Notes about these slides
•
Slides presented at Physics 500 / 400 Seminar @ U. New Mexico,
January 18, 2007 by Steve Koch.
•
I think I have attributed all images and data that aren’t from my own
publications or work, but it’s possible I’ve missed something. You
should probably check with me before propagating anything. In most
cases I can give you original drawing files if you want.
•
As noted on the acknowledgements slides, this work is highly
collaborative, thanks to everyone!
•
Slides are rough & subject to errors…please ask questions in the
discussion forums and talk about things!
Welcome to the Seminar on Biophysics and Medicine!
• Demo course web page
• Course requirements:
– Show up and ask questions!
– Grad students: 10 minute talk about research
• How should we do online discussion forum?
– Openwetware.org
• Demo Pub Med / Bookshelf
Studying protein-DNA interactions by
unzipping single DNA molecules:
What new information can we obtain?
Steve Koch, January 18, 2007, Physics 500/400 Seminar
Assistant Professor, Physics and Astronomy and CHTM
University of New Mexico
Outline
1. Single-molecule manipulation capabilities
provide new biological information
2. Optical Tweezers: Unzipping DNA molecules
to probe protein-DNA interactions
Computer Controlled
Electromagnet
Magnetic Field
F Gradient Force
3. Magnetic Forces: Improved efficiency for
DNA unzipping; eukaryotic RNA Polymerase;
molecular motors
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
TIR
Illumination
Non-magnetic Aspheric
4. I am looking for graduate students!
CCD Camera
Thank you to my wonderful collaborators!
Karen Adelman (NIH), Arthur La Porta (U.
Maryland),
Richard Yeh, Michelle D. Wang
Gayle Thayer, Jim Martin, George Bachand,
Alex Corwin, Maarten de Boer, Amanda Trent
Peter Goodwin, Jim Werner,
Dick Keller, Kim Rasmussen
Funding
DNA and proteins are structured polymers
Hemoglobin
Antibody
Michael Ströck, ribbon / atomistic model
Adenylate
kinase
Insulin
Glutamine
Synthetase
Gareth White, molecular surface representations of various proteins
DNA, polymer of 4 nucleotides, A,T,G, C
[Adenine-Thymine] [Guanine-Cytosine]
Proteins are polymers of 20 amino acids
Folding much more diverse than DNA
Typically the double-helical structure above,
Watson-Crick base pairing
More exposed chemical groups
Main purpose: storage of genetic information
Many purposes in the cell, including
structural, enzymatic, signaling
Proteins and DNA interact frequently in cells
DNA is a polymer 2 nanometers wide
(2 billionths of a meter) and up to
1 centimeter long!
In eukaryotes, DNA is wound around
histone proteins to form nucleosomes
3 billion basepair human genome
30,000 genes
12% encode DNA-binding proteins
DNA-binding proteins critical for
gene regulation
Gene regulation crucial for cell behavior
(All cell types in a human have the
same genome!)
From Molecular Biology of the Cell (Pub Med online)
Why single-molecule biophysics?
RNA Polymerase gives us one example
Biophysics Problems in biology are fascinating! Also increasingly complex.
Huge need for physics techniques (Instrumentation & Analysis)
http://opbs.okstate.edu/~petracek/Chapter%2026%20figures/Fig%2026-01a.JPG
Transcription central to gene regulation
http://www.uta.edu/biology/henry/classnotes/2457/
Example: Single-molecule manipulation was used to
discern the effect of a drug on RNA Polymerase
Karen Adelman (Wang Lab), NIEHS
Arthur La Porta (Wang Lab), U. Maryland
Adelman et al. Mol Cell. 14, 753 (2004).
Ensemble in vitro transcription assay
Adelman et al. Mol Cell. 14, 753 (2004).
(a drug)
The “ensemble” assays show that the drug
slows down transcription overall. But how?
Slower catalysis? or
Gel electrophoresis
Increased pausing?
