Classical B-DNA Research Articles

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichia coli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

DNA Quadruple Helices in Nanotechnology.

DNA has played an early and powerful role in the development of bottom-up nanotechnologies, not least because of DNA's precise, predictable, and controllable properties of assembly on the nanometer scale. Watson-Crick complementarity has been used to build complex 2D and 3D architectures and design a number of nanometer-scale systems for molecular computing, transport, motors, and biosensing applications. Most of such devices are built with classical B-DNA helices and involve classical A-T/U and G-C base pairs. However, in addition to the above components underlying the iconic double helix, a number of alternative pairing schemes of nucleobases are known. This review focuses on two of these noncanonical classes of DNA helices: G-quadruplexes and the i-motif. The unique properties of these two classes of DNA helix have been utilized toward some remarkable constructions and applications: G-wires; nanostructures such as DNA origami; reconfigurable structures and nanodevices; the formation and utilization of hemin-utilizing DNAzymes, capable of generating varied outputs from biosensing nanostructures; composite nanostructures made up of DNA as well as inorganic materials; and the construction of nanocarriers that show promise for the therapeutics of diseases.

Role of Alkali Metal Ions in G-Quadruplex Nucleic Acid Structure and Stability.

G-quadruplexes are guanine-rich nucleic acids that fold by forming successive quartets of guanines (the G-tetrads), stabilized by intra-quartet hydrogen bonds, inter-quartet stacking, and cation coordination. This specific although highly polymorphic type of secondary structure deviates significantly from the classical B-DNA duplex. G-quadruplexes are detectable in human cells and are strongly suspected to be involved in a number of biological processes at the DNA and RNA levels. The vast structural polymorphism exhibited by G-quadruplexes, together with their putative biological relevance, makes them attractive therapeutic targets compared to canonical duplex DNA. This chapter focuses on the essential and specific coordination of alkali metal cations by G-quadruplex nucleic acids, and most notably on studies highlighting cation-dependent dissimilarities in their stability, structure, formation, and interconversion. Section 1 surveys G-quadruplex structures and their interactions with alkali metal ions while Section 2 presents analytical methods used to study G-quadruplexes. The influence of alkali cations on the stability, structure, and kinetics of formation of G-quadruplex structures of quadruplexes will be discussed in Sections 3 and 4. Section 5 focuses on the cation-induced interconversion of G-quadruplex structures. In Sections 3 to 5, we will particularly emphasize the comparisons between cations, most often K(+) and Na(+) because of their prevalence in the literature and in cells.

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

Hidden Conformation Events in DNA Base Extrusions: A Generalized Ensemble Path Optimization and Equilibrium Simulation Study.

DNA base extrusion is a crucial component of many biomolecular processes. Elucidating how bases are selectively extruded from the interiors of double-strand DNAs is pivotal to accurately understanding and efficiently sampling this general type of conformational transitions. In this work, the on-the-path random walk (OTPRW) method, which is the first generalized ensemble sampling scheme designed for finite-temperature-string path optimizations, was improved and applied to obtain the minimum free energy path (MFEP) and the free energy profile of a classical B-DNA major-groove base extrusion pathway. Along the MFEP, an intermediate state and the corresponding transition state were located and characterized. The MFEP result suggests that a base-plane-elongation event rather than the commonly focused base-flipping event is dominant in the transition state formation portion of the pathway; and the energetic penalty at the transition state is mainly introduced by the stretching of the Watson-Crick base pair. Moreover to facilitate the essential base-plane-elongation dynamics, the surrounding environment of the flipped base needs to be intimately involved. Further taking the advantage of the extended-dynamics nature of the OTPRW Hamiltonian, an equilibrium generalized ensemble simulation was performed along the optimized path; and based on the collected samples, several base-flipping (opening) angle collective variables were evaluated. In consistence with the MFEP result, the collective variable analysis result reveals that none of these commonly employed flipping (opening) angles alone can adequately represent the base extrusion pathway, especially in the pre-transition-state portion. As further revealed by the collective variable analysis, the base-pairing partner of the extrusion target undergoes a series of in-plane rotations to facilitate the base-plane-elongation dynamics. A base-plane rotation angle is identified to be a possible reaction coordinate to represent these in-plane rotations. Notably, these in-plane rotation motions may play a pivotal role in determining the base extrusion selectivity.

The Conformer Substates of Nonoriented B-type DNA in Double Helical Poly(dG-dC)

A nonoriented hydrated film of poly(dG-dC) with ≈20 water molecules per nucleotide (called B by Loprete and Hartman (Biochem. 32, 4077–4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)2 dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on BIto-BII interconversion in the classical B-DNA forms are very similar to those in the B-form, and that the spectroscopic differences between the BI and BII features from classical B-DNA and those from the modified B-form are minor. Nonexponential kinetics of the BI→BII transition in the B-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (≈30 versus ≈10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is BII→BI transition on cooling from 290 K and BI→BII transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263–3274 (2002)).

