Molecular Biophysics

MOLECULAR BIOPHYSICS
Rudi Podgornik
Department of physics, Faculty of mathematics and physics,
University of Ljubljana and Department of theoretical physics, J.
Stefan Institute, 1000 Ljubljana, Slovenia.
1 Sunday, May 27, 12

Physical properties of the DNA molecule from the level of the base pairs all the
way to the organization of DNA in viruses and chromatin. Interactions within the
DNA molecule as well as between the DNA molecules in aqueous solutions at
various environmental conditions. Not chemistry, not biology, but physics, with
as few equations as is humanely possible for a theoretical physicist...

STRUCTURE OF DNA (X-ray scattering, Structure factor of a continuous single helix, Scattering
intensity of an orientationally averaged helix, Structure factor of a discrete helix, Scattering
intensity of a double helix, Details of B-DNA structure),
BASE-PAIR INTERACTIONS AND DNA MELTING (A model for primary stabilizing
interactions, The Peyrard-Bishop-Dauxois model of DNA melting, The DNA melting
temperature, Observing DNA melting),
MECHANICS AND STATISTICAL MECHANICS OF DNA (Elastic deformation energy, Elastic
equation of state, The Kirchhoff kinematic analogy, The Kratky-Porod model, Light scattering
from a Kratky-Porod filament in solution, Elastic response of a Kratky-Porod filament, The limit
of small stretching force, The limit of large stretching force, Extensible semiflexible chain, An
approximate elastic equation of state for DNA),
ELECTROSTATICS OF DNA and DNA -DNA interactions (Poisson-Boltzmann theory,
Counterion distribution, manning condensation, Salt screening, Strong coupling theory,
Correlation attraction, Osmotic stress method, Hydration force, Force equilibria with polyvalent
counterions),
DNA COLLAPSE AND DNA MESOPHASES (Collapse of a single DNA molecule, The DNA
toroidal globule, Nematic LC transition in a DNA solution, Elastic energy of a DNA hexagonal
columnar LC, Cell model of a DNA array, Osmotic pressure of a DNA array, Electrostatic part of
the osmotic pressure, Equation of state of a DNA array, Fluctuations and positional order in a
DNA array),
DNA ORGANIZATION IN CHROMATIN AND VIRUSES (Nucleosomes, Caspar-Klug theory and
elaborations, Continuum elasticity of viral capsids, Viral capsids under mechanical stress,
Osmotic encapsulation of DNA, The inverse spool model).

LECTURE 1
STRUCTURE OF DNA (X-ray scattering, Structure factor of a
continuous single helix, Scattering intensity of an
orientationally averaged helix, Single and double helix,
Scattering intensity of a double helix, Details of the double
helical B-DNA structure)


Thomas Young, hieroglyphs and interference
The double life of Dr. Thomas Younga (1773-1829)
Interference, 1820.
Light is a wave and not a
ow of particles as Newton
thought!
Hieroglyphs can be read,
1814.
Cartouches on the Rosetta
stone.
They are not pictograms but
have phonetic values!

Interference
Interferencof waves passing through two slits.
Interference of many sources of waves.
Surface or volume patterns.
Light is a wave. And therefore light waves interfer with one another. Just like waves on the
surface of water. Total amplitude is a sum of partial amplitudes of all waves.

Interference on multiple slits
The separation between the slits is reected in the interference pattern. The exact relationship is
given by the Fourier transform.
Huygens principle: every single slit acts as a source of a spherical wave so that the total
amplitude is:
The Fourier transform of the distribution of slits. Therefore the intensity of light is:
The intensity is given by the square of the Fourier transform of the slit distribution (numerically).

November 1895, Wilhelm Rntgen discovers X-rays at the University of Wurzburg. Thinks they are
longitudinal light waves.
Max von Laue 1912
discovers di!raction of
X-rays by crystals. Nobel
prize for physics 1915.
Wavelength ~ 1
X-rays
Interference of many sources of waves.
Surface or volume patterns.
Wilhelm Conrad Rntgen
(1845-1923)
Max Theodor Felix von Laue
(1879-1960)

Scattering of X-rays
Von Laue (1912) ZnS (zinc sulde crystal).
In 1912 William Lawrence Bragg (1890-1971) understands why light waves passing through crystals
produce intricate patterns.

Scattering formalism of X-rays
Let us look at the amplitude of the light waves. Each atom in the sample re-radiates the incoming
wave.
From a discrete distribution of atoms to a continuuous distribution of matter.
The total intensity of the scattered beam is then
The scattering geometry is as follows:
(For elastic scattering k does not
change and Q is thus a measure of
the scattering angle)
The total amplitude is thus the Fourier transform of the density.
Q

Scattering of X-rays on ordered arrangement of atoms
Like on a protein crystal. The volume pattern of the scattered X-rays can be captured on a two
dimensional lm. This pattern can be deconvoluted into atomic positions.
First succesful deconvolution into atomic structure of a protein by Max Ferdinand Perutz (1914-2002)
in John Cowdery Kendrew (1917-1997). Nobel prize for chemistry in 1962.
molecular models
because we have only 2D
pattern of waves.

Scattering of X-rays on DNA
Even if the order is not ideal, scattering experiment is feasible, but provides us with less information
on atomic positions. William Astbury (1898-1961) started scattering X-rays on DNA in 1943 but best
di!ractograms from 1947.
Maurice Hugh Frederick Wilkins (1916-2004).
Obtains the best quality di!ractograms up to 1951
until the work of Rosalind Franklin.
Wilkins meets Watson in Naples in1951.

Why scatter X-rays on DNA?
Erwin Schroedinger
(1887-1961)
Founder of QM.
In 1945 he published a book What is life?
Aperiodic crystal.
Secret of life.
This book inuenced many physicists.

Ordered samples of DNA
Wet spinning.
A. Rupprecht
(learn from spiders)
DNA ber spinning.
Orientationally ordered
DNA moelcules.
We need to prepare macroscopic samples with as much order as we can get. DNA bers!

DNA in concentrated solutions makes ordered phases. Not that difcult to observe.
Conmar Robinson in 1958 described
the cholesteric phase of poly(benzyl-
L-glutamate) in dimethylformamide
and in 1961 of DNA.
V. Luzzati et al. in 1961 observed
indications of ordered phases of DNA
in SAXS (small angle X-ray scattering)
R. Rill et a. in 1981 saw ordered
phases with short fragment DNA (146
bp about 50 nm)
R. Podgornik et al. (1997) discover
the line hexatic phase.
line hexatic phase
(1997)
cholesteric phase
(1961)
Ordered DNA mesophases.

Nature of DNA mesophases.
Durand, Doucet, Livolant (1992) J. Physique 2, 1769-178
Pelta, Durand, Doucet, Livolant (1996) Biophys. J., 71, 48-63.

The best X-ray scattering on DNA (Franklin and Gosling, 1952). Only diffractograms with enough order can
be analyzed quantitatively by applying the theory if X-ray scattering on helical molecules.
Diffractogram 51
Rosalind Elsie Franklin
(1920-1958)
Her X-ray diffraction produced the
most beautiful pattern that could
be analyzed quantitatively.
From her lab notebook.

B-DNA and A-DNA
They differ in the density or hydration of DNA within the ber. A more dense then B, but usualy in B
conformation.

PhD thesis of Francis Harry Compton Crick (1916-2004).
Advisor: W. Bragg. Finished at age 35!
X-rays
Alexander (Alec) Rawson Stokes,
(1919-2003): Bessels functions.
A continuous helix.
Stokes and Crick: scattering on helices

Helices
The + sign refers to a right handed helix, where the sign of the polar angle " is dened by the
right-handed screw, and the # sign refers to a left handed helix. Obviously in the rst case " and
with it the angular velocity vector points in the direction of the axis, i.e. z.
Kelvin dened chirality as: I call any geometrical gure, or group of points, chiral, and say it has
chirality, if its image in a plane mirror, ideally realized, cannot be brought to coincide with itself.
For a helix the scalar product between the tangent vector and a direction in space, in this case the
direction of the z axis, is a constant

Single, double, symmetric, asymmetric helices
We can of course take two helical curves inscribed on the same cylinder of radius a. Six
turns of a single and double continuous right-handed helix.
The two strands of the double helix are shifted by an angle of 3$/8 making the double helix
asymmetric with respect to the central axis. Take a plane perpendicular to the long axis of this cylinder
and cut the two helices. The intersection of this plane and the two helices denes two points which do
not coincide as long as the two helices are shifted by a nite angle.
Symmetric and asymmetric helices.

Rotational symmetries of helices
On rotating the helix around this axis for a single directed helical line we have to rotate by a
complete turn of 2$ for the helix to coincide with its position before the rotation. For a double helix
whose two single helical lines have antiparallel directions so that one climbs along the helix and the
other one descends we have to rotate only by half that angle, i.e. by $, for the two forms to coincide
A major di!erence between single and double helices.

Dyadic axis because of the double helix

Scattering form a continuous single helix
We take the equation of the helix in the form:
where P is the pitch of the helix describing its periodicity in the z-direction.
Here % is an arbitrary initial angle of the helix at z = 0. In a disordered sample all these angles are
equally probable and have to be averaged over, leading to:
How did the Bessels function come into this play?
Through its simple integral representation involving an integration over a polar angle
This is the dependence of the scattered beam intensity on the scattering vecor Q.
On a 2D screen this vector has two components: transverze and longitudinal.

Scattering form a continuous single helix: result
A very peculiar property emerges
from this formula:
St. Andreas cross!
The opening of this cross depends
on the radius of the helix and its
pitch (periodicity) and the
prsiodicity in the Qz direction
depends solely on the pitch of the
helix.

Is DNA a helix?
In 1953 this was not a simple questin to answer and the answer changed depending on the person
and time.
Franklin in Gosling (july 1952)
proclaim the death of the DNA helix
after an Intensive therapy of
besselized injections.
Aaron Klug (a later collaborator of
Franklin and Nobel laureate in 2001):
Rosalind Franklin started to write a
paper on the double helical structure
of DNA even before she learned of
the Watson-Crick model.

Much more then a single helix?
Three entangled chains with
bases sticking out and charged
phosphates in the middle.
His son Peter was in Cambridge at the same time as Watson and Crick.
Pauling is very good at molecular modeling. Protein structural motifs are his work. In the beginning
of 1953 Pauling proposes a model of DNA.
Linus Carl Pauling
(1901-1994)

DNA is a double helix!
Saturday
February 28, 1953.
A model is built in the Cavendish
laboratory in Cambridge consistent with all
the discernible features of the
diffractogram 51...
Francis Harry Compton Crick (1916-2004).
Important contributions to science: central
dogma, physics of viruses, neurobiology, origin of
life ...

Anatomy of DNA diffraction
Andreas cross diamonds
layers missing layers

Diffraction model of DNA
We will see that we can explain most of the features of the diffractogram 51 from simple properties of
scattering of X-rays on the asymmetric discrete double helix.
DNA = asymmetric discrete double helix

A.A. Lucas, Rosetta Stone of the genetic language
Get Full Text via UMLinks
AA Lucas - International Journal of Quantum Chemistry, 2002





We wish to suggest a structure for the salt of
deoxyribose nucleic acid (D.N.A.). This structure has
novel features which are of considerable biological
interest....
Nature, April 25, 1953.
Watson in Crick
F. Crick, 35.
J. Watson, 25.
Nobel prize (with M. Wilkins) for medicine, 1962.

Maurice Hugh Frederick Wilkins (19162004)
Worked on the Manhattan project
and continued in biophysics.
Wilkins, Stokes in Wilson

Who discovered the structure of DNA?
The Franklin-Gosling paper
one month before the W-C model.
The reference added by hand later!
Franklin in Gosling
Rosalind Franklin died in 1958.
Nobel prize can not be awarded posthumously, 1962.

DNAScattering@home project
Seti@home, Folding@home ... why not DNAScattering@home.
a
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s
i
g
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1
)




FINIS

counterions),
DNA array),

LECTURE 2
BASE-PAIR INTERACTIONS AND DNA MELTING (A model
for primary stabilizing interactions, The Peyrard-Bishop-
Dauxois model of DNA melting, The DNA melting temperature,
Observing DNA melting),
MECHANICS AND STATISTICAL MECHANICS OF DNA
(Elastic deformation energy, Elastic equation of state, The
Kirchhoff kinematic analogy)

Different views of the Watson-Crick double helix model
poynting to various properties of the double-helix. The most iconic science image of the 20. century.
The Watson-Crick double helix model

A double helix of phosphates. Phosphates outside - nitrogen bases inside.
Antiparallel backbones. Asymmetric positions of the two helices.

chromosome replication
transcription translation
Properties of DNA double helix biologically relevant

Twisted ladder of nitrogen bases
The four nitrogen-rich bases are: adenine, cytosyne, guanine and thymine (A,C,G,T). The molecular weights
(MW) of these four monophosphate-deoxyribonucleotides are not identical.
They do not all have the same molecular weight,
but they are close. The average MW of a bp
always composed of one purine and one
pyrimidine is 660.
3HB 2HB

Directionality of the ladder of nitrogen bases
The DNA backbone, formed by a sequence of phosphates and sugars, is oriented because on the
"incoming" side the phosphate is linked to the 5 carbon of the sugar ring while, on the "outgoing" side it
is linked to the 3 carbon of the sugar ring.

The nature of the Watson-Crick double helix
A directed phosphate backbone and nucleotides (purines - one nitrogen ring or pyrimidines - two nitrogen
rings) attached to both backbones interacting via hydrogen bonds.

Interactions holding DNA together
Primary interactions:
The primary interactions act perpendicular to the long axis of the molecule as well as between the
base pairs along the axial direction of the molecule. They are the
covalent bond interactions between the phosphate groups in the phosphate backbone running
along the long axis of the molecule
base stacking interactions between the bases in the stack along the axis of the molecule
hydrogen bond interactions between complementary bases perpendicular to the axis of the
molecule
Auxiliary interactions:
Though these are certainly the primary interactions that set the overall feature of the B-DNA
geometry, there are other interactions that further stabilize it and adjust all the important details of
the structure. These auxiliary interactions are composed of
water-DNA surface hydrogen bonding interactions between the polar moieties of the DNA surface
and the water molecules as well as the
counterion-mediated electrostatic interactions along the charged phosphate groups along the DNA
The auxiliary interactions are connected with the biological environment of DNA, i.e. an aqueous
solution with cationic counterions that ensure the overall electroneutrality of the system. Dehydration
of the DNA molecule such as severe osmotic stress on the order of a 1000 atm, leads invariably to a
degradation of the double-helical structure.

The phosphate-phosphate bond along the phosphate backbone has a length of 6 , basically determined by
the structural properties of phosphate and ribose sugars. The nitrogen bases are planar and interact with
strong hydrophobic attractions, that keep them at a separation of 3.4.
(poly(dA) single stranded DNA, base-stacking energy is large ! 3.2 " 4.0 kcal per mole of base pairs).
If DNA would be a ladder of stacked bases that would not satisfy the strong hydrophobic stacking
interactions. If the ladder is tilted, at an angle that could satisfy the phosphate-phosphate bond length as
well as the stacking separation between bases ( not observed ). Instead the bases twist at the same time,
making a helical conguration observed in DNA. This twisting should be about 32 degrees, that leads to
about 11 bases per turn of DNA, which is very close to 10 observed in X- ray scattering of B-DNA.
Why a double helical DNA why not simply a ladder of base-pairs?
The Calladine-Drew argument
Covalent and stacking interactions along the phosphate
backbone

Stacking interactions between bases
The base pairs in DNA are almost planar and there are strong attractive interactins between
them. Partly van der Waals and partly hydrophobic - hydrogen bond interactions.
Stacking of coins is similar to nitrogen base stacking in DNA.
One can make an experiment with connected coins and see that they stack nicely in a helix.

