Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

NMR Studies of the Interaction of Bleomycin with (dC-dG)3

Abstract

The interaction of bleomycin A₂ and Zn(II)-bleomycin A₂ with the oligonucleotide (dC-dG)₃ has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the ¹H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected.     

A one- and two-dimensional NMR study of the B to Z transition of (m5dC-dG)3 in methanolic solution

The deoxyribose hexanucleoside pentaphosphate (m5dC-dG)3 has been studied by 500 MHz 1H NMR in D2O (0.1 M NaCl) and in D2O/deuterated methanol mixtures. Two conformations, in slow equilibrium on the NMR time scale, were detected in methanolic solution. Two-dimensional nuclear Overhauser effect (NOE) experiments were used to assign the base and many of the sugar resonances as well as to determine structural features for both conformations. The results were consistent with the an equilibrium in solution between B-DNA and Z-DNA. The majority of the molecules have a B-DNA structure in low-salt D2O and a Z-DNA structure at high methanol concentrations. A cross-strand NOE between methyl groups on adjacent cytosines is observed for Z-DNA but not B-DNA. The B-DNA conformation predominates at low methanol concentrations and is stabilized by increasing temperature, while the Z-DNA conformation predominates at high methanol concentrations and low temperatures. 31P NMR spectra gave results consistent with those obtained by 1H NMR. Comparison of the 31P spectra with those obtained on poly(dG-m5dC) allow assignment of the lower field resonances to GpC in the Z conformation.     

Z-DNA is Formed by poly(dC-dG) and poly (dm5C-dG) at Micro or Nanomolar Concentrations of some Zinc(II) and Copper(II) Complexes

Abstract

We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm⁵C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4μM (poly(dC-dG)) and 44μM (poly(dm⁵C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2μM for poly(dm⁵C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.     

Z Helix-coil Transition of d(C-Br8G-C-G-C-Br8G) Studied by CD, 1H-NMR and IR Spectroscopies

Abstract

The conformation of díC-Bi⁸G-C-G-C-Br⁸G) in aqueous solution was studied by CD and ¹H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)₃ oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of diC-Bi⁸G-C- G-C-Br⁸G) demonstrating the duplex formation were observed up to 60°C. It is interesting to note that the significant highfield shifts of the dC H₅″ exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)₃ containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)₃ oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br⁸G-C-G-C-Br⁸G) is obtained directly from the coil form. However, IR data suggest that in the Z form of dlC-Bi⁸G-C-G-C-Bi⁸G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)₃ crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)₃ are compared to those of the B helix-coil transition observed for methylated d(C-G)₃ in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

31P-NMR analysis of the B to Z transition in double-stranded (dC-dG)3 and (dC-dG)4 in high salt solution

In 4M NaCl solutions (dC-dG)n (n = 3,4; approximately 9 mM) exist as a mixture o +/- B and Z forms. The low and high field components of two 31P NMR resonances originating from internal phosphodiester groups are assigned to the GpC and CpG linkages, respectively. Low temperatures stabilize the Z-forms, which completely disappear above 50 degrees C (n = 3) and 65 degrees C (n = 4). delta H = -44 and -17 kJ/mol for B to Z transition in the hexamer and octamer duplexes, respectively. Temperature dependent changes (0-50 degrees C range) in the spin-lattice relaxation times at 145.7 MHz are distinctly different for the 31P nuclei o +/- GpC and CpG groups. The relaxation data can be explained by assuming that the GpC phosphodiester groups undergo more local internal motion than do the CpG groups.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

Abstract

It has been shown for the first time that conformational junction between contiguous right- handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)₁₃ and (dC-dG)₁₆ blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease SI cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)_n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)₁₃ segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)_n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Conformational Transitions of Poly(dl-dC) in Aqueous Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i. e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni²⁺ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA- dT) and poly(dA-dC). poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dl-dC) as in the low-salt form (0.1M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl₂ in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

Anti-Syn Conformational Range of Pyrimidines with Deoxyribofuranose

Abstract

The ability of pyrimidine bases to adopt the syn conformation in DNA has been investigated. The distances between atoms on the sugar and base and the resulting steric energies have been calculated as a function of glycosidic torsion angle for the principal sugar puckers of the deoxyribose of cytosine. The results indicate that pyrimidines can assume both the anti and syn conformations for the ³E, ₄E, ₁E, ²E, ₃E sugar puckers and syn for the ₂E sugar pucker. For these sugar puckers the difference between the minimum energies of the anti and syn conformations is in the range of 0.1–2.0 kcal/mole, with the minimum syn energy being lower in the case of the ₄E, ₁E and ²E sugar puckers. It is particularly significant that cytosine can assume the syn conformation for the ³E sugar pucker commonly observed for the syn nucleotides in Z-DNA with both alternating pyrimidine/purine (APP) and non-APP sequences. The results of this investigation confirm that steric interactions resulting from putting a pyrimidine nucleotide in the syn conformation are not a major factor in the preference for APP base sequences in Z-DNA.     

