duplexes of the synthetic DNA polymer poly(dI-dC) with those of poly(dI-dbr5C) and poly(dG-dC) in the B and Z conformations. Cross peaks in the 2D NOE spectra arising from proton-proton dipolar interactions which are more or less independent of the DNA conformation are used to assign the spectra of these molecules. Other cross peaks are sensitive to the conformational details, and these are used to make deductions about the average conformation in solution. The proton-proton interactions that give rise to the cross peaks in the 2D NOE spectrum of poly(dI-dC) are indicative of a B family conformation and rule out the possibility of some alternative conformations, including A, Z, alternating B, and left-handed B-DNA. The spectra are similar to those obtained from B-form poly(dI-dbr5C) and poly(dG-dC) but different from Z-form poly(dG-dC). Taken together, these results indicate that the solution conformation of poly(dI-dC) is not unusual but more closely resembles that of other B-form DNAs.     

Experimental and calculated study of the vibrational modes of poly(dG-dC).poly(dG-dC) in B and Z conformations

Infrared spectra of the B and Z forms of poly(dG-dC).poly(dG-dC) are presented. Experimental assignments relative to certain vibration modes have been confirmed by calculation based on the GF-Wilson method. The calculated results show that only the geometry change between B and Z forms, is responsible for the observed modifications in the vibrational spectra.     

NMR relaxation and the structure of a synthetic DNA poly(dA-dT).poly(dA-dT)

1H-1H and 31P-1H nuclear Overhauser effects and 31P NMR spin-lattice relaxation times were measured for a synthetic DNA poly(dA-dT).poly(dA-dT) in a low-salt aqueous solution. The results have shown that all bases in the double helix are anti-orientated with respect to deoxyribose residues and that the sugar-phosphate backbone has an alternating architecture.     

1H NMR relaxation studies of protein-polysaccharide mixtures

NMR water proton relaxation was used to characterize the structure of plant proteins and plant protein-polysaccharide mixtures in aqueous solutions. The method is based on the mobility determination of the water molecules in the biopolymer environment in solutions through relaxation time measurements. Differences of conformation between pea globulin and alpha gliadin seem to control the water molecules mobility in their environment. As deduced from the study of complexes, the electrostatic interactions may also play a major role in the water molecule motions. The phase separation induced under specific conditions seems to promote the translational diffusion of structured water molecules whereas the rotational motion was more restricted.