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Measuring DNA Flexibility (Bending & Twisting) Magnetic Traps, WLC. How Mag. Traps Work Equipartion Theorem, Brownian Noise Worm Like Chain– model for extended biopolymer -- DNA (later look at AFM & Protein Folding) . --DNA has Linking Number =Twist and Writhe. polymers. DNA.

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Measuring dna flexibility bending twisting magnetic traps wlc
Measuring DNA Flexibility (Bending & Twisting)Magnetic Traps, WLC

  • How Mag. Traps Work

    Equipartion Theorem, Brownian Noise

  • Worm Like Chain– model for extended biopolymer -- DNA (later look at AFM & Protein Folding) .

    --DNA has Linking Number =Twist and Writhe


Dna structure

polymers

DNA

DNA Structure

  • Right-hand helix

  • One turn: 3.4 nm, ~10.5 bp

  • Twist angle between bps θ=36

Molecular Cell Biology, Lodish

Wikipedia

2nm

DNA will resist twisting


How stiff is dna longitudinally laterally magnetic trap movie web browser measureforce 3g2

N

S

Microscopy

Video camera CCD

How stiff is DNA, longitudinally, laterally?Magnetic Trap movie (Web-browser: Measureforce.3g2)

How to attach DNA: to glass; to paramagnetic bead

Set-up of Experimental system

Detect nanometer displacements with visible light

Experimental Set-up


Magnetic tweezers and dna
Magnetic Tweezers and DNA

Can be conveniently used to stretch and twist DNA.

With Super-paramagnetic bead, no permanent dipole.

More sophisticated experiments: Watch as a function of protein which interacts with DNA (polymerases, topoisomerases), as a function of chromatin: look for bending, twisting.

Dipole moment induced, and ma B. t = m x B = 0

U = - m. B : U ~ -moB2.

F = - U (Force is always the slope of the energy)

Δ

It is the gradient of the force, which determines the direction. The force is up, i.e., where B is highest.

  • DNA tends to be stretched out if move magnet up.

  • DNA also tends to twist if twist magnets

  • (since m follows B).

  • (either mechanically, or electrically move magnets)

Forces ranging from a few fN to nearly 100 pN: Huge Range


What is super paramagnetism
What is super-paramagnetism?

  • Three types of magnetic response to Bext (called the magnetization)

  • Diamagnetic: Electrons are paired up (as is normal). Electron spins cancel. Weak suspectibility, w slight repulsion. Bext 0, Bint  0. Most atoms (Copper, silver, gold….)

  • Paramagnetic: Have an unpaired electron (spin). Small suspectibility, w slight attraction. Feels greater effect of Bext. Bext 0, Bint  0. Includes magnesium, molybdenum, lithium, tantalum.

  • Ferromagnetic: Have an unpaired electron (spin), and align in domains (1012 to 1015 atoms). Large positive susceptibility, i.e. strong attraction to Bext. Retains Bint. Iron, nickel, cobalt.

  • Super-paramagnetic: small particles which are ferromagnetic but magnetism can flip randomly w temp. Average Bint is random; an external magnetic field is able to magnetize the nanoparticles, similarly to a paramagnet. However, their magnetic susceptibility is much larger than the one of paramagnets. (Important in maximum capacity in hard-drives– how small can you go & still get super-paramagnetism).

http://www.ndt-ed.org/EducationResources/CommunityCollege/MagParticle/Physics/MagneticMatls.htm

http://en.wikipedia.org/wiki/Superparamagnetism


Measuring dna flexibility bending twisting magnetic traps wlc
Using Brownian noise.To know F, you might need to know size of magnetic field and bead’s susceptibilityF=kxKnow F, measure x, get k.

Difficult.

Instead, analysis of bead’s Brownian motion.

DNA-bead system acts like a small pendulum

pulled vertically (z) from it’s anchoring point,

subject to Brownian fluctuations (x,y).

[Mag Traps.x y motion.force.swf]


Measuring dna flexibility bending twisting magnetic traps wlc

kB T l

F =

< x2 >

Force measurement- Magnetic Pendulum

The DNA-bead system behaves like a small pendulum pulled to the vertical of its anchoring point & subjected to Brownian fluctuations

Do not need to characterize the magnetic field nor the bead susceptibility.

Just use Brownian motion.

Equipartition theorem:

Each degree of freedom goes as x2 or v2 has ½kBT of energy.

Derive the Force vs. side-ways motion.

½ k < x2> = ½ kBT

F = k l

Note: Uvert. disp = ½ kl2

Udx displacement = ½ k(l2+dx2)

Therefore, same k applies to dx .

½ (F/ l) < x2> = ½ kBT

T. Strick et al., J. Stat. Phys., 93, 648-672, 1998


Measuring dna flexibility bending twisting magnetic traps wlc

Focal point

Z

Z

Magnetic Traps: Measuring DNA extension

Diffraction rings

Can get like 10 nm resolution

(with visible or IR (500-1000 nm light!)


Measuring dna flexibility bending twisting magnetic traps wlc

How to measure distances so accurately

center

The width of the distribution

(the standard deviation)

is ≈ 250 nm (l/2)

But this doesn’t tell you

about the

standard error of the mean

= sd/√N

width


Measuring dna flexibility bending twisting magnetic traps wlc

Force measurements- raw data

kB TL

F =

< x2 >

Measure < x2 >, L

and get F

Measure z, measure dx

Find F by formula.

Example: Take L = 7.8 mm

(4.04 pN-nm)(7800nm)/ 5772 nm = 0.097 pN

At higher F, smaller dx; so does dz.

Z = l

Lambda DNA = 48 kbp = 15 mm

At low extension, with length doubling, dx ~ const., F doubles.

At big extension (L: 12-14 mm),

Dx decrease, F ↑10x.

