DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix

Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond.     

2'-deoxyribonolactone lesion in DNA: refined solution structure determined by nuclear magnetic resonance and molecular modeling

The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].     

An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C-G-m5C-G-A-G-m5C-G) in solution. The B-duplex and hairpin forms

By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.     

NMR observation of a novel C-tetrad in the structure of the SV40 repeat sequence GGGCGG

We report the NMR structure of the DNA sequence d-TGGGCGGT in Na(+) solutions at neutral pH, containing a repeat sequence from SV40 viral genome. The structure is a novel quadruplex incorporating the C-tetrad formed by symmetrical pairing of four Cs via NH(2)&bond;O(2) H-bonds in a plane. The C-tetrad has a wider cavity compared to G-tetrads and stacks well over the adjacent G4-tetrad, but poorly on the G6 tetrad. The quadruplex helix is largely underwound by 8-10 degrees compared to B-DNA except at the C5-G6 step. To our knowledge this is the first report of C-tetrad formation in DNA structures, and would be of significance from the point of view of both structural diversity and specific recognition.     

Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site.     

A two B-Z junction containing DNA resolves into an all right-handed double-helix

Natural and artificial oligonucleotides are capable of assuming many different conformations and functions. Here we present results of an NMR restrained molecular modelling study on the conformational preferences of the modified decanucleotide d((m)C1G2(m)C3G4C5(L)G6(L)(m)C7G8(m)C9G10) .d((m)C11G12(m)C13G14C15(L)G (L)16(m)C17-G18(m)C19G20 ) which contains L deoxynucleotides in its centre. This chimeric DNA was expected to form a right-left-right-handed B-type double-helix (BB*B) at low salt concentration. Actually, it matured into a fully right-handed double helix with its central C(L)pG(L) core forming a right-handed Z-DNA helix embedded in a B-DNA matrix (BZ*B). The interplay between base-base and base-sugar stackings within the core and its immediately adjacent residues was found to be critical in ensuring the stabilisation of the right-handed helix. The structure could serve as a model for the design of antisense oligonucleotides resistant to nucleases and capable of hybridising to natural DNAs and RNAs.     

NMR studies of pH-dependent conformational polymorphism of alternating (C-T)n sequences.

Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).     

Ternary interactions of spermine with DNA: 4''-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.     

NMR structure determination of a modified DNA oligonucleotide containing a new intercalating nucleic acid

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C(1)T(2)C(3)A(4)A(5)C(6)X(7)C(8)A(9)A(10)G(11)C(12)T(13)):d(A(14)G(15)C(16)T(17)-T(18)G(19)X(20)G(21)T(22)T(23)G(24)A(25)G(26)). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used two-dimensional (1)H NMR spectroscopy to determine a high-resolution solution structure of the weak INA-INA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 A. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.     

Helix bending as a factor in protein/DNA recognition

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine–purine step: C–A/T–G, T–A, or C–G. Purine–purine steps, especially A–A, show the least tendency toward roll deformations. © 1998 John Wiley & Sons, Inc. Biopoly 44: 361–403, 1997     

Structure of a bent DNA: two-dimensional NMR studies on d(GAAAATTTTC)2

Intrinsic DNA bending is caused by specific DNA sequences. The decamer d(GA4T4C)2, when it repeats in a synthetic polymer or in kinetoplast DNA, results in a macroscopic bending of the molecule as a whole. We employed high-resolution two-dimensional NMR methods to examine the intrinsic structural properties of the d(GA4T4C)2 duplex in solution. Examination of the NOESY data at 50- and 100-ms mixing times indicated that the kinds of observed NOEs can originate if each of the ten nucleotidyl residues belongs to the B-DNA family, i.e., C2'-endo,anti. However, the degree of observed NOE intensities from the A-T junction as well as the observed AH2-AH2 cross-peaks from adjacent AT pairs could not be rationalized on the basis of a straight B-DNA model but could be explained by only a B-DNA model with some structural discontinuity at the A-T junction--the site of 2-fold symmetry in the molecule. In view of the fact that the degree of observed NOE intensities can be complicated by spin diffusion and by fine structural distortion, we have resorted to the use of quantitative theoretical NOESY simulation (which takes into account primary, secondary, and higher orders of NOE) to delineate the structural discontinuity at the A-T junction and to arrive at a structure for the duplex d(GA4T4C)2. We propose a "junction B-DNA model" which can quantitatively explain the 2D NOESY data at 100- and 50-ms mixing times. In this model the two structural blocks in the molecule, i.e., d(GA4).d(T4C) and d(T4C).d(GA4), are conformationally equivalent and are connected at the A-T junction where the base pairs are stably stacked, but the two local structural frames do not coincide in space. This model can create an overall bending of 10 degrees with a center of curvature 50 A away from the center of the duplex. It is the thesis of this paper that the observed bending in polymers with a repeat of d(GA4T4C)2 and the bending in natural DNAs where AnTn.AnTn repeats are present originate at the oligonucleotide repeat level.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Antitumor drug nogalamycin binds DNA in both grooves simultaneously: molecular structure of nogalamycin-DNA complex

The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, d[CGT(pS)ACG] and d[m5CGT(pS)Am5CG], were determined at high resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.     