Influence of D and L amino-acid residues on the conformation of peptides in solution: A carbon-13 nuclear magnetic resonance study of cyclo(prolyl-leucyl)

The ¹³C chemical shifts and spin-lattice relaxation times are reported for cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α-carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The ¹³C spin-lattic relaxation time(T₁) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L -L isomer as compared to the L -D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T₁ values. The differences in T₁ values and chemical shifts between cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu) appear to result from a difference in conformations of the two diketopiperazine rings.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

A Renaissance Man: In Memoriam of Jon Widom (1955–2011)

Abstract

Assignment of the ¹H and ³¹P resonances of a decamer DNA duplex, d(CGCTTAAGCG)₂ was determined by two-dimensional COSY, NOESY and ¹H- ³¹P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. The solution structure of the decamer was calculated by an iterative hybrid relaxation matrix method combined with NOESY-distance restrained molecular dynamics. The distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. The hybrid matrix-derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. J_H3′-P coupling constants for each of the phosphates of the decamer were obtained from ¹H-³¹P J-resolved selective proton flip 2D spectra. By using a modified Karplus relationship the C4′-C3′-03′-P torsional angles (?) were obtained. Comparison of the ³¹P chemical shifts and J_H3′-P coupling constants of this sequence has allowed a greater insight into the various factors responsible for ³¹P chemical shift variations in oligonucleotides. It also provides an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These correlations are consistent with the hypothesis that changes in local helical structure perturb the deoxyribose phosphate backbone. The variation of the ³¹P chemical shift, and the degree of this variation from one base step to the next is proposed as a potential probe of local helical conformation within the DNA double helix. The pattern of calculated ? and ζ torsional angles from the restrained molecular dynamics refinement agrees quite well with the measured J_H3′-P coupling constants. Thus, the local helical parameters determine the length of the phosphodiester backbone which in turn constrains the phosphate in various allowed conformations.     

B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

The Conformation of the d(ACCCGGGT) Duplex in Aqueous Solution

Abstract

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)₂ have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D₂O at 12°C. The observed NOE's between the base protons and their own H2′ protons and between the base protons and the H2′ protons of the 5′adjacent nucleotide and the observed coupling constants between the deoxyribose 1′ and 2′,2″ protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

A critical evaluation of models for complex molecular dynamics: application of NMR studies of double- and single-stranded DNA

The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120–160 base pairs (bp) long is examined by molecular-dynamics modeling using ¹³C-nmr spin-relaxation data obtained over the frequency range of 37–125 MHz. The broad range of ¹³C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T₁ values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τ_x = ～10^?6 s;τ_Z = ～1.8 × 10^?8s), which is in good agreement with values calculated from hydrodynamic theory (τ_x = ～1.8 × 10^?6 s; τ_Z = ～2.7 × 10^?8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C? H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τ_w = ～4 × 10^?9 s, θ_cone = ～21°) or as a bistable jump (τ_A = τ_B = ～2 × 10^?9 s, θ = ～15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C? H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τ_W = ～0.2 × 10^?9 s, θ_cone = ～50°), and overall motion is affected by becoming essentially isotropic (τ_x = τ_Z = ～5 × 10^?8 s) for the single-stranded molecules. Since ¹³C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.     

Thermodynamic and Structural Properties of r(ACC) as Revealed by Ultraviolet Electronic Absorption,Circular Dichroism, 1H-NMR Spectroscopy and Monte Carlo Simulations

Abstract

UV absorption, circular dichroïsm (CD) and ¹H NMR, associated with Monte Carlo (MC) molecular structure simulations have been applied to the study of the trinucleoside diphosphate: r(ACC).

The MC study which has been conducted as a function of temperature, is based on random variations of the nucleotide conformational angles, i.e. phosphodiester chain torsional angles and sugar pucker pseudorotational angles. All of the chemical bond lengths and valence angles remained fixed during the structural simulation, except those of the sugar pucker. Six different initial structures have been selected in order to explore the molecular conformational space as completely as possible. This simulation procedure led to distinct families of equilibrium conformations at 283,298 and 318 K.

The thermodynamical parameters such as variations in entropy, enthalpy and also melting temperature (ΔS⁰ _x, ΔH⁰ _x and T_m) of the stacking (X) equilibrium were obtained from UV absorption and circular dichroïsm (CD) spectra recorded over a 80K temperature range. Chemical shifts (δ), vicinal coupling constants(³J _k) and cross-relaxation rates (σ_k,l) of trimers were measured at 400.13 MHz over a range of concentrations (2–13mM) and temperatures (283–333K). Least-squares fitting of the experimental chemical shifts to simple models of association (A) and stacking equilibria allowed separation of the variations in the δ values (Δδ_x and Δδ_A) due to either phenomenon. The three NMR data sets (Δδ_x, ³J^k,l and σ_k.l) were then evaluated for the minima conformers obtained with the MC simulations. Theoretical values of Δδ_x were estimated using the results of an ab initio study while the coupling constant data were simulated with Karplus-type equations. Finally, the relaxation data were simulated from the distance matrices using treatment for cases of both slow conformational exchange accompanied by rapid small-amplitude fluctuations about the minima structures.

A consistent picture of the large amplitude deformations (torsional angle variation) of these trimers has emerged from the present study. Optimized conformational blends at 283, 296 and 318K were obtained by least-squares fitting of the experimental data to the theoretical ones, while considering the populations as adjustable parameters. As it would be expected, the right-handed helical conformation (A-RNA type) is found to be the major stacked species, in the temperature range of 283 to 318K. Limited evidence for bulged structures has been obtained, whereas novel reverse-stacked and half-stacked conformers also presented theoretical data compatible with the NMR observables of aqueous r(ACC).