B-X transition in synthetic and natural (dA-dT)n.(dA-dT)n sequences

AB-X transition of polyh(dA-dT).poly(dA-dT) was observed to occur in methanol-water mixtures with methanol concentrations higher than 50% in the presence of a specific combination of monovalent and divalent cations. In the presence of Na+, divalent cations induce denaturation of poly(dA-dT).poly(dA-dT) accompanied by condensation and/or aggregation, and effect similar to that observed previously with random sequence DNA (Votavová, Kucerová, Felsberg and Sponar, J. Biomol. Struct. Dyn. 4,477-489, 1986). In the presence of Cs+ cations a B-X transition was induced by addition of Ca2+ or Mn2+ but not Mg2+ or Ni2+ ions. Circular dichroism and ultraviolet spectroscopy demonstrate that the X conformation is a double stranded form of poly(dA-dT).poly(dA-dT) belonging presumably to the B family which, however has an altered base stacking. The X conformation of poly(dA-dT).poly(dA-dT) found in methanol-water mixtures is a condensed and/or aggregated form. In contrast, the X conformation characterized by similar CD spectra observed in high salt concentrations is not aggregated up to a concentration of 6 M CsF. In methanol-water mixtures (A+T)-rich bacterial DNA behaves essentially as a random sequence DNA revealing no detectable amount of the X form. On the other hand crab (Cancer pagurus) satellite and crab non-satellite DNAs containing varying amounts of (dA-dT)n.(dA-dT)n sequences were shown to undergo a B-X transition, at least partly, in both methanol-water mixtures and 6 M CsF solutions.     

Titration of poly(dA-dT) . poly(dA-dT) in solution at variable NaCl concentration

CD and uv absorption data showed that high molecular weight poly(dA-dT) . poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl]< or =0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH- ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments.     

The Z-conformation of poly(dA-dT).poly(dA-dT) in solution as studied by ultraviolet resonance Raman spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Conformational variability of poly(dA-dT).poly(dA-dT) and some other deoxyribonucleic acids includes a novel type of double helix

The article reviews data indicating that poly(dA-dT).poly(dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT).poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the 31P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the 31P NMR spectrum of the limiting alternating B conformation of poly(dA-dT).poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack. However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the 31P NMR resonance of poly(dA-dT).poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC).poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC).poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT).poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT).poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT).poly(dA-dT) X-DNA. It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

Conformation of the synthetic DNA poly(amino2dA-dT) duplex in high-salt and aqueous alcohol solutions.

It has previously been demonstrated by other workers that the duplex of a synthetic DNA poly(amino2dA-dT) undergoes a salt-induced conformational isomerization. We show in the present work using circular dichroism that the same isomerization is induced in poly(amino2dA-dT) by various alcohols. The isomerization was originally identified as the B-to-Z and then B-to-A conformational transition of DNA but we demonstrate that the high-salt or alcohol conformation of poly (amino2dA-dT) is the non Z-DNA zig-zag double helix we have previously observed with poly(dA-dT) and called X-DNA. X-DNA is a cesium cation specific conformation of poly(dA-dT) while no similar cation specificity is observed with poly(amino2dA-dT). Thus it appears that the extra amino group attached to A and cesium cations make the same thing; they probably dehydrate the double helix minor groove and relieve its conformational variability. Poly(amino2dA-dT) is exceptionally stable in X-DNA and conditions inducing it are mild, which opens the door to assess its molecular structure.     

Salt-induced conformational changes of poly(dA-dT).

Conformational changes of poly(dA-dT) . poly(dA-dT) induced by increasing ionic strength were studied using CD spectroscopy. It was found that a pronounced noncooperative inversion of the long-wavelength part of the CD spectrum of poly(dA-dT) . poly(dA-dT) occurred at high concentrations of CsF in solution. It was suggested that a great difference between the geometries of the purine and pyrimidine residues in the helix was characteristic of the structure of poly(dA-dT) . poly(dA-dT) in concentrated CsF solutions.     

Raman spectroscopic elucidation of DNA backbone conformations for poly(dG-dT).poly(dA-dC) and poly(dA-dT).poly(dA-dT) in CsF solution

Raman spectra have been recorded for poly(dG-dT) · poly(dA-dC) and poly(dA-dT) · poly(dA-dT) in low salt and at high concentrations of CsF. Poly(dG-dT) · poly(dA-dC) shows no change in the 682-cm^?1 guanine mode, demonstrating the absence of the Z-structure at high salt. The 790-cm^?1 phosphodiester symmetric stretch, however, shifts up 5 cm^?1 in 4.3M CsF, suggesting a slight conformational change, associated with ion binding or hydration changes. Poly(dA-dT) · poly(dA-dT) shows an additional broad band at 816 cm^?1, attributed to the phosphodiester modes associated with the C3′-endo deoxyribose units in the alternating B-structure. In this case, both the 841- and the 816-cm^?1 asymmetric phosphodiester stretches, associated with the C2′- and C3′-endo units, shift down on addition of CsF in a sequential manner. Correlation of this sequence with that previously observed for the two ³¹P-nmr resonances, establishes that the phosphodiester stretching frequencies depend on the conformation of the 5′-sugar, and not on the 3′-sugar.     

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.     

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.     

Kinetic investigation of unfolding and partial refolding of a crab satellite (dA-dT)n.