Example: Single-molecule manipulation was used to
discern the effect of a drug on RNA Polymerase
Karen Adelman (Wang Lab), NIEHS
Arthur La Porta (Wang Lab), U. Maryland
Adelman et al. Mol Cell. 14, 753 (2004).
Optical tweezers assay
Video of a similar experiment from Berkeley
http://alice.berkeley.edu/RNAP/
Adelman et al. Mol Cell. 14, 753 (2004).
Can monitor the length of transcription in real-time
Answer:
The drug increases pausing of RNA Polymerase
Using optical tweezers, we can apply and measure
forces on single tethered biomolecules
Optical Trap
“Laser tweezers”
Microsphere
Biomolecular
“Tether”
Coverglass
Wang Lab (Cornell) Tweezers
Richard Yeh
Opportunities for MEMS and nanophotonics!
Less costly, more accessible, more stable
Using optical tweezers, we can apply and measure
forces on single tethered biomolecules
Quadrant photodiode
to measure force
Microsphere
Biomolecular
“Tether”
Optical Trap
Standard methods for attaching
DNA to coverglass and bead
Dielectic particles (500 nm
polystyrene) attracted to laser focus
Piezoelectric stage moves
coverglass relative to trap center
Coverglass
piezoelectric stage
Infrared laser focused
through microscope objective
Newton’s third law
Force on bead = force on laser
collect exit light onto photodiode
to measure force, displacement
Using optical tweezers, we can apply and measure
forces on single tethered biomolecules
Microsphere
Biomolecular
“Tether”
Coverglass
Forces from < 1 pN to 100s pN
pN = piconewton, 1 trillionth of N
Length precision ~ 1 nm
Thermal energy
4 pN – nm = 1/40 eV
Kinesin
8 nm step, 6 pN stall
(molecular motor)
RNA Polymerase 0.3 nm step, 25 pN stall
DNA Unzipping 15 pN
We built one versatile optical tweezers for use in
several different biological systems
Richard Yeh (Wang Lab), Bechtel-Nevada
E. Coli RNA Polymerase Transcription
Karen Adelman et al.
PNAS 2002, Mol. Cell 2004
Single Nucleosome Disruption
Brent Brower-Toland, David Wacker et al.
PNAS 2002, JMB 2005
DNA Unzipping with Bound Protein
Koch et al.
Biophys. J. 2002, Phys. Rev. Lett 2003
DNA Unzipping: Mechanical force biases thermal
opening / closing fluctuations
F
Unzipping DNA first demonstrated:
Bockelmann, Essevaz-Roulet, Heslot 1997
F
F
F
singlestranded DNA
unzip
zip
doublestranded DNA
F
DNA Image: http://www.biophysics.org/btol/
F
Force to unzip DNA depends on sequence
F
This DNA Molecule has
17 nearly identical
~200 bp repeats
F
DNA Capped by hairpin
(allows reversal)
Characteristic Unzipping
Force Plateau
DNA is a flexible polymer, subject to Brownian motion
• Simulations
Polymer physics modeling lets us know
how many bases pairs have been unzipped
F
F
12
j
...
Force
Polymer
Extension
ssDNA Freely-Jointed Chain
(Smith et al. 1996 Science)
0.80 nm persistence length
580 pN
stretch modulus
0.54 nm contour length per nt
Statistical physics
Velocity Clamp
100 ms
Polymer physics modeling lets us know
how many bases pairs have been unzipped
F
F
12
j
...
Velocity Clamp
Force
Polymer
Extension
ssDNA Freely-Jointed Chain
(Smith et al. 1996 Science)
0.80 nm persistence length
580 pN
stretch modulus
0.54 nm contour length per nt
100 ms
unzip
zip
Intuitively, one expects a
binding protein to inhibit DNA unzipping
F
F
12
j
...
PDB: 1DC1 BsoBI dimer bound to DNA
Restriction enzymes
Bind and cut specific DNA sequences
Well-studied model system
No Mg++ in binding buffer (High EDTA)
prevents endonuclease activity.