Spring constant gets bigger.

Hard to stretch it when almost

all stretched out!

X

T. Strick et al., J. Stat. Phys., 93, 648-672, 1998


Measuring dna flexibility bending twisting magnetic traps wlc

Force vs. Extension for DNA

F=-kx works well at very low force;

at higher force, DNA is extended (> 50%), need FJC or better is WLC

At very low (< 100 fN)

and at high forces (> 5 pN),

the FJC does a good job.

In between it has a problem.

There you have to use WJC.

You measure the Persistence length

Strick, J. Stat. Physics,1998.


Measuring dna flexibility bending twisting magnetic traps wlc

Analysis: The Worm-Like Chain for elasticity

F= - kx

Hooke’s Law: You apply a force on something and it increases in length linearly. Proportional constant = k. Minus sign because it’s a restoring force.

Force related to fractional increase (x/L). What if force isn’t proportional to distance?

DNA is much longer than it is wide – λ DNA (virus DNA) is about 16.5 µm upon full extension, but the molecule’s diameter is only about 2 nanometers, or some four orders of magnitude smaller. Can expect some simplification where don’t need to get into details of molecular bonding.

http://biocurious.com/2006/07/04/wormlike-chains


Measuring dna flexibility bending twisting magnetic traps wlc

Towards the Worm-Like Chain for elasticity

try: The Freely jointed Chain (FJC):

The molecule as a chain of perfectly rigid subunits of length b joined

by perfectly flexible hinges. Segment-to-segment angle = q

You can think of b as the length of the repeating subunits, and is called the Kuhn length (= 2 x Persistence length).

Persistence length: you start out going in some direction: how long will you tend to keep going: for DNA—about 50 nm, or 150bp.

If the molecule is under an applied force f as above, the effective energy for the chain is given by

If there were no applied force, all configurations have equal energy (and therefore the system has large configurational entropy), and the chain orients itself in any which way—analogous to a random walk.

http://biocurious.com/2006/07/04/wormlike-chains


Measuring dna flexibility bending twisting magnetic traps wlc

Towards WLC: The FJC

When a force is applied, the molecule is elongated and the work being done is that to remove entropy in the system. An analytic force-extension relation for the freely jointed chain:

Hyperbolic cotangent: coth (x)= cosh(x)/sinh(x) = (ex + e-x)/(ex - e-x)

In the limit of low stretching (expand hyperbolic cotangent about f = 0)

coth(x) = 1/x + (1/3) x – 1/45 x3 +…

we get back Hooke’s law (show!)

(Indeed, all polymer models reduce to a linear relation in the limit of low force, and DNA acts Hookean at very low forces).

In the high-force limit the model predicts z approaches the contour (total) length Ltot as 1/f. Does not follow the force extension curve data for dsDNA or proteins at all.

coth(x) = 1 + e-2x ; as x infinity, coth goes to 1; zLtot = 1-f

http://biocurious.com/2006/07/04/wormlike-chains


Measuring dna flexibility bending twisting magnetic traps wlc

Two Models of DNA

(simple) Freely Jointed Chain (FJC)

& (more complicated) Worm-like Chain (WLC)

FJC: Head in one direction for length b, then turn in any direction for length b.

[b= Kuhn length = ½ P, where P= Persistence Length]

Idealized FJC:

Realistic Chain:

FJC: Completely straight, unstretchable. No thermal fluctuations away from straight line are allowed

The polymer can only disorder at the joints between segments

FJC: Can think of DNA as a random walk in 3-D.

WLC: Have a correlation length


Measuring dna flexibility bending twisting magnetic traps wlc

WLC: A slight extension of FJC

A more realistic model for dsDNA is actually quite similar to the freely jointed chain, but changes from discrete segments to a continuous elastic medium. This can be done because DNA is actually a rather stiff molecule, with successive segments displaying a sort of cooperativity—all pointing in roughly the same direction. The figure below is a cartoon of the wormlike chain (WLC) model, where now we define r(s) as the position as a fuction of the relaxed-state contour length, s. Also shown is the tangent vector t(s), which is the first derivative of r(s) with respect to a line segment ds.

There is now another added term to the effective energy of the chain which is related to the curvature (itself proportional to the square of the tangent vector), and the summation is now replaced by integration along the entire contour length:


Measuring dna flexibility bending twisting magnetic traps wlc

WLC: A slight extension of FJC

A new phenomenological parameter has entered the energy term, A, which is a measure of the persistence length of the chain, or how long a segment of the chain will have tangent vectors all pointing in nearly the same direction. Indeed, the tangent-tangent correlation function for the wormlike chain at zero stretching force is given by

or, the similarity in directionality for the chain decays as an exponential in the persistence length.

[didn’t have time to go over in class]

An analytic solution to WLC is not currently known, but the above equation has been solved numerically.

At low force it again displays a Hookean linear relation, but as the extension nears the contour length of the molecule, it scales not as 1/f as predicted by freely jointed chains, but as 1/f1/2, in significantly better agreement with the data.


Measuring dna flexibility bending twisting magnetic traps wlc

Analysis: The Worm-Like Chain for elasticity

[didn’t have time to go over in class]

Increase in end-to-end length (x) of polymer:

Worm Like Chain (WLC) of entropic elasticity.

Force related to fractional increase (x/L)

where A = Lp: persistence length, a measure of the chains bending rigidity

= 2x Kuhn Length

L = contour length

x = extension

Each unfolding event increases the contour length of the homopolymer by a constant value, ∆L.



Measuring dna flexibility bending twisting magnetic traps wlc

Class evaluation

1. What was the most interesting thing you learned in class today?

2. What are you confused about?

3. Related to today’s subject, what would you like to know more about?

4. Any helpful comments.

Answer, and turn in at the end of class.