Conformational analysis of d(C3G3), a B-family duplex in solution

NMR and circular dichroism studies of the duplex formed by the self-complementary DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs from standard B-form. An analysis of NMR coupling constants within the deoxyribose moieties yields a 70% or greater contribution from pseudorotation phase angles corresponding to the C3'-exo conformation, a conformation similar to the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton distances are consistent with a B-form structure, but some internucleotide distances are intermediate between A- and B-form structures. Circular dichroism spectra have B-form characteristics but also include an unusual negative band at 282 nm. The solution spectroscopic results are in contrast with X-ray crystallographic studies which find A-form structures for similar sequences.     

Ethanol-induced structural transitions of DNA on mica

The effect of ethanol on the structure of DNA confined to mica in the presence of Mg2+was examined by varying the ethanol concentration and imaging the DNA by atomic force microscopy. Contour length measurements of the DNA show a transition from all-B-form at 0% ethanol to all-A-form at >25% ethanol. At intermediate ethanol concentrations, contour lengths suggest that individual molecules of air-dried DNA are trapped with mixed compositions of A-form and B-form. The relative composition depends on the ethanol concentration. Fitting the length distributions at intermediate ethanol concentrations to a simple binomial model results in an upper bound estimate for the A-form and B-form domains of approximately 54 bp in the individual molecules. In addition to length changes, the apparent persistence length of DNA decreases with increasing ethanol concentration. At high concentrations of ethanol (>20%), DNA formed several higher order structures, including flower shaped condensates and toroids.     

The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Unzipping of A-Form DNA-RNA,A-Form DNA-PNA,and B-Form DNA-DNA in the α-Hemolysin Nanopore

Unzipping of double-stranded nucleic acids by an electric field applied across a wild-type α-hemolysin (αHL) nanopore provides structural information about different duplex forms. In this work, comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and the unzipping duration between the two helical forms. We observed that the B-form duplex blocks the channel 1.9 ± 0.2 pA more and unzips ∼15-fold more slowly than an A-form duplex at 120 mV. We developed a model to describe the dependence of duplex unzipping on structure. We demonstrate that the wider A-form duplex (d = 2.4 nm) is unable to enter the vestibule opening of αHL on the cis side, leading to unzipping outside of the nanopore with higher residual current and faster unzipping times. In contrast, the smaller B-form duplexes (d = 2.0 nm) enter the vestibule of αHL, resulting in decreased current blockages and slower unzipping. We investigated the effects of varying the length of the single-stranded overhang, and studied A-form DNA-PNA duplexes to provide additional support for the proposed model. This study identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA.     

The flexibility of A-form DNA.

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Linear dichroism studies of nucleic acids. III. Reduced dichoism curves of DNA in ethanol–water and in poly(vinyl alcohol) films

The uv linear dichroism of calf thylus DNA has been studied at different degrees of orientation both in flow-oriented ethanol–water solutions and in a stretched aqueous host of poly(vinyl alcohol) (PVA). The reduced dichroism (LD^R) curves in the region 250–290 nm for DNA in PVA films at 75 and 100% relative humidity (r.h.) are in fair agreement with the curves calculated for the A- and B-forms of DNA, based on the fiber structures and the π-π* transitions of the free bases. This suggests that DNA adopts its A and B conformations in PVA at 75 and 100% r.h. In ethanol, on the other hand, a deviation from the A-form spectrum shows that the conformation of DNA in the solution can differ from the fiber structure. At shorter wavelenghts, a positive contribution to LD^R is explained in terms of an out-of-plane polarized n-π* transition.     

Mechanical properties of high-G.C content DNA with a-type base-stacking

The sequence of a DNA molecule is known to influence its secondary structure and flexibility. Using a combination of bulk and single-molecule techniques, we measure the structural and mechanical properties of two DNAs which differ in both sequence and base-stacking arrangement in aqueous buffer, as revealed by circular dichroism: one with 50% G·C content and B-form and the other with 70% G·C content and A-form. Atomic force microscopy measurements reveal that the local A-form structure of the high-G·C DNA does not lead to a global contour-length decrease with respect to that of the molecule in B-form although it affects its persistence length. In the presence of force, however, the stiffness of high-G·C content DNA is similar to that of balanced-G·C DNA as magnetic and optical tweezers measured typical values for the persistence length of both DNA substrates. This indicates that sequence-induced local distortions from the B-form are compromised under tension. Finally, high-G·C DNA is significantly harder to stretch than 50%-G·C DNA as manifested by a larger stretch modulus. Our results show that a local, basepair configuration of DNA induced by high-G·C content influences the stretching elasticity of the polymer but that it does not affect the global, double-helix arrangement.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