Dramatic increase in unzipping force seen with
700 pM BsoBI endonuclease
F
F
12
j
...
Dramatic increase in unzipping force seen with
700 pM BsoBI endonuclease
F
F
12
j
...
Very obvious increased force
(Worked the first time!)
Dramatic increase in unzipping force seen with
700 pM BsoBI endonuclease
F
F
12
j
...
Very obvious increased force
(Worked the first time!)
Binding locations
match predictions
Arrows show
unoccupied sites
We have a new single molecule method for detecting
where, when, and what of protein binding
Detecting where a protein is bound allows singlemolecule, ordered, reversible restriction mapping
1. Define threshold force
2. Unzip many molecules
3. Histogram data F > 20 pN
(grayscale map)
Three different restriction enzymes
produce correct maps
Binding detected
where we expect;
not where we don’t
(Non-repetetive)
“Traditional” Genome Mapping Technology
High throughput restriction fingerprinting
•
Each lane is a separate BAC
HindIII digestion
•
Gels are digitized and then
processed to find overlaps
(Fingerprints remain
unordered)
•
Project ramped up to about
20,000 fingerprint maps per
week (about 1x coverage)
(120 per hour)
•
Difficulties with small and
closely spaced bands
Source: Nature 2001 Genome Issue “A physical Map…”
Slides after this we did not see today (1/18/07)
New possibilities enabled due to ordered,
non-catalytic, single-molecule method
Repetitive DNA not a problem
Can work with functional
binding proteins
(e.g. transcription factors)
In principle could map a
chromosome from single cell
Drawbacks
Resolution decreases with length
Not automated or easy yet!
Microelectromechanical
Systems (MEMS)
Detecting when a protein is bound permits
site-specific equilibrium constant measurement
“When” = site-specific
equilibrium association constant
Protein + DNAsite  proteinDNAsite
KA 
1
[protein  DNAsite]

[protein]
[DNAsite]
Measure this ratio
([protein] >> [DNA] for this assay)
1
0
9
8
Agreement in both magnitude and slope
10
12
Terry et al., 1983
Ha et8 al.,
Indicates
ion1989
pairs involved in EcoRI-DNA binding
UFAPA
-1
Equilibrium Association Constant,
KA (M )
2
Method has been validated using well-studied
EcoRI – pBR322 DNA system
10
11
10
10
10
Our method has
larger uncertainty
9
10
8
10
7
Need for increased efficiency
Terry et al., 1983
Ha et al., 1989
UFAPA
100
125
150 175 200
250
+
[Na ] (mM)
(Salt screens the electrostatic attraction of protein-DNA)
There are many benefits of this site-specific, singlemolecule equilibrium constant measurement
Remove complication of
non-specific DNA
Can measure KA even when
off-rate very high
very tricky with standard methods
Probe multiple sequences
simultaneously
12
Terry et al., 1983
Ha et al., 1989
UFAPA
-1
Equilibrium Association Constant,
KA (M )
situations with lower KA
10
10
11
10
10
10
9
10
8
10
7
100
125
150 175 200
+
[Na ] (mM)
MSH2-MSH6 (mismatch repair protein) binding
affinity, specificity, and ATP-dependent sliding
Wang Lab: J. Jiang et al., Mol. Cell 20, 771 (2005)
250
Analysis of forces can determine what is bound
Forces = What / “how strong”
33 pN threshold correct 90% of time
0.12
BsoBI Alpha (N=141)
BsoBI Beta (N = 35)
-1
Probability Density (pN )
0.10
Can potentially distinguish
binding species on a
molecule by molecule basis
Graph shows two different
Protein-DNA complexes
0.08
0.06
0.04
0.02
0.00
0
10
20
30
40
Unbinding Force (pN)
50
60
Magnetics and MEMS can provide complementary
single-molecule capabilities, speedier results
Computer Controlled
Electromagnet
Magnetic Field
F Gradient Force
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
TIR
Illumination
Non-magnetic Aspheric
CCD Camera
Koch, Thayer, Corwin, de Boer, APL 173901
Electromagnetic
Force Apparatus
Very compliant
Microfabricated Spring
Constructing electromagnetic “tweezers” for
parallel single-molecule experiments
Jim Martin, Gayle Thayer (Sandia) Peter Goodwin, Jim Werner, Dick Keller (LANL)
Computer Controlled
Electromagnet
Combination of proven
SM technologies
Magnetic Field
F Gradient Force
pN, nm sensitivity
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
TIR
Illumination
Non-magnetic Aspheric
CCD Camera
Many molecules in parallel
Ideal for many experiments:
protein – DNA / unzipping
Short molecular bonds
Transcription
At Sandia / CINT, prototyped magnetic tweezers
Computer Controlled
Electromagnet
Magnetic Field
F Gradient Force
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
TIR
Illumination
Fluorescence Microscopy
Non-magnetic Aspheric
CCD Camera
~ 1/10 millimeter
Movie links probably won’t work.