Stacking interactions
Base stacking interactions are longer ranged than covalent interactions and extend to several .
Mostly they are just van der Waals interactions.
Left: stacking energy between two guanine disks as a function of separation. The calculation is performed
with two different methods, adapted from M. Elstner et al. op.cit. Right: stacking energy between base-pairs
at a twist angle corresponding to the B-DNA geometry, .e. 36&, adapted from V.R. Cooper et al. op.cit. Only
the base sequence on one of the strands is shown.
0.6 kcal/mole is thermal energy
at room temperature.
These are strong interactions!

van der Waals interactions
Van der Waals interactions depend on polarizabilities of the molecules or their dielectric response functions
as functions of frequency.
Electromagnetic eld uctuations lead to uctuating interaction pressure:
Thermodynamic average of the stress tensor at the boundary yield the interaction forces.
Lifshitz in 1954 (E. M. Lifshitz, Dokl. Akad. Nauk. SSSR, (1954); (1955); Zh. Eksp.Teor. Fiz., (1955)) gured
out how to do this calculation and get the average of the EM stress tensor:

The Lifshitz theory of vdW interactions
Interaction free energy depends on the dielectric response fo the interacting bodies and the medium.

1
2
D
The van der Waals - London dispersion transform of the dielectric function.
this can be either calculated or measure. To asses the vdW interaction
between bases or base-pairs we need their spectra. Mostly in the UV
region.
For DNA one commonly thinks that the adsorption peak is at 260 nm
but it really depends on the BP composition. Slight variations between
different nitrogen bases.
van der Waals - London
dispersion transform

UV absorption spectrum of bases
Interaction free energy depends on the dielectric response of the interacting bodies and the medium.
What is plotted is the real, the imaginary and the vdW-London dispersion transform for different bases.
In an aqueous DNA solution a measurable absorption kicks in at about 320 nm leading to a peak at
approximately 260 nm and then a dip between 220 and 230, raising again to a very pronounced peak just
below 200 nm and then fading off into the far UV region.

Hydrogen bonded nitrogen base pairs
A purine interacts with a pyrimidine via hydrogen bonds. These are (almost) electrostatic interactions +
quantum mechanics.

The hydrogen bond on the other hand has a length of about 0.275 nm and a typical strength of
about 10-20 kB T at room temperature, depending also strongly on the nature of the hydrogen
bond.
Contrary to the covalent bond the hydrogen bond is quite soft and at room temperature one has to
input an energy of about 0.16 kB T to stretch the bond by about 0.01 nm
Hydrogen bonds
The covalent bond has a typical length of 0.154 nm and an energy of about 145 kB T at room
temperature. The covalent bond is quite rigid at room temperature and one has to input an energy of about
6 kB T to stretch the bond by about 0.01 nm.

Hydrogen bonds in water
Most water molecules form strong bonds with only two others. Recent results suggest the tidy water molecule
network depicted in most chemistry textbooks is inaccurate. Instead, water might actually form a loose
network of rings and chains. Recent x-ray experiments reinforce the prevailing view of waters structure.
Lei Liang et al. PR B 83, 024201 (2011) Ab initio investigation of hydrogen bonding and network structure in
a supercooled model of water
The dynamic interactions of water molecules. Individual H2O molecules are V-shaped, consisting of two
hydrogen atoms (depicted in white) attached to the sides of a single oxygen atom (depicted in red).
Neighboring H2O molecules interact transiently by way of hydrogen bonds (depicted as blue and white ovals).

The Peyrard-Bishop-Dauxois model
The form of the hydrogen bond and stacking interactions is very complicated. Can we approximate it with a
neat model? Indeed we can. The congurational part of the Hamiltonian (total energy) is:
M. Peyrard and A.R. Bishop, Phys. Rev. Letts. 62 2755-2758 (1989), T. Dauxois, M. Peyrard and A.R.
Bishop, 47 R44-R47 (1993).
Stacking:
Hydrogen bonds:

Basic equations of the Peyrard-Bishop-Dauxois model
The total energy in this model is then:
The static solution with no kinetic energy is obtained from:
Domain wall solution:
This solution is valid only at zero temperature. Domain wall energy grows with the system size!!!!!

DNA melting in the Peyrard-Bishop-Dauxois model
At a nitie (but low enough) temperature:
One can derive the following result for Gaussian uctuations around the domain wall solution:
For small T, the expression within the brackets
is negative. The DNA segment can reduce its free
energy by increasing n0, close the open base
pairs.
In contrast, for large T the term within the
brackets becomes positive and the DNA segment
can reduce its free energy by decreasing n0, i.e.
by opening the base pairs that were closed.
The Peyrard-Bishop-Duxois model therefore leads to a melting transition of DNA corresponding to separation
of the two strands of the doble helical DNA.

Elasticity of DNA
Most of the interactions holding the structure of DNA together are not innitely strong and allow for
deformations of the basic double helical structure.
Since the deformation energy doe not depend on the overall orientation of the DNA molecule, given by the
tangential direction, it has to depend on the changes of the tangential direction. Assume DNA is a curve in
space:
Frame of reference:
Since the di are unit vectors they can only rotate in
space as one moves along the molecule. This is what the
above equation says. Darboux vector.
We assume that this frame of reference is baked into the material of the molecule and deforms with it.

Elastic deformation (free) energy of DNA
Jean-Baptiste Biot, who assisted Laplace in revising Mchanique cleste for the press, says that Pierre-
Simon de Laplace himself was frequently unable to recover the details in the chain of reasoning, and, if
satised that the conclusions were correct, he was content to insert the constantly recurring formula, "Il
est ais voir que ... ("It is easy to see that...").
"It is easy to see that...":
The equations of elastic equilibrium are then equivalent to Eulers spinning top equations:
where #2,1 is the local curvature in the direction of d2 and d1.
This is referred to as the Euler-Kirchhoff kinematic analogy!

Euler-Kirchhoff kinematic analogy
Euler-Kirchhoff kinematic analogy in action (Nizette and Goriely, 2002):
HELIX HELIX

Elastic instabilities: buckling and torsional
Many solutions exist and Euler himself derived them for a planar case:
HELIX
Studied in detail by Max Born in his
PhD thesis!
Wehrner Heisenberg did his PhD thesis
on turbulence! (nearly unked)

Twisting and buckling of DNA
HELIX
Twisting DNA From 2009 APS March Meeting Paper.Twisting DNA coils around itself just like a hose or a
rubber band. Abstract: W40.00014 Image Credit: Bryan Daniels, Cornell University

The DNA plectoneme formation
Many solutions exist and Euler himself derived them for a planar case:
For 3D case - Plectonemic solutions of a twisted rod with no self-contact. The gure illustrates how
plectonemic solutions are constructed by combining non-planar localizing solutions (such as the Coyne
solution) with the constant curvature helical solutions. Plectoneme formation in twisted uctuating rods,
Prashant K. Purohit. (from Greek %&'( - thread, )*&#+o, - twisted.)
HELIX
Planar solutions do not always have the lowest
elastic energy.
At a critical value of the torque:
the solution escapes into the 3rd dimension.

Measuring DNA plectoneme formation
Magnetic tweezers used to stretch and turn DNA (N. Dekker lab graphics):
Measure the extension and the torque of the DNA molecule and infer the formation of a plectoneme.
When n = nb turns have been applied to the tube, a buckling transition allows the system to relax its
torsional constraint by forming a loop, causing the system to contract. As one continues to twist the tube
its extension decreases linearly as the number of loops grows regularly. When the critical torque is reached,
the formation of a plectoneme relaxes the torsional constraint and prevents the torque from increasing.
Each additional turn added to the system further lengthens the plectonemes, preventing the torque from
increasing.

Elastic buckling instability in DNA: Manning buckling
Manning (Cell Biophys, 7, 57-89 (1985): DNA condensation is Euler buckling due to self-
interactions.
Euler buckling instability: press on an elastic lament hard enough and it buckles.
Manning buckling instability: turn on attractions along the lament and it buckles too!
The role of compressional force is played by diminished (on addition of polyvalent counterions)
electrostatic interactions. No correlation effect at that time.
Becasue at that time it was thought that the
interactions along (or between) DNA moelcules can
only be repulsive, some mental gymnastics was
needed to get the instability. But the idea itself is
OK.

The Manning buckling
Assume instead that the interactions along DNA can become attractive. We will see later on how
this is possible (there are clear experimental indications in the Rau-Parsegian experiment).
Instead of pressing on the DNA rod (we ignore all thermal effects) and thus compressing it, we
simply turn on the attractive interactions along the backbone.
Basic equation:
V(r-r)
This is a mean-eld like calculation and interaction potential is attractive. No thermal uctuations.
Podgornik et al. (1999).

Elasticity of DNA so far
We now put together the elastic energy and the denition of the Darboux vector:
...obtaining:
with
This holds true for an ideally unstretchable chain. For a realistic chain however, it is always more or
less stretchable:
bending rigidity
stretching modulus
Rudi Podgornik, Per Lyngs Hansen, and V. Adrian Parsegian, Elastic moduli renormalization in self-
interacting stretchable polyelectrolytes, JCP 113 22 (2000).
K
C
=
1
4
! R
2 (Bending is just local stretching.
Landau and Lifshitz, 1995.)

Measuring the elastic moduli of DNA
Lowering the ionic strength increases the measured persistence length, but seems to reduce DNAs
elastic stretch modulus, contradicting the elastic rod model. Bustamante et al. (2000).
(Baumann, Smith, Bloomeld, Bustamante 1997)
Since variations in ionic
strength are involved, we
assume that the foul play is
due to electrostatics.
Elastic moduli can be measured in various ways. Here they stretch the DNA chain by optical tweezers:
What are the relevant
interactions?

Electrostatic Interactions along DNA
DNA contains a lot of structural charges due to the dissociated phosphates along the backbone.
One of the most charged molecules in nature. This has many important consequences.
it has lots of discrete structural (phosphate) charges (pH > 1)
it has lots of room to accommodate small counterions
helix
discrete charges
grooves
Charge:
2 e
0
/ 3.4
~ e
0
/ nm
2
Polipeptides: 0.6 e
o
/ nm Membranes: 0.1 - 1 e
0
/ nm
2


Electrostatic interactions and DNA elasticity
Elastic energy is not everything, we also have interactions along the DNA chain:
Interactions mediated by salt
as well as polyvalent counterions.
We do not know the explicit form of the total interaction potential when the bathing solution contains uni-
univalent salt (weak coupling) as well as polyvalent counterions (strong coupling). Th. Odijk (1977).
J. Skolnick and M. Fixman (1977)
This is the famous Debye-
Huckel interaction potential
that we will derive later.

Electrostatic renormalization of elastic moduli
L
L
a
a
What do electrostatic interactions do to the elastic moduli of DNA?
It is because of the interactions that:
In fact...
The rst part is the Odijk-Skolnick-Fixman result and the other one is ours. Elastic moduli depend on the
magnitude of the electrostatic interactions in a big way!

Quantitatively:
Wenner, Williams, Rouzina and Bloomeld (2002).
For ionic strengths: 1000, 500, 250, 100, 53.3, 25, 10, 2.6 mM.

a = 6.7 0.7 (Manning a = 7.2 )
L
P
~ 48 nm ~ 1200 pN
A constrained t : L
0
, K
c
, !(K
c
)
Mg
2+
and Co (NH
3
)
6
3+
yield lower persistence length then polyamines spermidine
3+
and putrescine
2+
.
What is it about the polyvalent cations? What are in fact the measured interactions?

FINIS

counterions),
DNA array),

LECTURE 3
DNA MESOPHASES (DNA and the phenomenology of DNA
mesophases, salient features of DNA mesophases, unusual
features of DNA mesophases, the screw-like order and
molecular pitch, DNA-DNA azimuthal correlations, angular
frustrations and the orthorhombic phase, organization of
eucaryotic genome, nucleosomes and nucleosomal core
particles (NCP), NCP mesophases, Equation of state of a DNA
array, Fluctuations and positional order in a DNA array)

Matter in Nature is (mostly?) ordered

Ideally ordered ... but for entropy
Molecular chaos. Molecular order.
Entropy Molecular interactions

The double helix structure of DNA
Difractogram 51
(Franklin and Gosling, 1952).
Nature, april 25, 1953.
Based on the CCV theory of diffraction by helical molecules and X-ray diffraction experiment.
(Rupprecht and RP, 1994)

it is a RH double helix
it has lots of discrete structural (phosphate) charges (pH > 1)
it has lots of room to accommodate small counterions
helix
discrete charges
grooves
Charge:
2 e
0
/ 3.4
~ e
0
/ nm
2
Polipeptides: 0.6 e
o
/ nm
Membranes: 0.1 - 1 e
0
/ nm
2

Structure
(B-form double-helix)
a ~ 1 nm
h ~ 0.17 nm
DNA pitch p ~ 3.4 nm
DNA length L ~ 50 nm to ~ 'm
DNA persistence length L ~ 50 nm
DNA in Nature: ordered or disordered?
p

Discovery of DNA mesophases
Livolant et al. 1997.
line hexatic phase
1997
cholesteric phase
1961
Conmar Robinson in 1958 described
the cholesteric phase of poly(benzyl-
L-glutamate) in dimethylformamide
and in 1961 of DNA.
indications of ordered phases of DNA
in SAXS (small angle X-ray scattering)
phases with short fragment DNA (146
bp about 50 nm)
R. Podgornik et al. (1997) discover
the line hexatic phase.

And in vivo (in viro)?
(R. Cavenoff (1995)) (Kleinschmidt et al. (1962))
E.Coli T2
DNA in organisms is always tightly packed. We therefore need to study large densities of DNA.

The relevance of high density mesophases
.. organisms exposed to prolonged stress can
circumvent their fate through formation of
highly ordered DNA assemblies... Minsky,
Nature 2002.
Cryo-electron microscopy of -15. The
genome is (hexagonally) packed in coaxial
coils in at least three outer layers. Jiang et
al. 2006.
D. Nelson, Nature (1995)
Type II SC:
Tension
Non-chiral
Magnetic eld
Temperature
London repulsion
DNA:
Bending
Chiral
Density
Ionic strength
Debye-Huckel repulsion

Methods of preparation
Osmotic
stress
(membrane)
condensing
agents
Osmotic stress
(PEG...PSI DNA)
Controlled
drying
Condensation
(polyamines...)
Dense ordered phases of DNA can be
prepared by a variety of methods used in
different experimental setups.
While there are no qualitative differences between the nature of the phases and their boundaries,
they have not been studied systematically.
osmoticant
The question is how to control and change the DNA density in solution.

