Four-stranded DNA helices: conformational analysis of regular poly(dT).poly(dA).poly(dA).poly(dT) helices with various types of base binding

The paper presents results obtained in conformational analysis of homopolymeric four-stranded poly(dT).poly(dA).poly(dA).poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

The molecular electrostatic potential of the B-DNA helix. VI. The regions of the base pairs in poly (dG.dC) and poly (dA.dT).

The evaluation of the electrostatic molecular potential at important nucleophilic sites of the purine and pyrimidine bases in poly (dG.dC) and poly (dA.dT) and of the evolution of the potential through the series free bases-nucleosides-nucleotides-single polynucleotide helices-double helices enables the interpretation of the evolution of the corresponding reactivity of the bases towards a series of electrophilic carcinogenic and mutagenic reactants.     

Temperature dependence of the Raman spectrum of DNA. II. Raman signatures of premelting and melting transitions of poly(dA).poly(dT) and comparison with poly(dA-dT).poly(dA-dT).

The temperature dependence of the Raman spectrum of poly(dA).poly(dT) (dA: deoxyadenosine; dT: thymidine), a model for DNA containing consecutive adenine.thymine (A.T) pairs, has been analyzed using a spectrometer of high spectral precision and sensitivity. Three temperature intervals are distinguished: (a) premelting (10 < t < 70 degrees C), in which the native double helix is structurally altered but not dissociated into single strands; (b) melting (70 < t < 80 degrees C), in which the duplex is dissociated into single strands; and (c) postmelting (80 < t degrees C), in which no significant structural change can be detected. The distinctive Raman difference signatures observed between 10 and 70 degrees C and between 70 and 80 degrees C are interpreted in terms of the structural changes specific to premelting and melting transitions, respectively. Premelting alters the low-temperature conformation of the deoxyribose-phosphate backbone and eliminates base hydrogen bonding that is distinct from canonical Watson-Crick hydrogen bonding; these premelting perturbations occur without disruption of base stacking. Conversely, melting eliminates canonical Watson-Crick pairing and base stacking. The results are compared with those reported previously on poly(dA-dT).poly(dA-dT), the DNA structure consisting of alternating A.T and T.A pairs (L. Movileanu, J. M. Benevides, and G. J. Thomas, Jr. Journal of Raman Spectroscopy, 1999, Vol. 30, pp. 637-649). Poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) exhibit strikingly dissimilar temperature-dependent Raman profiles prior to the onset of melting. However, the two duplexes exhibit very similar melting transitions, including the same Raman indicators of ruptured Watson-Crick pairing, base unstacking and collapse of backbone order. A detailed analysis of the data provides a comprehensive Raman assignment scheme for adenosine and thymidine residues of B-DNA, delineates Raman markers diagnostic of consecutive A.T and alternating A.T/T.A tracts of DNA, and identifies the distinct Raman difference signatures for premelting and melting transitions in the two types of sequences.     

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA

The interaction of the Cu(II) drugs CuL(NO₃) and CuL′(NO₃) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL′ is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL]⁺ and [CuL′]⁺) with [poly(dA–dT)]₂, [poly(dG–dC)]₂, and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 °C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA–dT)]₂, with a site size n = 6–7, whereas they act as external binders with [poly(dG–dC)]₂ and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL′]⁺ were slightly larger than with [CuL]⁺. The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL]⁺ and [CuL′]⁺, with a significative dose–response effect for [CuL′]⁺, which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH₂ group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.     

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

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.     

The complex of poly(dG).poly(dC) with arginine: stabilization of the B form and transition to multistranded structures

We have studied by X-ray diffraction fibers of complexes of poly(dG).poly(dC) with N-alpha-acetyl-L-arginine ethylamide. Although these polynucleotides favour the A form of DNA, in this complex it is never found, thus confirming that arginine prevents the appearance of this form of DNA. At high relative humidity the B form is present. Upon dehydration two new structures appear. One of them is a triple helix, most likely formed by poly(dC+).poly(dG).poly(dC). The other structure found also has features which indicate a multistranded conformation.     