See “zero force epi.avi” and “700 fN epi”
Zero Force
700 fN Force
Proof of principle for instrument succeeded for
4400 basepair double-stranded DNA tethers
Computer Controlled
Electromagnet
Magnetic Field
F Gradient Force
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
CCD Camera
Movie links probably won’t work.
See “TIR 1”
Current
Evanescent scattering signal
cycling magnet, 1.5 micron
dsDNA
Non-magnetic Aspheric
EWD Signal
TIR
Illumination
Frame number
MEMS Force Sensor: A direct way of measuring
forces on magnetic microspheres
Alex Corwin, Maarten de Boer, Gayle Thayer (Sandia)
Folded beam suspension
Differential Moire
displacment sensing
As low as 0.1 pN / nm
<1 pN sensitivity
50 microns
Standard processing (Sandia’s SUMMiT V™)
Adjustable spring constant (dynamic maybe)
Works in water (buffer)
Insensitive to temperature or buffer conditions
We can measure forces on single 3 micron beads to
characterize their polydispersity
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
0
Electromagnet pole
Single microsphere
Affix 2.8 micron bead to sensor
Position bead relative to magnet pole
4
Counts
Spring Deflection (nm)
10 microns
Microspheres glued with
Micromanipulator
Ramp current, measure displacement
2
Remove bead, repeat with new bead
0
600
700
800
20 A Deflection (nm)
2
4
6
8 10 12 14
Magnet Current (A)
16
18
20
We can measure forces on single 3 micron beads to
characterize their polydispersity
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
0
Electromagnet pole
2
This information critical for
biophysics experiments
0
600
700
800
20 A Deflection (nm)
2
4
6
Single microsphere
9% s.d. in saturated
moment of beads
(Literature: 41% – 72%)
4
Counts
Spring Deflection (nm)
10 microns
Microspheres glued with
Micromanipulator
8 10 12 14
Magnet Current (A)
16
18
20
We can also use a single bead as a micron-scale force
sensor to map electromagnet force field
Single bead affixed to edge of spring
We can also use a single bead as a micron-scale force
sensor to map electromagnet force field
Single bead affixed to edge of spring
Translate bead relative to magnet pole
Use of simple spring provides force
calibration, insensitive to:
unknown magnetite content
unknown electromagnet props.
temperature, buffer, etc.
We can also use a single bead as a micron-scale force
sensor to map electromagnet force field
FEMM
Spring
200
150
200
100
FEMM Force (pN)
Spring Force (pN)
300
Z= 160 m
(Closest to pole)
100
50
0
0
0
2
4
6
8
Magnet Current (A)
Z= 1000 m
10
Good agreement with FEMM Calculations http://femm.foster-miller.net
Absolute difference
Figure x due to magnetite content and properties, etc.