How do we make dense phases of DNA?
(1)
The Boyle experiment
Setting the osmotic
pressure and measuring
the density of DNA.
DNA equation of state!
Differrent ionic
conditions, different
DNA length, type of
ions.....
Osmotic stress method to investigate the equation of state as well as the order of the macromolecular
solutions (Parsegian & Rand)
DNA spherulite
PEG
Polyethylene glycol

Changing density: Boyle experiment - osmotic stress method
Osmotic stress method (Parsegian & Rand, 80)
pressure
osmotic pressure
Mechanical work:
pressing the
macromolecules together
with forces.
Chemical work:
displacing the solvent
molecules from between
the macromolecules
How to change the density? Either push the molecules together, or suck the solvent from the
space between the molecules. Equivalent!
" dV
#dN
PEG

Why does DNA make ordered phases?
S. Fraden, Puried TMV in aqueous buffer. Top
phase is isotropic, bottom is nematic.
Onsager (1949) excluded volume interactions.
Length to thickness ratio.
Khokhlov-Semenov et al. 1981
No order in exible polymer solutions.
For stiff polymers the role of the length is
played by the persistence length.
ds-DNA persistence length (0.2 M NaCl) 50nm
E~300 MPa (plexiglass)
(C. Holm and M. Deserno)
HA or ss-DNA

How do we make dense phases of DNA?
(2)
Condense DNA by ading
certain condensing
agents to the DNA
solution.
Mostly polyvalent
cations that modify the
interactions between
DNAs.
DNA condensation
Hud & Downing (2001)
Cobalt hexamine: [Co(NH3)6]
3+
Polyamines: spermidine
3+


Durand, Doucet, Livolant (1992) J. Physique 2, 1769.
Pelta, Durand, Doucet, Livolant (1996) Biophys. J., 71, 48
RP et al. COSB. 8 (1998) 309. COCIS. 3 (1998) 534.
160 380 440 670 1055 mg/ml
DNA mesophase zoo
15
blue phases
cholesteric
hexatic - deformed hexatic
columnar hexagonal - hexagonal
orthorhombic
The exact positions of the phase boundaries do depend on the method of preparation (osmotic
stress, controlled drying, condensation ) and the length of the DNA.
50 nm fragment (F. Livolant)
Mesophases at different DNA density.

Cholesteric phase
C. J. Barrett Durand, Doucet, Livolant (1992) J. Physique 2, 1769.
160 - 380 mg/ml
Cholesteric pitch:
(Stanley et al. BJ 89 (2005))
The pitch of the cholesteric phase ~ m.
(cholesteric droplets)

Hexatic - deformed hexatic
380 - 440 mg/ml
(Predicted by Toner, 1983 observed by RP et al. 1996.)
Long range BO order ~ 0.6 mm
Long range nematic order
Liquid like positional order, *
PO
No twisting of the hexatic order
(cholesteric-hexatic coupling)
A most mysterious phase!
Kamien and Levine, PRL (2000).

Columnar hexagonal - hexagonal
440 - 670 mg/ml
A 2D crystal. Scattering from a columnar hexagonal phase reveals hexagonal order in the
equatorial reections. Hexagonal phase is a real 3D crystal, order in all directions.
Relative displacements of 1/6 DNA pitch along z-axis. Livolant et al., Nature (1989).
2D 3D
Changes in DNA pitch as a function of density.
Livolant and Leforestier, Prog. Pol. Sci. (1996).
This is the range of densities of the
Franklin-Gosling experiment.

Orthorhombic
670 ! mg/ml
Orthorhombic phase is also a real 3D crystal. A distorted hexagonal lattice.
Relative displacement of m1 and m2 is ~ 1/3 DNA pitch along z-axis.
A (volume) discontinuous transition from the hexagonal to orthorhombic.
Here too the pitch of the molecule changes.
The orthorhombic lattice parameters also
change as a function of the DNA density.
Durand, Doucet, Livolant, J. Phys. France II
(1992).

DNA - a helical molecule
The CCV theory (1952).
Three possible types of uctuations in a
nematic composed of helical molecules.
Description of the order in a parent nematic phase of DNA molecules.
translation
rotation
screw-like
motion
The phase . is a
characteristic of each
molecule.
%
i
%
j
de Gennes macroscopic order parameter.
P=0 parent nematic phase
P=1 corresponds to a screw-like phase
Initial position and
initial phase.
(Manna, Lorman, RP and Zeks, 2007)

Modulation of the molecular pitch
Pitch of the B-DNA = 3.4 nm.
Renormalization of the phase of the order
parameter of drives the renormalization of
molecular pitch.
Of course if DNA would not be soft an enormous energy would be needed to accomplish this.
Taking the experimental data of F. Livolant one obtains the
torque needed to get these changes in pitch of
The measured energies of DNA twisting and bending are ~ 10-100 pN nm and the
measured overwinding torques are indeed of this magnitude (30 pN nm).
DNA is fortunately soft enough.
The chiral term introduces a renormalization of the phase of the order parameter.
Since it describes the helical molecule this means that its pitch is changed! Instead of twisting
the hexatic order, the screw phase overwinds the molecule. The solution of the puzzle.

A side benet of the screw-like phase
Experimentally seen displacement in the z-direction for 1/3 or 1/6 of the pitch.
There exists a coupling between the order parameter of the
screw-like order and the symmetry of the lattice.
Too technical to derive here, but displacements of both p/6 and p/3 can be derived for
hexagonal and orthorhombic symmetries. Exactly as seen in experiments!
Hexagonal Orthorhombic

Direct visualization of the screw-like phase
Ordered phases of bacterial agella (Barry et al. PRL 2006).
Three pitches: zero, 1.1 'm and 3.6 'm.
Corresponding order under Xed polarizers and uorescent
labeling.
- periodicity
- direction of molecular axis
- nature of uctuations
- modulation of the density
For DNA (because of the
difference of scale) this
experiment is not feasible.
Resonant X-ray scattering?
Screw-like uctuations observed directly.

Why the orthorhombic symmetry?
If DNAs were cylinders, they would pack hexagonally as do all cylindrical objects at high density.
IP = 78.5398163% IP = 90.6899682%
Also: why are there relative displacements in the z direction of nearest neighbors in hexagonal
and orthorhombic phases?
DNAs do not behave like featureless cylinders.
The orthorhombic packing of DNA at high densities is very unusual.

Interactions between helices can not be isotropic
Realistic geometric models of DNA with explicit positions of the charges on the phospahates.
explicit DNA structure
explicit counterions
explicit salt ions
different salt concentrations
Schematics of the
orientational effect. Strand
opposition.
A A R R R
Allahyarov et al., 2000 - 2004

A two-state interaction model
Critical distance R
0
; for R > R
0
(large interaxial distances): / = 0 (parallel orientation)
for R < R
0
(small interaxial distances) / 0 0 (equilibrium nonzero angle orientation)
/ mutual
azimuthal
orientation of the
DNAs
The interaction potential between two helices is not a monotonic function of separation any
more.
A two state model:
I. Intermediate densities : Parallel alignment of transverse polarization vectors
II. High densities : Preferred angle between transverse polarization vectors
The vectors joining axes with the points where 5- 3 strand hits the
plane are conventionally called transverse polarization vectors p

Azimutal interactions in constrained DNA nematics
Assume a DNA ber where molecules are not allowed to uctuate translationally.
Only azimutal
uctuations are
allowed.
A proper structural description of DNA on this level is:
We call this vectorial property the transverze polarization (TP) vector.
Lorman, RP and Zeks, PRL 2005.
The origin of the azimutal
angle can be chosen
arbitrarily, as the
interaction depends only
on the difference!

Intermediate density hexatic - TP coupling $ = 0
A. deformed hexatic in the direction of rst neighbors (excluding the central) ' = 0
B. deformed hexatic in the direction of second neighbors (excluding the central) ' = )/6
C. deformed hexatic in general direction 0 < ' < )/6.
We know very little about the angular dependence of the interactions between
DNA helices. The idea here would be that we assume some form and see what the
consequences would be. Then we modify or strengthen the assumptions.
First assume that the TP vectors wnat to be colinear.

High density hexagonal - TP coupling $ ! 0
Again line hexatic phase as the parent phase. But a bit more complicated.
The nearest neighbor TP vector are at a nite angle!
This is possible only in periodic systems in terms of TP. Crystalline order is thus a
consequence!
Start with e.g. A. structure
deformed hexatic
a. one dimensional crystal in e.g. x-direction, one angle of reorientation
b. one dimensional crystal in general direction, one angle of reorientation
c. two dimensional crystal in, two angles of reorientation
Freezing and crystallization of the bond orientational order at higher densities.
Angular correlations.
Azimutal frustrations of the
lattice.
Observed already by Franklin in 1952, but not understood until now!

Microscopic calculations
Starting point is the microscopic Kornyshev - Leikin potential with an explicit
angular dependence. Only crystalline and liquid phases are considered.
Phase diagram is obtained for equilibria between different crystalline and 2D uid phases
As a function of monovalent salt concentration.
(Harreis,Kornyshev, Likos, Loewen, Sutmann 2002)
Lattice symmetry is taken a priori.

Is this real?
Can one observe azimutal correlations predicted on the theory that is based on the two-
state KL model?
Kornyshev and Leikin (PRL, 2005) reanalyzed the old diffraction data by S.B. Zimmerman.
From almost contact to 40 separation.
- equatorial n=0 peaks change
with DNA density, positional
order
- for small inter-chain
correlations n 0 0 peaks should
show no density variation
Equatorial lines remain the same - B-DNA for all densities.
Strong azimutal
correlations
of the type A, 2a or 2c.

Chromatin
Of course this is the physicists very idealized conception of chromatin in eukaryots.
Nucleosomes: elementary particles of chromatin.
Sort of.
DNA condensed by histone proteins.

Nucleosomal core particles
The structural cascade of chromatin in eukaryots starts with the nucleosomal particle.
From DNA to chromosomes. Many levels of as yet poorly understood organization but with
interesting physics (Schiessel, 2002).
Viruses: toroidal packing just as in DNA collapse in vitro.
Bacteria (prokariotes): nucleoid, a loose DNA - protein gel
A histone octamer (4 X 2) of 4 core histones:
H2A, H2B, H3 and H4
147 bp DNA wrapped 1.75 times in a left-handed
helix, stable up to 0.75 M salt.
Existence of a polar dyadic axis.

The nucleosomal core particle (NCP)
The elementary particle of chromatin organization. Missing just linker DNA and H1 histone.
Expanded N-tails Folded N-tails
Folded vs. expanded N-tails
(N-terminals of histone core proteins)
Folded tails below 50 mM
and expanded from 50
mM and above.
(Mangenot et al. 2002)
Bridging interactions can lead to association of nominally equally charged colloids.
Important also at large densities of NCPs where they make condensed mesophases...
Muehlbacher, Schiessel and Holm, (2005).
RP and Licer (2006)

Intriguing texture and
order of
mesophases...
NCP mesophases in ionic solutions
S. Mangenot, A. Leforestier, D. Durand, and F. Livolant (2003-2005).
X-ray scattering plus optical texture serve to identify the phases. They come in very exotic
varieties....Superb work of F. Livolant et al.
inverted hexagonal phase
orthorhombic phase
lamellar phase

More NCP mesophases in ionic solutions
S. Mangenot, A. Leforestier, D. Durand, and F. Livolant (2003-2005).
X-ray scattering plus optical texture serve to identify the phases. They come in different
varieties: isotropic, lamellar, hexagonal, inverted hexagonal.

NCP mesophase phase diagram
Changing the osmotic pressure (i.e. density) and ionic strength of the solution.
Here the parent phase is assumed
to be columnar hexagonal.
Constructing the order parameter
based on the existence of dyadic axis:
And the density variation:
Three order parameters out of polarization vector:

Writing down the free energy from invariants of the three order parameters
that are dened via transverse polarization vector.
lamellar phase
inverted hexagonal phase
orthorhombic
phase
Free energy minimization.
(Manna, RP, Lorman and Zeks,
2007)
Lamellar phase of NCPs
Starting from a parent hexagonal phase transitions into several other phases of lower
symmetry are possible, e.g. a lamellar phase (very technical).
Duality between the P ordering and the u displacement (plus chirality).
Curies principle.

Inverted hexagonal and orthorhombic lattice
These last three phases have
not been observed (yet).
Lorman, RP, Zeks EPL (2005).
lamellar phase inverted hexagonal orthorhombic phase
Apart from the lamellar phase other phases and transitions between them are possible.
Phase diagram.
Nature of the 2D molecular displacement.

From observing to understanding DNA mesophases
Not that difficult to observe, but not simple to understand.
line hexatic phase
cholesteric phase
Many details still need to be worked out, but the big picture is most probably correct.

FINIS

counterions),
DNA array),

LECTURE 4
DNA MESOPHASES (some additional observations on the
nature of sf DNA mesophases, the smectic controversy, the
effects of stacking interactions on the nature of mesopahses)
STATISTICAL MECHANICS OF DNA (The Kratky-Porod
model, Light scattering from a Kratky-Porod filament in
solution, Elastic response of a Kratky-Porod filament, The limit
of small stretching force, The limit of large stretching force,
Extensible semiflexible chain, An approximate elastic equation
of state for DNA)

Liquid crystalline mesophases of DNA in solution
A zoo of mesophases is observed: (chiral) nematic, line hexatic, 2D hexagonal, 3D
hexagonal, orthorhombic. But differences for long and short DNA.
Ordinary mesophases of mesogenic molecules - liquid crystals:

Phase diagram of liquid crystals
Usually one has a smectic phase between the nematic and the crystalline phase.
In my previous talk I said nothing about the smectic DNA phases. Forgot? Not really,
because they are not there. Nice story, though.

Long and/or short DNA?
Short DNA wrapped around histone
octamer of the nucleosome core:
147 bp DNA wrapped 1.75 times in a
left-handed helix, stable up to 0.75
M salt. (S. Kruger)
Long genomic DNA obtained from CB
or salmon testes, protein free.
Can be up to 1 m long and
creates extremely viscoelastic
solutions. (D. Rau)
The beauty and advantage of DNA is that one can prepare absolutely monodisperse
solutions of sfDNA, where all the molecules are 146 bp long. This turns out to coincide
exactly wit the persistence length of DNA.
There are slight variations in the nature of sDNA/lDNA mesophases,
but poorly explored!

sf DNA mesophases (1)
Nucleosomal DNA is ideally monodisperse and 146 bp (~50 nm) long. This short fragment
DNA makes several mesophases identied by Strzelecka et al. Nature (1988). On the cover!
cholesteric-precholesteric cholesteric-smectic precholesteric
cholesteric
(170-220 mg/ml)
smectic
(220-275 mg/ml)
smectic
(275-309 mg/ml)

sf DNA mesophases (2)
Same density range studied later by Livolant et al. Nature (1989). Similar methods
(polarization microscopy and cryomicroscopy) but also with X-ray scattering.
Obviously a B-DNA pattern (we know this already), but also with a strong meridional arc (red
arrow) which revelas a hexagonal order with interhelical spacing 2.97 nm (380 mg/ml). More
(hexagonal) diffraction orders seen at even higher concentrations.
The nature of the phase is hexagonal columnar with no (smectic) order in the direction of the
long axis of the molecules but with pronounced (hexagonal) order in the plabe perpendicular to
their axis.

