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A study of the structure of highly concentrated phases of DNA by X-ray diffraction
Denys Durand, J. Doucet, F. Livolant
To cite this version:
Denys Durand, J. Doucet, F. Livolant. A study of the structure of highly concentrated phases of DNA by X-ray diffraction. Journal de Physique II, EDP Sciences, 1992, 2 (9), pp.1769-1783.
�10.1051/jp2:1992233�. �jpa-00247765�
Classification
Physics Abstracts
61.10 64.70 87.15
A study of the structure of highly concentrated phases of DNA
by X-ray diffraction
D. Durand (1. ~), J. Doucet (1, 3) and F. Livolant (4)
(1) L-U-R-E-, Laboratoire CNRS-CEA-MEN, Bit. 209D, Universit6 Paris-Sud, 91405 Orsay Cedex, France
(2) Laboratoire L£on Brillouin (CEA-CNRS), CE Saclay, 91191 Gif-sur-Yvette Cedex, France (3) Laboratoire de Physique des Solides, Bit. 510, Universit6 Paris-Sud, 91405 Orsay Cedex,
France
(4) Centre de Biologie Cellulaire (CNRS), 67 rue Maurice Giinsbourg, 94205 Ivry-sur-Seine Cedex, France
(Received18 March 1992, accepted in final form 5 June 1992)
R4sum4. L'ADN donne en solution aqueuse concentr6e plusieurs phases cristallines liquides et cristallines. Quand la concentration en ADN augmente, on observe la s6quence de phases
suivante : isotrope - cholest6rique - colonnaire hexagonale
- phases cristallines. Le but de ce travail £tart d'obtenir par diffraction des rayons X, des informations structurales sur les phases
tr~s concentr6es en particulier sur les phases cristallines form6es par des fragments d'ADN de 500A de longueur. Nous avons montr£ que dans la phase hexagonale ordonn6e h 2
dimensions, un orate longitudinal entre mot£cules d'ADN voisines s'installe progressivement, et donne lieu h une phase hexagonale ordonn6e h 3 dimensions. Quand la concentration en ADN cr&t encore, on observe une transition discontinue vers une phase de sym6trie orthorhombique.
Les pararn~tres structuraux caract6ristiques de ces diff6rentes phases ont 6t£ d6termin6s. Un r£sultat important est que le nombre de nuc16otides par tour d'h61ice d6croit continoment, quand
la concentration en ADN augmente, depuis 10,3 ± 0,1 h la transition cholest6rique
- hexagonale, jusqu'h 9±0,1 pour les dchantillons les plus concentr6s par ailleurs, la conformation des
mo16cules d'ADN semble ne subir aucun changement et reste de type B.
Abstract In aqueous solution, pure DNA forms multiple liquid crystalline and crystalline phases whose nature depends on the polymer concentration. The following phase sequence is
observed when the DNA concentration increases : isotropic
- cholesteric
- columnar hexag-
onal - crystalline phases. The aim of this work is to obtain structural information about the
highly concentrated phases formed by 500A long DNA molecules in particular about the
crystalline phases by means of X-ray diffraction. We show that in the two-dimensional (2D) ordered hexagonal phase a longitudinal order progressively appears between neighbouring DNA helices leading continuously to a three-dimensional (3D) ordered hexagonal phase. For higher
concentrations the specimens undergo a discontinuous transition towards an orthorhombic phase.
The characteristic structural parameters of these different phases have been determined. An
important result is that the number of nucleotides per helix rum decreases continuously, when the DNA concentration increases, from 10.3 ± 0.I at the cholesteric
- hexagonal transition down to 9 ± 0.I without any apparent change of the B conformation of the molecules.
1770 JOURNAL DE PHYSIQUE II N° 9
1. Introduction.
It has been well established for some time that in aqueous solution, DNA can form like
numerous polymers highly ordered liquid crystalline phases [1-18] above a critical
concentration depending on the length of the DNA molecules and nearly insensitive to the
supporting electrolyte concentration [18]. For instance, the critical DNA concentration is about 160 mg/ml for 500 A long DNA fragments in physiological salt conditions [I1, 12, 18].
The nature of the different liquid crystalline phases depends on the DNA concentration and the following phase sequence is observed :
cholesteric
germs columnar
isotropic - or
- cholesteric
- hexagonal
phase precholesteric phase phase
organization (for details see Refs. [17, 18]).
In the columnar hixagonal phase [14, 19] the DNA molecules are unidirectionally aligned
and form a hexagonal network in the plane perpendicular to their axis. The order is only two- dimensional : the columns of molecules are able to slide against each other, and each molecule
is free to rotate around its axis. For higher DNA concentrations more ordered structures are
observed [14].