Results directly applicable to biophysics
experiments using same bead / magnet system
I hope I have shown the potential of single-molecule
manipulation tools for biophysics experiments
Computer Controlled
Electromagnet
Magnetic Field
F Gradient Force
Magnetic Beads
Single molecule
tether (e.g. DNA)
Scattered
Evanescent
Light
TIR
Illumination
Non-magnetic Aspheric
CCD Camera
Split
photodiode
Chromatin
from live cell
And we have only
scratched the surface
of what can be done!
Your ideas can help!!!
Loose
chromatin
Light source
Nanotractor
Spring
High force
Chromatin
Pre-teaser
Adjustable
gap Fingers
Force sensing unit
Mechanical
Clamp
Thank you to my wonderful collaborators!
Karen Adelman (NIH), Arthur La Porta (U.
Maryland),
Richard Yeh, Michelle D. Wang
Gayle Thayer, Jim Martin, George Bachand,
Alex Corwin, Maarten de Boer, Amanda Trent
Peter Goodwin, Jim Werner,
Dick Keller, Kim Rasmussen
Funding
• Slides after this one are scratch work.
Optical tweezers can apply and measure
forces
Collect laser light to measure force
Non-magnetic Aspheric
Computer Controlled
Electromagnet
Illumination
TIR
Microsphere
Biomolecule
“Tether”
tether (e.g.
DNA)
Single molecule
Magnetic
Beads
Magnetic
Beads
Single
molecule
tether (e.g. DNA)
Force
TIR
Coverglass
Illumination
Electromagnet
Computer Controlled
Non-magnetic Aspheric
CCD Camera
•
Small dielectric particles (beads) are
attracted to the brightest spot of
the laser focus
•
Create single-molecule tethers by
Magnetic
Field
securing one end to the coverglass,
Light
F Gradient Force
Evanescent other end to bead.
Optical Trap
Scattered
•
Apply forces by moving the
Scattered
coverglass away from the center of
Evanescent the laser
F Gradient Force
Light
Magnetic
Field
•
piezoelectric stage
CCD Camera
Laser focused through microscope objective
Collection of laser light after
passing through bead and
calibration allow determination of
length of tether, and force applied
(pN)
– Thermal energy
1 kBT ~ 4 pN•nm
Same DNA, now in the presence EcoRI
F
12
F
•
•
j
...
80 pM EcoRI
Each repeat of the DNA has two
EcoRI binding sites separated by
11 bp
Same DNA, now in the presence EcoRI
F
12
F
•
•
•
•
j
...
80 pM EcoRI
Each repeat of the DNA has two
EcoRI binding sites separated by
11 bp
Standard deviation of “event” ~ 3
nt
Shows that UFAPA can get fairly
good relative resolution.
– Could have applications for probing
larger protein-DNA complexes.
Using optical tweezers, we can apply and measure
forces on single tethered biomolecules
Quadrant photodiode
to measure force
Microsphere
Biomolecular
“Tether”
Coverglass
Optical Trap
piezoelectric stage
Infrared laser focused
through microscope objective
Trap stiffness proportional
to laser intensity
Optical Tweezers
Closed-loop
piezo controller
Fiber-coupled diode-pumped
solid state laser (1064 nm)
Acousto-optic deflector
E-Series DAQ,
Labview
Nikon TE200
Optical Tweezers
Fiber-coupled diode-pumped
solid state laser (1064 nm, 5W)
Acousto-optic modulator
Closed-loop
piezo controller
Nikon TE200, inverted scope
N.A. 1.4, IR objective
E-Series DAQ,
Labview point by point
feedback (10 kHz)
Optical Tweezers
Collect laser light after bead
to measure force
Microsphere
Biomolecule “Tether”
Optical Trap
Key points:
Force
Can manipulate biomolecules
while measuring length and force
Coverglass
piezoelectric stage
Infrared laser focused
through microscope objective
100 s loop time
• Including real-time data analysis