Energetics of complex formation of the dna hairpin structure d(GCGAAGC) with aromatic ligands

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantrone and mutagens ethidium bromide and proflavin have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the hairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.     

NMR study of complex formation of aromatic ligands with heptadeoxynucleotide 5'-d(GCGAAGC) forming stable hairpin structure in aqueous solution

Complex formation of hairpin-producing heptadeoxynucleotide 5'-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional 1H NMR and two-dimensional correlation 1H-1H (2M-TOCSY, 2M-NOESY), 1H-31P (2M-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2M-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

On the origin of the decrease in stability of the DNA hairpin d(GCGAAGC) on complexation with aromatic drugs

Molecular dynamics simulations of drug-DNA complexes have been carried out in order to explain the experimentally observed decrease in thermal stability of the DNA hairpin d(GCGAAGC) on binding the aromatic drug molecules, daunomycin, ethidium bromide, novantrone and proflavine. This complexation behavior is in contrast to the stabilizing effect of the same aromatic drug molecules on DNA duplexes. Analysis of the energy parameters and the hydration properties of the complexes shows that the main factor correlating with the decrease in melting temperatures of the drug-hairpin complexes is the number of water bridges, with a reduction of at least 40% on ligand binding.     

Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings

The structure of the ¹³C,¹⁵N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond ¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C and ¹³C-¹⁵N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.     

Hybridization kinetics of oligodeoxyribonucleotides with a d(GCGAAGC) hairpin at the 3'-end

In order to protect them against enzymatic attack of serum in the antisense strategy, oligodeoxyribonucleotides can be protected on their 3'-side by the sequence d(GCGAAGC) which spontaneously forms a hairpin which is known for its extraordinary stability with regard to thermal denaturation or nuclease degradation (I. Hirao, G. Kawai, S. Yoshizawa, Y. Nishimura, Y. Ishido, K. Watanabe and K. Miura, Nucleic Acids Res. 22, 576-582 (1994)). By contrast, the hairpin does not prevent hybridization of the 5'-stem part of the oligonucleotide to a target DNA strand. As soon as this pairing occurs, the stability of the hairpin is disrupted. Its opening rate, followed by its pairing if possible, is of the same order than that of hybridization of the stem part.     

Contribution of enthalpy to the energetics of complex formation of aromatic ligands with DNA

The energy contributions of electrostatic, van der Waals interactions, hydrogen bonds, and interactions of charge transfer type to the enthalpy of complex formation of the double-stand DNA with the antitumor antibiotics daunomycin, nogalamycin, and novantron, as well as the mutagens ethidium bromide and proflavine have been calculated. According to the calculations, the van der Waals component (except for nogalamycin) is energetically favorable during complex formation of the antibiotics with DNA, and the contributions of H bonds and electrostatic interactions are unfavorable, with the probability of charge transfer in the complexes being low. It has been shown that the relatively low value of the experimental enthalpy of binding is the sum of components greater in absolute value and different in the sign, which is the cause of large errors in estimating the total enthalpy of complex formation of aromatic ligands with DNA.     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

Opening of the extraordinarily stable mini-hairpin d(GCGAAGC).

For the purposes of the antisense strategy oligodeoxyribonucleotides can be protected against serum and cell nuclease digestion by tagging at their 3'-end with a sequence naturally forming a very stable hairpin, d(GCGAAGC). This nuclease-resistant hairpin is also known for its high thermostability. We demonstrate in this study that attachment of d(GCGAAGC) at the 3'-end of an oligodeoxyribonucleotide does not hinder hybridization of the 5'-part of this oligonucleotide to a complementary DNA strand. Moreover, the hairpin is in equilibrium between a folded and an open structure, with an energy minimum in favor of pairing if it is possible, even with mismatches.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

The three-dimensional structure of a DNA hairpin in solution two-dimensional NMR studies and structural analysis of d(ATCCTATTTATAGGAT)

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two-dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5' and H5" resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter-residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation-matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles beta, gamma and epsilon were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi-conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B-type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained-molecular-mechanics as well as free-molecular-mechanics calculations are in accordance with those obtained by means of the analysis described above. The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T-A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B-type double-helical stem. The second base of the loop is folded into the minor groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.     

Kinetic and thermodynamic characterization of DNA duplex–hairpin interconversion for two DNA decamers: d(CAACGGGTTG) and d(CAACCCGTTG)

The duplex–hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50°C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (ΔH_DH = 26 ± 3 kcal/mole) for both decanters at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of ΔH_DH, and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G·G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAAC-CCGTTG) decamer was determined to form a duplex with two centrally located C·C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C⁺·C mismatch. At pH values greater than 6.4, the hairpin–duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5–7.0 mM and salt concentrations of 10–100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the ΔH_DH, dependence on sail indicate that an intermediate internally bulged duplex (no C⁺·C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation. © 1995 John Wiley & Sons, Inc.     

Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation

Differential scanning calorimetry (DSC), temperature-dependent uv-absorption spectroscopy, and temperature-dependent CD were used to monitor and characterize the salt-dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex-to-single-strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na⁺), a single, monophasic transition curve was observed. At lower salt (?0.01M Na⁺), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.     

