notes about these slides
DESCRIPTION
Notes about these slides. Slides presented at Physics 500 / 400 Seminar @ U. New Mexico, January 18, 2007 by Steve Koch. - PowerPoint PPT PresentationTRANSCRIPT
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 CHTMUniversity of New Mexico
Outline
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
1. Single-molecule manipulation capabilities provide new biological information
2. Optical Tweezers: Unzipping DNA molecules to probe protein-DNA interactions
3. Magnetic Forces: Improved efficiency for DNA unzipping; eukaryotic RNA Polymerase; molecular motors
4. I am looking for graduate students!
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
Michael Ströck, ribbon / atomistic model
DNA, polymer of 4 nucleotides, A,T,G, C[Adenine-Thymine] [Guanine-Cytosine]
Typically the double-helical structure above,Watson-Crick base pairing
Main purpose: storage of genetic information
Gareth White, molecular surface representations of various proteins
Antibody
Hemoglobin
Insulin
Adenylatekinase
Glutamine Synthetase
Proteins are polymers of 20 amino acids
Folding much more diverse than DNA
More exposed chemical groups
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 to1 centimeter long!
From Molecular Biology of the Cell (Pub Med online)
In eukaryotes, DNA is wound aroundhistone proteins to form nucleosomes
3 billion basepair human genome 30,000 genes 12% encode DNA-binding proteins
DNA-binding proteins critical forgene regulation
Gene regulation crucial for cell behavior(All cell types in a human have thesame genome!)
Why single-molecule biophysics? RNA Polymerase gives us one example
http://www.uta.edu/biology/henry/classnotes/2457/
http://opbs.okstate.edu/~petracek/Chapter%2026%20figures/Fig%2026-01a.JPG
Biophysics Problems in biology are fascinating! Also increasingly complex. Huge need for physics techniques (Instrumentation & Analysis)
Transcription central to gene regulation
Example: Single-molecule manipulation was used to discern the effect of a drug on RNA Polymerase
Karen Adelman (Wang Lab), NIEHSArthur La Porta (Wang Lab), U. Maryland
Ensemble in vitro transcription assay
The “ensemble” assays show that the drug slows down transcription overall. But how?
Slower catalysis? or Increased pausing?
Adelman et al. Mol Cell. 14, 753 (2004).
Gel electrophoresis
(a drug)
Adelman et al. Mol Cell. 14, 753 (2004).
Example: Single-molecule manipulation was used to discern the effect of a drug on RNA Polymerase
Karen Adelman (Wang Lab), NIEHSArthur La Porta (Wang Lab), U. Maryland
Optical tweezers assay
Answer:
The drug increases pausing of RNA Polymerase
Adelman et al. Mol Cell. 14, 753 (2004).
Video of a similar experiment from Berkeleyhttp://alice.berkeley.edu/RNAP/
Can monitor the length of transcription in real-time
Adelman et al. Mol Cell. 14, 753 (2004).
Optical Trap“Laser tweezers”
Microsphere
Biomolecular “Tether”
Coverglass
Using optical tweezers, we can apply and measure forces on single tethered biomolecules
Wang Lab (Cornell) TweezersRichard Yeh
Opportunities for MEMS and nanophotonics! Less costly, more accessible, more stable
Piezoelectric stage moves coverglass relative to trap center
Infrared laser focused through microscope
objective
piezoelectric stage
Quadrant photodiodeto measure force
Optical Trap
Microsphere
Biomolecular “Tether”
Coverglass
Using optical tweezers, we can apply and measure forces on single tethered biomolecules
Newton’s third lawForce on bead = force on lasercollect exit light onto photodiodeto measure force, displacement
Standard methods for attaching DNA to coverglass and bead
Dielectic particles (500 nm polystyrene) attracted to laser focus
Microsphere
Biomolecular “Tether”
Coverglass
Forces from < 1 pN to 100s pNpN = 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
Using optical tweezers, we can apply and measure forces on single tethered biomolecules
E. Coli RNA Polymerase TranscriptionKaren Adelman et al. PNAS 2002, Mol. Cell 2004
Single Nucleosome DisruptionBrent Brower-Toland, David Wacker et al. PNAS 2002, JMB 2005
DNA Unzipping with Bound ProteinKoch et al. Biophys. J. 2002, Phys. Rev. Lett 2003
We built one versatile optical tweezers for use in several different biological systems
Richard Yeh (Wang Lab), Bechtel-Nevada
F
F
F
F
F
double-stranded DNA
single-stranded DNA
unzip
zip
F
DNA Image: http://www.biophysics.org/btol/
Unzipping DNA first demonstrated:
Bockelmann, Essevaz-Roulet, Heslot 1997
DNA Unzipping: Mechanical force biases thermal opening / closing fluctuations
F
F
DNA Capped by hairpin(allows reversal)
Characteristic Unzipping Force Plateau
Force to unzip DNA depends on sequence
This DNA Molecule has17 nearly identical~200 bp repeats
DNA is a flexible polymer, subject to Brownian motion
• Simulations
F
F
1 2...j
Polymer physics modeling lets us knowhow many bases pairs have been unzipped
Velocity Clamp100 ms
ssDNA Freely-Jointed Chain (Smith et al. 1996 Science)
0.80 nm persistence length580 pN stretch modulus0.54 nm contour length per nt
Force
Polymer Extension
Statistical physics
F
F
1 2...j
Polymer physics modeling lets us knowhow many bases pairs have been unzipped
100 ms
unzip
zip
Velocity Clamp
ssDNA Freely-Jointed Chain (Smith et al. 1996 Science)
0.80 nm persistence length580 pN stretch modulus0.54 nm contour length per nt
Force
Polymer Extension
F
F
1 2...j
PDB: 1DC1 BsoBI dimer bound to DNA
Intuitively, one expects a binding protein to inhibit DNA unzipping
Restriction enzymesBind and cut specific DNA sequencesWell-studied model system
No Mg++ in binding buffer (High EDTA)prevents endonuclease activity.