Untenability of the heteronomous DNA model for poly(dA).poly(dT) in solution. This DNA adopts a right-handed B-DNA duplex in which the two strands are conformationally equivalent. A 500 MHz NMR study using one dimensional NOE

1D NOE 1H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA).poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion. The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H2O + 10% D2O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA).poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Determination of handedness of DNA double helices from NOE difference spectra: the structure of poly(dG-dC).poly(dG-dC) in low salt

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC).(poly)dG-dC) in low salt solutions exist as a right-handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1. 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2'H2" sugar protons; and in the difference NOE spectra in the H2'H2" region the signatures of the right and left-handed helices become imprinted.     

Synergistic effects in the melting of DNA hydration shell: melting of the minor groove hydration spine in poly(dA).poly(dT) and its effect on base pair stability.

We propose that water of hydration in contact with the double helix can exist in several states. One state, found in the narrow groove of poly(dA).poly(dT), should be considered as frozen to the helix, i.e., an integral part of the double helix. We find that this enhanced helix greatly effects the stability of that helix against base separation melting. Most water surrounding the helix is, however, melted or disassociated with respect to being an integral part of helix and plays a much less significant role in stabilizing the helix dynamically, although these water molecules play an important role in stabilizing the helix conformation statically. We study the temperature dependence of the melting of the hydration spine and find that narrow groove nonbonded interactions are necessary to stabilize the spine above room temperature and to show the broad transition observed experimentally. This calculation requires that synergistic effects of nonbonded interactions between DNA and its hydration shell affect the state of water-base atom hydrogen bonds. The attraction of waters into narrow groove tends to retain waters in the groove and compress or strain these hydrogen bonds.     

Vibrational normal modes and dynamical stability of DNA triplex poly(dA). 2poly(dT): S-type structure is more stable and in better agreement with observations in solution.

A normal-mode and statistical mechanical calculation was carried out to determine the vibrational normal modes, contribution of internal fluctuations to the free energy, and hydrogen bond disruption of DNA triplex poly(dA).2poly(dT). The calculation was performed on both the x-ray fiber diffraction model with a N-type sugar conformation, and a newly proposed model with a S-type sugar conformation. Our calculated normal modes for the S-type structure are in better agreement with observed IR spectra for samples in D2O solution. We also find that the contribution of internal fluctuations to free energy, premelting hydrogen bond disruption probability, and hydrogen bond melting temperatures for the Hoogsteen and Watson-Crick hydrogen bonds all show that the S-type structure is dynamically more stable than the N-type structure in a nominal solution environment. Therefore our calculation supports experimental findings that the triplex d(T)n.d(A)nd(T)n most likely adopts a S-type sugar conformation in solution or at high humidity. Our calculations, however, do not preclude the possibility of an N-type conformation at lower humidities.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Identification of the translocatable element IS1 in a molecular chimera constructed with plasmid pBR322 DNA into which a bacteriophage MS2 DNA copy was inserted by the poly(dA).poly(dT) linker method.

Analysis of the plasmid DNA derived from a colony of bacteria carrying pMS2-7 (preceding paper, Devos et al., 1979) revealed the presence of an additional, smaller plasmid DNA, identified as pMS2-701. It was shown that pMS2-701 also contained the nearly full-length MS2 DNA copy, but the extra DNA insertion that was present to the right of the MS2 DNA insert in pMS2-7 was absent. Transformation of Escherichia coli with a DNA preparation containing both plasmid DNAs allowed the recloning of the pMS2-701 plasmid. Upon further subculturing, however, the pMS2-7 type plasmid containing the extra DNA insertion reappeared. Furthermore, the proportion of pMS2-7 relative to pMS2-701 increased in the course of successive subcultures. The extra DNA insertion in pMS2-7 was identified as the translocatable element IS1³ by mapping of restriction sites and by nucleotide sequence analysis. The boundaries between IS1 and pMS2-7 DNA revealed that IS1 had been inserted between the N-proximal part of the ampicillin gene and the poly(dA)-poly(dT) linker, and that a repetitious sequence of nine base-pairs in length had been generated by the translocation process.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.