Energetics of the B-H transition in supercoiled DNA carrying d(CT)x.d(AG)x and d(C)n.d(G)n inserts.

We have studied the B-H transition in the d(AG)x inserts of varying length under superhelical stress. The new data and previously published results for the d(G)31 insert are treated within a phenomenological model of the B-H transition, making it possible to obtain, for the first time, the energy parameters of the B-H transition in the d(AG)x and d(G)n sequences.     

DNA octaplex formation with an I-motif of water-mediated A-quartets: reinterpretation of the crystal structure of d(GCGAAAGC)

The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and reasonably refined including the disordered residues. The two DNA strands form a base-intercalated duplex, and the four duplexes are assembled according to the crystallographic 222 symmetry to form an octaplex. In the central region, the eight strands are associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium cations link the four duplexes to support the octaplex formation. Based on these structural features, a proposal that folding of d(GAAA)n, found in the non-coding region of genomes, into an octaplex can induce slippage during replication to facilitate length polymorphism is presented.     

Contribution of changes in translational, rotational, and vibrational degrees of freedom to the energy of complex formation of aromatic ligands with DNA

A method for calculation and analysis of the contribution of changes in translational, rotational, and vibrational degrees of freedom to the energy of complex formation of aromatic compounds with DNA duplex has been developed. The results of calculations of the thermodynamic parameters (ΔG, ΔH, ΔS) indicate that changes in the translational and rotational degrees of freedom destabilize, and changes in the vibrational degree of freedom stabilize the complexes, the energy contribution from the movements under consideration being predominantly of entropic character. It is shown that the energy components of changes in translational, rotational, and vibrational degrees of freedom are in the main comparable with the experimentally determined thermodynamic parameters, which requires consideration of these components in the energy analysis of complex formation of aromatic molecules with DNA. It has been found that the total contribution of changes in translational, rotational, and vibrational degrees of freedom to the Gibbs energy of complexing of aromatic molecules with DNA can be assumed to be on the average the same for different ligands and equal to 8.2 kcal/mol.     

Three-dimensional structure of a DNA hairpin in solution: two-dimensional NMR studies and distance geometry calculations on d(CGCGTTTTCGCG)

The three-dimensional structure of d(CGCGTTTTCGCG) in solution has been determined from proton NMR data by using distance geometry methods. The rate of dipolar cross-relaxation between protons close together in space is used to calculate distances between proton pairs within 5 A of each other; these distances are used as input to a distance geometry algorithm that embeds this distance matrix in three-dimensional space. The resulting refined structures that best agree with the input distances are all very similar to each other and show that the DNA sequence forms a hairpin in solution; the bases of the loop region are stacked, and the stem region forms a right-handed helix. The advantages and limitations of the technique, as well as the computer requirements of the algorithm, are discussed.     

Energetics of OCP1-OCP2 complex formation

OCP1 and OCP2, the most abundant proteins in the cochlea, are putative subunits of an SCF E3 ubiquitin ligase. Previous work has demonstrated that they form a heterodimeric complex. The thermodynamic details of that interaction are herein examined by isothermal titration calorimetry. At 25 degrees C, addition of OCP1 to OCP2 yields an apparent association constant of 4.0 x 10(7) M(-1). Enthalpically-driven (DeltaH=-35.9 kcal/mol) and entropically unfavorable (-TDeltaS=25.5 kcal/mol), the reaction is evidently unaccompanied by protonation/deprotonation events. DeltaH is strongly dependent on temperature, with DeltaC(p)=-1.31 kcal mol(-1) K(-1). Addition of OCP2 to OCP1 produces a slightly less favorable DeltaH, presumably due to the requirement for dissociation of the OCP2 homodimer prior to OCP1 binding. The thermodynamic signature for OCP1/OCP2 complex formation is inconsistent with a rigid-body association and suggests that the reaction is accompanied by a substantial degree of folding.     

Effect of DNA structure on the formation of collagen-DNA complex

Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms.     

A proton NMR study of a DNA dumb-bell structure with hairpin loops of only two nucleotides: d(CACGTGTGTGCGTGCA).

One- and two-dimensional nuclear magnetic resonance (NMR) experiments were used to study the conformation of the DNA hexadecanucleotide d(CACGTGTGTGCGTGCA) in aqueous solution. NMR spectra were recorded for the compound in D2O and in H2O/D2O (90/10) over the temperature range 1 degree C-60 degrees C. Assignments of imino proton resonances and of non-exchangeable proton resonances (except for some H4', H5' and H5" resonances) are given. The 1H-NMR spectra indicate that below about 20 degrees C, the compound exists as a single monomolecular species. Between 20 degrees C and 55 degrees C the oligonucleotide occurs as a mixture of structures in fast exchange on the NMR time scale, except for the temperature region 30 degrees - 34 degrees C, where substantial line broadening indicates intermediate exchange; above 60 degrees C the single strand predominates. The imino proton spectra, chemical shift values, and scalar coupling and NOE data reveal that the monomeric form, which is exclusively present below 20 degrees C, consists of a structure with a B-DNA double helix region of six base pairs, both ends of which are closed by hairpin loops of only two nucleotides, giving the molecule a dumbbell-like structure: [sequence: see text].     

An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres

The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.