Double-helical DNA and RNA circular dichroism. Calculations on base-sugar-phosphateehlix interactions

Theoretical calculations of the near ultraviolet (uv) circular dichroism of double-helical DNA and RNA models were performed in order to evaluate the effects, on the calculated circular dichroism, of including the interactions of near uv quantum transitions of the nucleic acid bases with classical polarizable bonds of the sugar-phosphate backbone. Double-helical models (A-form, B-form, and C-form DNA and RNA-11) from X-ray diffraction data were used in the calculations. The results indicate that the contributions to the circular dichroism in the near uv region, of these types of interactions, provide calculated spectra that are slightly altered from calculated spectra when only base–base transition interactions were considered.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Electric dichroism of chromatin

The linear dichroism of sheared calf thymus chromatin, oriented in solution by a pulsed electric field, has been measured. The limiting value of this dichroism is considerably less negative than that of calf thymus DNA, but does not approach the positive values predicted if chromatin were uniformly supercoiled in the manner suggested by X-ray studies. The decay of the dichroism of chromatin, after termination of the electric pulse, is similar to that of DNA, indicating that chromatin retains a high degree of chain flexibility. These data, as well as previous flow dichroism studies (Ohba, 1966; Smart &; Bonner, 1971), suggest that chromatin is not predominantly supercoiled in solutions of low ionic strength. Evidence for a structurally heterogeneous model for chromatin is discussed.     

Solution conformation of DNA

Optical observations on linear B-form DNA by the method of electric linear dichroism show that the value of the limiting reduced dichroism is molecular weight-dependent, increasing with molecular weight to a limit of about ?1.41 ± 0.02 in aqueous solution. These data and the rotational relaxation times obtained from the decay of the dichroism when the orienting field is instantaneously removed, imply the existence of a non-linear tertiary equilibrium structure for DNA. The data indicate that the essential B-form parameters of the double-stranded DNA are retained in this tertiary structure, and are not consistent with a DNA structure in which the base-pairs have a 34 ° propeller-like twist (Hogan et al., 1978). The interpretation of the dichroism data is supported by a clear demonstration that the magnitude of dichroism change in ethanol corresponds to that expected for the B to A-form structural transition as determined from X-ray diffraction. We propose that the tertiary structure of B-DNA is a helical coil and suggest the limits of the structural parameters of the coil consistent with the observations.     

The characteristic CD spectra of DNA from young rat liver.

The circular dichroism spectroscopy was used to study conformational changes of DNA upon aging. DNA from 8 weeks old rat liver showed A-form unexpectedly in physiological saline. The characteristic spectrum gradually changed upon aging and that of 30 months old rat liver DNA showed normal B-form.     

Electric properties and structure of DNA-restriction fragments from measurements of the electric dichroism

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.     

Linear dichroism studies of nucleic acids. II. Calculation of reduced dichroism curves of A-and B-form DNA

We have calculated the uv linear dichroism for the A- and B-forms of DNA using π-π* transition moments and band components determined from the free DNA bases. The reduced dichroism (LD^R) as a function of wavelength is estimated is in the 220–300-nm region, for both the oriented-gas model and a simple exciton model. For B-form DNA, LD^R is obtained to ?1.48S (S being the orientation factor) over the whole wavelenth region by both models. For A-form DNA, LD^R is not constant, but changes monotonically from about ?1.15S at 220 nm to about ?1.35S to ?1.45S at 300 nm, depending on base combination and degree of interaction (?1.35S for the oriented gas). It is emphasized that a common assumption of a single “effective” transition moment of the principal band at 260 nm may not generally be made because of the extensive overlap of differently polarized bands. The possibility of using the reduced dichroism curve for characterizing the secondary structure of DNA is discussed.     