Even shorter DNA (sDNA) mesophases?
"END-TO-END ADHESION AND LIQUID CRYSTAL CONDENSATION OF 6-20 BASE PAIR DNA
DUPLEXES," M. Nakata, G. Zanchetta, B.D. Chapman, C.D. Jones, J.O. Cross, R. Pindak, T. Bellini,
and N.A. Clark, Science 318, 1276-1279 (2007).
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G.Zanchetta, Ph.D. thesis (Uni Milan, 2008) and Michi Nakata.
Packing and stacking of sDNA

sDNA phases
sDNA, N = 20, 16, 14, 10, 8, 6 bp.
In spite of the challenges presented by the extremely small sDNA sample quantities
available, these techniques pro- vided unambiguous evidence for chiral nematic (N) and
uniaxial columnar (CU ) liquid crystal phases in the sDNA solutions. At higher
concentration, more ordered columnar (C2) and crystal-like (X) phases were also found.
G.Zanchetta, Ph.D. thesis (Uni Milan, 2008).

Experimental c (DNA concentration) - N (oligomer length) phase behavior for sDNA and lDNA,
along with the theoretical behavior. (A) The solid red triangles and solid red curve bound the
measured I-N phase coexistence for lDNA (N ~ 100). The solid red circles and red dotted line give
the measured N !CU phase boundary of lDNA. For N < 20, phase transitions from our data are
marked by red open symbols and the range of each phase indicated by colored columns.
Theoretical phase boundaries for these transitions with DNA effective diameter D = 4.0 nm
(heavy black lines/labels) and with DNA chemical diameter D = 2.4 nm (heavy orange lines/
labels). OBF I-N line is shown in black and TBB N-CU line in dashed black.
sDNA phases: theory and experiment

Onsagers theory
Here c fa d" the number of rods per unit volume pointing in a small solid angle d"
around a direction labelled with the unit vector a.
The basic model is Onsagers treatment of monodisperse repulsive hard rods (length L, diameter
D), which, if they are sufficiently anisotropic in shape, nematic order for volume fraction # > #IN =
4D/L $24/N (D!2nm,L!N/3nm for B-DNA). The complete computer simulated phase diagram for
hard rods by Bolhuis and Frenkel quantitatively confirms this prediction for L/D > 4.7 (N ~ 28 bp),
and also shows that for L/D < 4.7 there should be no LC phases at any #.

Kratky-Porod chain
DNA usually lives at or around the room temperature and is a quintessential soft object. If we look
at the elastic free energy of an e.g. teardrop configuration, we see that for sufficiently long DNAs
it becomes comparable and eventually smaller then the thermal energy.
For a torsionally relaxed filament, viz. a filament whose elastic energy does not depend on the
torsional deformation, with symmetric cross section we just found out that the elastic energy is
given as

Statistical properties of the chain
Start with the following definition of the separation between beginning and end of the chain:
What we want to know is:
average size of the chain
orientational persistence of the chain
orientational correlations along the chain

The three statistical properties are interrelated as one can show by writing explicitly :
We need this.

Two limits:
Flexible filament with short range orientational correlations:
Stiff filament with long range orientational correlations:
(long range)
(short range)

The Kratky-Porod chain interpolates between both limits:
Exponential orientational correlation functions:
Orientational persistence of the chain:
The average size of the chain:
A relatively simple model that allows for some cool results. For short chains stiff rod and for long
chains flexible fillament. Depends on the value of the persistence length.

How big is the persistence length of DNA?
O. Kratky, G. Porod (1949), "Rntgenuntersuchung gelster Fadenmolekle." Rec. Trav. Chim.
Pays-Bas. 68: 1106-1123.

Light scattering from DNA solutions
Similar to X-ray scattering but on a differen length scale. X-rays % = 0.154 nm whereas
light is between 400 and 700 nm. A difference in factor of 3500!
We remember the scattering intensity and the form factor of the molecule:
We will now use what we know about the statistical behavior of DNA to evaluate the scattering
intensity. Stiff chain vs. flexible chain.
Stiff chain Flexible chain

How to calculate the scattering intensity? Gaussian limit
For a Gaussian distribution:
The scattering intensity is then given by the Debye function:
... and the average size of the chain is then:
This is of course an approximation valid for a Gaussian chain, and a Gaussian chain
is a chain with short range correlations:
"

How to calculate the scattering intensity? Rod limit
In the rod limit we have:
...and the average size of the chain is then:
This is of course an approximation valid for a rod-like chain, and a rod-like chain
is a chain with long range correlations:
"

The Peterlin approximation
He assumed that he can use:
This result interpolates between the two limits: Gaussian and rod-like.
"
with
"

Persistence length from light scattering of DNA solutions
A. Peterlin, Nature 171, 259 - 260 (1953). Peterlin, A. , J. Chem. Phys., 47, 669 (1950); 48, 13
(1951); J. Polymer Sci., 8, 173 (1952).

Stretching single molecules
A single DNA molecule can be probed with small forces. Force versus extension data (red crosses) for *
phage dsDNA (48,502 bp) pulled by magnetic beads in 10 mM Na+ buffer (Smith, Finzi, Bustamante:
Science 1992, 258:1122).
Ten years of tension: single-molecule DNA mechanics, Carlos Bustamante, Zev Bryant & Steven B.
Smith, NATURE 421 (2003).
1 kBT = 4 10
21
J = 4 pN nm

http://phet.colorado.edu/en/simulation/stretching-dna
Stretching experiment@home
As an assignment everybody should play with this java applet !
a
s
s
i
g
n
m
e
n
t

(
2
)

Stretching ss-DNA and ds-DNA
Single stranded DNA
Double stranded DNA
So what do we see in a typical experiment? It all depends ...
In order to understand anything we need to compare with theory, see where it ts,see where it
deviates and then try to gure out why.

Twisting single molecules
A single DNA molecule overwound and under a constant stretching force. The twist is converted into a
plectoneme with writhe but the length of DNA decreases. Under sufcient torsional strain, a twisted
DNA molecule buckles to form plectonemes, shortening the measured extension. b, Extension as a
function of turns introduced into the molecule remains nearly constant until the buckling transition is
reached (1), after which the molecule contracts linearly (2). Underwinding at 1 pN does not cause
reduced extension because the strained molecule forms alternative underwound structures (for
example, melted DNA) in preference to buckling.

Single chain (Baumann, Smith, Bloomeld, Bustamante 1997).
To extract elastic properties must be tted to theory.
Elastic equation of state

Elastic equation of state in the Kratky-Porod model
We now use the Kratky-Porod model to evaluate the elastic equation of state with thermal
uctuations for a single elastic lament. The model is:
The equation of state is formally obtained from:
Various limits of this expression can be considered depending on the magnitude of the stretching force:
(A) Small f:
(B) Large f:
(C) Large f:
The Marko-Siggia interpolation formula.
(J. F. Marko and E. D. Siggia, Stretching DNA,
Macromolecules 28 8759 (1995).)
Fitting the experiment to the theory allows to estimate the length and the elastic moduli of DNA.

Approximate Forms of the Elastic equation of state for ds-DNA
Elasticity of DNA without twist constraint. (A) For forces less than kBT/Lp ~0.1 pN, random-walk
uctuations are being suppressed; (B) for forces between 0.1 and 10 pN the small curvature
uctuations reduce the length of the DNA by a fraction ~ (kBT/A f )1/2; (C) for forces between 10
and 60 pN, simple linear elasticity of DNA is observed, as the double helix starts to be lengthened;
(D) at about 65 pN, a force plateau is observed, corresponding to coexisting regions of normal
and overstretched DNA; (E) nally, above 70 pN, DNA is entirely converted to its overstretched form.
DNA under high tension: Overstretching, undertwisting, and relaxation dynamics
John F. Marko PRE (1998).

Elastic equation of state for ss-DNA
The problem here is that ss-DNA can self-pair up and thus becomes locally a ds-DNA
complicating the nature of the elastic response. One can not assume a priori that:
Overstretching B-DNA: The Elastic Response of Individual ds and ss DNA Molecules,
Smith et al.: Science 271 795 (1996).
different ss-DNA
ds-DNA
ds-ss-DNA
ss-DNA
Stretching, electrostatic, pairing and stacking energies (Y. Zang et al. BJ (2001)). Different salts and
different base sequences - different equations of state.

DNA persistence length
Apparent persistence length of DNA from the stretching experiments. Should be a constant...
Remember light scattering experiments from 1953:
(not bad!)
E~300 Mpa (plexiglass)
CNT 10
8
#tubules 0.1 M NaCl 10
7
TMV 0.1 M NaCl 10
6

F-actin 0.1 M NaCl 10000
Schizophyllan water 200
Xanthan 0.1 M NaCl 120
ds-DNA 0.2 M NaCl 50
Spectrin 0.1 M NaCl 15
ss-DNA 0.2 M NaCl 3
Hyaluronic acid 0.2 M NaCl 1
Long Alkanes 0.5
DNA is neither incredibly stiff nor is it totally oppy. Somwhere in between. The golden mean.

Overstretching transition of DNA
Force-extension curves for single molecules of dsDNA (red triangles) and ssDNA (right black line). A
theoretical curve for dsDNA is shown as the left black line. The overstretching transition appears to
be a transition from dsDNA to ssDNA.
If one end of the DNA molecule is allowed to rotate freely, at about 65 pN, a cooperative overstretching
transition occurs, stretching the molecule to 1.7 times its contour length (Cluzel et al. 1996; Smith et
al. 1996). Model of overstretched DNA as a new double-stranded form of DNA, referred to as S-DNA,
was proposed (Cluzel et al. 1996). While these models did predict an overstretching transition, the
predicted transition was less cooperative and at a higher force than that observed experimentally
(Konrad and Bolonick 1996; Lebrun and Lavery 1996; Olson and Zhurkin 2000). Rouzina and Bloomeld
(Rouzina and Bloomeld 2001a; Rouzina and Bloomeld 2001b) have proposed an alternative model for
DNA overstretching as a force-induced melting process.

Overstretching DNA into P-DNA or s-DNA
For positively super- coiled DNA (1 > +0.037) stretched by 3 pN, we observe a similar coexistence of
B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that
this structure has 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has
tightly interwound phosphate backbones and exposed bases in common with Paulings early DNA
structure. For untwisted DNA s-DNA as a new form of stretched DNA.
ALLEMAND et al, Proc. Natl. Acad. Sci. USA (1998).
Cluzel et al. Science (1998). Force: a new structural control parameter?
David Bensimon, Structure (1996) 4:885.

Overstretching and melting of DNA
The connection between the two stresses by Rouzina nad Bloomeld. Slat dependence very
indicative. DNA Overstretching Transition: Ionic Strength Effects
Olli Punkkinen, Per Lyngs Hansen, Ling Miao, and Ilpo Vattulainen, BJ 89 (2005) 967978.
Fluorescence data directly demonstrate that overstretching comprises a gradual conversion from
double-stranded to single- stranded DNA, irrespective of attachment geometry. These conversions
initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as
energetically favorable nucleation points for melting. van Mameren et al. PNAS (2009) 106 18231.

Persistence length of DNA with polyvalents
Experimental points for Lp for solutions with the following multivalent cations: E, CoHex3+ (Baumann
et al., 1997; Baumann and Bloomeld, unpublished); F, CoHex3+ (Porschke, 1986); 2, Mg2+
(Porschke, 1986); #, spermidine3+ (Baumann et al., 1997); , spermine4+ (Porschke, 1986).
Mg2+ and Co (NH3)63+ yield lower persistence length then polyamines spermidine3+ and
putrescine2+. 2+ do not condense DNA!
Repulsive interactions increase the persistence length and attractive interactions decrease it.
Renormalization of the bending rigidity. A full non-linear theory (Hansen et al. 1999)
collapse
?

Experiments by Brian Todd on
DNA stretching in the presence of polyvalent counterions.
A single strand of DNA is stretched between an immobilized bead and a bead pulled to the right.
Bathed in a solution containing the condensing agent, the DNA is initially stretched by a relatively
large force. The force prior to condensation is well characterized by the KP model (dashed line). At
the condensation force the bead-bead separation abruptly decreases. Over the period of a couple
minutes, condensation of DNA completes, bringing the magnetic beads into contact.
Cobalt hexamine: [Co(NH3)6]
3+
DNA condensation with magnetic tweezers

Reentrant DNA condensation
Brian A. Todd and Donald C. Rau, Interplay of ion binding and attraction in DNA condensed by
multivalent cations, Nucleic Acids Research 36 501(2008).
Neither overcharging (Besteman et al. 2007) nor signicant alterations in the condensed state are
required to describe the resolubilization of condensed DNA. Indirect measurement of interactions?

What are the interactions between DNA molecules?
In various congurations differen segments of DNA come close together and we need to know
what are the interactions that they feel at these separations.
How are we to measure these interactions and how are we to understand them?

FINIS

counterions),
DNA array),

LECTURE 5
DNA -DNA INTERACTIONS (Osmotic stress method,
Hydration force, Electrostatic interactions, Salt screening,
Poisson-Boltzmann theory, Counterion distribution, Manning
condensation, Strong coupling theory, Correlation attraction,,
Force equilibria with polyvalent counterions)

Measuring DNA-DNA interactions
0.34 nm /bp
a ~ 1 nm
DNA length from 50 nm to ~ m
Structure (B-form)
" dV # dN = 0 (mechanical energy= osmotic energy)
Osmotic stress method (Parsegian & Rand, 70).
This method allows the direct measurement of
osmotic pressure. And from osmotic pressure
one can deconvolute the interactions....
The Boyle experiment with real gases. Could one do a Boyle experiment with non-ideal solutions?

Osmotic stress method
LeNeveu, Rand, Parsegian (1976)Nature 259,601.
Equivalence of osmotic pressure of the DNA subphase and the osmoticant subphase (PEG
300-20000, DEX 110000 etc.) C
DNA
from abs at 260 nm, gravimetry, X-ray scattering....
Osmoti cpressure of osmoticnt in solution, osmotic pressure of DNA solution. In chemical
equilibrium they must be the same (exchangeable water). Changing one changes the other. What
we really have here is an osmotic balance.
The result is osmotic pressure of DNA vs. DNA density in a very wide range of densities.
DNA equation of state 3 = 3(CDNA)
Very different types of dependence signaling very different physics. The smaller the density the
less details we need to know about DNA charge distribution. DNA becomes a charged cylinder.

DNA mesophases and interactions
A B
L
L
L
P
L
!
!
"
c
!
A B
x-ray beam
(Livolant, Leforestier, Rill, Robinson, Strzelecka, Podgornik, Strey )

DNA equation of state
(Rau, Parsegian, Podgornik, Strey, Lindsay, Raspaud, Livolant )

Regimes of the DNA equation of state
High pressure - high density:
The interaction is dominated by a strong decaying force independent of the salt
concentration, but dependent on the salt type. The decay length is on the order of the H2O
molecule size.
Intermediate pressure - intermediate density:
The interaction is dominated by a salt concentration dependent force, also dependent on
the salt type. The decay length is a multiple of the Debye length.
Small pressure - small density:
The interaction is dominated by the free counterions in solution. It is not given by the ideal
gas law for DNA segments.
Small to medium pressure - small to medium density:
The interaction is a competition between a long range repulsion and a short range
attratction.
The equation of state depends crucially on the type and valency of the ionic composition of the
bathing solution. Lots of regimes and subregimes still not entirely elucidated. Complicated.
Monovalent and polyvalent divide.

