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Single-molecule DNA Manipulations. Course Overview. Day 1: Techniques & Basic Results Day 2: Twisting DNA molecules Day 3: DNA and RNA polymerases Day 4: DNA topoisomerases Day 5: DNA packaging. Day 1: Techniques & Basic Results. Historic overview & Introduction

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Single-molecule DNA Manipulations

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Single molecule dna manipulations l.jpg

Single-molecule DNA Manipulations


Course overview l.jpg

Course Overview

  • Day 1: Techniques & Basic Results

  • Day 2: Twisting DNA molecules

  • Day 3: DNA and RNA polymerases

  • Day 4: DNA topoisomerases

  • Day 5: DNA packaging


Day 1 techniques basic results l.jpg

Day 1: Techniques & Basic Results

  • Historic overview & Introduction

  • Measurement techniques, calibration, noise

  • Stretching nucleic acids

  • Single-molecule DNA sequencing?

  • Zero-force experiments (fluorescence measurements)


Length energy and force scales l.jpg

Bacteria

~eV

~kBT

(4 x 10-21 J)

or 4 pN nm

(1.6 x 10-19 J)

Length, energy- and force-scales

Energies


Rotation of the f1 atpase l.jpg

Rotation of the F1-ATPase

Noji et al., Nature (1997) 386: 299-302.


Single molecule paradigm l.jpg

Object localized in space

Real-time readout

“Synchronized”

Reversible

Object freely diffusing

Offline readout

“Unsynchronized”

Irreversible

Single-molecule paradigm

Single-molecule assay

Bulk biochemical assay


Instruments l.jpg

Fixed-position

Atomic Force Microscope

Micropipette

Optical Tweezer

Fixed-force

Magnetic Trap

Instruments

Feedback loop can convert one into the other


Atomic force microscope l.jpg

Atomic Force Microscope

Cantilevers:

~50-100 mm long, 30 mm wide, 0.2 mm thick

High spatial positionning accuracy (0.1 nm)

Very stiff (5-100 pN/nm) cantilever

High-force instrument (~10 pN-1 nN)

High bandwidth (~kHz in water)

Large size and high bandwidth lead to large noise:

5-10 pN rms noise with 1 kHz bandwidth

High noise is due to size ( ~0.5 pN/Hz-1/2) smaller cantilevers being developped

  • Measurement modes (imaging, stretching)


Micropipette l.jpg

Micropipette

Weaker cantilever (~2 pN/mm)

Larger cantilever, lower bandwidth

 lower force noise than AFM

High forces achievable

Rotation also possible (Bustamante, Heslot)


Optical trap l.jpg

Optical Trap

Relatively stiff (~0.1-1 pN/nm)

High Forces (~100 pN)

Small beads (~0.5 mm)

High bandwidth (~kHz)

Low force noise

Rotation possible but difficult

Bead trapped at beam waist

Higher dielectric than water needed


Magnetic tweezer l.jpg

Magnetic Tweezer

Very weak stiffness of “trap” (pN/mm)

 results in a “constant force” mode

 requires a stiff tether

Force depends on bead size:

1 mm dia bead ~ 1 pN

2.8 mm dia bead ~ 15 pN

4.5 mm dia bead ~ 80 pN

Bandwidth depends on bead size

Low force noise, ultra stable and very low drift

Rotation easy


Other techniques l.jpg

Other techniques

  • Flow fields

    • Force changes along DNA

    • Costly in protein

  • Electric fields

    • Force changes along DNA.


Force calibration l.jpg

Force Calibration

  • Calibration against flow field (F=6phrv)

  • Micropipettes/AFM cantilevers can be calibrated using a set of levers of decreasing stiffness

  • Trap stiffness in all cases easily determined by analyzing Brownian motion


Brownian motion analysis l.jpg

Fx= Fsinq ~ Fq ~ F

dx

__

l

F

F

__

__

l

l

1

1

_

_

<dx2> = kBT

kx<dx2> = kBT

2

2

Brownian Motion Analysis

(Tethered bead in a harmonic potential)

_

Fx = dx = kxdx

Equipartition of energy:

kBT l

____

F =

<dx2>


Excellent low force measurement technique l.jpg

Excellent low-force measurement technique


Signal to noise l.jpg

Signal-to-noise

Thermal agitation causes the mean force to fluctuate with a variance

<dF2> = 4kBT 6phr Df

For a ~1 mm diameter bead in water (h = 10-3 poise) at room temperature,

dF ~ 10 fN/Hz1/2

The detector (bead, cantilever) undergoes rms fluctuations

in its mean position:

dz = dF/kz

(for DNA in our experiments k ~ 10-8 to 10-7 N/m)

dz ~ tens of nm with ~1 s averaging

  • To reduce noise, three approaches (each with its own problems):

