Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na⁺. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.     

Effect of dangling ends on the stability of a DNA double helix.

The effect of 3' and 5' dangling ends has been studied on the stability of a self-complementary double-helix of d(ATGCGCAT) in 1 mol dm-3 NaCl-phosphate buffer. It was shown that the effect on the DNA was smaller than that on r(AUGCAU).     

Influence of base sequence on the stability of the double helix of DNA.

On the basis of published measurements of the melting transitions of synthetic polydeoxyribonucleotides with known sequences we have determined the parameters of the interplane (stacking) interactions of base pairs in DNA over the range of ionic strengths from 0.01 to 0.1 M Na+. We found that deviations of the stacking-interaction energy from the mean value of 7-8 kcal/mole were extremely small and did not exceed 0.2 kcal/mole. We report an analysis of the influence of the heterogeneity of the stacking interactions on the melting parameters of polynucleotides with random sequences (models of natural DNA's). Inclusion of this effect does not significantly distort the linear dependence of the melting temperature on the relative content of G-C pairs and insignificantly affects the width of the helix-coil transition in DNA under normal conditions. However it is the heterogeneity of the stacking interactions that plays the crucial role in the melting of DNA under conditions where the difference between the relative stabilities of the A-T and G-C pairs tends to zero, as in concentrated solutions of tetraethylammonium and tetramethylammonium salts.     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Effect of low-molecular amines on DNA conformation and stability of the double helix]

Interaction of low-molecular amines (cystamine, cysteamine, cystaphose, asparagine, beta-alanine) with DNA was studied. The amines change the positive circular dichroism (CD) band of DNA as well as temperature and range width of melting. Effect of amines on DNA depends on ionic strength of the solvent, concentration and structure of the ligand. Monamines cause destabilization of DNA double helix followed by stabilization as ligand concentration increases. At concentrations stabilizing the double helix DNA conformation undergoes transition from the B- to C-form. The results obtained enable to relate the stabilizing effect of low-molecular amines and conformational B leads to C-transition to the non-specific interaction of ligand amino groups with DNA phosphates, and the destabilizing effect of monoamines of low concentrations to their interaction with bases, mainly in the denaturated sites of DNA. It is proposed that a stronger effectiveness of amines as compared to monovalent metals in the conformational shift of DNA towards the C-form is due to the additional effect of disturbance of hydrophobic interactions in DNA double helix.     

Elongational flow studies on DNA in aqueous solution and stress-induced scission of the double helix.

Elongational flow techniques are used to investigate the birefringent response and flow-induced molecular scission of monodisperse phage-DNA samples in aqueous solution. A 4-roll mill apparatus was used to characterize the solutions at low stain rates, epsilon less than or equal to 300 s-1, and the opposed jets apparatus used to study fracture of the DNA molecules at strain rates up to 15 x 10(3) s-1. The molecular weight values were measured before and after fracture in elongational flow using the high-resolution technique of pulsed field gel electrophoresis (PFGE). The birefringent response incorporates both rigid and flexible components. The birefringence is nonlocalized and rises gradually to a plateau value, similar to rigid-rod behavior. In addition a certain minimum value in the strain rate is necessary, an onset value epsilon 0, before the signal appears, indicating a flexible component. This behavior is consistent with a hinged-rod model and is similar to that observed for the protein collagen molecule at elevated temperature. We propose that this type of behavior is likely for multistrand rope-like macromolecules where localized separation or partial untwisting of the intertwined chains occurs, creating temporary hinges, in accordance with biochemical evidence for sequence-specific sites of flexibility. Results are presented on the entanglement effects at high concentrations. We have calculated rotational diffusion rates as a function of concentration and molecular weight. Using PFGE to measure the molecular weight profiles, our fracture studies at high strain rates demonstrate chain halving and quartering in accordance with the predictions of the thermally activated barrier to scission theory for single-chain polymers.     

Effect of the size of the ligand molecule on the stability of the DNA double helix

The influence of ligand length on helix-coil transition parameters in the presence of different ligand concentrations has been considered theoretically. A decrease of this influence with an increase of ligand length when the binding constant has a constant value was shown. When binding free energy is proportional to the ligand length, i. e. when K = K0m (K-binding constant, m-ligand length) the length effect is unambigous. In the presence of low-ligand concentration the stability of DNA double helix increases with their length, whereas in the presence of high concentrations it decreases.     

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.     

Cytosine, the double helix and DNA self-assembly

DNA self-assembly has crucial implications in reading out the genetic information in the cell and in nanotechnological applications. In a recent paper, self-assembled DNA crystals displaying spectacular triangular motifs have been described (Zheng et al., 2009). The authors claimed that their data demonstrate the possibility to rationally design well-ordered macromolecular 3D DNA lattice with precise spatial control using sticky ends. However, the authors did not recognize the fundamental features that control DNA self-assembly in the lateral direction. By analysing available crystallographic data and simulating a DNA triangle, we show that the double helix geometry, sequence-specific cytosine–phosphate interactions and divalent cations are in fact responsible for the precise spatial assembly of DNA.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

Tau could protect DNA double helix structure

The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 degrees C and that of plasmid from 75 to 85 degrees C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated with tau were markedly slower than those of DNA and DNA/bovine serum albumin used as controls during thermal denaturation. In contrast, rates of decrease in the DNA absorbance with tau were faster than those of controls when samples were immediately transferred from thermal conditions to room temperature. It revealed that tau prevented DNA from thermal denaturation, and improved renaturation of DNA. Circular dichroic spectra results indicated that there were little detectable conformational changes in DNA double helix when tau was added. Furthermore, tau showed its ability to protect DNA from hydroxyl radical (.OH) attacking in vitro, implying that tau functions as a DNA-protecting molecule to the radical.     

Geometric features of the DNA double helix in the supercoiled state

The geometric features of the DNA molecule in the supercoiled state were considered. A model of the supercoiled structure of the DNA molecule was constructed; the model takes into account its natural helicity. The force factors arising in the molecule at various superhelix angles were calculated.     

NMR study of the effect of sugar-phosphate backbone ethylation on the stability and conformation of DNA double helix

Temperature variation studies of the imido proton NMR and 31P NMR resonances of the self-associated d(C-C-A-A-G-A-T-T-G-G) and d[C-C-A-A-G-p(Et)-A-T-T-G-G] duplexes (both the R and S diastereoisomers) and the heteroduplexes formed with their complementary strand d(C-C-A-A-T-C-T-T-G-G) were carried out in aqueous solution. Results demonstrate that phosphate backbone ethylation did not disrupt the interstrand hydrogen bonding involved in double-helix formation but perturbed the helix. The S isomer perturbed the duplex more than the R isomer. The line broadening patterns and faster fraying motion in the alkylated duplexes compared to those in the nonalkylated duplexes indicate that the perturbation introduced in the middle propagates along the backbone to the end of the duplex.     

Na2CO3 influence on DNA double helix stability: strong anion destabilizing effect

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.