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• 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

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)

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

Video of a similar experiment from Berkeleyhttp://alice.berkeley.edu/RNAP/

Can monitor the length of transcription in real-time

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

Coverglass

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

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

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

double-stranded DNA

single-stranded DNA

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

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

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

Polymer Extension

Statistical physics

1 2...j

Polymer physics modeling lets us knowhow many bases pairs have been unzipped

100 ms

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

Polymer Extension

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.

1 2...j

Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease

1 2...j

Very obvious increased force(Worked the first time!)

1 2...j

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[protein

[protein]

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

Terry et al., 1983 Ha et al., 1989 UFAPA

[Na+] (mM)

100100 125 150 175 200200 250

[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

[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

0.12 BsoBI Alpha (N=141) BsoBI Beta (N = 35)

Figure 5 Koch SJ and Wang MD

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

TIR Illumination

Magnetic Beads

ElectromagneticForce Apparatus

Very compliantMicrofabricated Spring

Koch, Thayer, Corwin, de Boer, APL 173901

Constructing electromagnetic “tweezers” for parallel single-molecule experiments

TIR Illumination

Magnetic Beads

TIR Illumination

Magnetic Beads

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

TIR Illumination

Magnetic Beads

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

TIR Illumination

Magnetic Beads

TIR Illumination

Magnetic Beads

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 6 8 10 12 14 16 18 200

50100150200250300350400450500550600650700750800

Figure 2

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

0 2 4 6 8 10 12 14 16 18 200

50100150200250300350400450500550600650700750800

Figure 2

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

Translate bead relative to magnet pole

Use of simple spring provides forcecalibration, insensitive to:

unknown magnetite contentunknown electromagnet props.temperature, buffer, etc.

0 2 4 6 8 100

300 Spring

Figure x

Magnet Current (A)

ce (pN

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

TIR Illumination

Magnetic Beads

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”

CoverglassTIR Illumination

Magnetic Beads

TIR Illumination

Magnetic Beads

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

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

Coverglass

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”

Coverglass

Key points:

Can manipulate biomoleculeswhile measuring length and force

100 s loop time• Including real-time data analysis

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