Structure of aggregates of water and Li+, Na+, or K+ counterions with nucleic acid in solution

The position and orientation of water molecules hydrating fragments of DNA in the B and Z conformations are analyzed with the help of computer simulations. Monte Carlo studies are carried out at room temperature, high relative humidity (500 water molecules per pitch) and in the presence of counterions such as Li⁺, Na⁺, and K⁺. Differences in hydration patterns and in the counterionic structures were found by compairing B-DNA with Z-DNA double helices and B-DNA helices with different base-pair distributions. The present extension of our similations to Z-DNA and to Li⁺ and K⁺ counterions permits some general conclusions concerning nucleic acids in solution.     

Molecular dynamics investigation of the interaction between DNA and distamycin

The complex of the minor groove binding drug distamycin and the B-DNA oligomer d-(CGCAAATTTGCG) was investigated by molecular dynamics simulations. For this purpose, accurate atomic partial charges of distamycin were determined by extended quantum chemical calculations. The complex was simulated without water but with hydrated counterions. The oligomer without the drug was simulated in the same fashion and also with 1713 water molecules and sodium counterions. The simulations revealed that the binding of distamycin in the minor groove induces a stiffening of the DNA helix. The drug also prevents a transition from B-DNA to A-DNA that was found to occur rapidly (30 ps) in the segment without bound distamycin in a water-free environment but not in simulations including water. In other simulations, we investigated the relaxation processes after distamycin was moved from its preferred binding site, either radially or along the minor groove. Binding in the major groove was simulated as well and resulted in a bound configuration with the guanidinium end of distamycin close to two phosphate groups. We suggest that, in an aqueous environment, tight hydration shells covering the DNA backbone prevent such an arrangement and thus lead to distamycin's propensity for minor groove binding.     

A 5-nanosecond molecular dynamics trajectory for B-DNA: analysis of structure, motions, and solvation.

We report the results of four new molecular dynamics (MD) simulations on the DNA duplex of sequence d(CGCGAATTCGCG)2, including explicit consideration of solvent water, and a sufficient number of Na+ counterions to provide electroneutrality to the system. Our simulations are configured particularly to characterize the latest MD models of DNA, and to provide a basis for examining the sensitivity of MD results to the treatment of boundary conditions, electrostatics, initial placement of solvent, and run lengths. The trajectories employ the AMBER 4.1 force field. The simulations use particle mesh Ewald summation for boundary conditions, and range in length from 500 ps to 5.0 ns. Analysis of the results is carried out by means of time series for conformationalm, helicoidal parameters, newly developed indices of DNA axis bending, and groove widths. The results support a dynamically stable model of B-DNA for d(CGCGAATTCGCG)2 over the entire length of the trajectory. The MD results are compared with corresponding crystallographic and NMR studies on the d(CGCGAATTCGCG)2 duplex, and placed in the context of observed behavior of B-DNA by comparisons with the complete crystallographic data base of B-form structures. The calculated distributions of mobile solvent molecules, both water and counterions, are displayed. The calculated solvent structure of the primary solvation shell is compared with the location of ordered solvent positions in the corresponding crystal structure. The results indicate that ordered solvent positions in crystals are roughly twice as structured as bulk water. Detailed analysis of the solvent dynamics reveals evidence of the incorporation of ions in the primary solvation of the minor groove B-form DNA. The idea of localized complexation of otherwise mobile counterions in electronegative pockets in the grooves of DNA helices introduces an additional source of sequence-dependent effects on local conformational, helicoidal, and morphological structure, and may have important implications for understanding the functional energetics and specificity of the interactions of DNA and RNA with regulatory proteins, pharmaceutical agents, and other ligands.     

A molecular dynamics simulation of the (dG)6 . (dC)6 minihelix including counterions and water

The results of a 60 ps molecular dynamics (MD) simulation of (dG)6.(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to fluctuate with time, showing the dynamic nature of the base-pairing interaction. These fluctuations gave rise to unusual hydrogen-bonding patterns. Good intrastrand base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B-DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B-DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n.(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.     

Structure of hydrated Na+ ions around a region of A- or B-DNA helix

A molecular-dynamics simulation was used to carry out an introductory study of the hydration of a section of a rigid single A- or B-DNA helix with one Na⁺ counterion per nucleotide. Four Na⁺ ions and four nucleotides and periodic boundary conditions were used to mimic an infinite helix. The atoms of the helix and the Na⁺ ions were assumed to be Lennard-Jones spheres that also carried charges. Stillinger four-point charge model water molecules were used. We carried out five calculations, for 26 and 46 water molecules in B-DNA and 20, 32, and 46 in A-DNA fragments. The arrangements of the Na⁺ ions are found to have some similarities to those obtained by Clementi and Corongiu. In the calculations with 46 water molecules, we found that two Na⁺ ions can be bridged by about two water molecules and form a hydrated bound pair, which in turn forms a bridge between the guanine N7 and a near phosphate group. These bound pairs may be important in stabilizing the helix structure of DNA molecules.     

