10
DNA concentrated aqueous solutions in vitro form liquid crystals, likewise observed
in vivo for some cellular systems [8-11], the investigation of these anisotropic
systems is acquiring increasing importance. In fact, in the last two decades a
considerable attention has been addressed to the study of DNA liquid crystals, which
represent an experimental model very promising able to allow the explanation of
important aspects of the DNA biological activity. In particular, studies on DNA liquid
crystalline phases were performed successfully to get information about the
structure, the function and the genes regulation [12] and to test new biosensors and
potential applications in the gene therapy [13-15].
The different techniques employed to investigate DNA anisotropic solutions have
shown that the ordering and the stability of these systems depend on different
parameters such as the temperature, the ionic strength, the DNA concentration, the
molecular weight and the water content in the case of DNA fibers [16-22]. Among
the employed various techniques of investigation, NMR spectroscopy is providing
remarkable contributions of knowledges at an atomic level for the study of dynamic
and structural properties of DNA chains (rods) and of counterion environment that
surrounds the polyion.
In particular, the solid state
31
P NMR spectroscopy has been used for studies on
DNA backbone conformation and dynamic properties in oriented fibers [23-26];
moreover by means of the analysis of
31
P NMR lineshapes of DNA liquid crystalline
solutions phase diagrams have been determined for the isotropic-anisotropic phase
transition [21,27-29]. Several
23
Na NMR experiments, performed using a wide range
of temperatures, of DNA concentrations and ionic strengths, have provided
11
information about the counterion-DNA interaction [30-34]. Some works have been
also addressed to the study of the degree of the DNA magnetic ordering by
analyzing
2
H NMR spectra of deuterated oligonucleotides in the solid state [22,35-
38] or of the deuterated water [39].
A notable attention has been dedicated to the characterisation of ordered DNA
aqueous systems and of relative physiological counterions, but investigations on the
interaction between the DNA in the liquid crystalline state and exogenous agents are
lacking. In this regard, some investigations performed by means of the optical
microscopy are noteworthy and have shown the ability of some organic ligands, such
as daunomicine [40] and ethidium bromide [41], some platinum (II) coordination
complexes [42] and magnesium ion [17] to affect the DNA packing in liquid crystals.
These research themes are acquiring great interest, therefore they need a particular
intensification of the study on the modifications induced by external agents on the
DNA spatial organisation and on the surrounding counterion atmosphere.
By then it is assessed that intercalators, such as antibiotics and dyes, and
particular divalent metal ions are activators or inhibitors of important processes,
such as the replication, the transcription and the translation of the genetic code.
Therefore, it is of great interest to analyse in detail the structural variations induced
on DNA liquid crystals by these agents and to reveal possible correlations between
the DNA structure and its biological activity.
To get these objectives, methodologies of experimental investigation based on
the use of the multinuclear NMR spectroscopy have been employed, allowing the
acquisition of set of information on the structural and dynamic properties of DNA-
12
exogenous agents systems. To this aim, DNA liquid crystalline samples, at different
temperatures and concentrations, have been analysed by means of
31
P,
23
Na,
2
H and
17
O NMR Spectroscopy. In particular, various spectroscopies have contributed to
monitor simultaneously DNA structure and physical state (
31
P NMR), interactions
between DNA and sodium counterions (
23
Na NMR), and the degree of order of
examined liquid crystals and of the water (
2
H and
17
O NMR). Moreover, the
employment of the Polarized Light Optical Microscopy has provided complementary
information for the characterisation of the DNA liquid crystalline phases. In the
present work the following exogenous agents have been analysed: the classical
intercalator ethidium bromide (EB) [43,44], divalent metal ions Mg
2+
, Mn
2+
, Ni
2+
and
Cd
2+
, and the intercalating platinum complex Chloro(2,2’:6’,2’’-
Terpyridine)Platinum(II) Chloride [Pt(Terpy)Cl]Cl (PtTCC).
13
Chapter 1
DNA structural properties
1.1 DNA structure
The DNA is a macromolecule formed by a high number of
deoxyiribonucleotides, each constituted by a base, a sugar and a phosphate group.
DNA bases bring the genetic information, while sugars and phosphate groups have a
structural role. The sugar is the deoxyribose, which differently from ribose is without
an oxygen atom in the 2' position (Fig. 1.1).