Studies of the concentrated phases of DNA in vitro are of interest since the local
concentration of DNA in vivo can also be quite large it can reach values up to 800 mg/ml as
estimated by Kellenberger et al. [20] and liquid crystal formation may possibly play some
role in packaging DNA in certain systems. Various works have evidenced that condensed chromatin can present the same geometry as DNA molecules in liquid crystals (for a review
see Ref. [17]). For example, a cholesteric organization can be recognized in Dinoflagellate
chromosomes [21, 22], in bacterial nucleoids [23] and in special kinds of mitochondria [24]. A
hexagonal packing of DNA molecules is also observed in some virus capsids [25, 26].
Studies of DNA liquid crystals are also of intrinsic interest in terms of liquid crystalline properties of polyelectrolytes [27]. DNA is a good example of a linear polyelectrolyte, even of
rigid rodlike ones when the DNA fragments are sufficiently short [28-30]. Little information is available about the behaviour of such macromolecules. A strong polyelectrolyte, like DNA, is surrounded by a counterion layer which determines the effective polymer dimensions [31].
Thus it is expected that the ordering properties of DNA will be strongly influenced by the
polyionic character of this system.
Until now, a lot of experiments espec1ally polarizing and electron microscopy studies
were performed on the liquid crystalline phases of DNA, but much less is known about the structural features at the molecular scale of these phases as well as of the more concentrated
phases. The aim of the present work is to obtain structural information about the very highly
concentrated phases of DNA in particular the crystalline phases in order to get a better
insight into the nature and geometry of the intermolecular interactions. The appropriate technique for such a study is X-ray diffraction. The results obtained for the crystalline phases
will be compared to those given by X-ray measurements on DNA fibers.
2. Material and methods.
2.I DNA PREPARATION. DNA fragments with a most probable length of 500 A (146 base
pairs) were isolated from nucleosome cores obtained by digestion of calf thymus chromatin
with micrococcal nuclease after removal of Hl histones (for a detailed description of the
procedure used to isolate and characterize the DNA fragments, see Ref. [ll]). Concentrated solutions of DNA (m 200 mg/ml) were prepared in two types of saline buffers : 1) 0.25 M
ammonium acetate, 10mM sodium cacodylate and 0.5mM EDTA (pH =7); 2)
0.25MNaCl, 0.5mM EDTA and 10mM sodium cacodylate. In such conditions the
organization of the DNA molecules is cholesteric the transition to the columnar hexagonal phase occurs after slow evaporation of the water. Specimens of the cholesteric phase and of the columnar hexagonal phase were introduced into quartz capillaries of I mm diameter. In the latter case the DNA fragments were flow-aligned with their axis oriented parallel to the
capillary axis. The more concentrated phases were obtained by evaporation of the water.
2.2 METHOD. X-ray diffraction experiments were performed using a synchrotron source
(station D43) at LURE (Univ. Paris-Sud). The X-ray beam was monochromatized by a bent
germanium crystal which selected a wavelength of 405 A. X-ray diffraction pattems of the specimens were obtained on films with the sample-film distance fixed at 100, 125 or 250 mm.
3. Results and discussion.
X-ray diffraction pattems were recorded at constant temperature (20 °C) for a great number of different DNA concentrations from the cholesteric phase up to the more concentrated state
(typically about 055 mg/ml for DNA in ammonium acetate buffer and 840 mg/ml for DNA in Nacl solution). Results are very similar for the two types of buffer. For clarity, only the characteristic parameters corresponding to the ammonium acetate salt will be given below.
The phase sequence as a function of the concentration is the following :
cholesteric
- hexagonal
- orthorhombic
3.I THE CHOLESTERIC PHASE. The texture of the samples in the cholestedc phase was
controlled optically with a polarizing microscope. Typical « fingerprint » pattems [8, 16, 17]
were observed between crossed circular polarizers, with a cholesteric pitch of the order of 2 ~Lm.
For this phase the X-ray diffraction pattems are characterized by a broad and very intense
ring in the inner part (Fig. I). Its diameter increases when the water content is lowered. At
larger diffraction angles, several broad and much less intense rings are visible. They are
characteristic of the pseudo-periodicity of the DNA structure along the helix axis which is due to base pair stacking [32]. The most intense of these rings corresponds to a spacing of 3.36 A
and represents the periodicity of the base pairs. In the small angle region, one can deduce from such a pattem the variation of the scattered intensity, I(s), as a function of s, where
s =
2 sin 9/A, 2 9 being the scattering angle and A the wavelength of the X-rays. Figure 2 shows the I (s) curves obtained for two different concentrations in the cholesteric phase. They
are characterized by an intense peak appreciably broader than the experimental resolution. A concentration increase moves the position of the maximum of the scattered intensity towards
higher s-values simultaneously the width of the peak decreases. The value of the maximum
seems to vary very slowly with the concentration but no quantitative results can be obtained
with our apparatus. Finally, preliminary experiments have shown that the behaviour of I (s) is very sensitive to the salt concentration : at fixed DNA concentration a decrease of the
salt concentration results in a narrowing of the peak.