8.2
8.0
7.8
7.6
7.4
7.2
2.0 1.8 1.6 1.4 1.2 1.0
C
DNA
[M]
Large densities - large pressure
The equation of state becomes almost universal for a single type of salt. Does not depend on the
concentration of the salt anymore. Similar to what is observed in other systems - either other
polyelectrolytes or e.g. lipid multilayers. Hydration force.
2
9.0
8.5
8.0
7.5
7.0
6.5
6.0
log
!
[dynes/cm
2
]
25 20 15 10 5 0
Surface separation, []
rescaled charge density
HPC schizophyllan

Na-DNA Na-Xanthan
TMA-DNA (raw)

TMA-DNA (rescaled)
DDP bilayers

Commonality of forces among
charged, neutral, cylindrical
and planar molecules in salt
solution and distilled water.
Charges: DNA 1e/1.75 ,
xanthan 4 e/ 15 , DDP
1e/55
(Leikin et al. 1993)

Hydration forces: Counterion entropy:
F ! P
2
e
"D / #
F !"
kT
l
PO
4
ln V
Hydration force
Hydration force is dominant in the regime of high density mesophases. Orientational,
positional and bond orientational order.
Hydration force is still poorly understood. It is not due to entropy counterions at large DNA
density, since this interaction is long range whereas hydration force is very short range - 0.1
- 0.4 nm decay length.
We have a good theory for the hydration force in ice, but ice is not water (though it is
similar) - Gruen and Marcelja, 1984. There the origin is reorientation of water molecules
close to polar interfaces.

Intermediate densities - intermediate pressure
Equation of state in the regime of lower density with less ordered mesophases.
The interaction free energy decays exponentially with intersurface separation, the decay length
depends on the salt concentration, but it is not equal to the Debye decay length! Diffraction
intensity shows broad lines - thermal disorder.

Conformational uctuations
Surprisingly the PB limit for nite salt does not work. What are we missing in this picture?
Orientational order
L
p
~ 50 nm
K
C
= k
B
T L
p
DNA is a exible molecule.
E ~ 300 Mpa (plexiglass)
At room temperature big conformational uctuations.
Cell model of a DNA molecules in an array. Effects of nite density

Conformational uctuations - hard connement
Thermal motion can be observed as soon as molecules have enough room to wiggle around their
equilibrium positions.
Consequences
Assume that the length of DNA before it hits the tube is L
||
(Odijk deection length) then:
This is the Odijk interaction (1986). Similar to Helfrich interaction between surfaces.
Long range interaction (short range ( thermal undulations ( long range)
D ~ 3.5 nm
L
P
~ 50 nm
L
||
~ 8.5 nm

Electrostatic interactions - point charges
Take a symmetric uni-uni valent electrolyte. In equilibrium the average % is zero, and the average
charge density &:
Electroneutrality
But in the presence of an external eld
Together with Poisson equation this
yields the Debye - Hueckel equation
Debye length ~ 3.05 /4M
Screening.

Electrostatic interactions - line charges
Osmotic pressure.
For line charges a similar argument is valid, start with the density of co and counterions
in thermal equilibrium with a mean electrostatic potential:
The dominant behavior is exponential with Debye screening length (inverse '0). On the same
level one can also get the charge density
Again a symmetric electrolyte but for a line charge the solution is different (just geometry):
The cell model, simulates the nite density
of the cylindrical objects (DNA) in solution.
R

Experimental counterion distribution
25-mer of DNA in aqueous solution
with Na
+
, Rb
+
, Mg
2+
, St
2+
. Das et
al. PRL (2003).
0.4 M Na
+
(triangles), Rb
+
(squares), Mg
2+
(diamonds), Sr
2+
(circles).
Rb
+
(open squares), Sr
2+
(open circles)
plus DELPHI.
Good t for mono and di-valents.
One can detect the distribution of counterions also experimentally. Very close to predictions.
Good t for monovalents as well as for (some) divalents. Not bad at all.

Conformational uctuations - soft connement
Instead of a hard wall now assume a soft interaction potential V(r)
The Odijk length and the Odijk interaction now come out as:
Now assume (this from the PB theory):
Fluctuation renormalization of interactions! (Podgornik et al. 1989)
We do not see direct electrostatic interactions, we only see uctuation modied interactions.

DNA in monovalent (NaCl) salt solution. Paradigmatic behavior for all monovalent salts but only
monovalent salts. In polyvalent salts the situation is completely different.
Only for counterion-only case
does one see direct effects of
electrostatics because they are so
long range.
Renormalized value of the decay
length due to thermal undulations of
the DNA molecules in the array:
!
(r)
= 4 !
D.
Factor 4 due to elasticity
(fourth derivative) as well as the 1D
nature of DNA (linear polymer).
Conformational uctuations - experiment
Electrostatics can only be seen indirectly, as modied by the presence of conformational
uctuations.

In Manning theory E = 1 - 0.75 = 0.25 . Measured E seems to be twice as large. Similar result in EF
experiments (Schellman and Stigter, 1977). Big differences between Na, Li, K, Rb and 4 Me (NH
3
).
A very pleasing analogy
between multilamellar lipid
arrays and hexagonal DNA
arrays in terms of the
interplay between thermal
uctuations and interactions.
However, no vdW interactions
in DNA arrays, except
Conformational uctuations - consequences

Atom uctuations around equilibrium positions in a crystal?
Are the conformational uctuations of DNA around equilibrium positions similar to the harmonic
uctuations of atoms around equilibrium positions in crystals? NO, they are not.
Worse t to data. DNA at this densities is not a crystal.
(Long) DNA liquid xtals are always positionally disordered. ( is ~ 4 to 7 lattice spacings!
Harmonic uctuations around mean positions R
n,m
.

Odijk (1993) proposed a similar theory. Applied to DNA data by Kassapidou and van der
Maarel (1998).
DNA is uid not crystalline
Odijks theory, central quantity
is u - magnitude of uctuations:
For ('u) 1 reduces to previous result.
For ('u) 1 algebraic decay.
It is impossible to understand the behavior of DNA at these concentrations by making an analogy with
the befavior of crystals at nite temperature. There is no positional order.

Low densities - low pressure
Very low DNA densities and also low salt concentrations. Ideally we have only counterions.
In this regime the overwhelming contribution to the interactions and consequently to the
equation of state of DNA are the free counterions. Their number is much bigger then the number of
DNA molecules in solution and thus their entropy is overwhelming.
At ultralow concentrations, however, that is below 0.001 M DNA one should be able to see
the ideal gas of DNA molecules itself. But only at those vanishing concentrations.

Low salt - Donnan limit
No salt and low salt.
So we delimit ourselves to the case of low added salt concentration
which is usually the case in these experiments. Instead of having only
the counterions in solution we have to consider also the effect of a small
concentration of salt:
As opposed to
Here n
0
is the salt concentration. This is an approximate solution. The concentration of
the counterions at the wall of the cell is calculated via the counterions only model.
In this dilute limit we can disregard the entropy of the DNA fragments and just consider the free
counterions.
Text

Osmotic coefcient is never really
Independent of the DNA density, it
has a long range tail:
Manning
Onsager-Oosawa-Manning does not work!
It really is a limiting law.
The PB equation for monovalent salts
works almost perfectly (quantitatively).
Raspaud et al. 2000.
Short fragment nucleosomal DNA + buffer + salt.
Comparison with experiment
Measured ratio between the ideal osmotic pressure of counterions and with electrostatic.

For polyvalent counterions one obtains discontinuous jumps in the force curve -
an indication of the underlying SC attractions. At least a three component system: uni-uni valent salt plus
polyvalent counterions. Thus a tug-of-war between attractions and repulsions.
The repulsive part probably dependent on the ionic strength at larger separations but independent at
smaller separations, and the attractive part on the amount of polyvalent counterions.
CoHex
Small to medium pressure - small to medium density
Polyvalent counterions - Mn the only divalent, but otherwise tri- and more valent counterions.
I show the case of Co(Hex)
3+.

Rau et al., 1997. Podgornik et al. 2000
Monovalent counterions. Polyvalent counterions.
What one observes (large to small density of DNA) for monovalents is a strong hydration repulsion
followed by the uctuation enhanced electrostatic repulsion.
Electrostatics can only be observed masqued by uctuations.
For polyvalents one observes an (correlation) attractive force mixed with repulsion (~ 0.1 kT/ bp)
just like vdW equation of state!
Fundamental di!erences in the measured equation of state i.e. osmotic pressure.
Monovalent vs. polyvalent counterions

Polyvalent counterions and DNA condensation
For large enough polyvalent counterion concentration DNA precipitates out of solutions making a
condensed phase with a xed density or a toroid. In most vertebrate sperm cells DNA is condensed
by arginine-rich proteins into thousands of toroidal structures, each measuring ~ 100 nm in
outside diameter. The DNA of some bacteriophages also is packaged into a single toroid, or spool,
with similar dimensions. Thus, the toroid represents a fundamental morphology selected by
nature for the high-density packaging of DNA (Hud & Downing 2001).
Very few - luckily for us - (cat)ions induce DNA
condensation. Mn
2+
, Cd
2+
, Co(NH
3
)3+
, polyamines
such as sperimidine
3+
, spermine
4+
, protamine
21+
,
polylysine
N+

Condensation is a complex phenomenon still
not completely understood.
Polyvalent counterions are necessary for
condensation
Best condensing agents appear to bind into the major
or minor groove.
Almost all divalent cations condense ssDNA
but not dsDNA.
Electrostatics is an important but not the
only factor in DNA collapse (CoHex more
efcient then spermidine!). Thats why it is a very
tough problem.
Theoretical models of DNA collapse are few though we have a pretty good idea what they need
to entail.

DNA toroid formation
For su)ciently large polyvalent counterion
concentration collapse of DNA.
Depending on the details the collapse can be
into a toroidal structure.
Hud & Downing, 2001.
Toroid formation
in the bulk.
95-185 nm, 35-85 nm, 2.7 nm Chattoraj et al. (1978).

Viral equation of state and DNA encapsidation.
How to explain quantitatively the osmotic DNA encapsidation data and connect them with the bulk
equation of state? By applying the inverse spool theory.
The inverse spool model is characterised by interactions and curvature:
or the two corresponding (free) energy densities:
We assume here that DNA conguration is described by the (continuum) density and director elds.
A continuum theory.

Continuum nematic nanodrop model of packing.
elastic constants
(bare & interaction)
persistence length of DNA, ~ 50 nm.
DNA elastic constant.
Frank elastic energy: splay twist bend
Assume that DNA inside the capsid is like an ordinary polymer nematic (it sure looks like it).
All three elastic constants are density dependent, but for long semiexible polymers only one elastic
constant is important. The other two are negligible (for various reasons).
The elastic free energy part is then:

The osmotic pressure.
The total free energy is composed of bulk DNA (interaction) free energy, elastic free energy
and chemical potential since DNA can exchange between inside and outside of the capsid.
From this it follows (rst integral of the EL equation) that:
The stress tensor as well as the osmotic pressure can be derived from the rst integral of the EL
equation (following LL).
Similar to the rst integral in the PB theory of electrostatic interactions:
total osmotic pressure = osmotic pressure of bulk DNA + elastic free energy
total pressure = osmotic pressure of ions - electrostatic eld energy
The total osmotic pressure has to be a constant.
Same as for ordinary LC but here elastic constants are density dependent!

2
Free toroid formation
Previous mesoscopic theory of DNA nanodrop can be simply generalized
(Ubbink & Odijk 1996):
Siber, Lo6dorfer & Podgornik, 2010. (a variation on the UO theme)
For spontaneous aggregation of DNA the total osmotic pressure has to be p=0!
In order for this equation to be solvable, p0 has to have an attractive branch.

FINIS

counterions),
DNA array),

LECTURE 6
ELECTROSTATICS OF DNA (Poisson-Boltzmann theory,
Counterion distribution, Manning condensation, Salt
screening, Strong coupling theory, Correlation attraction,
Force equilibria with polyvalent counterions)

Charges and electrostatic interactions
Coulombs law and Poisson equation:
Equal sign charges - repulsion, different sign charges - attraction.
This form of CL is only valid for two charges in 3D. For 2 or 1 dimensional
systems the Coulomb law is modied:
Where now and ) are now the surface charge density and the linear charge density.
2D 1D

Below this separation (Bjerrums length - 7.1 in water) the + and -
would tend to associate (Bjerrum pairs). Entropy!
Apart from ES energy, we also have thermal effects.
kT (entropy) and electrostatic interactions
Minimize the free energy: a stable minimum or not?
1D
2D
3D
1D
3D
2D
Condensation.

1948 - annus mirabilis for colloid science: DLVO theory
Verwey & Overbeek
(1948)
Deryaguin & Landau
(1941)
disjoining pressure
DLVO theory of colloid stability:
Electrostatics plus van der Waals.
Gouy (1910)
Chapman (1913)
Debye & Huckel (1913)

The weak coupling (PB) theory: collective description
+
Electrostatic energy: collapse to the surface. Entropy: uniform distribution of charges
The two together: non-homogeneous ionic prole
Non-equilibrium free energy = (electrostatic energy) - k (ideal gas entropy)
Weak coupling limit = minimization of the collective free energy.
plus electroneutrality

The Poisson - Boltzmann equation in cylindrical geometry
This leadds to the Poisson - Boltzmann equation and the electroneutrality boundary
condition. This is the basis of the cell-modell of DNA on the mean-eld level.
Minimization of the free energy (electrostatic eld energy plus ideal entropy)
To solve the Poisson - Boltzmann eq. in cylindrical
geometry one has to introduce a neutralizing cell.
Has to satisfy BC at both boundaries.
Poisson Boltzmann
Inhomogeneous electrostatic potential!
T
h
e

c
e
l
l

m
o
d
e
l
.


Analytical solutions for counterion-only case
The Manning parameter.
Solution of the cylindrical cell model
Approximate form of the solution

Counterion condensation
Onsager-Oosawa-Manning counterion condensation.
Onsager in 1967 observed an anomaly in the partition function of a Coulomb gas in 2D.
Manning realised in 1969 this is relevant for polyelectrolytes.
In the 70 Oosawa, Manning and Dolar realised that the FKL solution gives the same behavior.
For DNA:
For HA e.g. Q ~ 0.72.
The osmotic coefcient and the fraction of condensed charge. Osmotic coefcient quanties
the non-ideality of the system. It also counts the numer of ions that are osmotically active.
On the average 3 out of 4 phosphates are neutralized by counterions.
The rest are acting as free, osmotically active counterions.
The concentration of counterions at the outer cell boundary gives the osmotic pressure in the
system. A fundamental insight of the PB theory.

Equivalence of osmotic pressure (PEG 20000 and
DEX 110000) C
DNA
from absorbance at 260 nm.
Experiments
Raspaud et al. 2000.
The counterion-only cell-model of DNA does not fare bad in th elimit of low densities of DNA and low
salt. The bold lines are the numerical solutions of the PB equation and the correposnding osmotci
pressure.

