Applications of combined atomic

force/fluorescence microscopy to medical

research and drug discovery

Martin Guthold

Department of Physics

Oct. 16, 2009

Outline:

1.What is AFM – an imaging and manipulation tool

2.Imaging applications

3.Force Spectroscopy

4.Fiber Manipulations

5.Drug Discovery

Potential Establishment of a Nanoimaging NCRR:

I. Leveraging current instrumentationI.1 Two combined AFM-inverted optical microscopes (existing)

Veeco Topometrix AFMs (out of production/support); optical microscopes: Zeiss Axiovert 200 & Zeiss Observer-D1. This AFM is versatile, great for manipulating and imaging nanofibers, though higher noise.

I.2 Veeco Nanoscope IIIa, Multimode (existing)(Great for high resolution imaging of DNA, protein-DNA complexes; atomic resolution on hard surfaces), cannot sit on top of optical microscope. Currently used for numerous imaging applications.

II. New instrumentation (NCRR, or U 54)II.1 Veeco Bioscope, or Asylum MFP-3D-CF (~ $ 200,000)

High resolution, versatile, can sit on top of optical microscope, can be used for manipulations, force spectroscopy, lateral manipulations, etc

II.2 Zeiss confocal microscope (~ $ 300,000 – 400,000)Versatile, high-powered optical microscope, NanoSelection from cells, cell imaging, FRET experiments, etc., single molecule fluorscence experiments

Schematic of an AFM

Sample

Laser

Photodetector

Cantilever

Force-controlled by feedback

Substrate

Piezo-electric transducer

20m30 nm

Gold atoms

Imaging applications

• Protein-DNA complexes

• Two DNA-drug interactions – Acramtu (Bierbach)– Cisplatin (Scarpinato)

• DNA- coated carbon-nanotubes for radiation heat therapy (Gmeiner)

Transcription complexesE. coli RNA polymerase bound to one and two PR promoters

“Wrapping of DNA around the E.coli RNA polymerase open promoter complex”

C. Rivetti, M. Guthold, C. Bustamante. The EMBO Journal (1999) 18, 4464–4475, doi:10.1093/emboj/18.16.4464

Veeco Nanoscope IIIa

Loop formation in transcription regulation (activation and repression)

Contacts between RNAP·σ54 and NtrCD54E,S160F mediated by DNA-looping. The DNA loop had the expected length of about 130 nm.

K. Rippe M. Guthold, et al. , J. Mol. Biol. (1997), 270, 125-138

“Interconvertible Lac Repressor-DNA Loops Revealed by Single-Molecule Experiment.”

O. K.Wong, M. Guthold ,PLoS Biology; (2008), 6, pe232, 15pages

Veeco Nanoscope IIIa

on

off

k

k

D

A B A B

A BK

A B

Some AFM imaging studies

• DNA, proteins, and Protein-DNA complexes

(Determining the binding constant)

1. Association constant of UvrD dimerization (Ratcliff et al. “A Novel Single-Molecule Study To Determine Protein−Protein Association Constants” J. Am. Chem. Soc. (2001) 123 (24), 5632–35

2. Protein-DNA binding constants Yang Y. “Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions” Nucl. Acids Res. (2005) 33, 4322-34

Can determine specific and non-specific binding!

Images of free DNA and MutS-DNA complexes

PT-ACRAMTU monoadduct cisplatin intrastrand cross-linkPT-ACRAMTU monoadduct cisplatin intrastrand cross-link

Comparison of PT-ACRAMTU’s monoadduct and cisplatin’s cross-link formed in dsDNA based on solution NMR and biophysical data. Figure from Ulrich Bierbach.

Characterizing drug-induced changes in DNA, protein-DNA complexes

(Prof. Bierbach, WFU-CCC)

Acramtu

Characterizing drug-induced changes in DNA, protein-DNA complexes

(Prof. Bierbach, WFU-CCC)

Acramtu, potentially a new cancer therapeutic,

- lengthens DNA,

- does not kink it,

- slightly stiffens DNA

- may ecape DNA repairFigure 2: Comparison of unmodified DNA (left) to Acramtu-drugged DNA.

Figure 3: Histograms of contour length data

Acramtu lengthened the DNA by ~23%, at a dosage of 1 Acramtu per 5 base pairs.

