X-ray and NMR studies of the DNA oligomer d(ATATAT): Hoogsteen base pairing in duplex DNA

We present and analyze the structure of the oligonucleotide d(ATATAT) found in two different forms by X-ray crystallography and in solution by NMR. We find that in both crystal lattices the oligonucleotide forms an antiparallel double helical duplex in which base pairing is of the Hoogsteen type. The double helix is apparently very similar to the standard B-form of DNA, with about 10 base pairs per turn. However, the adenines in the duplex are flipped over; as a result, the physicochemical features of both grooves of the helix are changed. In particular, the minor groove is narrow and hydrophobic. On the other hand, d(ATATAT) displays a propensity to adopt the B conformation in solution. These results confirm the polymorphism of AT-rich sequences in DNA. Furthermore, we show that extrahelical adenines and thymines can be minor groove binders in Hoogsteen DNA.     

In search of a Hoogsteen base paired DNA duplex in aqueous solution

When the oligodeoxynucleotides d(A)6 and d(T)6 are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC in equilibrium with H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)6. We show that (i) addition of extra d(A)6 does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix-drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, 31P NMR spectra, 1H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded. Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

Considering the oligonucleotide secondary structures in thermodynamic and kinetic analysis of DNA duplex formation

The influence of secondary structures of DNA oligonucleotides on thermodynamics and kinetics at the formation of their bimolecular complexes (duplexes) has been studied. The models considering inherent secondary structures of duplex components and their influence on quantitative thermodynamic and kinetic characteristics of the duplexes have been developed. The values of thermodynamic impacts given by individual structural elements of the double helix have been shown to depend on hairpin structuring of the duplex components. The «concentration» method to consider oligonucleotides intramolecular structure with thermodynamic parameters of bimolecular duplex formation has been proposed. According to stop-flow measurements, the observed values of association and dissociation constants are influenced by the presence of inherent structures in duplex components. The influence observed is increased with the lowering of the sample temperature. The analysis of experimental data involving the developed models provides the possibility to determine «proper» kinetic constants for the helix-to-coil transition. The difference between observed and calculated rate constants can amount up to two or more orders of magnitude.     

NMR spectroscopic study of a DNA duplex with mercury-mediated T-T base pairs

Recently, we reported that T-T mismatches can specifically recognize Hg(II) (T-Hg(II)-T pair formation). In order to understand the properties of the T-Hg(II)-T pair, we recorded NMR spectra for a DNA duplex, d(CGCGTTGTCC).d(GGACTTCGCG), with two successive T-T mismatches (Hg (II)-binding sites). We assigned 1H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg(II) by using 1D 1H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

Structural and dynamic studies of a non-self-complementary dodecamer DNA duplex.

A non-self-complementary dodecamer duplex d(CCTAAATTTGCC).d(GGCAAATTTAGG) has been investigated in solution by high resolution 1H NMR. Almost complete resonance assignment of both non-exchangeable and exchangeable protons, has been achieved. The duplex is essentially B-type, with distortions apparent at the AT and TA steps. These distortions and their affects on dynamics have been probed by the measurement of base-pair lifetimes, and observation of water of hydration. Base-pair opening rates were derived from measurements of T1's and effects on linewidths of the T and G imino protons on addition of an exchange catalyst. Our results are generally in line with observations reported for other systems, but we see only a slight drop in the A.T base-pair lifetime on moving out from the central region. This observation is reinforced by the detection of DNA-water nOe's for residues distributed throughout the dodecamer sequence.     

New thermodynamic characterization and transition mechanism of DNA duplex formation.

A new transition mechanism of DNA duplex association was proposed and a segregated transition model (STM) was further derived. The experimental results in various molar ratios showed that the duplex association transition is imperfect and the thermodynamic properties and self-transition behavior of single strands exert a significant influence on DNA duplex formation.     

An NMR structural study of deaminated base pairs in DNA.

The structurally aberrant base pairs TG, UG and TI may occur in DNA as a consequence of deamination of 5-methylcytosine, cytosine and adenine respectively. Results of NMR spectroscopic studies are reported here for these deaminated base pairs in a model seven base pair long oligonucleotide duplex. We find that in all three cases, the DNA helix is a normal B form and both mispaired bases are intrahelical and hydrogen bonded with one another in a wobble geometry. Similarly, in all three cases, all sugars are found to be normal C2' endo in conformation. Symmetric structural perturbations are observed in the helix twist on the 3' side of the mispaired pyrimidine and on the 5' side of the mispaired purine. In all three cases, the amino group of the G residue on the 3' side of the mispaired pyrimidine shows hindered rotation. Although less thermodynamically stable than helices containing only Watson-Crick base pairs, these helices melt normally from the ends and not from the mispair outwards.     

Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation.

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12 bp, were selected to evaluate the heat capacity changes and temperature-dependent thermodynamic properties of duplex formation. Direct comparison reveals that the temperature-independent thermodynamic parameters could provide a reasonable approximation only when the temperature of interest has a small deviation from the mean melting temperature over the experimental range. The heat capacity changes depend on the base composition and sequences and are generally limited in the range of -160 to approximately -40 cal.mol-1.K-1 per base pair. In contrast to the enthalpy and entropy changes, the free energy change and melting temperature are relatively insensitive to the heat capacity change. Finally, the 16 NN-model free energy parameters and one helix initiation at physiological temperature were extracted from the temperature-dependent thermodynamic data of the 41 RNA/DNA hybrids.     

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4₁22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C₂:C₂⁺ (singly-protonated between the Watson– Crick sites), G₃:G₃ (between the minor groove sites), A₄:A₄ (between the major groove sites) and A₅:A₅ (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Deuterium NMR study of structural and dynamic properties of horseradish peroxidase

High field deuterium NMR spectra have been recorded for various horseradish peroxidase complexes reconstituted with hemins possessing specific 2H labels. The line width of the 2H NMR signals of deuteroheme reconstituted-horseradish peroxidase (HRP) and its cyano complex for the immobilized skeletal 2-2H and 4-2H labels yield the overall protein rotational correlation time (22 ms at 55 degrees C), which is consistent with expectations based on molecular weight. Meso-2H4 labels yield broad (1.3 kHz) signals just upfield from the diamagnetic protein envelope for HRP, and in the central portion of the protein envelope for the CN- ligated resting state HRP. Meso-2H4-labeled mesohemin-reconstituted HRP exhibits a similar signal but shifted further upfield by approximately 10 ppm. The net upfield meso-H hyperfine shifts confirm a five-coordinate structure for resting state HRP. 2Ha resonances for essentially rotationally immobile vinyl groups were detected in both resting state HRP and CN- ligated resting state HRP. Heme methyl-2H-labeling yields relatively narrow lines (approximately 80 Hz) indicative of effective averaging of the quadrupolar relaxation by rapid methyl rotation. Thus the 2H line width of rapidly rotating methyls in hemoproteins can be used effectively to determine the overall protein tumbling rate. Preliminary 2H experiments in meso-2H4-labeled compound I do not support large pi spin density at these positions on the porphyrin cation radical, and argue for a a1u rather than a a2u orbital ground state.     

The interaction of polyamines with DNA: a 23Na NMR study.

The interaction between a variety of polyamines, both naturally occurring and synthetic, and calf thymus DNA has been studied using 23Na NMR. The relaxation behaviour of 23Na reflects the extent of interaction of Na+ with DNA phosphate groups and therefore the extent of charge neutralisation of DNA phosphate groups (P) by polyamine amino and imino groups (N) in solutions of DNa, polyamine and Na+. The studies reveal that whereas spermine and spermidine are capable of expelling nearly all of the Na+ ions from DNA at N/P approximately 1, diamines such as putrescine and homologues of spermine and spermidine are capable of neutralising only roughly 50% of DNA phosphates. The results provide a challenge to current models of DNA-polyamine interactions.     

NMR study of self-paired parallel duplex of d(AAAAACCCCC) in solution.

The oligonucleotide d(A5C5) in solution forms a parallel self-duplex at neutral and low pH values. H2O NMR spectra at pH 5.1 indicate the presence of five imino resonances at lower temperatures; and the structure is stable up to 60 degrees C. These signals can arise only from the hemiprotonated C+.C pairs [Westhof, E. and Sundaralingham, M. (1980) Biochemistry 77, 1852-1856; Westhof, E. and Sundaralingham, M. (1980) J. Mol. Biol. 142, 331-361] and constitute the first direct observation of C+.C hemiprotonated pairs in solution. The cross peaks from H1's and more than five distinct AH8's in 500 MHz 1H 2D-NOESY spectra indicate that there are two conformationally different and energetically similar A-tracts. There is good qualitative agreement between NOESY data and two theoretically derived structures in which A-tracts are reverse Watson-Crick and reverse Hoogsteen base-paired, respectively.     