From the position (s~) of the maximum off (s) one can deduce an approximate value of the
mean interhelices distance, a~, using the formula a~
= I.117/~, which is an extension of the
Bragg law suitable for such a liquid crystalline phase (after Ref. [33]). With increasing DNA concentration a~ is found to decrease from about 49 A to 32 A.
1772 JOURNAL DE PHYSIQUE II N° 9
*
Fig. I. Pattem of the cholesteric phase recorded at wavelength 1.405 A. The
very strong ring in the inner part indicates the existence of a short-range order with a mean interhelices distance a~ » 35.5 A. The most extemal ring represents the periodicity of the base pairs (d~~
= 3.36 A).
lsl la-u-) 3
~
- FWHM
0 0.025 0.050 o.075 o-loo
s
k~l
Fig. 2. Scattered intensity profile I(s) as a function of s = 2 sin 9/A, obtained using a microden- sitometer in the cholesteric phase for two values Cl(- -) and C~(--) of the DNA concentration, with
C~~C
j. The horizontal bar represents the instrumental resolution during this experiment ; FWHM (full width at half maximum)
= 0.0012 A-I The maximum at about 0.075 A-I is due to the DNA
molecular conformation and is not due to intermolecular interactions like the mean peak around 0.025 A-1
The broad peak described above is thought to reflect a local ordered arrangement of the DNA molecules superimposed on the cholesteric order. Robinson et al. [34] have observed a similar peak in concentrated solutions of PBLG ~poly-~Gbenzyl-L-glutamate) and proposed
that a local hexagonal order perpendicular to the molecular axis is present in the cholesteric phase. A single peak is also observed in the scattering of semi-dilute solutions of highly charged synthetic [35, 36] and biological [37, 38] polyelectrolytes, and especially in isotropic
solutions of short DNA fragments (150-160 base pairs) [30, 39]. For flexible chains no definite
interpretation of this scattering peak has yet been given in terms of intermolecular solution
structure [40]. It seems, however, that for « rodlike » polyelectrolytes, such as DNA [30] or chondroitin sulfate [38], there are indications of some local hexagonal alignment of molecules in the isotropic phase responsible for this scattering peak.
Other experiments are now necessary to completely elucidate the nature and the origin of the short-range order observed in this cholesteric phase. In particular, a quantitative study of
I(s) as a function of DNA concentration, salt concentration and counterion type must be undertaken.
3.2 CHOLESTERIC
- HEXAGONAL TRANSITION. -When the content of water is decreased,
the X-ray small angle diffraction pattems change suddenly : a very strong and narrow ring (with a width equal to the instrumental resolution width) appears superimposed on the broad
ring specific of the cholesteric phase. This indicates a discontinuous transition from the cholesteric to the two-dimensional hexagonal phase (the justification for such a structure will be given in Sect. 3.3. I). The diameter of this narrow ring provides the value of the parameter of
the hexagonal long-range lateral order: a~=2/(s/);
at the transition one finds:
a~ = 31.5 A. In the high angle region the ring corresponding to a spacing of 3.36 A remains unchanged.
Direct measurements of the DNA concentration in our samples have not been performed, mostly because of experimental difficulties. However, it is possible to evaluate it in the
hexagonal and more concentrated phases- from the parameters of the two-dimensional latice perpendicular to the axis of the molecules. The concentration C defined as :
C
= weight of DNA/volume of solution
is given by : C
= MDNA/"h (~)
where M~~~ is the molecular weight of a base pair (b.p.), « the area of the unit cell of the two-dimensional lattice and h the axial translation per residue (commonly called « rise »).
Equation (I) allows us to obtain an approximate value of the DNA concentration at the cholesteric
- hexagonal transition : C~
= 380 mg/ml.
Finally, we would like to stress an experimental observation. When the critical concen- tration C~ is approached, the X-ray pattems of the cholesteric phase of some specimens undergo modifications : reinforcements appear simultaneously on the ring corresponding to the spacing between two b.p. in the direction of the capillary, and the perpendicular to this direction on the ring in the small angle region. It indicates that, by approaching the transition towards the hexagonal phase, the DNA molecules align spontaneously with the cholesteric
pitch axis perpendicular to the capillary. Other experiments are required to clarify this point.
