•Single Molecule: Detection and Manipulation

Ilan Tsafrir Tamar Arzi

Physics of Complex Systems - Weizmann Institute of Science

•Interaction Between Membranes and Polymers

Joel Stavans’ group:

Jaffar Ali Ido Braslavsky Roee Amit

Single Molecule: Detection and Manipulation

Tools:

•Optical Tweezers•Confocal fluorescence microscopy•Evanescent illumination•Molecular biology techniques

(Ensemble (1018( <=> Single molecule (100

Averages <=> Dynamics, Individual behavior

(/(0

zaeII

Optical Tweezers

Small particles (micron scale(

can be trapped by the intensity gradient of focused light

Noninvasive Techniques in Cell Biology: 375 (1990(

F=-a(

Force (pN(

Microscope Objective

Infrared Light

BeadBead

Pulling on a DNA moleculewith Optical Tweezers

Bead

0 2 4 6 8 10 12 14 16 18

0

1

2

3

4

5

For

ce

[pN

]

Extension [m]

0 2 4 6 8 10 12 14 16 18

0

1

2

3

4

5

Fo

rce

[pN

]

Extension [m]

DNA : Force - Extension Curve

Approximation of the Worm-like Chain Model prediction:

P

Tk

L

X

4

1-

(-4(1

1F B

2LX

• Looping of DNA Opher Gileadi (Weizmann, Molecular Genetics(

Stephen Quake (Applied Physics, Caltech(

• Collapse of DNAStephen Quake

• Single Molecule Enzymatic ActivityDavid Bensimon (ENS, Physics(

Single Molecule: Detection and Manipulation

Transcription Regulation by looping of DNA

DNA

Looping of DNA

Enhancer site

Spontaneous

Gelles, Science 267:378 (1995(

Looping dynamics: Time scales of bound and unbound statesDependence on lengthDependence on Persistence length Science 233:889(1986(

TIBS 20:500 (1995(

P(L(

L

Looping - influence of bending protein

Protein Induced

Asymmetric

IHF

IHF

IHF - Integrating Host Factor This protein induces a U-Turn in DNAIHF - blue/red DNA - green/yellow

Evanescent Illumination(/(

0zaeII

BeadBead

Bead

Microscope Objective

Summary:

• Macromolecules are interesting as a complex physical system while understanding their physical properties could contribute to the understanding of their function as a biological ingredient.

• The experimental tools enable us to monitor and manipulate single molecules.