Crystallographic study of one turn of G/C-rich B-DNA

The DNA decamer d(CCAGGCCTGG) has been studied by X-ray crystallography. At a nominal resolution of 1.6 A, the structure was refined to R = 16.9% using stereochemical restraints. The oligodeoxyribonucleotide forms a straight B-DNA double helix with crystallographic dyad symmetry and ten base-pairs per turn. In the crystal lattice, DNA fragments stack end-to-end along the c-axis to form continuous double helices. The overall helical structure and, notably, the groove dimensions of the decamer are more similar to standard, fiber diffraction-determined B-DNA than A-tract DNA. A unique stacking geometry is observed at the CA/TG base-pair step, where an increased rotation about the helix axis and a sliding motion of the base-pairs along their long axes leads to a superposition of the base rings with neighboring carbonyl and amino functions. Three-center (bifurcated) hydrogen bonds are possible at the CC/GG base-pair steps of the decamer. In their common sequence elements, d(CCAGGCCTGG) and the related G.A mismatch decamer d(CCAAGATTGG) show very similar three-dimensional structures, except that d(CCAGGCCTGG) appears to have a less regularly hydrated minor groove. The paucity of minor groove hydration in the center of the decamer may be a general feature of G/C-rich DNA and explain its relative instability in the B-form of DNA.     

Structural polymorphism of telomeric DNA regulated by pH and divalent cation

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.     

Structural model for an oligonucleotide containing a bulged guanosine by NMR and energy minimization

We present three-dimensional structural models for a DNA oligomer containing a bulged guanosine based on proton NMR data and energy minimization computations. The nonexchangeable proton resonances of the duplex 5'd(GATGGGCAG).d(CTGCGCCATC) are assigned by nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy connectivities, and the NMR spectrum is compared with that of a regular 8-mer of similar sequence, 5'd(GATGGCAG).d(CTGCCATC). Experimental proton-proton distances are obtained from NOESY spectra acquired with mixing times of 100, 150, and 200 ms. A refined three-dimensional structure for the bulge-containing duplex is calculated from regular B DNA starting coordinates by using the AMBER molecular mechanics program [Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., Profeta, S., & Weiner, P. (1984) J. Am. Chem. Soc. 106, 765-784]. We compare structures obtained by building the helix in three and four base pair increments with structures obtained by direct minimization of the entire nine base sequence, with and without experimental distance constraints. The general features of all the calculated structures are very similar. The helix is of the B family, with the extra guanine stacked into the helix, and the helix axis is bent by 18-23 degrees, in agreement with gel mobility data for bulge-containing sequences [Rice, J. A. (1987) Ph.D. Thesis, Yale University].     

NMR studies on the binding of antitumor drug nogalamycin to DNA hexamer d(CGTACG).

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.     

Structural studies of a stable parallel-stranded DNA duplex incorporating isoguanine:cytosine and isocytosine:guanine basepairs by nuclear magnetic resonance spectroscopy.

Isoguanine (2-hydroxyladenine) is a product of oxidative damage to DNA and has been shown to cause mutation. It is also a potent inducer of parallel-stranded DNA duplex structure. The structure of the parallel-stranded DNA duplex (PS-duplex) 5'-d(TiGiCAiCiGiGAiCT) + 5'-d(ACGTGCCTGA), containing the isoguanine (iG) and 5-methyl-isocytosine (iC) bases, has been determined by NMR refinement. All imino protons associated with the iG:C, G:iC, and A:T (except the two terminal A:T) basepairs are observed at 2 degrees C, consistent with the formation of a stable duplex suggested by the earlier Tm measurements [Sugiyama, H., S. Ikeda, and I. Saito. 1996. J. Am. Chem. Soc. 118:9994-9995]. All basepairs are in the reverse Watson-Crick configuration. The structural characteristics of the refined PS-duplex are different from those of B-DNA. The PS duplex has two grooves with similar width (7.0 A) and depth (7.7 A), in contrast to the two distinct grooves (major groove width 11.7 A, depth 8.5 A, and minor groove width 5.7 A, depth 7.5 A) of B-DNA. The resonances of the amino protons of iG and C are clearly resolved and observable, but those of the G and iC are very broad and difficult to observe. Several intercalators with different complexities, including ethidium, daunorubicin, and nogalamycin, have been used to probe the flexibility of the backbone of the (iG, iC)-containing PS-duplex. All of them produce drug-induced UV/vis spectra identical to their respective spectra when bound to B-DNA, suggesting that those drugs bind to the (iG, iC)-containing PS-duplex using similar intercalation processes. The results may be useful in the design of intercalator-conjugated oligonucleotides for antisense applications. The study presented in this paper augments our understanding of a growing number of parallel-stranded DNA structures, including the G-quartet, the i-motif, and the unusual homo basepaired parallel-stranded double helix. Their possible relevance is discussed.