F
F
1 2...j
Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease
F
F
1 2...j
Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease
Very obvious increased force(Worked the first time!)
F
F
1 2...j
Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease
Very obvious increased force(Worked the first time!)
Binding locations match predictions
Arrows showunoccupied sites
We have a new single molecule method for detecting where, when, and what of protein binding
Detecting where a protein is bound allows single-molecule, 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
(Non-repetetive)
not where we don’t
Binding detected where we expect;
“Traditional” Genome Mapping TechnologyHigh throughput restriction fingerprinting
Source: Nature 2001 Genome Issue “A physical Map…”
• Each lane is a separate BACHindIII 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
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 cellDrawbacks Resolution decreases with length Not automated or easy yet!
Microelectromechanical Systems (MEMS)
Detecting when a protein is bound permitssite-specific equilibrium constant measurement
“When” = site-specific equilibrium association constant
Protein + DNAsite proteinDNAsite
][DNA
]DNA[protein
[protein]
1
site
siteAK
Measure this ratio([protein] >> [DNA] for this assay)
Method has been validated using well-studiedEcoRI – pBR322 DNA system
100100 125 150 175 200200 250
107
108
109
1010
1011
1012
Terry et al., 1983 Ha et al., 1989 UFAPA
Equ
ilibr
ium
Ass
ocia
tion
Con
stan
t, KA
(M-1)
[Na+] (mM)
100100 125 150 175 200200 250
107
108
109
1010
1011
1012
Terry et al., 1983 Ha et al., 1989 UFAPA
Equ
ilibr
ium
Ass
ocia
tion
Con
stan
t, KA
(M-1)
[Na+] (mM)
Agreement in both magnitude and slope
Indicates 8 ion pairs involved in EcoRI-DNA binding
Our method haslarger uncertainty
Need for increased efficiency
(Salt screens the electrostatic attraction of protein-DNA)
100100 125 150 175 200200 250
107
108
109
1010
1011
1012
Terry et al., 1983 Ha et al., 1989 UFAPA
Equ
ilibr
ium
Ass
ocia
tion
Con
stan
t, KA
(M-1)
[Na+] (mM)
There are many benefits of this site-specific, single-molecule equilibrium constant measurement
Remove complication ofnon-specific DNAsituations with lower KA
Can measure KA even when off-rate very highvery tricky with standard methods
Probe multiple sequences simultaneously
MSH2-MSH6 (mismatch repair protein) bindingaffinity, specificity, and ATP-dependent slidingWang Lab: J. Jiang et al., Mol. Cell 20, 771 (2005)
Analysis of forces can determine what is bound
Forces = What / “how strong”
0 10 20 30 40 50 600.00
0.02
0.04
0.06
0.08
0.10
0.12 BsoBI Alpha (N=141) BsoBI Beta (N = 35)
Figure 5 Koch SJ and Wang MD
Pro
babi
lity
Den
sity
(pN
-1 )
Unbinding Force (pN)
Can potentially distinguish binding species on a molecule by molecule basis
Graph shows two differentProtein-DNA complexes
33 pN threshold correct 90% of time
Magnetics and MEMS can provide complementary single-molecule capabilities, speedier results
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
ElectromagneticForce Apparatus
Very compliantMicrofabricated Spring
Koch, Thayer, Corwin, de Boer, APL 173901
Constructing electromagnetic “tweezers” for parallel single-molecule experiments
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
Combination of proven SM technologies
pN, nm sensitivity
Many molecules in parallel
Ideal for many experiments:protein – DNA / unzippingShort molecular bondsTranscription
Jim Martin, Gayle Thayer (Sandia) Peter Goodwin, Jim Werner, Dick Keller (LANL)
At Sandia / CINT, prototyped magnetic tweezers
Zero Force 700 fN Force
~ 1/10 millimeter
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
Fluorescence Microscopy
Movie links probably won’t work. See “zero force epi.avi” and “700 fN epi”
Proof of principle for instrument succeeded for 4400 basepair double-stranded DNA tethers
Evanescent scattering signal cycling magnet, 1.5 micron dsDNA
Frame number
Curr
ent
EW
D S
ignal
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
Movie links probably won’t work. See “TIR 1”
50 microns
Folded beam suspension
As low as 0.1 pN / nm
Differential Moiredisplacment sensing
<1 pN sensitivity
Standard processing (Sandia’s SUMMiT V™)
Adjustable spring constant (dynamic maybe)
