Single Molecule Studies of DNA Mechanics

with Optical Tweezers

Mustafa Yorulmaz

Koç University,

Material Science and Engineering

March 25, 2008 Koç University, Material Science and Engineering

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Outline Motivation Methods Models Aims Basics of optical tweezers Physics beyond optical trapping Experimental part Force-extension curves Results Conclusions

March 25, 2008 Koç University, Material Science and Engineering

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Motivation

Physical and chemical studies of DNA were performed in bulk previously and measurements were averaged which made it difficult to resolve the time-dependent stresses and strains.

Now, it is possible to make direct measurements of mechanical properties of DNA.

The nature of interactions between proteins and DNA can be investigated with this process.

Knowledge of DNA’s stretching and twisting properties leads to knowledge of DNA replication and transcription.

March 25, 2008 Koç University, Material Science and Engineering

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Techniques

There are two techniques in general that are used to study the mechanical properties of DNA; Optical tweezers

Apply and sense forces on micron-sized dielectric particles in an aqueous environment

Atomic force microscopy Force extension curve can be obtained through the use

of a tip at the end of a flexible cantilever of known force constant

March 25, 2008 Koç University, Material Science and Engineering

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Models

Freely jointed chain (FJC) model The molecule is made up of

rigid segment

Worm-like chain (WLC) model The molecule is treated like a

flexible rod of length L curves smoothly

The stiffer the chain, the longer the persistence length

/s Pe

(1995)( )

1 1

4(1 / ) 4B

p o o

Marco Sigga WLC

k T xF

L x L L

. . .(1995)( )

2coth 1

2p B

oB p o

Smith et al FJC

FL k T Fx L

k T FL K

In order to understand the physics of DNA, two models were used;

March 25, 2008 Koç University, Material Science and Engineering

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Aims

The force-extension (F-x) regimes of DNA were studied to investigate the elastic behaviors of DNA

Experimental F-x relationships were fit to the entropic elasticity theories based on FJC and WLC models

Persistence lengths which characterize the elastic behavior of DNA were obtained through these fits.

March 25, 2008 Koç University, Material Science and Engineering

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Basics of optical tweezers Optical Tweezers are examples of optical trapping which is a

subfield of laser physics. They are used to trap and manipulate particles (in range from m

to nm) and also to measure the forces (in range from 1 to 100 pN) on these particles

A strongly focused laser beam is used for trapping the particles. The particles that are of interest has to show dielectric property.

* http://www.physics.uq.edu.au/lp/tweezers/papers/nieminen2001jqsrt70/Y. Kimuraab and P. R. Bianco Analyst, 2006, 131, 868-874

March 25, 2008 Koç University, Material Science and Engineering

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Physics beyond optical trapping Trapping occurs due to radiation

pressure of laser which results by the momentum change of light.

There are two forces that let the particle to be hold in trap: Scattering force: due to reflection of

light (to push object) Gradient force: due to refraction of

light (to pull object)

nparticle>nmedium so that Fgradient>Fscattering

Using high NA objectives and high power lasers maximizes the trapping force.

Schematic diagram showing the force on a dielectric sphere due to both reflection and refraction of two rays of light.

March 25, 2008 Koç University, Material Science and Engineering

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Illustrative examples from our laboratory: Manipulation of microspheres

Thanks to Ahmet Faruk Coşkun

March 25, 2008 Koç University, Material Science and Engineering

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Experimental part

Two ends of DNA were attached to dielectric spheres.

Two ends of DNA were attached to the trapped dielectric sphere and glass substrate

M. D. Wang et al., Biophys. J. 72, 1335

One end of the DNA is attached to the glass substrate and the other end is attached to the dielectric particle which is trapped by the optical tweezer

The dual beam optical tweezer is used for stretching the DNA

http://www.the-scientist.com/article/display/15545/

March 25, 2008 Koç University, Material Science and Engineering

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Force-extension curves

F-x curve for -phage dsDNA. The data are fit to WLC models and FJC model. Persistence length P=53 nm.

Bustamante, C., S. B. Smith, J. Liphardt, and D. Smith, 2000, Curr. Opin. Struct. Biol. 10, 279

F-x curve of DNA which is stretched between two dielectric particles is shown. We observe the overstretching over 60 pN

Bustamente, C., Bryant Z., Smith S. B., Nature 421 423-427

March 25, 2008 Koç University, Material Science and Engineering

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Force-extension curve

Data of F-x of a single DNA were fit to WLC and FJC model. Contour length of the DNA is Lo=~1314 nm and persistence length is found as Lp= ~43 nm for WLC models and Lp=~3.94 nm for FJC model

M. D. Wang et al., Biophys. J. 72, 1335

March 25, 2008 Koç University, Material Science and Engineering

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Results

Persistence lengths are calculated under different buffer conditions of 10mM Na+ and Mg2+ or spermidine3+ to be ~0.47 nm and ~0.40 nm respectively: Entropic elasticity.

There was no further decrease in persistence length: Intrinsic elasticity.

DNA behaves like an ideal entropic springs for low-to-moderate extensions beyond their rest length.

Overstretching of DNA was observed above extension forces for >6o pN.

March 25, 2008 Koç University, Material Science and Engineering

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Conclusions

Use of optical tweezers is very useful for determining the mechanical properties of single molecules of DNA.

This relationship informs us about the biological interactions.

March 25, 2008 Koç University, Material Science and Engineering

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Thank you for your listening..

Questions??

March 25, 2008 Koç University, Material Science and Engineering

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Mechanical Properties can be obtained

B pk TL

22

( )2 2B p B pk TL L k TL

ER L

Bending rigidity of the polymer represented as an elastic rod

Energy required to bend a segment. It reveals information about interactions between protein and DNA as DNA is bending protein convert some part of its binding energy into mechanical work.