Proton nuclear magnetic resonance investigations of the nucleation and propagation reactions associated with the helix-coil transition of d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpCpApT have been monitored in the presence of 0.1 M phosphate at neutral pH in aqueous solution. While the resonance positions of the terminal and internal AT base pairs shift upfield and broaden as average resonances with increasing temperature (helix and coil exchange several times prior to exchange with water from the coil form), those of the central GC base pairs broaden in the absence of upfield shifts (exchange with water occurs each time helix converts to coil). The line-width changes at the AT base pairs monitor the lifetime of the coil state at these positions prior to exchange with water while the line-width changes at the GC base pairs monitor the lifetime of the helix prior to dissociation to strands. This permits the separation of the propagation reaction at the AT base pairs from the nucleation reaction at the GC base pairs during helix formation. The experimental data have been quantitatively analyzed to yield (at 20 degrees) a nucleation formation rate of approximately 10(3) sec-1 for the GC base pairs (bimolecular rate constant of approximately 6 times 10(6) l. mol-1 sec-1) and a dissociation rate of 6 times 10(2) sec-1 at these same base pairs (unimolecular dissociation to strands). The unimolecular propagation reactions at the terminal and terminal base pairs are associated with reaction rates greater than 10(4) sec-1. These values are consistent with a slow formation of a stable nucleus at the GC base pairs followed by a rapid propagation reaction at the AT base pairs. The line width of the (GC) central base pairs in the presence of phosphate is a direct measure of the lifetime of the total helix and yields an activation energy of 45 kcal for helix to coil conversion measured over a narrow temperature range. The exchange from the coil form with water is catalyzed by 0.1 M phosphate with a rate constant kHPO2-/4 = 0.2 times 10(6) 1. mol-1 sec-1.     

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 the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex

One- and two-dimensional nuclear magnetic resonance (NMR) experiments have been undertaken to investigate the conformation of the d(C1-G2-C3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) self-complementary dodecanucleotide (henceforth called O6meG.C 12-mer), which contains C3.O6meG10 interactions in the interior of the helix. We observe intact base pairs at G2.C11 and G4.C9 on either side of the modification site at low temperature though these base pairs are kinetically destabilized in the O6meG.C 12-mer duplex compared to the G.C 12-mer duplex. One-dimensional nuclear Overhauser effects (NOEs) on the exchangeable imino protons demonstrate that the C3 and O6meG10 bases are stacked into the helix and act as spacers between the flanking G2.C11 and G4.C9 base pairs. The nonexchangeable base and H1', H2', H2', H3', and H4' protons have been completely assigned in the O6meG.C 12-mer duplex at 25 degrees C by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) experiments. The observed NOEs and their directionality demonstrate that the O6meG.C 12-mer is a right-handed helix in which the O6meG10 and C3 bases maintain their anti conformation about the glycosidic bond at the modification site. The NOEs between the H8 of O6meG10 and the sugar protons of O6meG10 and adjacent C9 exhibit an altered pattern indicative of a small conformational change from a regular duplex in the C9-O6meG10 step of the O6meG.C 12-mer duplex. We propose a pairing scheme for the C3.O6meG10 interaction at the modification site. Three phosphorus resonances are shifted to low field of the normal spectral dispersion in the O6meG.C 12-mer phosphorus spectrum at low temperature, indicative of an altered phosphodiester backbone at the modification site. These NMR results are compared with the corresponding parameters in the G.C 12-mer, which contains Watson-Crick base pairs at the same position in the helix.     

Bulged adenosine influence on the RNA duplex conformation in solution

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.     

Parallel-stranded DNA with mixed AT/GC composition: role of trans G.C base pairs in sequence dependent helical stability

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs

Adjacent GxU wobble base pairs are frequently found in rRNA. Atomic structures of small RNA motifs help to provide a better understanding of the effects of various tandem mismatches on duplex structure and stability, thereby providing better rules for RNA structure prediction and validation. The crystal structure of an RNA duplex containing the sequence r(GGUAUUGC-GGUACC)2 has been solved at 2.1 A resolution using experimental phases. Novel refinement strategies were needed for building the correct solvent model. At present, this is the only short RNA duplex structure containing 5'-U-U-3'/3'-G-G-5' non-symmetric tandem GxU wobble base pairs. In the 14mer duplex, the six central base pairs are all displaced away from the helix axis, yielding significant changes in local backbone conformation, helix parameters and charge distribution that may provide specific recognition sites for biologically relevant ligand binding. The greatest deviations from A-form helix occur where the guanine of a wobble base pair stacks over a purine from the opposite strand. In this vicinity, the intra-strand phosphate distances increase significantly, and the major groove width increases up to 3 A. Structural comparisons with other short duplexes containing symmetrical tandem GxU or GxT wobble base pairs show that nearest-neighbor sequence dependencies govern helical twist and the occurrence of cross-strand purine stacks.     

High base pair opening rates in tracts of GC base pairs

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.     

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.     

Sequence-specific recognition of deoxyribonucleic acid. Chemical synthesis and nuclear magnetic resonance assignment of the imino protons of lambda OR3 operator deoxyribonucleic acid

Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.     

Proton exchange and local stability in a DNA triple helix containing a G.TA triad

Recognition of a thymine-adenine base pair in DNA by triplex-forming oligonucleotides can be achieved by a guanine through the formation of a G.TA triad within the parallel triple helix motif. In the present work, we provide the first characterization of the stability of individual base pairs and base triads in a DNA triple helix containing a G.TA triad. The DNA investigated is the intramolecular triple helix formed by the 32mer d(AGATAGAACCCCTTCTATCTTATATCTGTCTT). The exchange rates of imino protons in this triple helix have been measured by nuclear magnetic resonance spectroscopy using magnetization transfer from water and real-time exchange. The exchange rates are compared with those in a homologous DNA triple helix in which the G.TA triad is replaced by a canonical C⁺.GC triad. The results indicate that, in the G.TA triad, the stability of the Watson–Crick TA base pair is comparable with that of AT base pairs in canonical T.AT triads. However, the presence of the G.TA triad destabilizes neighboring triads by 0.6–1.8 kcal/mol at 1°C. These effects extend to triads that are two positions removed from the site of the G.TA triad. Therefore, the lower stability of DNA triple helices containing G.TA triads originates, in large part, from the energetic effects of the G.TA triad upon the stability of canonical triads located in its vicinity.     

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.     

High resolution NMR study of CAP binding site 22mer in H2O solution

High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

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).     

Behavior of the imino protons of the lambda OR3 17mer studied by high resolution NMR

Imino proton resonances of lambda OR3 17mer were observed with time-shared Redfield pulse method by using a JEOL 500 MHz NMR instrument. They show gradual broadening and disappearance with the elevation of temperature indicating the stepwise melting of the duplex. By the selective irradiation at each peak position nuclear Overhauser effects were observed between the imino and adenine C2H protons and between imino protons themselves. Combining these data, fifteen imino proton resonances could be assigned to each base pair except two terminal AT base pairs. Based on the assignment it can be said that AT rich regions near the terminal melt first, followed by the melting of the inside GC rich core. The two AT base pairs in the middle of the GC core are resistant to heat. Spin lattice relaxation times were also observed and the results are consistent with the melting profile.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.