Crab (dA-dT)n was isolated from the testes of Cancer borealis by a procedure involving separation of DNA and segregation of the satellite fraction by Hg2+ binding/Cs2SO4 density gradient ultracentrifugation. The titration of crab (dA-dT)n samples at 10 degrees indicated a sharp absorbance change at pH 11.98 in agreement with the pHm value observed for synthetic poly(dA-dT) under identical conditions. The reversal of the titration, however, resulted only in about 50% recovery of the original absorbance (at 260 nm) in marked contrast to the complete reversibility of the synthetic material. pH-jump experiments were carried out for the purpose of characterizing the rates and mechanisms of conformational transitions brought about by changes in the solution environment. It was found that the disintegration of the putative native structure of crab (dA-dT)n starts with a very fast reaction (occurring within the 6-msec deadtime of the instrument and comprising 65% of the total absorbance change) and it is completed via a slower first-order reaction (k = 66 sec minus 1). It is postulated that the first process is due to the rapid untwisting of end regions and, perhaps, some short hairpin-like helical branches present on the macromolecules. The second reaction is believed to be the end-to-end type unwinding of the double-helical backbone of crab (dA-dT)n. In the presence of low concentration (3 mug/ml) of Hg2+ ions the overall rate of disintegration process decreased drastically. pH jumps from pH values above pHm to values below were used to study the rates of absorbance changes corresponding to the refolding of the strands of denatured crab (DA-dT)n. A concentration independent process consisting of two phases was observed. The first phase was a gradual nonexponential process spanning the first second of the reaction, and the other, a very slow first-order process characterized by the rate constant value of 0.053 sec minus 1. It is proposed that the first part of the process (involving about 24% of nucleotide residues) is an intramolecular formation of helical hairpins (frequently interrupted by mismatching bases) and the second part is a manifestation of some association of the extant unpaired bases during the folding of the branched structure. Refolded crab (dA-dT)n samples when subjected again to pH greater than pHm in the stopped-flow apparatus displayed not the disintegration pattern of the native crab (dA-dT)n but rather that of synthetic poly(dA-dT. The marked facility of crab (dA-dT)n macromolecules for rapid conformational transitions induced by slight changes in the solution environment might be relevant to the biological function of this DNA.     

Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+

The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).     

The CD of ligand-DNA systems. 2. Poly(dA-dT) B-DNA.

A systematic theoretical study of the CD of [poly(dA-dT)]2 and its complexes with achiral small molecules is presented. The CD spectra of [poly(dA-dT)]2 and of poly(dA):poly(dT) are calculated for various DNA structures using the matrix method. The calculated and experimental spectra agree reasonably well for [poly(dA-dT)]2 but less well for poly(dA):poly(dT). The calculated CD spectrum of [poly(dA-dT)]2 fails to reproduce the wavelength region of 205-245 nm of the experimental spectrum. This discrepancy can be explained by a magnetic dipole allowed transition contributing significantly to the CD spectrum in this region. The induced CD of a transition moment of a molecule bound to [poly(dA-dT)]2 is also calculated. As was the case for [poly(dG-dC)]2, the induced CD of a groove bound molecule is one order of magnitude stronger than that of an intercalated molecule. The calculations also show considerable differences between pyrimidine-purine sites and purine-pyrimidine sites. Both signs and magnitudes of the CD induced into ligands bound in the minor groove agree with experimental observations.     

Right-handed and left-handed helices of poly(dA-dC) X (dG-dT).

The secondary structures of poly(dA-dC) X (dG-dT) were studied using CD and IR spectroscopies. We give spectroscopic evidence of secondary structure transitions of poly(dA-dC) X (dG-dT) from a B to a Z-like helix, induced by transition metal ions (Ni2+) in presence of high concentrations of Cs+ and Na+. In the presence of Na+, the B in equilibrium Z transition occurs at any temperature, whereas premelting conditions are required in presence of Cs+. For these two alkali ions the Z-like form is only induced by Ni2+ ions through their specific interactions at N7 of purines, under conditions of low water activity due to the high alkali salt concentration. We also show that the CD spectrum obtained in presence of Cs+ ions and characterized by a negative band at 275 nm, cannot be interpreted in terms of Z-like left-handed helix but reflects a modified B right-handed helix.     

Conformation of poly(dG-dC)·Poly(dG-dC) and Poly(dA-dT)·Poly(dA-dT) interacting with the antitumor drug cis-[Pt(NH32]Cl2

The interaction of cis-dichlorodiammine platinum(II) with poly(dG-dC)·poly(dG-dC) and poly(dA-dT) ·poly(dA-dT) was studied by circular dichroism. Significant conformational changes were induced in both alternating polymers: in the case of poly(dG-dC) ·poly(dG-dC) the spectra were not conclusive in terms of a well defined conformation, even if the presence of left-handed helices could be suggested. For poly(dA-dT)·poly(dA-dT) the data were interpreted in terms of a dimer-helix → single hairpin helix transition induced by the metal. The results obtained are discussed with reference to the antitumor activity of the drug.     

A polycationic amine that induces unique conformational changes in poly(dA-dT) in low salt

Circular dichroism was used to examine alterations in the secondary structure of poly(dA-dT) X poly(dA-dT) upon binding polymer X, a polycationic CD probe for aspects of DNA structure. Stable complex formation is evidenced by increasing Tm and the appearance of large extrinsic bands in the greater than 300 nm, region which increase proportionally with r (ratio of polymer charge to DNa phosphate), in the range 0.0 to 0.32. At relatively low values of r (less than .32), CD spectra of the poly(dA-dT) X poly(dA-dT)-polymer X complex show a gradual non-cooperative inversion in the long wavelength portion (275 nm) of the intrinsic band in low salt solutions suggesting structural and conformational flexibility in poly(dA-dT) X poly(dA-dT) and further implicating polymer X as a potential probe for variations in DNA secondary structure. The dinucleotide repeat configuration of poly(dA-dT) X poly(dA-dT) is presumed to play a role in the observed intrinsic CD changes. NMR data support an "alternating B" conformation for the complex.