The Poisson-Boltzmann equation
(effect of salt)
Osmotic pressure calculated
from the solution of the PB
equation:
In cylindrical cell geometry there are no analytical solutions at nite dilution.
At innite dilution: Tracy - Widom (1997) analytical solution.
At best we remain with the linearized solution or the complete numerical solution.
The dominant behavior is exponential with Debye screening length (inverse '0).
Comparison with experiments shows that the measure screening length is always larger then th
eDebye length, in fact it is 4 times larger. The factor of 4 comes from conformational
uctuations of DNA governed by the elastic bending energy (fourth derivative).

Developments in the 80s colloid science:
Oosawa derives attractive interactions
between DNAs (late 60s early 70)
Simulation of DLVO interactions (early
80s - electric bilayer simulation Torrie
and Valleau)
Fundamental paper by Gulbrand et al.
(1984)
Established that for planar surfaces the
Interactions with divalent counterions
can be attractive!
They dubbed it the correlation effect because
it stemms from a correlation term in the
stress tensor.
1984 - annus horribilis for colloid science
Figure from my PhD thesis in 85.

Hexagonal array of DNA with poly-counterions:
(Lyubartsev and Nordenskiold, 1995)
A pair of DNAs with poly-counterions:
(Gronbech-Jensen et al. 1997)
1990s - getting worse and worse...
Attractions seem to be everywhere!

A historical guide to the correlation effect
Gulbrand, Jonsson, Wennerstrom and Linse
(1984)
Lyubartsev and Nordenskiold (1995)
Gronbech-Jensen et al. (1997)
(MC simulations)
Oosawa (1971)
(counterion uctuations)
Kjellander and Marcelja (1984-1986)
(inhomogeneous integral equations closure)
Podgornik et al. (1988-1991)
Attard et al. (1988)
Ha and Liu (1997)
Podgornik and Parsegian (1999)
(Gaussian uctuations)
Netz and Moreira (2000-2001)
Naji and Netz (2003-2004)
(General analysis of Coulomb uids)
Ninham and Parsegian (75)
(screened van der Waals
interactions)
Rouzina and Bloomeld (1996)
(checkerboard model)
Shklovskii et al. (1999-2002)
Lau and Pincus (2001)
Levin (2002)
(Wigner crystal model)
Kornyshev and Leikin (1997-2002)
(Debye-Hueckel-Bjerrum model)

Collective description
(Poisson - Boltzmann N description)
Screened Debye-Hueckel

vs.
Single particle description
(Strong Coupling 1 description)
Weak coupling limit
(Poisson - Boltzmann)
!$ 0
Strong coupling limit
(Netz - Moreira)
!$ "
Z
Ratio between the Bjerrum and the Gouy - Chapman lengths. Bulk versus surface
interactions.
Bjerrum length Gouy - Chapman length
Coulombs law
and
kT
Coupling parameter
Final reconciliation: Coulomb and Boltzmann
For DNA: ! = 2.8, 22 and 76.

+
Electrostatic energy
without mobile counterions
of a single counterion
Z
Strong coupling limit = virial expansion to rst order.
+ Z
Z
Z
+
of two counterions
Strong coupling theory: one particle description
Netz (2000)
Electrostatic energy of a single counterion plus entropy of a trapped counterion.

Interaction between planar symmetrically charged surfaces
Weak coupling ( = Poisson - Boltzmann + Gaussian ucts)
Strong coupling
Total pressure (PB+ ucts)
Poisson - Boltzmann (analytic)
Fluctuational (analytic)
(a lot easier to evaluate)
Netz and Moreira (2000-2001).

WC and SC present interaction limiting laws
The exact interactions is always between these two limits.
Netz and Moreira (2000).
Extensive simulations completely corroborate this picture.
In their respective regimes of validity the SC and WC (= PB + Gaussian flucts)
are quantitative.
Weak coupling
Strong coupling

Interaction between planar asymmetrically charged surfaces
Weak- and strong-coupling electrostatic interactions between asymmetrically
charged planar surfaces M. Kandu7, M. Trulsson, A. Naji, Y. Burak, J. Forsman, and R.
Podgornik (PRE 78, 061105 2008).
By suitably normalizing the results one can concentrate exclusively on the interval "1
< 8 < 1. All other cases can be mapped onto this interval with appropriate rescaling of
the parameters. The values 8 = 1 and 8 = "1 represent exceptional points in the
parameter space: 8 = 1 is the standard symmetric case (11 = 12) already amply
treated in the literature, and 8 = "1 represents the antisymmetric case (11 = "12)
with no counterions between surfaces that reduces to the trivial case of a planar
capacitor.
New parameter describing asymmetry:
D = 2 a

Weak coupling ( = Poisson - Boltzmann + Gaussian ucts)
Depending on 8 the PB interaction (p0) can be repulsive, attractive or zero. Within the
mean-eld theory two surfaces of equal sign always repel, that is at all separation
distances D. When the surfaces bear charges of opposite sign 8 < 0, they attract at
large separations and a repulsion emerges only at sufciently small separations.
The Gaussian uctuations around the mean-eld always give rise to (small) attractive
contribution (p2) to the total interaction.
Strong coupling
Asymmetrically charged surfaces: WC and SC
The SC pressure can become attractive for both like charged and oppositely charged
surfaces which contrasts with the mean-eld theory that does not allow attraction
between like-charged surfaces. An explicit analytic form.

Rescaled interaction pressure, p, as a function of the rescaled intersurface distance, for
three different values of the asymmetry parameter 8 = 0.5, 0 and "0.5 (left to right).
Solid lines represent the PB prediction, dotted lines show the second-order weak-
coupling pressure (PB plus second-order corrections), and the dashed lines are the SC
prediction. Symbols correspond to MC simulations data at three different coupling
parameters 9 = 0.32 (open quares), 8.6 (lled squares), and 86 (open circles). Insets
show details at small pressures along with the PB theory result as well as the second-
order WC result for 9 = 0.32.

An exact model vs. WC and SC
One-dimensional counterion gas between charged surfaces: Exact results compared
with weak- and strong-coupling analyses. David S. Dean, Ron R. Horgan, Ali Naji, and
Rudolf Podgornik, THE JOURNAL OF CHEMICAL PHYSICS 130, 094504 (2009).
A. Lenard, J. Math. Phys. 26, 82 (1961).
S. Edwards and A. Lenard, J. Math. Phys. 3, 778 (1962).

Thus, it may be expected that in the limit N ( :, the mean-eld theory corresponding to
this system becomes exact! On the other hand, the SC description may be expected to
follow simply for N = 1. One should bear in mind that the 1D system considered here
corresponds to a 3D system of mobile charged plates (membranes) conned between two
xed planar charged walls and thus, such a 1D system with even a single counterion
would have a completely meaningful thermodynamic behavior.
Coupling parameter in 3D in 1D:

Rescaled interaction pressure, p, as a function of the rescaled distance, L, between
charged boundaries in the 1D system of counterions. We show the results for three
different values of the asymmetry parameter 8 = 1.0, 0.5, and -0.5 (lefttop to right).
Thick solid lines represent the PB prediction and the dashed lines are the SC prediction.
Symbols correspond to MC simulations data and thin solid lines are the exact results at
different numbers of counterions as indicated in the graphs.

And contains the bare as well as the image contributions due to dielectric inhomogeneity.
The external charge is assumed to be of the form:
Planar Greens function.
Image effects of the dielectric discontinuities in the system.
The interaction potential is:
Interactions and dielectric images.

Strong-Coupling Electrostatics in the Presence of Dielectric InhomogeneitiesY. S. Jho, M.
Kanduc, A. Naji, R. Podgornik, M. W. Kim, and P. A. Pincus (PRL 2008).
Note that while the weak-coupling results are less sensitive to the dielectric jump
effects, a remarkable dependence on dielectric missmatch is found in the SC regime.
Dielectric images in the SC vs. WC

We investigate completely general distribution of charges in counterions.
I will not deal with exible counterions.
Weak and strong coupling theory: multipoles
When the multipoles become large enough the flexibility effects can not be ignored any more.
multipolar effects should merge into polyelectrolyte effects.
large stiff multipoles long flexible polyelectrolytes
Dipolar part: Abrashkin A, Andelman D and Orland H, PRL 99 077801 2007.
Quadrupolar part: Maset S and Bohinc K J. Phys. A 40 11815 2007.
Spermidine (charge 3+)

For some multivalent ions, the separations between charges in an ion are too large to be
ignored, leading to breakdown of the pointlike picture of ions assumed in the PB theory.
Yong Woon Kim, Juyeon Yi and P. A. Pincus PRL 101, 208305(2008)
Pressure as a function of the plate separation D (a) for small dumbbell ions of d = 0.5 with
various values of Xi. Solid and dashed lines denote SC predictions for respective dumbbell sizes.
For comparison, pressures for the equivalent system consisting of pointlike ions and having the
same surface charge are plotted together in (a): MC simulations (lled symbols), PB (solid), and
SC (dashed) solutions.
Stiff dumbbells.
Dumbbell counterions

Yong Woon Kim, Juyeon Yi and P. A. Pincus PRL 101, 208305(2008)
Pressure as a function of the plate separation D for large dumbbell ions at Xi = 0.05 (open
symbols) and at Xi = 10^4 (lled symbols). Solid and dashed lines denote PB and SC predictions
for respective dumbbell sizes. For comparison, pressures for the equivalent system consisting of
pointlikeions and having the same surface charge are plotted together: MC simulations(lled
symbols), PB (solid),and SC (dashed) solutions.

Multipole expansion for charge density of pointlike counterins (plus dielectric inhomogeneities):
First part is just the standard primitive model. Point counterions.
Dipolar part: Abrashkin A, Andelman D and Orland H, PRL 99 077801 2007.
Quadrupolar part: Maset S and Bohinc K J. Phys. A 40 11815 2007.

A reformulation of the model of the electrostatic interations between (bio)colloids.
Multipolar counterions
(quadrupolar moment for a rod)

The Poisson - Boltzmann theory
The generalized PB equation for the case of counterions with multipolar structure.
Just as in the standard PB theory there exists a pressure equation
(rst integral of the PB equation)
Positional as well as orientational prole:
Orientational OP.

The Poisson - Boltzmann theory: numerics
(c) Dimensionless
pressure-distance curves
obtained from the rst-
order approximation.
Quadrupolar contributions
are attractive. No image
effects! (planar geometry)
This is all for point quandrupoles. Thus only multipolar and not
steric and entropic effects, which are easy to understand in terms of
bridging and steric hindrance (Maset S and Bohinc K J. Phys. A 40 11815 2007).
An approximate (perturbation) solution.
Mean-eld results for various
t in rst-order approximation.
(a) Counterion density
prole. By increasing t in the
mean-eld limit counterions
are depleted from the center
toward both surfaces. (b)
Order parameter prole.

The strong coupling theory: numerics
(c) Quadrupolar pressure
difference. Contribution is
repulsive at small
separations and becomes
attractive at larger
separations. At very large
separations it goes to zero.
One can conclude at this point that in the dielectrically homogeneous case the higher
order multipoles are completely irrelevant on the SC level. Only the monopolar term
survives in the partition function. In a dielectrically homogeneous case
multipolar moments play absolutely no role in the strong coupling limit!
An exact solution.
Strong coupling results for
9=50 and ;=0.95 and
various t. (a) Counterion
density prole. (b) Order
parameter prole.
Counterions are less ordered
at the center. Order is
increased with increasing t.

Helical molecules: WC vs. SC
Charge can be quantied
by the Manning parameter:
Or with the surface charge density:
Weak coupling (= Poisson - Boltzmann)
No Gaussian uctuations. Too complicated to evaluate plus give a small effect.
Strong coupling
In both cases the interactions depend not only on the separation but also on the
mutual orientation angle (rst calculated by Kornyshev and Leikin). This creates and
additional degree of freedom and leads to angular frustrations in the condensed phase.
Orthorhombic phase at large densities is a consequence of this angular frustration.

On the mean-eld level the force between charged helices is always repulsive and
decays with increasing interaxial distance. In a symmetric system on the mean-
eld level this is well known. Force per unit lengthseparation dependence for two
helices (ds-DNA parameters) at various mutual azimuthal angles /0 and valencies.
The corresponding Manning parameter for q=1 is Q=4.1 and for q=2 it is Q=8.2.
Weak coupling
On the PB level the angular dependence of the interaction is small.

ds-DNA parameters of a=1 nm and H=3.4 nm in the presence of monovalent
counterions. The corresponding Manning parameter is Q=4.1 and the coupling parameter
9=3. The inset shows force per unit lengthseparation curves for orientation /0=) for
different cylinder radii. Note the scale compared to WC.
Strong coupling

Bound state
Collapse transition into a bound state for ds-DNA parameters.
(a) Bound state interaxial separation as a function of mutual orientation. For
polyvalent ions the rapid jump appears at a certain angle, corresponding to a phase
transition.
(b) Corresponding free energydistance plot for divalent counterions (q = 2) at
different orientations. Two minima (for the rst three cases) are clearly visible which
are responsible for the phase transition.
Different from the KL theory: no specific interactions, no counterions distribution put
in by hand. Valid for DNA covered CNTs and possibly also DNA itself (?).

Debye-Hueckel interaction:
smeared charge
smeared counterions
Manning condensation
Kornyshev-Leikin interaction:
explicit charge
explicit counterions
linearized PB
Kornyshev - Leikin, 1998, 2000, 2002.
Molecular theories of electrostatics
Debye-Hueckel-Bjerrum theory
DH equation. Microscopic charge density.

Kornyshev-Leikin interactions
Orientational dependence.
Optimal angle.
Non-monotonic interactions (Kornyshev-Leikin, 1997).
(*/2)

The holy grail
an indication of the underlying SC attractions. At least a two component system: uni-
uni valent salt plus polyvalent counterions. Thus a tug-of-war between attractions and
repulsions.
The repulsive part dependent on the ionic strength and the amount of polyvalents.
CoHex

Multicomponent strong coupling: SC-DH
Strngly coupled polyvalent counterions and weakly coupled uni-uni valent salt.
Very rich behavior for different parts of parameter space.
Coupling parameter for polyvalent counterions. Fixed charge to counterion
charge ratio. Explicit asymptotic results for small (left) and large (right) separations.
(&=0 DH salt only, and &=1 full compensation)
(a << 1) (a >> 1)
(SC-DH = dressed counterions)

MC vs. SC-DH approximation
Good correspondence in the SC regime.

The holy grail
Non-monotonic
interaction pressure, like
in the experiment with CoHex and salt.
Existence of critical pressure.
Can we reach quantitative agreement?

Is there a connection between the two: it seems to me that there is
(Kanduc et al. , 2009).
One can derive a duality relation between bridging free energy and SC free energy. It is of the form,
that one can prove as an exact theorem:
This would mean that the SC electrostatics and PE bridging
interactions correspond to the same theoretical framework.
In itself this is an interesting conclusion.
WC for one theory is SC for another one - like the concept of duality in string theory:)
Duality: SC and PE bridging.

FINIS

counterions),
DNA array),

LECTURE 7
DNA ORGANIZATION IN CHROMATIN AND
INTRODUCTION TO VIRUSES (Nucleosomes, N-tail
configuration, polyelectrolyte bridging interactions, second
virial coefficient of NCP solutions, NCP aggregation)

Attractions in polyvalent salts
an indication of the underlying SC attractions. At least a two component system: uni-
uni valent salt plus polyvalent counterions. Thus a tug-of-war between attractions and
repulsions.
For sufficiently large concentration of the polyvalent salt there is a minimum in the
osmotic pressure corresponding to an equilibrium interhelical separation.
CoHex
Attractive interactions lead to unstable regions of the equation of state. Like in teh van der Waals gas.