Orientational and rotational velocity correlation functions for water-hydrating B- and Z-DNA double helices

The molecular dynamics simulations reported earlier for the structure and dynamics of water molecules hydrating B- and Z-DNA double helices are analyzed for the orientational correlation functions and the proton rotational velocity autocorrelation functions. The spectra of the rotational velocity autocorrelation functions obtained from the simulation results are compared with the neutron inelastic scattering experiments on hydrated Na-DNA samples. The results predict a small frequency component associated with water molecules bound to the double helices that disappears for waters away from the double helix.     

A molecular dynamics study of conformational changes and hydration of left-handed d(CGCGCGCGCGCG)2 in a nonsalt solution.

Twelve dinucleotides (one complete turn) of left-handed, flexible, double-helix poly(dG-dC) Z-DNA have been simulated in aqueous solution with K+ counterions for 70 ps. Most of the d(GpC) phosphates have rotated in accordance with a ZI----ZII transition. The ZII conformation was probably partly stabilized by counterions, which coordinate one of the anionic oxygens and the guanine-N7 of the next (5'----3' direction) base. The presence of base-coordinating ions close to the helical axis rotated and pulled about half of the d(CpG) phosphates further into the groove. These ions also gave rise to rather large deviations from the crystal structure (ZI) with their tendency of pulling the bases closer toward the helical axis. A flipping of the orientation about the glycosyl bond from the +sc to the -sc region was observed for one guanosine, also leading to deviations from the crystal structure. Many bridges containing one or two water molecules were found, with a dominance for the latter. They essentially formed a network of intra- and interstrand bridges between anionic and esterified phosphate oxygens. A "spine" of water molecules could be distinguished as a dark zig-zag pattern in the water density map. The lifetime of a bridge containing one water was about twice as long as that of a two-water bridge and it lasted 5-15 times longer than a hydrogen bond in water. The lifetimes were also calculated for a selection of bridge types, in order of decreasing stability: O1P/O2P ... W ... O'4 much greater than O1P/O2P ... W ... guanine-N2 greater than O1P/O2P ... W ... O1P/O2P. The reorientational motion of water molecules in the first hydration shell around selected groups was slowed down considerably compared to bulk water and the decreasing order of correlation times was guanine-N2 greater than O'4 greater than O'3/O'5 greater than O1P/O2P.     

On the competition between water,sodium ions,and spermine in binding to DNA: a molecular dynamics computer simulation study

The interaction of DNA with the polyamine spermine(4+) (Spm(4+)), sodium ions, and water molecules has been studied using molecular dynamics computer simulations in a system modeling a DNA crystal. The simulation model consisted of three B-DNA decamers in a periodic hexagonal cell, containing 1200 water molecules, 8 Spm(4+), 32 Na(+), and 4 Cl(-) ions. The present paper gives a more detailed account of a recently published report of this system and compares results on this mixed Spm(4+)/Na(+)-cation system with an molecular dynamics simulation carried out for the same DNA decamer under similar conditions with only sodium counterions (Korolev et al., J. Mol. Biol. 308:907). The presence of Spm(4+) makes significant influence on the DNA hydration and on the interaction of the sodium ions with DNA. Spermine pushes water molecules out of the minor groove, whereas Na(+) attracts and organizes water around DNA. The major binding site of the Spm(4+) amino groups and the Na(+) ions is the phosphate group of DNA. The flexible polyamine spermine displays a high presence in the minor groove but does not form long-lived and structurally defined complexes. Sodium ions compete with Spm(4+) for binding to the DNA bases in the minor groove. Sodium ions also have several strong binding sites in the major groove. The ability of water molecules, Spm(4+), and Na(+) to modulate the local structure of the DNA double helix is discussed.     

