NMR investigation of mithramycin A binding to d(ATGCAT)2: a comparative study with chromomycin A3

The binding of mithramycin A to the d(A1T2G3C4A5T6) duplex was investigated by 1H NMR and found to be similar to that of its analogue chromomycin A3. In the presence of Mg2+, mithramycin binds strongly to d(ATGCAT)2. On the basis of the two-dimensional NOESY spectrum, the complex formed possesses C2 symmetry at a stoichiometry of two drugs per duplex (2:1) and is in slow chemical exchange on the NMR time scale. NOESY experiments reveal contacts from the E-pyranose of mithramycin to the terminal and nonterminal adenine H2 proton of DNA and from the drug hydroxyl proton to both G3NH2 protons, C4H1' proton, and A5H1' proton. These data place the drug chromophore and E pyranose on the minor groove side of d(ATGCAT)2. NOE contacts from the A-, B-, C-, and D-pyranoses of mithramycin to several deoxyribose protons suggest that the A- and B-rings are oriented along the sugar-phosphate backbone of G3-C4, while the C- and D-rings are located along the sugar-phosphate backbone of A5-T6. These drug-DNA contacts are very similar to those found for chromomycin binding to d(ATGCAT)2. Unlike chromomycin, the NOESY spectrum of mithramycin at the molar ratio of one drug per duplex reveals several chemical exchange cross-peaks corresponding to the drug-free and drug-bound proton resonances. From the intensity of these cross-peaks and the corresponding diagonal peaks, the off-rate constant was estimated to be 0.4 s-1. These data suggest that the exchange rate of mithramycin binding to d(ATGCAT)2 is faster than that of chromomycin.     

The three-dimensional structure of the 4:1 mithramycin:d(ACCCGGGT)(2) complex: evidence for an interaction between the E saccharides

Mithramycin and chromomycin, two antitumor drugs, each having an identical aglycone and nearly identical disaccharide and trisaccharide side chains, have differing binding properties to a small oligonucleotide, d(ACCCGGGT)(2) (M. A. Keniry et al., Journal of Molecular Biology, 1993, Vol. 231, pp. 753-767). In order to understand the forces that induce four mithramycin molecules to bind to d(ACCCGGGT)(2) instead of two drug molecules in the case of chromomycin, the structure of the 4:2:1 mithramycin: Mg(2+):d(ACCCGGGT)(2) complex was investigated by (1)H-nmr and restrained molecular dynamics. The resulting three-dimensional model showed that in order to accommodate the close approach of one neighboring mithramycin dimer, the inwardly directed CDE saccharide chain of the neighboring mithramycin dimer undergoes a conformational change such that the E saccharide no longer spans the minor groove but reorients so that the hydrophilic face of the E saccharides from the two dimers oppose each other. Two hydrogen bonds are formed between the hydroxyl groups of the two opposing E saccharide groups. The results are interpreted in terms of the differences in stereochemistry and functional group substitutions between mithramycin and chromomycin. A mithramycin dimer is able to self-associate on an oligonucleotide template because it has two hydroxyl groups on the same face of its terminal E saccharide. A chromomycin dimer is unable to self-associate because one of these hydroxyl groups is acetylated and the neighboring hydroxyl group has a stereochemistry that cannot permit close contact of the hydroxyl group with a neighbouring chromomycin dimer.Copyright 2000 John Wiley & Sons, Inc.     

Interactions of chromomycin A3 and mithramycin with the sequence d(TAGCTAGCTA)2

Anti-cancer antibiotics, chromomycin A3 (CHR) and mithramycin (MTR) inhibit DNA directed RNA synthesis in vivo by binding reversibly to template DNA in the minor groove with GC base specificity, in the presence of divalent cations like Mg2+. Under physiological conditions, (drug)2Mg2+ complexes formed by the antibiotics are the potential DNA binding ligands. Structures of CHR and MTR differ in their saccharide residues. Scrutiny of the DNA binding properties reveal significant differences in their sequence selectivity, orientation and stoichiometry of binding. Here, we have analyzed binding and thermodynamic parameters for the interaction of the antibiotics with a model oligonucleotide sequence, d(TAGCTAGCTA)2 to understand the role of sugars. The oligomer contains two potential binding sites (GpC) for the ligands. The study illustrates that the drugs bind differently to the sequence. (MTR)2Mg2+ binds to both sites whereas (CHR)2Mg2+ binds to a single site. UV melting profiles for the decanucleotide saturated with the ligands show that MTR bound oligomer is highly stabilized and melts symmetrically. In contrast, with CHR, loss of symmetry in the oligomer following its association with a single (CHR)2Mg2+ complex molecule leads to a biphasic melting curve. Results have been interpreted in the light of saccharide dependent differences in ligand flexibility between the two antibiotics.     

