Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.     

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.     

UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.     

Netropsin specifically recognizes one of the two conformationally equivalent strands of poly(dA).poly(dT). One dimensional NMR study at 500 MHz involving NOE transfer between netropsin and DNA protons

Recent observations that the heteronomous structural model for poly(dA).poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985], has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands? Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA).poly(dT) would be in closer proximity to the imino protons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, C11-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin-poly(dA).poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA).poly(dT) even though dA/dT strands are conformationally equivalent.     

Differences in melting behavior between homopolymers and copolymers of DNA: Role of nonbonded forces for GC and the role of the hydration spine and premelting transition for AT

Melting measurements of the mono-base-pair DNA polymers showed that the melting temperature T_m of the B-DNA homopolymer poly (dA ) · poly (dT) is higher than that of the copolymer poly [d(A-T)]. On the other hand, the T_mof the B-DNA homopolymer poly (dG) · poly (dC) is lower than that of the copolymer poly [d (G-C)]. From a structural point of view, the cross-strand base-stacking interaction in a DNA homopolymer is weaker than that in a DNA copolymer with the same base pair. One would then expect that all the DNA homopolymers are less stable than the copolymer with the same base pair. We find that the inversion of the melting order seen in the AT mono-base-pair DNA polymers is caused by the enhanced thermal stability of poly (dA) · poly (dT) from a well-defined spine of hydration attached to its minor groove. In this paper we employ the modified self-consistent phonon theory to calculate base-pair opening probabilities of four B-DNA polymers: poly(dA)-poly(dT), poly(dG) · poly(dC), poly[d(A-T)], and poly[d(G-C)] at temperatures from room temperature through the melting regions. Our calculations show that the spine of hydration can give the inverted melting order of the AT polymers as compared to the GC polymers in fair agreement with experimental measurements. Our calculated hydration spine disruption behavior in poly(dA) · poly(dT) at premelting temperatures is also in agreement with experimentally observed premelting transitions in poly (dA) · poly (dT). The work is in a sense a test of the validity of our models of nonbonded interactions and spine of hydration interactions. We find we have to develop the concept of a strained bond to fit observations in poly (dA) · poly(dT). The strained-bond concept also explains the otherwise anomalous stability of the hydration chain. © 1993 John Wiley & Sons, Inc.     

Netropsin Specifically Recognizes One of the Two Conformationally Equivalent Strands of Poly (dA)·Poly (dT). One Dimensional NMR Study at 500 MHz Involving NOE Transfer Between Netropsin and DNA Protons

Abstract

Recent observations that the heteronomous structural model for poly(dA)·poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985)), has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands?

Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA)-poly(dT) would be in closer proximity to the imino protrons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, Cll-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin·poly(dA)-poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA)-poly(dT) even though dA/dT strands are conformationally equivalent.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 1. Static geometry of DNA double helices

The static geometry of the phosphodiesters in oriented fibers of DNA and a variety of polynucleotides was investigated by solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. The structural parameters of the phosphodiester backbone expressed by two Euler angles beta and gamma were estimated on the basis of the NMR spectra of natural DNA, poly(dA).poly(dT), poly(rA).poly(dT), and poly-(rA).poly(rU). The Euler angles were calculated by using the known single crystal structures of a decamer, r(GCG)d(TATACGC), and a dodecamer, d(CGCGAATTCGCG). The distribution pattern of the Euler angles was quite different between these two oligonucleotides due to the different types of conformation, and it was fully consistent with the 31P NMR results, showing that the conformation of the B form DNA is very heterogeneous while that of the A or A' form is much more invariable with regard to the base composition. The structural parameters were also calculated by using various structures determined by the X-ray fiber diffraction studies, and they were evaluated on the basis of the 31P NMR data. Notably, poly(dA).poly(dT) fibers exhibited abnormal 31P NMR spectra which were very broad in line width and were not appreciably perturbed by hydration; a coiled double-helical structure is proposed as the most plausible model for this polymer.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex.

The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair.     

Premelting thermal fluctuational base pair opening probability of poly(dA).poly(dT) as predicted by the modified self-consistent phonon theory.

We employ a mean field, modified, self-consistent phonon theory to evaluate the single base-pair opening rate and the probability of a base pair in the amino proton exchangeable state for the homopolymer poly(dA).poly(dT) at temperatures below the helix-coil transition region. Our calculated premelting single base-pair opening probabilities are in general agreement with several available experimental estimates from imino proton exchange and formaldehyde-induced DNA melting measurements. These calculated opening probabilities, however, are in disagreement with the prediction of the helix-coil transition theory. Possible reasons for the differences are discussed, especially the possible different definition of a meaningful open state in the premelting region. The premelting open state of the modified self-consistent phonon approximation theory seems to be appropriate to describe a solvent-accessible open configuration that is sufficient to facilitate important chemical reactions such as imino proton exchange and formaldehyde reaction with the bases. This can be compared with the completely unstacked open state of the helix-coil transition theory originally defined in the helix-coil transition region. We propose that the amino proton exchangeable state is different from the open state associated with melting and only involves the breaking of the amino interbase H bond. The agreement between the calculated and experimentally estimated probability of a base pair in the amino proton exchangeable state seems to support this hypothesis.     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.     

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. Δθ/δT, the rate of change of ellipticity θ with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions Δθ/δT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (Δθ/ΔT ~ 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the θ values at 0°C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH⁺₄ to (C₂H₅)₄,N⁺ the Δθ/ΔT values first decreased, going through zero with TMA⁺ io and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O₂ of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.