Crystal structure of an antiparallel DNA fragment with Hoogsteen base pairing.

We report here an alternative double-helical structure of the DNA molecule. It has been found in the d(ATA(Br)UAT) and d(ATATAT) sequences by single-crystal x-ray crystallography. This sequence is found not only in TATA boxes, but also in other regulatory regions of DNA. Bases of the two antiparallel strands form Hoogsteen pairs, with adenines in the syn conformation. The structure is related neither to those found in triple helices nor to parallel DNA duplexes. Its conformational parameters are very similar to those of duplex DNA in the B form. Both forms may coexist under physiological conditions, although the Hoogsteen pairing greatly influences the recognition sites on DNA. Our results demonstrate that an alternative to the classical B-DNA double helix is possible.

Structure of Poly d(AI)·Poly d(CT) in Two Different Packing Arrangements

X-ray diffraction analysis of poly d(AI)poly·d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 Å and 32.4 Å in the two cases, almost 1.5 Å shorter than in classical B-DNA. The resulting B-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3. 0 Å resolution.

Solution structure of a conserved DNA sequence from the HIV-1 genome: restrained molecular dynamics simulation with distance and torsion angle restraints derived from two-dimensional NMR spectra.

The three-dimensional solution structure of a trisdecamer DNA duplex sequence, d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), from a conserved region of HIV-1 genome's long terminal repeat, has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances derived from two-dimensional nuclear Overhauser enhancement (2D NOE) experiments, using the iterative complete relaxation matrix algorithm MARDIGRAS, and torsion angles for sugar rings, estimated from stimulated fitting of double-quantum-filtered correlation (2QF-COSY) spectra, were obtained [Mujeeb, A., Kerwin, S. M., Egan, W. M., Kenyon, G. L., & James, T. L. (1992) Biochemistry 31, 9325-38]. These structural restraints have now been employed as the basis for structure refinement using restrained molecular dynamics (rMD) to search conformational space for structures consistent with the experimental restraints. Specifically, upper and lower bounds on the restraints were incorporated into the AMBER (version 4.0) total potential energy function of the system, the bounds being used to define the width of a flat-well penalty term in the AMBER force field. Confidence in the time-averaged structure obtained is engendered by convergence to essentially the same structure (root-mean-square deviation approximately 0.9 A) when two quite different DNA models, A-DNA and B-DNA (RMSD approximately 6.5 A), were employed as starting structures and when various initial trajectories were used for the rMD runs. The derived structure is further supported by the total energy calculated, the restraint violation energy, the restraint deviations, and the fit with experimental data. For the latter, the sixth-root residual index indicated a good fit of the determined structure with experimental 2D NOE spectral intensities (R1x < 0.07), and the RMS difference between vicinal proton coupling constants calculated for the derived structure and experimental coupling constants were also in reasonable agreement (JRMS = 0.9 Hz). While the structure of the trisdecamer is basically in the B-DNA family, some structural parameters manifest interesting local variations. The helix parameters indicate that, compared with classical B-DNA, the structure is longitudinally more compressed. Local structural variations at the two TG steps in particular together create bending into the major groove of the duplex. Comparison of the two-CTTG-tetrads in the duplex reveals that they have similar structures, with the TT moieties being almost identical; however, the -CTTG-pur sequence has a larger roll and slide for the -TG- step than for the -CTTG-pyr sequence, in accord with published X-ray crystallographic conclusions.

Methylation of cytosine influences the DNA structure.

In the past decade it has become increasingly clear that DNA has the potential to adopt a variety of unusual secondary structures which deviate from the classical right-handed B-DNA form. Intrinsic properties of the DNA molecule (primary base sequence and base modifications, degree of supercoiling) determine whether such an unusual structure is possible. Environmental factors (like ionic strength or pH of the medium, temperature, solvent polarity, interactions with proteins or drugs) have a strong influence on whether the structure is actually formed or not.

Structure of poly d(A).poly d(T).

On the basis of the x-ray data from polycrystalline and well oriented fibers of the sodium salt of poly d(A).poly d(T) (Arnott et al, Nucl. Acids Res. 11, 4141-4155 (1983), a revised B'-DNA model incorporating B-like adenine and thymine strands is shown to give a much better x-ray agreement (R = 0.25) than the previously assigned model consisting of mixed sugar conformations in the two strands. The narrowing of the minor and the widening of the major grooves are promiscuous features of B'-DNA, which are common to all poly d(purine).poly d(pyrimidine) duplexes with two hydrogen bonded base-pairs and are in marked contrast with classical B-DNA. Due to modest propeller (-15 degrees), the cross strand diagonal hydrogen bonds (0.37 nm) in this duplex are not as strong as those in A,T-rich oligonucleotide crystal structures.