Hydration of counterions interacting with DNA double helix: a molecular dynamics study

In the present work, molecular dynamics simulations have been carried out to study the dependence of counterion distribution around the DNA double helix on the character of ion hydration. The simulated systems consisted of DNA fragment d(CGCGAATTCGCG) in water solution with the counterions Na⁺, K⁺, Cs⁺ or Mg²⁺. The characteristic binding sites of the counterions with DNA and the changes in their hydration shell have been determined. The results show that due to the interaction with DNA at least two hydration shells of the counterions undergo changes. The first hydration shell of Na⁺, K⁺, Cs⁺, and Mg²⁺ counterions in the bulk consists of six, seven, ten, and six water molecules, respectively, while the second one has several times higher values. The Mg²⁺ and Na⁺ counterions, constraining water molecules of the first hydration shell, mostly form with DNA water-mediated contacts. In this case the coordination numbers of the first hydration shell do not change, while the coordination numbers of the second one decrease about twofold. The Cs⁺ and K⁺ counterions that do not constrain surrounding water molecules may be easily dehydrated, and when interacting with DNA their first hydration shell may be decreased by three and five water molecules, respectively. Due to the dehydration effect, these counterions can squeeze through the hydration shell of DNA to the bottom of the double helix grooves. The character of ion hydration establishes the correlation between the coordination numbers of the first and the second hydration shells.

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Graphical Abstract Hydration of counterions interacting with DNA double helix

     

Molecular dynamics simulations of B-DNA: An analysis of the role of initial molecular configuration,randomly assigned velocity distribution,long integration times,and nonconstrained termini

Molecular dynamics simulations of three DNA sequences using the AMBER 3.0 force field were performed with implicit inclusion of water through a distance-dependent dielectric constant and solvated counterions. Simulations of the self-complementary DNA dodecamer d(CGCGAATTCGCG) were started from a regular B-DNA structure and the x-ray single crystal B-DNA structure. Although mean convergence during the 89-ps calculation was confirmed, localized differences in backbone torsionals and base-pair helicoidals were observed. A nanosecond simulation of the nonself-complementary 14 base-pair DNA d(GGCGGAATTGGCGG) indicates that most structural parameters stabilize within the first 100–200 ps, while isolated features show low-frequency oscillations throughout the calculation. The lack of harmonic constraints on the ends of the molecules was shown not to perturb the structural dynamics of the internal oligonucleotide beyond the external 2 base pairs. Comparison of three simulations of the nonself-complementary 14 base-pair DNA d(GGCGAAATTCGCGG), identical in all respects other than the assignment of initial Maxwellian atomic velocity distributions, revealed the inherent systematic variability. The three calculations result in nearly superimposable global structures, with localized variations in torsionals and helicoidals. Our results provide a basis for performing a comparative analysis of the effect of DNA sequence on localized structure. © 1993 John Wiley & Sons, Inc.     

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA.

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.     

DNA and its counterions: a molecular dynamics study

The behaviour of mobile counterions, Na⁺ and K⁺, was analysed around a B-DNA double helix with the sequence CCATGCGCTGAC in aqueous solution during two 50 ns long molecular dynamics trajectories. The movement of both monovalent ions remains diffusive in the presence of DNA. Ions sample the complete space available during the simulation time, although individual ions sample only about one-third of the simulation box. Ions preferentially sample electronegative sites around DNA, but direct binding to DNA bases remains a rather rare event, with highest site occupancy values of <13%. The location of direct binding sites depends greatly on the nature of the counterion. While Na⁺ binding in both grooves is strongly sequence-dependent with the preferred binding site in the minor groove, K⁺ mainly visits the major groove and binds close to the centre of the oligomer. The electrostatic potential of an average DNA structure therefore cannot account for the ability of a site to bind a given cation; other factors must also play a role. An extensive analysis of the influence of counterions on DNA conformation showed no evidence of minor groove narrowing upon ion binding. A significant difference between the conformations of the double helix in the different simulations can be attributed to extensive α/γ transitions in the phosphate backbone during the simulation with Na⁺. These transitions, with lifetimes over tens of nanoseconds, however, appear to be correlated with ion binding to phosphates. The ion-specific conformational properties of DNA, hitherto largely overlooked, may play an important role in DNA recognition and binding.     

Simulations of the solvent structure for macromolecules. I. Solvation of B-DNA double helix at T = 300 K

Monte Carlo simulations are reported for a system of 447 water molecules enclosing a B-DNA double-helix fragment with 12 base pairs and the corresponding sugar and phosphate units. From a detailed analysis on the interaction energies and probability distributions (at a simulated temperature of 300 K), the water molecules can be partitioned into clusters strongly interacting with (1) the phosphates, (2) the sugars, (3) the sugars and the bases, and (4) the base pairs. In addition, transgroove and interphosphate filament of hydrogen-bonded water molecules have been detected. From simulations performed with variable numbers of water molecules, a theoretical isotherm has been obtained, with the characteristic sigmoidal shape, known from absorption–desorption experiments on related systems. The expected main features for the structure of water molecules solvating B-DNA with Na⁺ counterions are briefly discussed at the end of the paper.     