The mechanism of attractions
Non-monotonic
interaction pressure, like
in the experiment with CoHex and salt.
Existence of critical pressure.
Can we reach quantitative agreement?
The attraction has a relative long range and its effects can be detected up to 3.5 nm separations.

V(r-r)
Manning buckling with correlation attractions
Models (1), (2) and (3)
give similar results.
This is a mean-eld like calculation.
No thermal uctuations.
Just elasticity.
Euler buckling instability with intersegment attractive interactions. Incipient condensation
of DNA.
Shape equation of the elastic lament (DNA):

Buckling and thermal uctuations
Thermal uctuations? Odijk (1998) - harmonic theory. Renormalization of the bending
rigidity. A full non-linear theory (Hansen et al. 1999) takes us to:
Interaction has a repuslive (WC) and an attractive part (SC).
collapse
?
WLC
Liverpool, Golestanian, Kardar (2000). Ha and Thirumalai (2002), Nguyen, Rouzina and Shklovski (1999).

Baumann et al. 1997-2000.
Mg
2+
and Co (NH
3
)
6
3+
yield lower persistence length then
polyamines spermidine
3+
and putrescine
2+
. 2+ do not condense DNA!
Elastic softening and attractive interactions
Elastic softening by
Itself wil lprobably not drive
DNA collapse.
(hydration, helix secondary structure )
Force plateau when
Enough poly+ to condense DNA.
Attractions of 0.01-0.33 kT/bp
Consistent with Rau et al.

Elastic, Euler-like, states are important for DNA collapse. Stiff polymers have a different
Collapse pathway (originates in the buckling transition) then exible polymers.
There might be a whole slew of Euler-like intermediate states that lead to DNA collapse.
Much more ordered collapsed state then in Khan and Jonsson.
Stevens BJ (2001). This collapse is very different from a exible chain.
DNA condensation simulations

Khan and Jonsson, (1999): polyvalent counterions can collapse a exible chain but they
can not give rise to an ordered toroidal structure.
A new problem: interactions and polyelectrolyte exibility.
Swedish polyelectrolyte (no hard cores etc).
Collapsed Extended
The collapse of a featureless exible polyelectrolyte chain look very different from a
DNA collapse. In this case one has a compact glubule without any ordered structure.
Very similar to generic polymer collapse..
Flexible polyelectrolyte condensation simulations

Euler (elastic) intermediates are
clearly seen also in simulations of
Schnurr, Gittes and MacKintosh
PRE (2002).
toroidal
racquet-like
We understand well only one side of the transition.
The destabilization of the persistence length leading
to a 1st order transition.
?
Simulations of stiff polyelectrolyte chains show that collapse into an ordered structure is an interplay
of attractive interactions and elastic rigidity of the chain.

Coil - racket - torus. Montesi et al. 2004.

Eucariotic Genome
~ 1400 nm
~ 700 nm
~ 300 nm
~ 30 nm
~ 10 nm
NCP
Multi.level structure of chromatin. Viruses: toroidal packing just as in DNA collapse in vitro. Bacteria
(prokariotes): nucleoid, a loose DNA - protein gel
nucleosome

Nucleosome Core Particle (NCP)
Lger et al., Nature, 1997, 2002 at 1.9 resolution
A histone octamer (4 X 2) of 4 core histones: H2A, H2B, H3 and H4
147 bp DNA wrapped 1.75 times in a left-handed helix, stable up to 0.75 M salt
A highly charged hairy particle
(- 2*147 + 134 (220) = - 165)
Excluding the histone buried charges
(Bertin et al., 2004)
Longest tail ~ 25 aa.

Nucleosome Core Particle (NCP)
Lger et al., Nature, 1997, 2002 at 1.9 resolution

A histone octamer (4 X 2) of 4 core histones: H2A, H2B, H3 and H4
147 bp DNA wrapped 1.75 times in a left-handed helix, stable up to 0.75 M salt
A highly charged hairy particle
(- 2*147 + 134 (220) = - 165)
Excluding the histone buried charges
(Bertin et al., 2004)
Longest tail ~ 25 aa.

High density NCP mesophases
(Mangenot, Livolant, Leforestier, Durand, de Frutos, Raspaud, 2001 - 2003)
Isotropic
Lamellar (lipid bilayer like)
Hexagonal
Inverted hexagonal

NCPs in salt solutions at varying osmotic pressure
NCP in ionic solutions make ordered phases. Similar to ordered phases of phospholipids and DNA.

Equation of state of nucleosomal core particles
!h
reservoir
semi-permeable
membrane
solute
3.5 mM
25 mM
75 mM
210 mM
(NaCl)
Standard second virial coefcient:
Manometric osmotic stress method
(Raspaud, 2003)
DNA
NCP
Using osmotic stress technique is more difcult in this case. Can not have large samples of
material. One can nevertheless use osmotic balance in a different context (Raspaud 2003).

NCP in fact come in two different varieties: with folded N-tails and with expanded N-tails, depending
on ionic conditions (stable until 0.75 M NaCl). Mangenot et al. 2002
Two conformations of the NCP
Folded tails below 50 mM and expanded from 50 mM and above.
D

~

D

~

D

~

Polyelectrolyte bridging
Polyelectrolyte bridging:
another case of coupling between
conformations and interactions.
Podgornik (2004)
Electrostatic interactions effect
polymer structural parameters
(persistence length, stretching modulus)
as well as conformations
(DNA condensation).
Model system: a charged exible chain
and oppositely charged macroions.
Flexible polyelectrolytes can have different congurations in ionic solutions and can also interact in
different ways.
Coupling between polyelectrolyte congurations and interactions: bridging interaction.

Polyelectrolyte bridging and A2
Can polyelectrolyte bridging really be the culprit? (Podgornik, 2005) Can polyelectrolyte bridging
interactions mediate by the NCP N-tails leads to attractions that would modify the second virial
coefcient as seen in the experiment-
Indeed:
- mediates strong attraction
- range dependent on the salt
concentration
- deminishes the A2
However:
- attractive potential not
monotonic
- has a clearly dened cutoff
- does not make the A2 non-
monotonic
Task: compute polyelectrolyte mediated interactioons in different limits and try to make some
conclusions regarding the A2 based on a detailed study of the PE mediated interactions.
Analytical theory (Podgorni, 2005) and extensive Monte Carlo simulations.

Podgornik and Jonsson (1993).
The bridging and non-bridging congurations show up also in the interactions between macroions. In
the case of bridging of polyelectrolyte chains one has a relatively long range attractive interactions.
The details of these interactions depend on the charges adn ionic strength of the solution.
N = 20, 30. No salt ions.
Two charged macroions with a componsating one, or many polyelectrolyte chains.
N = 20, 30 monomers and size of the macroion ~ 40 .
Bridging between small macroions: simulations

Analytical calculations of the bridging attraction
60 mM 1 mM
One can apply straightforward polyelectrolyte theory to calculate the magnitude and spatial
dependence of bridging attractions. Same as simulations.
Charges on the chain and on the macroions: N = 100 and M = 30
Chain conformation determined by the macroions.
Non-bridging
Bridging

A rich parameter space of bridging attraction
Not always attractive. Dependnig on the length of the chain and the charge on the
macroion they can be either repulsive or attractive.
Charges on the chain and on the macroions: N = 50 and M = 40
60 mM
1 mM
Chain conformation determined by inter and intra.
Dzubiella, Moreira and Pincus, 2003
Chodanowski and Stoll, 2001
Formation of a polymer brush corona.

Simulation of NCP N-tail bridging interactions
Muellbacher et al. (2006).
Nikolay Korolev, Alexander P. Lyubartsev, and Lars Nordenskiold, 2006.

Other cases of non-monotonic A2?
Are there any similar cases in other systems? YES, A2 of proteins in solution:
lysozyme (Tessier et al. 2002)
b-lactoglobulin (Piazza et al. 2002)
apoferritin (Petsev et al. 2000)
In these cases there are no dangling N-tails that could account for bridging attractions. Other types
of interactions (patchiness, charge disorder etc.) can also account for some aspects of attracctive
interactions.
Allahyarov et al. (2003):

- Nucleic acids : DNA or RNA;
single stranded vs double
stranded; linear vs circular;
one or more pieces
(segmented genome)
- Capsid: helical, icosahedral
(complex protection,
attachment, enzymatic)
- Envelope derived from host
membrane lipids and virus
proteins
What are viruses?
1. Acellular (nucleic acid with protein capsid +/- membrane envelope)
2. Obligate intracellular parasites
3. No ATP generating system
4. No Ribosomes or means of Protein Synthesis
- crystallization of a virus rst reported in the 1930s.
- rst atomic resolution structure of a virus was 1978, tomato bushy stunt virus.
Although some viruses are very fragile & are essentially unable to survive
outside the protected host cell environment, many are able to persist for
long periods, in some cases for years in hostile conditions.

TMV
Hepatitis A
HIV
Bacterophage T7
(bacterial virus)
Ebola
inuenza
Rhinovirus
(common cold)

Bacteriophages.
capsid
DNA
- assemble the particle utilizing only the information available from the
components which make up the particle itself (capsid + genome).
- form regular geometric shapes, even though the proteins from which they are
made are irregularly shaped.
The three families of tailed dsDNA viruses (phages) that infect bacteria.
a, Myoviruses, contractile tails, are typically lytic and often have relatively broad host ranges.
b, Podoviruses, short non-contractile tail, are also typically lytic and have very narrow host ranges.
c, Siphoviruses, long non-contractile tails. Relatively broad host range, and many are capable of
integrating into the host genome. Scale bar, 50 nm Curtis A. Suttle Nature (2005).

Lateral DNA transfer, F. Bushman
(2002).
Marine phages - the most abundant
viruses in the world. 10^7 per milliliter!
Phage mediated lysis of marine bacteria
might be the limiting rate of carbon
xation from the atmosphere.
Types of bacteriophages

A hot dog and viruses on the side, please!

Viruses (bacteriophages) or death!
Let bacteriophages ght
bacteria for us!

FINIS

counterions),
DNA array),

LECTURE 7
DNA ORGANIZATION IN VIRUSES (Caspar-Klug theory and
elaborations, Continuum elasticity of viral capsids, Viral
capsids under mechanical stress, Osmotic encapsulation of
DNA, The inverse spool model).

Bacteriophages.
capsid
DNA
- assemble the particle utilizing only the information available from the
components which make up the particle itself (capsid + genome).
- form regular geometric shapes, even though the proteins from which they are
made are irregularly shaped.
The three families of tailed dsDNA viruses (phages) that infect bacteria.
a, Myoviruses, contractile tails, are typically lytic and often have relatively broad host ranges.
b, Podoviruses, short non-contractile tail, are also typically lytic and have very narrow host ranges.
c, Siphoviruses, long non-contractile tails. Relatively broad host range, and many are capable of
integrating into the host genome. Scale bar, 50 nm Curtis A. Suttle Nature (2005).

Physical principles of viral shapes.
Crick & Watson, Nature (1956).
Crick &Watson (1956), were the rst
to suggest that virus capsids are
composed of numerous identical
protein sub-units arranged either in
helical or cubic (=icosahedral)
symmetry after seeing EMs.
Caspar & Klug, (1962).
Principle of quasi-equivalence.
Triangulation number T.
10 (T-1)

HIV-1. Welker et al. 2000.
Ganser et al. 1999.
Not always icosahedral: HIV cores.
12 vefold defects needed to close the
shape (7 top + 5 bottom).
Quantization of cone angles:
112.9 (P=1), 83.6 (P=2), 60
(P=3)38.9 (P=4), 19.2 (P=5)

A zoo of icosahedral viruses, Baker et al. 1999.
Each has a di!erent triangulation number.

A pronounced di!erence in the
details of the shape between small
and large viruses.
spherical vs. faceted
Why?
Shape universality and size variability.

Continuum theory of viral shapes. Fppl - von Karman equations (1907)
2D elasticity curvature energy
Fppl - von Karman number:
Larger values of * > 154
lead to pronounced faceting.
The triangulation indices are (6,6).
Horribly non-linear,
di)cult to solve.
Lidmar et al., 2003.
Continuum theory of viral shapes.

(2,2) (4,4)
(6,6) (8,8)
* = 45, 176, 393, 694.
* = 8000000.
Sharpening of the edges.
Systematic solutions...
Solutions of the continuum theory of viral shapes.

Fitting the solution of Fppl - von Karman equation to real virus shape
Bacteriophage HK97 (full virus and cross-section on the r.h.s.) The best t occurs at * = 1480.
Comparison with the real world of viruses.

Siber and Podgornik, 2008.
Stability and collapse of viral capsids.
Osmotically stressing viral capsids.
At a critical value of the osmotic pressure.
Evilevitch 2008.
Two dimensionless parameters:

(Kleinschmidt et al., 1962)
Bacteriophage T2
P ~ 50 atm
+ ~ 100 mg/ml
(Champagne at 5-6 atm)
~ 630 m long
~ 1 mm thick
pack into 25 cm
6000 times compaction.
Osmotic shock & DNA packing density.

Three-dimensional reconstruction of the full-length "29 packaged genome. Surface rendering of the
reconstructed density maps, for the symmetrical renement, at two slightly di!erent isosurface values,
A and B on the gure. Adapted from Comolli et al., Virology 371 267 (2008).
Organization of ds-DNA inside the viral capsid seems to be ordered.
At those densities (in the bulk) one should have a nematic or hexatic-like order with ~25 separation.
DNA packing not disordered.

Direct experimental observation of DNA packing
Cryo-electron microscopy, epsilon15. Jiang et al. 2006.
Di!raction ring corresponds to 25 . Model of packing from densitometry traces.
Earnshaw & Harrison, Nature 1977. Scattering of X-rays from P22 phage heads.

Models of viral packing
a.) concentric shell or toroidal winding (Earnshaw & Casjens 1980)
b.) spiral fold model (Black et al. 1985)
c.) liquid crystal model with local parallel packing (Lepault et al. 1987)
d.) ball of yarn Earnshaw et al. (1987)
Cryomicrographs of
T4 bacteriophages.
Di!raction o! the DNA
within the capsid.
(Lepault et al. 1987)
INVERSE SPOOL

Review by Angelescu and Linse (2008).
Various computer models give similar results for DNA packing within bacteriophages.
Plus analytic calculations for simple models. Slosar and Podgornik, 2006.
Computer simulations of DNA packing inside the capsid
Simulation of a sti! chain within a spherical enclosure.

Inverse spool model
Kindt et al. 2001. Klug and Ortiz, 2003. Odijk and Slok, 2003.
Most formulations are on the level of a continuous self-interacting DNA chain. Di!erent
authors di!er on the details of the free energy expression for the DNA inside the inverse spool.
But the spool itself is assumed together with the depletion hole in the middle.
Simulations are set up a bit di!erently.
authors authors
One can set up a model of the DNA packing within the capsid on various levels.
A mechanical (forces and elasticity) or nanomechanical theory of viral packing:
Quantitative theory started with Grosberg in 79.
D. Marenduzzo et al. (2009).