Figure 1.1 Deoxyribose structure.
The nitrogenous base can be a residue of the purine, Adenine (A) or Guanine (G), or
a pyrimidine, Thymine (T) or Cytosine (C) (Fig. 1.2).
Figure 1.2 DNA nitrogenous bases: Thymine, Adenine, Cytosine and Guanine.
OH
1'
2'
O
3'
4'
5'
OH
OH
5
6
4
N
H
1
NH
3
2
O
O
CH
3
5
4
6
N
3
N
1
2
N
H
9
8
N
7
NH
2
N
3
2
4
NH
1
5
6
NH
2
O
5
4
6
N
3
NH
1
2
NH
9
8
N
7
NH
2
O
Thymine (T) Adenine (A) Cytosine (C) Guanine (G)
14
The DNA backbone is constituted by deoxyribose units bound to phosphate
groups: phosphodiesteric bonds bind oxydriles 3' and 5' of two adjacent sugars
forming linear polymers of nucleotides (Fig. 1.3). The bases sequence constitutes
the DNA variable part. Given that one extremity of the chain has a 5’-OH group and
the other a 3'-OH group, not bound to a nucleotide, the DNA chain shows a certain
polarity. Bases sequence is written in the direction 5'→ 3' by convention.
Figure 1.3 Structure of a nucleotide, base (G) + deoxyribose + phosphate, (a) and
of a part of the DNA chain (b).
In 1953 the DNA double helical tridimensional structure was discovered by
James Watson and Francis Crick [2] analysing X rays diffraction pictures of DNA
fibers obtained by Rosalind Franklin [1]. In the proposed double helical model
polynucleotide chains run in opposite directions with a helical winding around a
common axis. Phosphate groups and sugars are outside the helix, while purine and
pyrimidine bases are inside (Fig. 1.4a). The plane of sugars forms an angle of almost
5
4
6
N
3
NH
1
2
N
9
8
N
7
NH
2
O
1'
2'
O
3'
4'
5'
O
OH
OH
OH
O
P
base
O
O
O
O
-
OP
base
O
O
O
O
-
OP
base
O
O
O
-
OP
OH
(a) (b)
15
90° with the one of the bases, which is perpendicular to the helix axis. The diameter
of the helix is 20 Å. Adjacent bases are separated by 3.4 Å along the helix axis and
form an angle of 36° (torsion angle). In this way the helical structure repeats itself
every 10 residues of each chain, with ranges of 34 Å (helical pitch). Base pairs are
linked by hydrogen bonds: the adenine is always coupled with the thymine and the
guanine is always coupled with the cytosine.
Figure 1.4 Scheme of DNA polynucleotide chains.
The right handed winding of polynucleotide chains generates two grooves along
the helix, a deep and broad one (major groove) and a narrow one (minor groove),
aligned with phosphate groups (Fig. 1.4b). The atoms O2, in cytosine and in
thymine, and N3, in adenine and guanine, face the minor groove and the atoms on
the opposite sides of the bases are exposed in the major groove.
major groove
minor groove
(a) (b)
16
The double helical structure can assume different conformations such as A, B
and Z (Fig. 1.5), whose structure is dictated by the sugar conformation and bases
orientation respect to the sugar. DNA polymorphism depends on both double helix
bases sequence both phisical conditions such as the water activity and the salt
concentration. The B conformation, that proposed by Watson and Crick, is the one
which prevails in physiological conditions. Generally, the B conformation is favoured
by the presence of counterions such as alkaline metals, like Na
+
ions, and by a
relative humidity at least of 92%. The relative stability of A and B DNA
conformations depends on factors like the temperature and the ionic strength
[45,46]. The A conformation of the DNA shows a double helix right handed with a
shorter pitch (29 Å), a lower number of residues per turn of the helix (11), a
narrower and deeper major groove and a superficial and wider minor groove respect
to B-DNA. The main differences of the Z respect to the B conformation are: the
double helix is left handed, the number of base pairs per turn of the helix is 12, the
diameter of the helix is 18 Å and the separation between base pairs is 3.7 Å.
Figure 1.5 Structures of A, B and Z DNA conformations.