The conformation of the d(ACCCGGGT) duplex in aqueous solution

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 degrees 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.     

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.     

NMR studies of the interaction of the antibiotic nogalamycin with the hexadeoxyribonucleotide duplex d(5'-GCATGC)2

1H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)2 and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled us to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule. Nogalamycin intercalates at the 5'-CA and 5'-TG steps with the major axis of the anthracycline chromophore aligned approximately at right angles to the major axes of the base pairs. The nogalose sugar occupies the minor groove of the helix and makes many contacts with the deoxyribose moieties of three nucleotides along one strand of the duplex in the 5'-TGC segment. The charged dimethylamino group and hydroxyl functions of the bicyclic sugar lie in the major groove juxtaposed to the guanine base, the bridging atoms of the bicyclic sugar making contacts with the methyl group of the thymine. Thus the antibiotic is not symmetrically disposed in the intercalation site but is in close contact in both grooves with atoms comprising the 5'-TGC strand. The intercalation cavity is wedge-shaped, the major axes of the base pairs forming the site being tilted with respect to one another. All base-pair hydrogen-bonding interactions are maintained in the complex, and there is no evidence for Hoogsteen pairing. The free duplex adopts a regular right-handed B-type conformation in which all glycosidic bond angles are anti and all sugar puckers lie in the C2'-endo range. In the complex the glycosidic bond angles and the sugar puckers deviate little from those observed for the duplex alone. The presence of two bound nogalamycin molecules substantially slows the "breathing" motions of the base pairs forming the intercalation cavity, and the observation of two downfield-shifted resonances in the 31P NMR spectrum of the complex suggests a pronounced local helix unwinding at the drug binding site. The footprinting data of Fox and Waring [Fox, K.R., & Waring, M.J. (1986) Biochemistry 25, 4349-4356] imply that the highest affinity binding sites of nogalamycin have the sequence 5'-GCA (or 5'-TGC).(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational variation of the central CG site in d(ATGACGTCAT)2 and d(GAAAACGTTTTC)2. An NMR, molecular modelling and 3D-homology investigation.

The determination of the solution structure of two self-complementary oligomers d(ATGACGTCAT)2 (CG10) and d(GAAAACGTTTTC)2 (CG12), both containing the 5'-pur-ACGT-pyr-3' sequence, is reported. The impact of the base context on the conformation of the central CpG site has been examined by a combined approach of: (a) 2D 1H-NMR and 31P-NMR; (b) molecular mechanics under experimental constraints; (c) back-calculations of NOESY spectra and iterative refinements of distances; and (d) 3D-homology search of the central tetrad ACGT within the complete oligonucleotides. A full NMR study of each fragment is achieved by means of standard 2D experiments: NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Sugar phase angle, epsilon-zeta difference angle and NOE-derived distances are input as experimental constraints to generate molecular models by energy minimization with the help of jumna. The morass program is used to iteratively refine the structures obtained. The similarity of the two ACGTs within the whole oligonucleotides is investigated. Both the decamer and the dodecamer adopt a B-like DNA conformation. However, the helical parameters within this conformational type are significantly different in CG12 and CG10. The central CpG step conformation is not locked by its nearest environment (5'A and 3'T) as seen from the structural analysis of ACGT in the two molecules. In CG12, despite the presence of runs of A-T pairs, CpG presents a high twist of 43 degrees and a sugar phase at the guanine of about 180 degrees, previously observed in other ACGT-containing-oligomers. Conversely, ACGT in CG10 exhibits strong inclinations, positive rolls, a flat profile of sugar phase, twist and glycosidic angles, as a result of the nucleotide sequence extending beyond the tetrad. The structural specificity of CG10 and its flexibility (as reflected by its energy) are tentatively related to the process of recognition of the cyclic AMP response element by its cognate protein.     

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+)

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.     

