single molecule: detection and manipulation ilan tsafrir tamar arzi physics of complex systems -...
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•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
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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
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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
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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]
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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
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• 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
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Transcription Regulation by looping of DNA
DNA
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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
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Looping - influence of bending protein
Protein Induced
Asymmetric
IHF
IHF
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IHF - Integrating Host Factor This protein induces a U-Turn in DNAIHF - blue/red DNA - green/yellow
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Evanescent Illumination(/(
0zaeII
BeadBead
Bead
Microscope Objective
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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.