Soloution conformation of an olionucleotide containing a G.G mismatch determined by nuclear manetic resonance and molecular mechanics

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.

Analysis of the relative contributions of the nuclear Overhauser interproton distance restraints and the empirical energy function in the calculation of oligonucleotide structures using restrained molecular dynamics

The relative contributions of the interproton distance restraints derived from nuclear Overhauser enhancement measurements and of the empirical energy function in the determination of oligonucleotide structures by restrained molecular dynamics are investigated. The calculations are based on 102 intraresidue and 126 interresidue interproton distance restraints derived from short mixing time two-dimensional nuclear Overhauser enhancement data on the dodecamer 5'd(CGCGPATTCGCG)2 [Clore, G.M., Oschkinat, H., McLaughlin, L.W., Benseler, F., Scalfi Happ, C., Happ, E., & Gronenborn, A.M. (1988) Biochemistry 27, 4185-4197]. Eight interproton distance restraint lists were made up with errors ranging from -0.1/+0.2 to -1.2/+1.3 A for r less than 2.5 A and from -0.2/+0.3 to -1.3/+1.4 A for r greater than or equal to 2.5 A. These restraints were incorporated into the total energy function of the system in the form of square-well potentials with force constants set sufficiently high to ensure that the deviations between calculated distances and experimental restraints were very small (average interproton distance rms deviation of less than 0.06 A). For each data set, six calculations were carried out, three starting from classical A-DNA and three from classical B-DNA. The results show that structural changes occurring during the course of restrained molecular dynamics and the degree of structural convergence are determined by the interproton distance restraints. All the structures display similar small deviations from idealized geometry and have the same values for the nonbonding energy terms comprising van der Waals, electrostatic, and hydrogen-bonding components. Thus, the function of the empirical energy function is to maintain near perfect stereochemistry and nonbonded interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the beta-form of poly d(A).poly d(U).

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.

Structure of the α-form of poly[d(A)] · poly[d(T)] and related polynucleotide duplexes

The α-form of poly[d(A)] · poly[d(T)], observed in fibers at high (> 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2′- endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (β) form of poly[d(A)] · poly[d(T)]in which one strand has C-2′- endo rings and the other C-3′- endo. Since the orientations of the bases in poly[d(A)] · poly[d(T)]are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 °) at the junctions between general sequence tracts and the oligo [d(A)] · oligo[d(T)]tracts that occur in some native DNAs. The conclusions about the structure of α-poly[d(A)] · poly[d(T)]are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)] · poly[d(U)]and poly[d(A-I)] · poly[d(C-T)].

Structure refinement of oligonucleotides by molecular dynamics with nuclear overhauser effect interproton distance restraints: Application to 5′ d(C-G-T-A-C-G) 2

The solution structure of the self-complementary DNA hexamer 5′ d(C-G-T-A-C-G) 2 is refined by restrained molecular dynamics in which 192 interproton distances, determined from pre-steady-state nuclear Overhauser enhancement measurements, are incorporated into the total energy of the system in the form of effective potentials. First the method is tested by applying an idealized set of distance restraints taken from classical B-DNA to a simulation starting off from A-DNA and vice versa. It is shown that in both cases the expected transition between A- and B-DNA occurs. Second, a set of restrained molecular dynamics calculations is carried out starting from both A- and B-DNA with the experimental interproton distances for 5′ d(C-G-T-A-C-G) 2 as restraints. Convergence to the same B-type structure is achieved with the interproton distances equal to the measured values within experimental error. The root-mean-square atomic difference between the two average restrained dynamics structures (<1 Å) is approximately the same as the root-mean-square fluctuations of the atoms.

The molecular electrostatic potential, steric accessibility and hydration of Dickerson's B-DNA dodecamer d(CpGpCpGpApApTpTpCpGpCpG).

The paper presents the results of computation of the electrostatic potential and steric accessibility of the B-DNA self-complementary dodecamer CGCGAATTCGCG following the geometry of the recent single crystal structure of Dickerson et al. This structure shows significant variations from classical B-DNA; their influences on the calculated properties are discussed. The results are related to general features of hydration of the crystal. A particularly significant general finding concerns the greater negative potential in the center of the oligonucleotide helix than at its extremities. This will be a general feature of such short helices, independent of their base sequence. It may have important implications for the reactivity of DNA oligomers.