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels.

Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into duplexes of different sizes were found to display a helix length dependence and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on the dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry. 34:9265-9276). This sensitivity to size, which reflects global tumbling, is now reported for the more flexible five-atom-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into nucleic acids of different sizes. The DUAPs containing B-DNA systems were simulated with the same dynamic cylinder model, giving S = 0.20 +/- 0.01 for the more flexibly tethered spin label. This shows that S is dependent on tether length but not on global motion. An analysis with the same motional model of the B-Z transition in a (dG-dC)n polymer containing the five-atom-tethered six-membered ring cytidylate spin label (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry. 29:8522-8528) revealed an increase in S from 0.15 +/- 0.01 to 0.26 +/- 0.01 in response to the B- to Z-DNA transition. This indicates that S is not only sensitive to tether length, but also to conformational changes in DNA. Both the DUAP- and the DCAT-labeled systems were also simulated with a base disk model. From the DUAP spectral series, the perpendicular component of the correlation time tau perpendicular describing the spin-labeled base diffusion was found to be sensitive to global tumbling, confirming earlier results obtained with DUMTA. The DCAT polymer results demonstrated that tau perpendicular monitors a conformational change from B- to Z-DNA, indicating that tau perpendicular is also sensitive to local base dynamics. These results confirm that the dynamics of five-atom-tethered nitroxides are coupled to the nucleic acid dynamics and, as with two-atom-tethered spin labels, can be characterized by S and tau perpendicular. The analyses of both spin-labeled systems provide good evidence for spin-labeled base motions within double-stranded DNA occurring on the nanosecond time scale, and establish that both labels can be used to monitor changes in global tumbling and local order parameter due to variations in DNA conformation and protein-DNA interactions.     

Structural rigidity of paranemic crossover and juxtapose DNA nanostructures

Crossover motifs are integral components for designing DNA-based nanostructures and nanomechanical devices due to their enhanced rigidity compared to the normal B-DNA. Although the structural rigidity of the double helix B-DNA has been investigated extensively using both experimental and theoretical tools, to date there is no quantitative information about structural rigidity and the mechanical strength of parallel crossover DNA motifs. We have used fully atomistic molecular dynamics simulations in explicit solvent to get the force-extension curve of parallel DNA nanostructures to characterize their mechanical rigidity. In the presence of monovalent Na⁺ ions, we find that the stretch modulus (γ₁) of the paranemic crossover and its topoisomer JX DNA structure is significantly higher (∼30%) compared to normal B-DNA of the same sequence and length. However, this is in contrast to the original expectation that these motifs are almost twice as rigid compared to the double-stranded B-DNA. When the DNA motif is surrounded by a solvent with Mg²⁺ counterions, we find an enhanced rigidity compared to Na⁺ environment due to the electrostatic screening effects arising from the divalent nature of Mg²⁺ ions. To our knowledge, this is the first direct determination of the mechanical strength of these crossover motifs, which can be useful for the design of suitable DNA for DNA-based nanostructures and nanomechanical devices with improved structural rigidity.     

Triplex hydration: nanosecond molecular dynamics simulation of the solvated triplex formed by mixed sequences

A theoretical model for the hydration pattern and motion of ions around the triple helical DNA with mixed sequences d(GACTGGTGAC)d(GTCACCAGTC)*d(GACTGGTGAC) in solution, during MD simulation, using the particle mesh Ewald sum method, is elaborated here. The AMBER 5.0 force field has been used during the simulation in solvent. The simulation studies support a dynamically stable atmosphere around the DNA triplex in solution over the entire length of the trajectory. The results have been compared with Hoogsteen triplexes and examined in the context of the observed behaviour of hydration in crystallographic data of duplexes. The dynamical organization of counterions and water molecules around the triplex formed by mixed sequences is described here. It has been observed that cations prefer to bind between two adjoining purines of the second and the third strands. The idea of localized complexes (mobile counterions in unspecific electronegative pockets around the DNA triplex with water molecules) may have important implications for understanding the specificity of the interactions of nucleic acids with proteins and other ligands.     