3.3 THE HEXAGONAL PHASES. All the results presented below concem oriented specimens
with the DNA molecules parallel to the capillary axis : either the samples have been
introduced in the capillary after transition to the hexagonal phase and then flow-aligned, or they aligned spontaneously in the capillary in the cholesteric state and retain their orientation
during the transition to the hexagonal phase.
3.3.1 2D-ordered phase. Figure 3a shows a X-ray pattem characteristic of the columnar
hexagonal phase just after the transition. Such a pattem has been previously described in reference [14]. The sharp arc, with a strong equatorial reinforcement, reveals the long-range periodic lateral arrangement of the DNA molecules. Another very weak reflection is
observed in the equatorial region (not visible on this picture) : the ratio of the spacings of both
previous reflections is I : / in unit
s (s
=
2 sin 9/A). When the DNA concentration
increases, two new weak equatorial arcs appear in the ratios I : / and I
: / with the strong
reflection. The existence of these three weak reflections with spacing ratios 1: /:
1774 JOURNAL DE PHYSIQUE II N° 9
Fig. 3. -a) Pattem of the 2D-ordered hexagonal phase (Cm395mg/ml) recorded at wavelength
A
= 1.405 A. This pattem is characteristic of DNA in the B form with a strong meridional arc at 3.36 A
in its outer part. One observes in the inner part :I) The typical crosslike intensity distribution suggesting
a helical structure. ii) A strong equatorial reinforcement of the sharp arc revealing the hexagonal lateral order with interhelix distances of 30.9 A. b) Simulation of the inner part of the previous pattem using
the atomic coordinates given by Chandrasekaran and Amott [34] for the B form of calf thymus DNA and taking into account the disorientation of the helices axis with respect to the capillary axis.
/
: /
~gests a two-dimensional hexagonal lattice. We have checked that the absence of
the « : 4
» reflection, which is normally observed for such a lattice, is due to its very weak molecular structure factor. It is recalled that at the cholesteric
- hexagonal transition, the parameter a~ of the hexagonal lattice is found to be equal to 31.5 A then
a decrease of a~ is observed when the water content is lowered.
The inner part of figure 3a also displays regions of strong intensity forming a cross pattem.
Such features are characteristic of the helical structure of the DNA molecule : the scattered
intensity is located on layers corresponding to the helix pitch periodicity P [41]. Just after the cholesteric
- hexagonal transition no Bragg reflections are observed on these layers, which is indicative of the absence of longitudinal order between neighboring DNA molecules. Finally,
a strong diffuse arc near the meridian, located at 3.36 A, is present in the outer part of the pattem. This arc is usually observed for DNA chains in the B conformation and reveals the
step-like structure of the helix with a rise of h
= 3.36 A and the base pairs nearly perpendicular
to the helix axis.
It is possible to simulate numerically such a pattem using the atomic coordinates given by
Chandrasekaran and Amott [42] for the B conformation of calf thymus DNA (the same
coordinates have been used for the other simulations of the B conformation presented in this
paper). The result is illustrated in figure 3b where we have taken into account the disorientation of the helices axis with respect to the capillary axis. In fact, the DNA molecules
are not rigorously parallel to the capillary and we have shown that a good description of the diffuse arcs observed in figure 3a is obtained if we assume a Gaussian distribution of
orientation with a standard deviation of 10°. The main interest of the comparison between the
experimental (Fig. 3a) and simulated (Fig. 3b) pattems results from the fact that the
simulations include two parameters: the interhelix distance a~ and the helix pitch
P, and in particular allows the determination of P in this liquid crystalline hexagonal phase.
Just after the transition from cholesteric to hexagonal, P is found to be equal to 34.6 ± 0.3 A
which corresponds to 10.3 ±1 nudeotides per helix tum. This point will be discussed in details in section 4,1.
3.3.2 Evolution towards the 3D-ordered phase.-At increasing DNA concentration, the
inner part of the X-ray pattems changes and becomes more structured. The diffuse intensity
observed along the first layer condenses progressively into a sharp arc (Fig.4a) which becomes more intense with decreasing water content. This Bragg reflection is observable when the hexagonal parameter a~ becomes smaller than about 29.5A. For higher DNA
concentrations the same type of sharp arc occurs along the second layer and finally along the third one (Fig. 4b). We can conclude that a longitudinal order progressively appears between neighbouring DNA helices leading continuously to a 3D-ordered structure. The three-
dimensional lattice seems to be well established when the intermolecular distance has reached
a value close to 25.5 A.
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