NMR with 13C, 15N-doubly-labeled DNA: The shape Antennapedia homeodomain complex with a 14-mer DNA duplex

Nearly complete ¹H, ¹³C and¹⁵ N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly ¹⁵N-labeled Antennapedia homeodomain. The DNA was either fully ¹³C,¹⁵N-labeled or contained uniformly ¹³C, ¹⁵N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via¹ H–¹H NOEs observed in 3D¹³ C-resolved NOESY; and (iii) assignment of the imino and amino groups via ¹H–¹H NOEs and¹⁵ N–¹H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that ¹H signals of the protein were filtered out in ¹³C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding ¹³C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.     

Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide). An NMR study and molecular dynamic simulations with explicit water molecules.

We investigated the behaviour of a 15mer DNA duplex, [5'd(CAGAGTCACTGGCTC)3']. [5'd(GAGCCAG)3' + 5'd(GACTCTG)3'] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pKa of approximately 4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G.C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G.A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn).A+(anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three-dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.     

The crystal structure of an alternating RNA heptamer r(GUAUACA) forming a six base-paired duplex with 3'-end adenine overhangs

The crystal structure of an alternating RNA heptamer r(GUAUACA) has been determined to 2.0 Å resolution and refined to an R_work of 17.1% and R_free of 18.5% using 2797 reflections. The heptamer crystallized in the space group C222 with a unit cell of a = 25.74, b = 106.58, c = 30.26 Å and two independent strands in the asymmetric unit. Each heptamer forms a duplex with its symmetry-related strand and each duplex contains six Watson–Crick base pairs and 3′-end adenosine overhangs. Therefore, two kinds of duplex (duplex 1 and duplex 2) are formed. Duplexes 1 stack on each other forming a pseudo-continuous column, which is typical of the RNA packing mode, while duplex 2 is typical of A-DNA packing with its termini in abutting interactions. Overhang adenine residues stack within the duplexes with C3′-endo sugar pucker and C2′-endo sugar pucker in duplexes 1 and 2, respectively. A Na⁺ ion in the crystal lattice is water bridged to two N1 atoms of symmetry-related A7 bases.     

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), and CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA.TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A.T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in two different structures are reported.     

Hybridization of the bridged oligonucleotides with DNA: thermodynamic and kinetic studies

Hybridization properties of oligonucleotides containing non-nucleotide inserts designed on the basis of synthetic abasic sites, oligomethylene diols or oligoethylene glycols have been characterized. The influence of the inserts which generate extrahelical anucleotidic bulges on thermodynamics, kinetics of hybridization of bridged oligonucleotide with DNA has been studied by UV-melting and stopped-flow techniques. Circular dichroism spectrometry data show that anucleotidic bulges in the middle of the duplex does not alter the B-form helix conformation. Nevertheless, the insert induces destabilization of the duplex structure, caused mostly by the considerable enhancement of the dissociation rates. Free energy increments for the extrahelical anucleotidic bulges can be described in the nearest-neighbor approximation. The thermodynamic effect of the insert lengthening obeys a simple Jacobson-Stockmayer entropy extrapolation. Independently of the insert type, the free energy term is directly proportional to the logarithm of the number of bonds between the oligonucleotide fragments. The behavior of hydrophobic inserts formed by 10-hydroxydecyl-1-phospate units is an exception to the rule.     

Transition characteristics and thermodynamic analysis of DNA duplex formation: a quantitative consideration for the extent of duplex association

Transition characteristics and thermodynamic properties of the single-stranded self-transition and the double-stranded association were investigated and analyzed for 9-, 15- and 21-bp non-self-complementary DNA sequences. The multiple transition processes for the single-stranded self-transition and the double-stranded association were further put forth. The experimental results confirmed that the double-stranded association transition was generally imperfect and the thermodynamic properties of the single-stranded self-transition would exert an influence on a duplex formation. Combining ultraviolet melting experiments in various molar ratios, the extent of duplex association was estimated for three double-stranded DNAs. In our experimental range, the extent of duplex association decreases with increasing the number of base pairs in DNA sequences, which suggest that the short oligonucleotides may proceed in a two-state transition while the long oligonucleotides may not. When the extent of duplex association was considered, the true transition enthalpies of a duplex formation derived from UV and differential scanning calorimetry measurements were in good agreement.