The solution structure of the Ga(III)-bleomycin A2 complex resolved by NMR and molecular modeling; interaction with d(CCAGGCCTGG)

The solution structure of the Ga(III)-bleomycin A2 complex (GaBLM) has been determined using 2D NMR methods in combination with molecular dynamics calculations. Complete assignment of the amide and amine protons, observation of 80 NOEs and measurement of 15 (3)JH(-H) coupling constants provided us with a well-defined structure using a restrained simulated annealing protocol. On the basis of distance and dihedral angle constraints agreement, along with potential energy considerations, the favored model is a five-coordinate complex with the primary amine of beta-aminoalanine holding the axial position of a distorted tetragonal pyramid. The disaccharide moiety of GaBLM is not a ligand, sharing the same side of the equatorial plane with the axial amine ligand. Titration of the self-complementary oligonucleotide d(CCAGGCCTGG) with GaBLM results in the formation of only one 1:1 complex in slow exchange on the NMR time scale. Our data indicate that the bithiazole moiety intercalates between the C6*G15 and C7*G14 base pairs, in a similar mode to that reported by earlier studies. Structural implications and comparisons to other metallo-bleomycins are discussed.     

Interactions of polyamines with the DNA octamers d(m5CG)4 and d(GGAATTCC): A 1H‐NMR investigation

The interaction of two DNA octamers, d(m⁵CG)₄ and d(GGAATTCC), with the polyamines spermine⁴⁺ and spermidine³⁺, has been studied by means of ¹H‐nmr nuclear Overhauser effect (NOE) difference measurements. The experiments were performed at 10°C and for a polyamine charge to DNA charge (i.e., phosphate) ratio of 0.4, where the solution of d(m⁵CG)₄ contains about 50% Z‐form of the DNA. The results show that the polyamine intramolecular NOEs for the protons on the propyl chains are similarly negative with the two oligonucleotides, while those on the butyl chain show slightly more negative NOE with d(m⁵CG)₄ than with d(GGAATTCC). The fully N‐methylated analogues of spermine (Me₁₀Spn⁴⁺) and spermidine (Me₈Spd³⁺) as well as the diamines 1,3‐diaminopropane (DAP²⁺) and 1,4‐diaminobutane (putrescine²⁺) have been studied for the ability to transform d(m⁵CG)₄ from the B‐ to the Z‐form. ¹H‐nmr spectra showed the order spermine⁴⁺ > spermidine³⁺ > Me₁₀Spn⁴⁺ > Me₈Spd³⁺ > 1,3‐diaminopropane²⁺ > putrescine²⁺, with spermine showing the largest relative amount of Z‐DNA. ¹H‐nmr pulsed‐gradient self‐diffusion measurements of the triamines showed a large difference in the interaction of Spd and Me₈Spd with the two different duplexes. With the same duplex (either of the two), however, no difference between Spd and Me₈Spd can be seen. Within a two‐state model this is interpreted as a larger fraction of bound polyamines with d(m⁵CG)₄ than with d(GGAATTCC). © 1999 John Wiley & Sons, Inc. Biopoly 49: 41–53, 1999     

Structural investigation of the hedamycin:d(ACCGGT)2 complex by NMR and restrained molecular dynamics

Hedamycin, a member of the pluramycin family of drugs, displays a range of biological responses including antitumor and antimicrobial activity. The mechanism of action is via direct interaction with DNA through intercalation between the bases of the oligonucleotide and alkylation of a guanine residue at 5'-PyG-3' sites. There appears to be some minor structural differences between two earlier studies on the interaction of hedamycin with 5'-PyG-3' sites. In this study, a high-resolution NMR analysis of the hedamycin:d(ACCGGT)2 complex was undertaken in order to investigate the effect of replacing the thymine with a guanine at the preferred 5'-CGT-3' site. The resultant structure was compared with earlier work, with particular emphasis placed on the drug conformation. The structure of the hedamycin:d(ACCGGT)2 complex has many features in common with the two previous NMR structures of hedamycin:DNA complexes but differed in the conformation and orientation of the N,N-dimethylvancosamine saccharide of hedamycin in one of these structures. The preferential binding of hedamycin to 5'-CG-3' over 5'-TG-3' binding sites is explained in terms of the orientation and location of the N,N-dimethylvancosamine saccharide in the minor groove.     

NMR studies of the interaction of bleomycin with (dC-dG)3.

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.     

Impact of C5-cytosine methylation on the solution structure of d(GAAAACGTTTTC)2. An NMR and molecular modelling investigation.

The solution structures of d(GAAAACGTTTTC)2 and of its methylated derivative d(GAAAAMe5CGTTTTC)2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1H NMR and 31P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Back-calculations of NOESY spectra and simulations of double-quantum-filtered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program JUMNA. The MORASS software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.     

1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects

Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.     

Structural comparison of the B-DNA dodecamers d(CGCGTTAACGCG) and d(CGCGAATTCGCG) with T2A2 and A2T2 tracts.