Works in water (buffer)
Insensitive to temperature or buffer conditions
MEMS Force Sensor: A direct way of measuring forces on magnetic microspheres
Alex Corwin, Maarten de Boer, Gayle Thayer (Sandia)
We can measure forces on single 3 micron beads to characterize their polydispersity
Microspheres glued withMicromanipulator
10 microns
Electromagnet pole Single microsphere
Affix 2.8 micron bead to sensor
Position bead relative to magnet pole
Ramp current, measure displacement
Remove bead, repeat with new bead600 700 800
0
2
4
0 2 4 6 8 10 12 14 16 18 200
50100150200250300350400450500550600650700750800
Figure 2
Spr
ing
Def
lect
ion
(nm
)
Magnet Current (A)
Counts
20 A Deflection (nm)
We can measure forces on single 3 micron beads to characterize their polydispersity
Microspheres glued withMicromanipulator
10 microns
Electromagnet pole Single microsphere
600 700 8000
2
4
0 2 4 6 8 10 12 14 16 18 200
50100150200250300350400450500550600650700750800
Figure 2
Spr
ing
Def
lect
ion
(nm
)
Magnet Current (A)
Counts
20 A Deflection (nm)
9% s.d. in saturated moment of beads(Literature: 41% – 72%)
This information critical for biophysics experiments
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 forcecalibration, insensitive to:
unknown magnetite contentunknown electromagnet props.temperature, buffer, etc.
0
50
100
150
200
0 2 4 6 8 100
100
200
300 Spring
Figure x
Spr
ing
For
ce (
pN)
Magnet Current (A)
FEMM
FE
MM
For
ce (pN
)
We can also use a single bead as a micron-scale force sensor to map electromagnet force field
Good agreement with FEMM Calculations http://femm.foster-miller.net
Absolute difference due to magnetite content and properties, etc.
Results directly applicable to biophysics experiments using same bead / magnet system
Z= 160 m(Closest to pole)
Z= 1000 m
I hope I have shown the potential of single-molecule manipulation tools for biophysics experiments
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
Adjustable gap Fingers
Split photodiode
Light source
Mechanical Clamp
Loose chromatin
Nanotractor
High forceChromatin Pre-teaser
Chromatin from live cell
Force sensing unit
Spring
And we have onlyscratched the surfaceof what can be done!
Your ideas can help!!!
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
• Small dielectric particles (beads) are attracted to the brightest spot of the laser focus
• Create single-molecule tethers by securing one end to the coverglass, other end to bead.
• Apply forces by moving the coverglass away from the center of the laser
• 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•nmLaser focused through microscope objective
piezoelectric stage
Collect laser light to measure force
Optical Trap
Microsphere
Biomolecule “Tether”
Force
CoverglassTIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
TIR Illumination
Magnetic Beads
Computer ControlledElectromagnet
Magnetic FieldGradient ForceF
Single moleculetether (e.g. DNA)
CCD CameraNon-magnetic Aspheric
ScatteredEvanescent Light
F
F
Same DNA, now in the presence EcoRI
1 2...j
• 80 pM EcoRI• Each repeat of the DNA has two
EcoRI binding sites separated by 11 bp
F
F
Same DNA, now in the presence EcoRI
1 2...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. E.g., nucleosomes, transcription PICs
Infrared laser focused through microscope
objective
piezoelectric stage
Quadrant photodiodeto measure force
Optical Trap
Microsphere
Biomolecular “Tether”
Coverglass
Using optical tweezers, we can apply and measure forces on single tethered biomolecules
Trap stiffness proportional to laser intensity
Optical Tweezers
Nikon TE200
Closed-loop piezo controller
E-Series DAQ,Labview
Fiber-coupled diode-pumpedsolid state laser (1064 nm)
Acousto-optic deflector
Optical Tweezers
Nikon TE200, inverted scopeN.A. 1.4, IR objective
Closed-loop piezo controller
E-Series DAQ,Labview point by pointfeedback (10 kHz)
Fiber-coupled diode-pumpedsolid state laser (1064 nm, 5W)
Acousto-optic modulator
Optical Tweezers
Infrared laser focused through microscope objective
piezoelectric stage
Collect laser light after beadto measure force
Optical Trap
Microsphere
Biomolecule “Tether”
Force
Coverglass
Key points:
Can manipulate biomoleculeswhile measuring length and force
100 s loop time• Including real-time data analysis