The role of methylation in the intrinsic dynamics of B- and Z-DNA

Methylation of cytosine at the 5-carbon position (5 mC) is observed in both prokaryotes and eukaryotes. In humans, DNA methylation at CpG sites plays an important role in gene regulation and has been implicated in development, gene silencing, and cancer. In addition, the CpG dinucleotide is a known hot spot for pathologic mutations genome-wide. CpG tracts may adopt left-handed Z-DNA conformations, which have also been implicated in gene regulation and genomic instability. Methylation facilitates this B-Z transition but the underlying mechanism remains unclear. Herein, four structural models of the dinucleotide d(GC)(5) repeat sequence in B-, methylated B-, Z-, and methylated Z-DNA forms were constructed and an aggregate 100 nanoseconds of molecular dynamics simulations in explicit solvent under physiological conditions was performed for each model. Both unmethylated and methylated B-DNA were found to be more flexible than Z-DNA. However, methylation significantly destabilized the BII, relative to the BI, state through the Gp5mC steps. In addition, methylation decreased the free energy difference between B- and Z-DNA. Comparisons of α/γ backbone torsional angles showed that torsional states changed marginally upon methylation for B-DNA, and Z-DNA. Methylation-induced conformational changes and lower energy differences may contribute to the transition to Z-DNA by methylated, over unmethylated, B-DNA and may be a contributing factor to biological function.     

Internal dynamics of d(CGCAAATTTGCG)2: a comparison of NMR relaxation measurements with a molecular dynamics simulation

We report the analysis of a 250 ps molecular dynamics simulation of the dodecamer d(CGCAAATTT-GCG)₂ immersed in a rectangular box of 3469 water molecules with 22 Na⁺ counterions. The internal dynamics of the molecule were investigated by studying the relevant autocorrelation functions related to the ¹³C-NMR relaxation parameters of the C1′-H1′ bonds of the sugar rings. The calculated effective correlation times τ _e (∼13 ps) and the order parameter S² (∼0.82) of the Lipari and Szabo formalism (Lipari and Szabo 1982a, b) are in satisfactory agreement with those determined previously by NMR (Gaudin et al. 1995, 1996). ¹H-¹H NOE buildups have also been measured experimentally and agree with those computed from the simulation. These results validate the simulation, and a more detailed analysis of the internal dynamics of the dodecamer was undertaken. Analysis of the distributions and of the autocorrelation functions of the glycosidic angle flucuations χ shows that the rotational motion of the sugar rings about their glycosidic bond conforms to a restricted diffusion mechanism. The amplitude of the motions and the diffusion constant are 20° and 17.10⁹ rad²s^–1 respectively. These values are in good agreement with ¹³C NMR data. Furthermore the simulation allows us to rule out another model also consistent with the experiment, consisting of a two-state jump between a syn and an anti conformation. Received: 19 November 1996 / Accepted: 17 March 1997     

Temperature-dependent molecular dynamics and restrained X-ray refinement simulations of a Z-DNA hexamer

Molecular dynamics simulations of the Z-DNA hexamer 5BrdC-dG-5BrdC-dG-5BrdC-dG were performed at several temperatures between 100 K and 300 K. Above 250 K, a strong sequence-dependent flexibility in the nucleic acid is observed, with the guanine sugar and the phosphate of GpC sequences much more mobile than the cytosine sugar and phosphate of CpG sequences. At 300 K, the hexamer is in dynamic equilibrium between several Z forms, including the crystallographically determined ZI and ZII forms. The local base-pair geometry, however, is not very variable, except for the roll of the base-pairs. The hexamer molecular dynamics trajectories have been used to test the restrained parameter crystallographic refinement model for nucleic acids. X-ray diffraction intensities corresponding to observed diffraction data were computed. The average structures obtained from the simulations were then refined against the calculated intensities, using a restrained least-squares program developed for nucleic acids in order to analyse the effects of the refinement model on the derived quantities. In general, the temperature dependence of the atomic fluctuations determined directly from the refined Debye-Waller factors is in reasonably good agreement with the results obtained by calculating the atomic fluctuations directly from the Z-DNA molecular dynamics trajectories. The agreement is best for refinement of temperature factors without restraints. At the highest temperature studied (300 K), the effect of the refinement on the most mobile atoms (phosphates) is to significantly reduce the mean-square atomic fluctuations estimated from the refined Debye-Waller factors below the actual values (less than (delta r)2 greater than congruent to 0.5 A2). Analysis of the temperature-dependence of the mean-square atomic fluctuations provides information concerning the conformational potential within which the atoms move. The calculated temperature-dependence and anharmonicity of the Z-DNA helix are compared with the results observed for proteins. The average structures from the simulations were refined against the experimental X-ray intensities. It is found that low-temperature molecular dynamics simulations provide a useful tool for optimizing the refinement of X-ray structures.     

Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support

We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.