Bacteriophage / 29
authors authors
(A) Model capsid for f29, constructed by elongating a
pentakisdodecahedron and inserting 20 additional faces plus a
cylinderical core that is 146 long and 108 in diameter (Tao et
al., 1998).
(B) Space-lling model for the f29 capsid; each triangular face of
the polyhedron was lled with pseudoatoms with radii of 8 . THis
is to insure that DNA is well conned within the capsid.
(C) A typical conformation resulting from a 70 ms MD simulation of
packing DNA inside f29. Individual pseudoatoms (six base pairs) are
shown in this representation, clearly illustrating the folded toroidal
geometry. A rainbow coloring scheme was used to distinguish
between the head (red) and tail (blue) of the DNA. Note that the
capsid and connector are not shown. Other conformations are
shown in Figure S1.
It should be emphasized that MD is used because we
have found that it is the most efcient algorithm for equili-
brating successive structures along the packaging trajec-
tory (Arsuaga et al., 2002; LaMarque et al., 2004; Locker
and Harvey, 2006).146 long and 108 in diameter (Tao et al.,
1998).

Simulations of packing of phi29
authors authors
The capsid is not shown in the simulation.
(Petrov and Harvey, 2007)
The 500 ns MD trajectory of Phi29
bacteriophage during the force calculations at
100% packed.
Phi29 in polyhedral capsid with electrostatics

authors authors
Simulations of packing of phi29
The capsid is not shown in the simulation.
(Petrov and Harvey, 2007)
The 500 ns MD trajectory of epsilon15
bacteriophage during the force calculations at
100% packed.
epsilon15 in icosahedral capsid with electrostatics

But can be more complicated
authors authors
(A) This structure developed from a folded toroid. (B) Compactied bifolded toroid, which is
distorted to the extent that it conforms to the shape of the T2 protein capsid. Unfolded T2 toroid is
shown. (C) This fully extended T2 toroid is too large to t within a T2 head. (inferred from
experiments Hud, 1995).
Simulations: Christopher Forrey and M. Muthukumar, 2006,
Anton S. Petrov and Stephen C. Harvey, 2008

Implementations of the inverse spool model
- T. Odijk, Biophys. J. 75, 1223 (1998).
- T. Odijk, Philos. Trans. R. Soc. London, Ser. A 362, 1497 (2004).
- J.Kindt,S.Tzlil,A.Ben-Shaul,W.M.Gelbart,Proc.Natl. Acad. Sci. U.S.A. 98, 13671 (2001).
- P.K. Purohit, M.M. Inamdar, P.D. Grayson, T. Squires, J. Kondev, R. Phillips, Biophys. J. 88 851(2005).
- S.Tzlil, J.T.Kindt, W.M.Gelbart, A.Ben-Shaul,Biophys. J. 84, 1616 (2003).
- W.S. Klug, M.T. Feldmann, M. Ortiz, Comput. Mech. 35, 146 (2005).
- W.S. Klug, M. Ortiz, J. Mech. Phys. Sol. 51, 1815 (2003).
- Zhidong Li, Jianzhong Wu, and Zhen-Gang Wang, Biophys.J. 94 737746 (2008).
- R. Metzler and P.G. Dommersnes, Eur Biophys J 33 497505 (2004).
- A.Evilevitch,L.Lavelle,C.M.Knobler,E.Raspaud,W.M. Gelbart, PNAS U.S.A. 100, 9292 (2003).
- P. Grayson, A. Evilevitch, M.M. Inamdar, P.K. Purohit, C.M. Knobler, R. Phillips, W.M. Gelbart, Biophys.
J. 88, 230A (2005).
....
... plus simulations reviewed by D. Angelescu and P. Linse, Soft Matter 4 1981-1990 (2008).
The standard formulation (with several variations) is usually of this type:
Total (free) energy = interaction energy + bending energy
The bending energy is the lesser unknown and is standardly assumed to be of the form:

The interaction part of the free energy
authors authors
All (almost) existing formulations rely on a model or empirical expressions for the interaction free
energy, assuming there is no doubt regarding the elastic free energy.
But what one actually measures and
knows experimentally is the osmotic
pressure that can be measured in the
osmotic Boyle experiment.
For us the goal was then:
Reformulate the inverse spool model on
the level of the thermodynamic
observables i.e.
osmotic pressure.
The fitting form from: P. Grayson et al., Biophys. J. 88, 230A (2005). ... and then apply the principles of
equilibrium thermodynamics, obtain the chemical potential and the equilibrium packing fraction.
D = D(+)

Equivalence of osmotic
pressure
(PEG or DEX etc.)
Podgornik et al. 2001.
Osmotic Boyle experiment in vitro - bulk equation of state.
Di!erent regions of DNA density acually correspond to di!erent mesophases.
The equation of state of DNA in the bulk is its osmotic pressure as a function of DNA density
for any given (temperature, ionic strength, nature of salts...) condition (osmotic stress technique,
Parsegian et al. 80).

Conmar Robinson in 1958
described the cholesteric phase of
poly(benzyl-L-glutamate) in
dimethylformamide and in 1961 of
DNA.
indications of ordered phases of
DNA in SAXS (small angle X-ray
scattering)
phases with short fragment DNA
(146 bp about 50 nm)
line hexatic phase
(1996)
cholesteric phase
(1961)
DNA mesophases.

Nature of DNA mesophases.
Durand, Doucet, Livolant (1992) J. Physique 2, 1769-178
Pelta, Durand, Doucet, Livolant (1996) Biophys. J., 71, 48-63.

Rau et al., 1997. Podgornik et al. 2000
Monovalent counterions. Polyvalent counterions.
What one observes (large to small density of DNA) for monovalents is a strong hydration repulsion
followed by the uctuation enhanced electrostatic repulsion.
Electrostatics can only be observed masqued by uctuations.
For polyvalents one observes an (correlation) attractive force mixed with repulsion (~ 0.1 kT/ bp)
just like vdW equation of state!
Monovalent vs. polyvalent counterions
Fundamental di!erences in the measured equation of state i.e. osmotic pressure.

Hexagonal array of DNA with
poly-counterions:
(Lyubartsev and Nordenskiold, 1995)
A pair of DNAs with poly-counterions:
(Gronbech-Jensen et al. 1997)
Attractions set in when counterion valency is above 2, leading to an equilibrium spacing between DNA.
Polyvalent counterion-mediated attractions
Z

Non-monotonic interaction pressure, like in the experiment with CoHex and salt.
Existence of critical pressure. Can we reach quantitative agreement?
Dressed counterions theory
What is the origin of the vdW-like iso-ionic strength curves in the equation of state? In many
component systems of highly asymmetric electrolytes one gets vdW-form osmotic pressure curves
(Kanduc et al. 2010) due to an interplay of correlation attraction and mean-eld repulsion.

Osmotic Boyle experiment in viro - viral equation of state.
Compressing the DNA in a capsid by an osmotic balance. Similar to the osmotic stress experiment
for the bulk DNA. Idea and experiment from the Gelbart lab.
Changing the DNA density in the capsid and measuring the pressure. Osmotic pressure within
the capsid as a function of DNA density: (osmotic) equation of state in viro or in capsida.
D.Marenduzzo et al. (2009).

E. Nurmemmedov, M. Castelnovo, C. E. Catalano and A. Evilevitch, Quart. Rev. Biophys. 40 327 (2007).
Ejection % for EMBL3, lambda c160 and lambda c 221
bacteriophages. Approximate length of the genome is
37.7 kbp and 48.5 kbp. Main features of the experiment
are captured by the inverse spool model.
Boyle experiment.
Again equivalence of osmotic
pressure
(PEG or DEX etc.)
Evilevitch et al. 2008
The energetics of genome packing.
Encapsidated DNA osmotic pressure - viral equation of state.

The two equations of state.
DNA in the bulk (in vitro) and DNA in the capsid (in viro or in capsida).
Osmotic pressure and its dependence on the DNA density, i.e. the equation of state, appear to be
the unifying view of both packing problems.
E!ectively the same method - the osmotic stress method - (with slight variations) can be used in
order to determine both of them. The question now is : what is the connection between the two?
We thus need a theory of packing of DNA within the capsid - inverse spool model.

Viral equation of state and DNA encapsidation.
How to explain quantitatively the osmotic DNA encapsidation data and connect them with the bulk
equation of state? By applying the inverse spool theory.
The inverse spool model is characterised by interactions and curvature:
or the two corresponding (free) energy densities:
We assume here that DNA conguration is described by the (continuum) density and director elds.
A continuum theory.

Continuum nematic nanodrop model of packing.
elastic constants
(bare & interaction)
persistence length of DNA, ~ 50 nm.
DNA elastic constant.
Frank elastic energy: splay twist bend
Assume that DNA inside the capsid is like an ordinary polymer nematic (it sure looks like it).
All three elastic constants are density dependent, but for long semiexible polymers only one elastic
constant is important. The other two are negligible (for various reasons).
The elastic free energy part is then:

The osmotic pressure.
The total free energy is composed of bulk DNA (interaction) free energy, elastic free energy
and chemical potential since DNA can exchange between inside and outside of the capsid.
From this it follows (rst integral of the EL equation) that:
The stress tensor as well as the osmotic pressure can be derived from the rst integral of the EL
equation (following LL).
Similar to the rst integral in the PB theory of electrostatic interactions:
total osmotic pressure = osmotic pressure of bulk DNA + elastic free energy
total pressure = osmotic pressure of ions - electrostatic eld energy
The total osmotic pressure has to be a constant.
Same as for ordinary LC but here elastic constants are density dependent!

Cylindrically symmetric spooling inside viral capsid. The total amount of DNA within
the capsid is xed as:
Packing symmetry and viral loading curve.
From this we obtain the osmotic loading or osmotic encapsidation curves.
The inverse method (from elasticity theory) : assume a director prole and its symmetry.
Siber et al. (2008).

Equilibrium local osmotic pressure and the inverse spool.
interaction pressure curvature pressure total pressure
Inverse spool! Derived from nanomechanics. Quadratic depletion at the center.
No need to assume the depletion at the core (Odijk & Slork).
Osmotic pressure (measurable) as opposed to chemical potential is the main variable.
Depletion of the polymer (DNA) at the center of the capsid due to high bending energy.
The total osmotic pressure for an inverse spool (cylindrical symmetry) is then given by:
Two asymptotic forms of the solution.

DNA density prole inside capsid.
DNA density prole for monovalent and polyvalent salt. extracted from the bulk DNA equation of state.
Monovalent and polyvalent salt density proles show marked di!erences. Density jumps in the
polyvalent case. The di!erence should be experimentally observable.

Bulk osmotic pressure: mono vs. polyvalent counterions
Big di!erences also in the encapsidation curves.
In many component
systems of highly
asymmetric
electrolytes one gets
vdW-form osmotic
pressure curves.

Viral DNA equation of state: loading curve.
Encapsidated fraction as a function of external osmotic pressure. This is now the connection between
the in vitro and in viro.
Small (almost negligible) e!ects of DNA elasticity as embodied in the persistence length.

Free DNA toroid formation
For su)ciently large polyvalent counterion
concentration collapse of DNA.
Depending on the details the collapse can be
into a toroidal structure.
Hud & Downing (2001)
* phage w CoHex.
Toroid formation in solution, 70s. Evdokhimov et a. (1972) was the only one (?) who observed
torus formation with PEG.
95-185 nm, 35-85 nm, 2.7 nm
L
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o
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i
c

p
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e
s
s
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e
)

(
d
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2
Free DNA toroid formation - calculation
Previous mesoscopic theory of DNA nanodrop has to be upgraded leading to a minimization of
the elastic plus surface energy (Ubbink & Odijk 1996):
Siber, Losdorfer & Podgornik (2010). , = 0.15, 0.5, 20 (outer, middle and inner toroid). The outer radii
are approximately 160 nm, 120 nm and 50 nm, respectively.
For spontaneous aggregation of DNA the total osmotic pressure has to be p=0!
In order for this problem to be solvable, the interaction osmotic pressure has to have an attractive
branch - attractive interactions.

2
Encapsidated DNA toroid formation
In T5 bacteriophage: Leforestier & Livolant, 2009.
Livolant, 2009.
Formation of DNA toroids inside conning bacteriophage capsids. A careful examination of Figure 5
in Jeembaeva et al. (2008), led us to recognize the presence of toroids in partially DNA-lled Lambda
phage particles.
This problem is more complicated since there are additional interactions between the surface (inside)
moieties of the capsid proteins and the DNA toroid. Not much is known about these interactions.

T5 bacteriophage capsids containing a ds DNA fragment of about 17-m (50,000bp - initial 38,7 -m)
after partial ejection of their genome. In the presence of monovalent cations (a,a) the DNA chain
occupies the entire volume of the capsid (a). DNA toroids are formed by addition of polyamines
(Spm4+) (b-b)) or PEG (c-c) in the bu!er (15% solution of PEG 6000, osmotic pressure 3.2 atm). The
polycations can go through the capsid whereas PEG stays outside. Scale bar= 20nm.
Condensation in viro
2.88 nm
3 nm

Details of the experiment.
2.88 nm
3 nm
Receptor + 100mM NaCl, 1mM CaCl2 and 1mM MgCl2 ... 5 mM Spm

Calculating the shape of DNA condensed in viro
We add the surface free energy to the total free energy. We assume that the capsid DNA
toroidal condensate interactions are very short range:
with the surface free energy (. can be positive or negative) and the total length of DNA:
By minimizing the total free energy we get the shape of the DNA condensate with appropriate
constraints. A straightforward generalization of the Odijk-Ubbink calculation. Ubbink, J. and Odijk
T.,Europhys. Lett. 33, 353 (1996)

a) The shapes of DNA toroids in the [,, .] parameter space. Blue dashed lines in a) indicate the inner
surface of the spherical container with R = 40 nm. The volume of the DNA in these calculations was
xed to V = 1.4 10^4 nm^3. b) Three-dimensional rendering of the DNA toroid with , = 1, . = 0 c)
Three-dimensional rendering of the DNA toroid with , = 10, . = 100.
Deformation of DNA condensed in viro

2
Shape of a collapsed encapsidated DNA aggregate
Because the available volume inside the viral capsid is smaller then the natural size of
the collapsed DNA toroid, the aggregate has to interact sterically with the capsid walls!
Attractive interactions Repulsive interactions

DNA - T5 capsid interactions drastically change the shape of
condensed DNA. From convex toroidal to non-convex toroidal.
(Spm) (PEG)
Spm vs. PEG condensed DNA in viro

Fundamental difference between PEG and Spm
Why?
The semi-permeable capsid is rigid and the volume of the capsid is kept to a good
approximation a constant. After being collapsed, DNA occupies only a fraction of the capsid
volume and two compartments are created inside the capsid (left): one containing DNA with
its associated water and counterions and another one devoid of DNA. In Rau, Parsegian et
al. experiments (right) no semi-permeable membrane and therefore only one compartment:
the DNA solution. The existence of the second compartment could possibly be a
consequence of the negative hydrostatic pressure inside the capsid caused by its
impenetrability to the external PEG but we can not say anything more definitive at this
point.

EST MODUS IN REBUS SUNT
CERTI FINES DENIQUE

Molecular Biophysics

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average size of the chain

orientational persistence of the chain

orientational correlations along the chain

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