  • Average longer

  • Smaller detector

  • Stiffer (i.e. shorter) DNA or polymer


Polymer springs l.jpg

l

3

_

k BT

___

_

2

x

l0

Polymer Springs

  • Polymer elasticity characterized by

    • Persistence length x = A/kBT

    • Relative extension l/l0

  • Simplest case: random walk (Freely-Jointed Chain, FJC)

    • Fully flexible joints between persistence-length units (no bending energy)

    • Entropic elasticity at low force: F=

    • High force is like aligning spin with mag.field


Freely jointed chain vs worm like chain l.jpg

Freely-Jointed Chain vs. Worm-like chain

A: entropic

B: enthalpic

C: “overstretch”

1st measurement: Finzi et al., Science (1992) 258:1122-6.


Effect of ionic conditions on x l.jpg

Effect of ionic conditions on x

Fit is to Poisson-Boltzmann model for uniformly charged cylinder

Baumann et al., PNAS (1997) 94:6185-90.


B s transition and ssdna l.jpg

BS transition and ssDNA

C

B

D

A

Cluzel et al., Science (1996) 271:792-4.

Cui et al., “ “ “ 795-9.


Protein elasticity unfolding titin l.jpg

Protein elasticity:unfolding titin

Rief et al., Science (1997) 276 :1109-12.


Stretching ssdna l.jpg

Stretching ssDNA

Dessinges et al., PRL (2002) 89, 248102.


Base pairing disrupted by salt or chemical modification l.jpg

Base-pairing disrupted by salt or chemical modification

  • = 0.8 nm

  • Y ~ 200 Mpa

  • Electrostatics+pairing

  • Self-avoiding

ssDNA FJC:

Dessinges et al., PRL (2002) 89, 248102.


Single molecule sequencing l.jpg

Single-molecule sequencing?

  • l-Exonuclease

  • a-hemolysin/nanopores (D. Branton Harvard/Rowland)

  • Optical waveguides (W. Webb, Cornell U)

Human genome: ~3 Gbp…how to sequence in one hour??


Digestion of dna by l exonuclease l.jpg

Digestion of DNA by l-Exonuclease

“Direct” assay

“Conversion” assay

Perkins et al., Science (2003) 301: 1914-8.

Van Oijen et al., Science (2003) 301:1235-8.


Constant force digestion rate l.jpg

Constant-force digestion rate

Perkins et al., Science (2003) 301: 1914-8.


Digestion rate is force independent l.jpg

Digestion rate is force-independent

Perkins et al., Science (2003) 301: 1914-8.


Conversion of dsdna to ssdna l.jpg

Conversion of dsDNA to ssDNA

Van Oijen et al., Science (2003) 301:1235-8.


Waveguide sequencing l.jpg

Waveguide Sequencing

Levene et al., Science (2003) 299: 682-6.


Folding unfolding of structured rna l.jpg

Folding/unfolding of structured RNA

Liphardt et al., Science (2001) 292: 733-7.


Folding unfolding of structured rna31 l.jpg

Folding/unfolding of structured RNA

DG ~ F1/2Dx = 14pN x 20 nm = 280 pNnm = 70 kBT

(or, ~170 kJ/mol)

Liphardt et al., Science (2001) 292: 733-7.


Effect of an external potential on rates l.jpg

Effect of an external potential on rates

Energy landscape

(no external force)

Energy landscape

(including external force)

Energy

Dln

Dld

Potential energy

from external force

Reaction coordinate (distance, l) along stretching force

a(F) = a0 exp(FDln/kBT)

b(F) = b0 exp(-FDld/kBT)


Unzipping dna l.jpg

Unzipping DNA

Essevaz-Roulet et al. PNAS (1997) 94 11935-11940

Spatial resolution ~ 50 nm

Needle stiffness ~ 1.7 pN/mm

If Eu ~ 2kBT and Dxu ~ 2x0.3nm, we expect Fu ~ 13 pN


Unzipping signal and force flips l.jpg

Unzipping signal and force flips

Essevaz-Roulet et al. PNAS (1997) 94 11935-11940


Tirf based detection of dna hybridization l.jpg

TIRF-based detection of DNA hybridization

Singh-Zocchi et al., PNAS (2003) 100: 7605-10.


Hybridization signal l.jpg

Hybridization signal

Singh-Zocchi et al., PNAS (2003) 100: 7605-10.


Combining manipulation and visualization actin myosin interaction l.jpg

Combining manipulation and visualization: actin/myosin interaction

Ishijima et al. Cell (1998) 92:161-71.


Combining manipulation and visualization dna unzipping l.jpg

Combining manipulation and visualization: DNA unzipping

Lang et al., J Biol. (2003) 2 :6


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