Dissecting DNA Dynamics

with Optical Tweezers

Kristopher Hite
Hansen Lab
2/5/08
 Optical trap Origins

Arthur Ashkin circa 1988 at Bell Labs Steven Chu

“I started in 1970 with my discovery Winner - 1997 Nobel prize in

that I could optically trap small latex Physics
spheres. And I went on to propose that
we could do the same thing with Director - Lawrence Berkeley
atoms.” National Laboratory
 Light can manipulate micron-sized
particles
• Dielectric particles ranging from plastic beads to single

atoms can be held in 3 dimensions by focused laser beams

• Momentum transfer associated with bending light is basic

principle that enable optical tweezers.

• First demonstrated with a single focused laser beam in 1986

Momentum transfer - enables optical trap.

200 mW
~800 nm
Newton’s third Law: explains why the bead is trapped at
the waist of the focused laser beam
 Pulling on DNA

• A practical application for optical tweezing

• Attaching one polystyrene bead to each

end of a dsDNA molecule.

– Trap one bead in the optical tweezers and tug

on the other with a micropipette

Polystyrene beads
Coated with Steptavidin

~2.5 μm
Laminar Flow
 λ-phage DNA – 48.5
kbp

Ex
Fully extended = 16.4 μm
Assuming 3.38 Å/bp
Claudet, C. and J. Bednar,
Pulling the chromatin.
Eur Phys J E Soft Matter, 2006. 19(3): p. 331-7.
 Why go through this trouble?

• Can precisely measure force by

measuring bead displacement.
• Look at the ceiling above the objective
• Can calibrate how much force is required
to eject the bead from the trap
• Using Stoke’s law
 Measuring trap stiffness
Stoke’s Law
F = 6πrµv
– F is the frictional force,
– r is the radius of the spherical 
object,
– μ is the fluid viscosity
– v is the particle's velocity.
 Pulling on DNA
• Applications
– DNA strand alone.
• dsDNA, or ssDNA
– DNA complexed with…
• DNA binding proteins
• Dyes
– Chromatin
– Claudet, C. Et. al. 2006
 Pulling on DNA
• DNA strand alone.
• dsDNA, or ssDNA
– Smith, S.B., Y. Cui, and C. Bustamante,
Overstretching B-DNA: the elastic
response of individual double-
stranded and single-stranded DNA
molecules. Science, 1996. 271(5250): p.
795-9.
65 pN force applied to dsDNA causes transition to stable and
reversible elongated form.

Smith, S.B., et. al. 1996

dsDNA rapidly and reversibly snaps
into alternate elongated
conformation
• Under stretching force of 65 pN dsDNA
– undergoes cooperative transition into a stable
form with a rise/bp = 5.8 Å
– Is 70% longer than B-form DNA
– Can rapidly restore normal contour length
when relaxed below 65 pN
– Ionic strength of aqueous buffer affects force
required for transition
 Transition affected by ionic strengths of solution

150 mM
NaCl

5 mM
NaCl
2.5 mM
NaCl

0.625 mM
NaCl

Smith, S.B., et. al. 1996

 DNA elongation by protein
Binding

• RecA protein
– E. coli protein (human homolog Rad51)
– Binds ssDNA and dsDNA
– Lengthens dsDNA from 3.4 Å to 5.1 Å
• Bennink ML, Schärer OD, OD, Kanaar R, Sakata-Sogawa K, Schins JM,
Kanger JS, de Grooth BG, Greve J. Single-molecule manipulation of
double-stranded DNA using optical tweezers: interaction
studies of DNA with RecA and YOYO-1.Cytometry. 1999 Jul
1;36(3):200-8
 RecA protein elongates dsDNA as it binds without externally applied force

Bennink ML, et. al. 1999

When external force is applied –
On rate increases
Off rate decreases

Bennink ML, et. al. 1999

 Optical tweezers applied to
nucleoprotein complexes

• Kinetics of binding can be measured on a

single DNA molecule
• Activation energy of DNA binding can be
changed by applying external force
 Acknowledgements
• Dr. Robert Woody
• Dr. P. Shing Ho

• Hansen lab