Mechanism of binding site conformational switching in the CD44-hyaluronan protein-carbohydrate binding interaction

The transmembrane protein CD44, which has been implicated in cancer biology and inflammation, mediates cell adhesion through multimeric interactions with the linear extracellular glycosaminoglycan hyaluronan (HA; in megadaltons). Affinity switching of CD44 from a low-affinity state to a high-affinity state is required for normal CD44 physiological function; crystal structures of the CD44 hyaluronan binding domain complexed with HA oligomers point to a conformational rearrangement at a binding site loop, leading to the formation of direct contact between the oligomer and an arginine side chain as a molecular basis for affinity switching. Here, all-atom explicit-solvent molecular dynamics simulations were used to characterize the dynamics and thermodynamics of oligomeric hyaluronan (oHA) and its two crystallographic complexes with the CD44 hyaluronan binding domain: the “A-form,” which lacks arginine-HA close contact, and the “B-form,” which has direct arginine side-chain-HA contact. From the simulations, the conformational properties of oHA are essentially unaltered in going from the unbound state to either the A-form or the B-form bound state, with the oligomer retaining its flexibility when bound and with only two of the eight monosaccharides in the oligomer maintaining uninterrupted contact with the protein. Biased simulations revealed that altering the backbone conformation of a tyrosine residue in the arginine loop can induce the A-form → B-form conformational transition and that a large free-energy barrier prevents ready interconversion between the two forms, thereby suggesting that the tyrosine backbone forms a molecular switch.     

Conformational transitions of alternating purine-pyrimidine DNAs in perchlorate ethanol solutions.

Conformational transitions of poly(dA-dC).poly(dG-dT), poly(dA-dT).poly(dA-dT), and other alternating purine-pyrimidine DNAs were studied in aqueous ethanol solutions containing molar concentrations of sodium perchlorate, which is a novel solvent stabilizing non-B duplexes of DNA. Using CD and UV absorption spectroscopies, we show that this solvent unstacks bases and unwinds the B-forms of the DNAs to transform them into the A-form or Z-form. In the absence of divalent cations poly(dA-dC).poly(dG-dT) can adopt both of these conformations. Its transition into the Z-form is induced at higher salt and lower ethanol concentrations, and at higher temperatures than the transition into the A-form. Submillimolar concentrations of NiCl2 induce a highly cooperative and slow A-Z transition or Z-Z' transition, which is fast and displays low cooperativity. Poly(dA-dT).poly(dA-dT) easily isomerizes into the A-form in perchlorate-ethanol solutions, whereas high perchlorate concentrations denature the polynucleotide, which then cannot adopt the Z-form. At low temperatures, however, NiCl2 also cooperatively induces the Z'-form in poly(dA-dT).poly(dA-dT). Poly(dI-dC).poly(dI-dC) is known to adopt an unusual B-form in low-salt aqueous solution, which is transformed into a standard B-form by the combination of perchlorate and ethanol. NiCl2 then transforms poly(dI-dC).poly(dI-dC) into the Z'-form, which is also adopted by poly(dI-br5dC).poly(dI-br5dC).     

Electric dichroism and bending amplitudes of DNA fragments according to a simple orientation function for weakly bent rods

The linear dichroism is calculated for DNA fragments in their thermal bending equilibrium. These calculations are given for relatively short fragments, where bent molecules can be described by an arc model. Using the measured value of 350 A for the persistence length, the limit dichroism (corresponding to complete alignment) decreases due to thermal bending, e.g., for a fragment with 100 base pairs to 80% of the value expected for straight molecules. Thermal bending should lead to a strong continuous decrease of the dichroism with increasing chain length, which is not observed, however, in electric dichroism experiments due to electric stretching. The influence of the electric field on the bending equilibrium is described by a contribution to the bending energy, which is calculated from the movement of charge equivalents against the potential gradient upon bending. The charge equivalents, which are assigned to the helix ends, are derived from the dipole moments causing the stationary degree of orientation. By this procedure the energy term inducing DNA stretching is given for induced, permanent, and saturating induced dipole models without introduction of any additional parameter. The stationary dichroism at a given electric field strength is then calculated according to an arc model by integration over all angles of orientation of helix axes or chords with respect to the field vector, and at each of these angles the contribution to the dichroism is calculated by integration over all helices with different degrees of bending. Orientation functions obtained by this procedure are fitted to dichroism data measured for various restriction fragments. Optimal fits are found for an induced dipole model with saturation of the polarizability. The difference between orientation functions with and without electric stretching is used to evaluate dichroism bending amplitudes. Both chain length and field strength dependence of bending amplitudes are consistent with experimental amplitudes derived from the dichroism decay in low salt buffers containing multivalent ions like Mg2+, spermine, or [CoNH3)6]3+. Bending amplitudes can be used to evaluate the persistence length from electrooptical data obtained for a single DNA restriction fragment. Bending and stretching effects are considerable already at relatively low chain length, and thus should not be neglected in any quantitative evaluation of experimental data.