The x-ray structure of the deoxy oligonucleotide dodecamer d(CGCGTTAACGCG) recently determined in our laboratory shows that the helical parameters of the central TTAA segment are significantly different compared to the central AATT in d(CGCGAATTCGCG). The roll in the central TA step of the T2A2 dodecamer opens towards the minor groove while the AT step of the A2T2 dodecamer opens towards the major groove. Also, the roll angles at the steps 4 and 8 (GT and AC in T2A2) and (GA and TC in A2T2) are in opposite directions. The high cup and helical twist angles at the central base-pair of T2A2 decreases the base stacking interactions compared to A2T2. Tilt angles within the tetranucleotide segments TTAA and AATT have opposite signs. In spite of the local differences caused by the sequence inversion (TTAA----AATT), the two dodecamers exhibit similar overall bending. The top third is more bent than the bottom third relative to the central segment. This asymmetric bending in the two dodecamers is mainly due to crystal packing interactions.     

Deuterium NMR investigation of backbone dynamics in the synthetic oligonucleotide [d(CGCGAATTCGCG)]2

Backbone dynamics in the [5',5"-2H2]2'-deoxythymidine labeled duplex dodecamer [d-(CGCGAAT*T*CGC)]2 have been investigated by solid-state 2H NMR. Quadrupolar echo line shapes, spin-lattice relaxation, and quadrupolar echo decay times were obtained over hydration levels ranging from W = 0.0 to 25.2 (moles of H2O/mole of nucleotide). Variation of the line shape with changing hydration level was analyzed by using models employed in previous investigations of dodecamer base and sugar dynamics. Both fast local motions and a slower helix motion were present within the oligonucleotide. The fast motion was modeled as a four-site libration whose amplitude increased with hydration level. The root mean square amplitude of this librational model was 2-6 degrees larger than the amplitude observed in either the furanose ring or base labeled material for the entire range of hydration levels investigated. The observed line shape was inconsistent with a rapid three-site trans-gauche isomerization. A slow motion about the helix axis was observed at low water levels and increased in rate and amplitude with hydration. This motional model is in agreement with previous oligonucleotide studies.     

A solid-state deuterium NMR investigation of conformation and order in magnetically oriented [d(CGCGAATTCGCG)]2

Solid-state deuterium NMR has been used to investigate the oriented liquid crystal phase of the hydrated oligonucleotide [d(CGCGAATTCGCG)]2. Previous investigations have shown that the helix axis is aligned perpendicular to the magnetic field, while at reduced temperatures motion about the helix axis is eliminated. Synthetic oligonucleotide samples incorporating different labeled nucleosides, [2"-2H]-2'-deoxyadenosine, [methyl-2H]thymidine, [8-2H]-2'-deoxyguanosine, and [8-2H]-2'-deoxyadenosine, permitted investigation of both the base and sugar conformation and ordering in the aligned phase. From line-shape analysis of the purine-labeled samples the orientation of the base C8 C-D bond with respect to the helix axis was determined to be theta = 90 degrees with a distribution of sigma total = 20 degrees, which is comparable to the orientation of theta = 90 degrees, sigma total = 15 degrees, with an oriented fraction P = 0.7 found for the C3 symmetry axis of the methyl-labeled dodecamer. The orientation of the sugar 2" C-D bond with respect to the helix axis is theta = 22 degrees with a distribution of sigma total = 15 degrees in agreement with the expected C2'-endo sugar conformation. The fraction of C3'-endo was also investigated and from analysis of line shape cannot exceed 20%. These results, though preliminary in nature, illustrate the application of this aligned phase for structural investigations.     

NMR studies of tris-intercalation: solution structure and interaction of d(CTTCGCGCGAAG) with an acridine trimer

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of 1H and 31P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H5', H5"), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O1'endo range, those of the internal guanosines being closer to C2'endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the 3T4C5G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3'-5')G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.     

Proton NMR study of the B----Z transition of d(CGm5CG)2 and d(CGm5CGCG)2: theory and experiment.

The magnetic shielding produced by the double helix in a B-DNA and a Z-DNA conformation is calculated for each non exchangeable proton of the oligodeoxynucleotides d(CGm5CG)2 and d(CGm5CGCG)2. The differences between the values obtained for the two helical forms are in good agreement with the variations of chemical shift measured when the salt concentration of the solution is changed from 0.1 M to 2 or 4 M. The analysis of the theoretical chemical shift variations shows that the large upfield shift observed for some of the protons of the cytidine residues is due to the sum of the ring current and local magnetic anisotropy effects of the guanines of the two nearest neighbours residues.