A B Z
17
1.2 DNA liquid crystals
A liquid crystal is a molecular system, whose aggregation state assumes an
intermediate configuration (mesomorphism) between the threedimensional lattice of
the solid crystal and the amorphous state typical of the liquid. The phase transition
of the majority of crystals occurs directly from the anisotropic solid state to the
isotropic liquid one, nevertheless for many crystalline solids it is observed the
formation of one or more birefringent mesophases, which are more or less fluid and
show various degrees of organization.
A common characteristic of liquid crystalline phases is the presence of a non
locale spatial arrangement, characterized by an intermediate degree of order
between the solid state and the liquid state, a considerable motional freedom and
anisotropic physical properties. Generally, in liquid crystals moleculs tend to align
themselves along a preferential direction, which is called the director.
The birefringency, or optical anisotropy, is a property that is often observed
in liquid crystals. In these systems the light polarized along the director axis shows a
different refractive index respect to that in the direction perpendicular to the director
itself.
Liquid crystalline phases obtained by varying the temperature are called
thermotropic, while those produced by the influence of a solvent are denominated
lyotropic. Generally, thermotropic liquid crystalline mesophases are formed by
elongated rod shaped molecules (“rods”). Thermotropic liquid crystals can be divided
into three main categories: nematics, smectics and cholesterics (Fig. 1.6).
18
Figure 1.6 Nematic (a), smectic (b) and cholesteric (c) phases.
The nematic mesophase has a single optical axis and is, so that, an uniaxial
phase, without a positional order, in which molecules diffuse rapidly but not
isotropically and tend to be oriented parallelly along the director of the phase. The
smectic mesophase shows in addition a long range positional order. Smectic liquid
crystals are formed by molecules which arrange themselves in a layers structure and
can be both uniaxial both biaxial.
The cholesteric mesophase, called in this way because the cholesterol was
one of the first compounds in which the formation of this fase has been observed,
shows a molecular disposition similar to that of the nematic phase. It is formed by
optically active chiral molecules. Molecules are aligned in parallel and their
orientation rotates continuously around a fixed direction, which is called cholesteric
axis (C) (Fig. 1.7). For clarity, this continuous helical structure can be represented by
a series of parallel and equidistant planes, as shown in Figure 1.7. The director of
the phase, which represents the average local orientation of DNA fragments, is
(a)
(b) (c)
19
oriented perpendicularly along the cholesteric axis around which rotates by constant
shifts. The distance between planes in which molecules show the same orientation
and direction is called cholesteric pitch (P), the distance covered by the director to
do a complete turn of the helix. It is important to note that, in the representation in
Figure 1.7, reproduced planes do not have any physical meaning and serve only to
give a picture of the cholesteric phase structure.
Figure 1.7 Molecular ordering in a cholesteric mesophase.
In vivo the DNA molecule, at high concentrations, can form ordered liquid
crystalline phases. As an example, DNA cholesteric liquid crystalline phases have
been observed in some bacterial nucleoids [49] and in chromosomes of
dinoflagellates [50-53], while DNA columnar hexagonal packings have been met in
bacteriophages and in spermatozoa heads [54].
In 1961 Robinson, for the first time, observed by means of polarized light
optical microscopy that DNA concentrated aqueous solutions spontaneously formed
B
0
20
cholesteric liquid crystalline phases [8]. Because the DNA spontaneous ordering in
vitro takes place at concentrations similar to those in vivo, these DNA condensed
systems represent simple investigation models useful for the comprehension of DNA
condensation processes in biological systems and of their role in the DNA function
for the preservation of the genetic material.
DNA linear fragments in aqueous solution can form different liquid crystalline
phases depending on the polymer concentration. The sequence of these phases can
be schemed in the following way [11]:
Figure 1.8 DNA mesophases observed in vivo and in vitro as a function of the DNA
concentration.
These phases sequences have been obtained from DNA saline solutions, for
which it has been often observed the coexistence of more phases, as indicated by
the arrows reproduced in the Figure 1.8. In fact, the formation of biphasic e
triphasic systems is strictly dependent on the length of DNA fragments, the DNA
concentration, the ionic strength of the solution and the temperature. The transition
from the isotropic phase (diluted) to the cholesteric phase (more concentrated) can
follow two different evolutions. In the first case it is observed the formation of small
cholesteric germs
pre cholesteric stages
decondensed chromosomes
isotropic phase
chromatin
cholesteric phase
mithocondrial DNA
DNA concentration
columnar
hexagonal phase
bacteriophages
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