Fluorescence%20Correlation%20Spectroscopy%20technique%20and%20its%20applications%20to%20DNA%20dynamics

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Fluorescence Correlation Spectroscopy technique
and its applications to DNA dynamics Oleg
Krichevsky Ben-Gurion University in the Negev
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Outline

• Tutorial on FCS
• The basic idea of the technique
• Instrumentation
• Standard applications
• - measurements of concentrations
• - diffusion kinetics
• - binding assay
• DNA dynamics

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3) Polymer conformational dynamics - flexible
polymers (ssDNA) - semi-flexible polymers
(dsDNA) - semi-rigid polymers (F-actin)

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• Tools
• specific fluorescence labeling
• attaching fluorophores at precise positions
• Fluorescence Correlation Spectroscopy (FCS)

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Fluorescence Correlation Spectroscopy
(FCS) Magde, Elson Webb (1972) Rigler et al
(1993)
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General Properties of FCS Correlation Function
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Correlation function for simple diffusion
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Principles of confocal setup
Sampling volume 0.5 fl (Ø 0.45 x 2 mm) Incident
light power 10 - 50mW 0.1-300 molecules per
sampling volume on average
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• Enhancements and variations of the standard
  setup
• Two-color FCS (Schwille et al)
• Two-photon FCS (Berland et al)
• Scanning FCS (Petersen et al)

References and technical details in G. Bonnet
and O.K., Reports on Progress in Physics,
65(2002), 251-297
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Standard applications

1. Amplitude of G(t) ? concentration of moving
   molecules
2. Decay ? diffusion kinetics (in vitro and in vivo)
3. Binding assay

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FCS as a Binding Assay
Protein
Few nm
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Methyltransferase Lambda-DNA (methyltransferase
courtesy of Albert Jeltsch and Vikas Handa)
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with Grégore Altan-Bonnet Noel Goddard Albert
Libchaber Rockefeller University
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DNA hairpin fluctuations Molecular beacon
design TyagiKramer (1996)
to (k-)
tc (k)
5 - Rh6G CCCAA (Xn) TTGGG DABCYL 3
(n12-30) Signal/background Io/ Ic 50-100
I (kHz)
T (oC)
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FCS on Molecular beacons two processes two
characteristic time scales
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structural fluctuations
diffusion
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Control
Beacon
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Correlation functions of beacon control
Ratio of the correlation functions pure
conformational kinetics
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Conformational kinetics at different temperatures
Gconf
t (ms)
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The experimental procedure
1) Melting curves I(T)
I
T
2) FCS on beacons 3) FCS on controls
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Characteristic time scales of opening and closing
of T21 loop hairpin
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Different lengths of T-loops
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The loops of equal length but different sequence
T21 vs. A21
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Stacking interaction between bases
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Opening and closing times of different poly-A
loops
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Placing a defect in a poly-A loop
no defect
PNAS 95, 8602-8606 (1998) Phys. Rev. Letters
85, 2400-2403 (2000)
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In some simple situations we have some
understanding of the sequence-dependence of
hairpin closing kinetics

• In a number of other situations we have no
  undersanding
• poly-C loops
• short poly-T loops (below 7 bases(

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The experimental construct
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Phys. Rev. Letters 90, 138101 (2003)
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Conformational dynamics of polymers in good
solvents on the model of dsDNA and ssDNA
molecules
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Diffusion of dsDNA 6700bp
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Polymer Statistics Freely Jointed Chain model
Random Walks in Space
b
Ree
Ree
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Polymer conformational dynamics
Rouse (1953) Zimm (1956)
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Theory
b2
t
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Rouse theory of Polymer Dynamics
b
g
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Rouse modes
n
0
N
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Rouse model connectivity friction of polymer
segments
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Rouse model is nice but wrong
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Zimm model Rouse model hydrodynamic
interactions
Diverge with N gt cannot be neglected even for
distant monomers
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Zimm model Rouse model hydrodynamic
interactions
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From polymer coil diffusion measurements
Zimm model is right Rouse model is wrong
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Real polymers limited flexibility
b - Kuhn length defines polymer flexibility b
several monomers flexible polymer b gtgt monomer
size semi-flexible or stiff polymer Polymer can
be considered as flexible at the length scale gt b
dsDNA semi-flexible, b100nm340bp, dsDNA width
d2nm ssDNA flexible, b1-5nm2-10bases
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Results 2400 bp fragment
r2 (mm2)
Why no Zimm behavior?
2400bp 7b
t (ms)
small polymer
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Interpretation of g the friction of cylinder
with length b100nm and diameter d2nm
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Why not Zimm-model behavior? dsDNA is
semi-flexible, the hydrodynamic interactions are
weak
Korteweg-Helmholtz theorem when inertia can be
neglected, the flow is organized to have minimal
viscous losses
Rouse model
Zimm model
Rouse regime below
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For dsDNA b100nm, d2nm
Rouse regime from b2 (0.01 mm2) to 18b2 (0.2 mm2)
or R2ee
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Above r2c Zimm behavior 23000bp
Zimm regime No free parameters, No polymer
parameters
Best power fit gives power 0.64
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For flexible polymer No Rouse regime, Zimm
regime only
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Single-stranded DNA
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Theory for semi-flexible polymers parameters
b,d. Harnau, Winkler, Reineker (1996)
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Conclusions

• 1) First measurements of individual monomer
  dynamics within large polymer coil
• 2) There is a large range of dsDNA dynamics
  unaffected by hydrodynamic interactions (Rouse
  model)
• 3) The dynamics of ssDNA is dominated by
  hydrodynamic interactions (Zimm theory)

Phys. Rev. Lett. 92, 048303 (2004)
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Thanks to my group Roman Shusterman Sergey
Alon Tatiana Gavrinyov Carmit Gabay
And to friends and collaborators Grégoire
Altan-Bonnet Noel Goddard Albert Libchaber Didier
Chatenay Rony Granek David Mukamel Albert
Jeltsch Vikas Handa Dina Raveh Anna Bakhrat