Investigating Mismatch Repair Protein-DNA complexes(with Profs. Scarpinato & Salsbury)

• Goals:– Characterize cis-platin-DNA

complex

– Characterize Msh2, Msh2/6 binding to DNA

– Does DNA conformation/flexibility (bending) influence DNA repair vs. cell death decision?

– Protein-DNA binding constant

– Effect of protein-binding drugs on Msh2/6 binding

– Binding affinity:

AFM images of 414 bp cis-platinated DNA fragment.

on

off

k

k

D

P DNA P DNA

P DNAK

P DNA

Veeco Nanoscope IIIa

End-to-end distance

Contour length

Smooth DNA molecule

A B C D

Atomic Force Microscopy of 414 bp DNA fragment of unplatinated (top panel) and cisplatinated (single 1,2 GpG crosslink) (bottom panel) homoduplexes. AFM images (A), contour length (B), end-to-end distance (C), and model of smooth and kinked DNA molecule (D).

End-to-end distance

Contour length

Kinked DNA molecule

500 nm

80 nm

500 nm

80 nm

Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes

(Gmeiner)

•Ghosh, S., Dutta, S., Gomes, E., Carroll, D., D’Agostino, Jr., R., Olson, J. Guthold, M., Gmeiner, W. H. “Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes”, ACS Nano (2009) 3, 2667-73

- MWNT can be encased with single-stranded DNA (d(GT)40

- DNA-encasement results in well-dispersed MWNTs.

- Encased MWNT are soluble in aqueous solvent.

- AFM analysis gives diameter and length information.

In vitro heating experiments with DNA-encased MWNTs

(a, c) DNA-encased

(b, d) non-DNA-encased MWNTs

DNA encased nanotubes show higher heating efficiency, upon irradiation with 1064 nm laser

(e, f) Conditions suitable for a 5 °C temperature increase upon irradiation of DNA-encased MWNTs.

•Ghosh, S.,“Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes”, ACS Nano (2009) 3, 2667-73

Tumor ablation by DNA-encased MWCT

Tumor xenografts were generated by subcutaneous injection of 3 × 106 PC3 cells suspended in 200 μL of 1:1 PBS/Matrigel in both flanks of 12 male nude mice

Tumors injected with DNA-encased MWNTs and irradiated with a nIR laser at 1064 nm were completely eradicated within six days; completely healed over by day 24.)

Control groups showed no effect

•Ghosh, S.,“Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes”, ACS Nano (2009) 3, 2667-73

G-quadruplexes and Resolvase(with Profs. Vaughn & Akman)

Goals:

– Characterize conformation of G-

quadruplexes (one-stranded, two

stranded, four stranded) – are

they parallel, antiparallel,

kinked?

– What is the conformation of

resolvase-G-quadruplex

complexes (monomer, dimer,

multimer; kinking?

First AFM images of resolvase

Veeco Nanoscope IIIa

Ligand binding forces and how they related to the koff rate.

• Protein-ligand is spanned between the tip and the substrate.

• The tip is then retracted, and, thus, applying a force to the bonds under investigation.

• If the force is measured as a function of the pulling rate, it is termed force spectroscopy.

Atomic Force Microscopy (Force Spectroscopy)

Connection between rupture force and off-rate k-1

Assume a two-state model for the reaction.

1

1

k

k

A B

bound unbound

Bell model: an applied force lowers the activation energy.

1

B

G

k T1 1k (0 ) e

Dissociation rate without an applied force:

1

B

F x

k T1 1k ( F ) k (0 )e

Dissociation rate with applied force:

G. Bell (1978) Science 200, 616-627; E. Evans & K Ritchie (1997) Biophys. J. 72, 1541-55

B

B11

1

k T rF ln

k Tx k (0 )x

Connection between rupture force and off-rate, k-1

F … rupture force

T … temperature

k-1 … off-rate

x-1 … width of potential

kB …Boltzmann constant

The rupture force is related to the

off-rate

Experiment: Measure rupture force as a function of pulling rate. (here done of two different proteins).

For this treatment, we assume the reaction proceeds far from

equlibrium.

The faster you pull the higher the rupture force.

Data from F. Schwesinger et al. (2000) PNAS 97, 9972-77, First done by Rief at al. Science (1997) 276, 1109-12

Integrin-ligand binding forces

Force, F (pN) cantilever deflection

T-B photodiode signal

Tip travel, z (nm)

Sample moving up

protein

ligand

Sample moving up

Sample moving down

Sample moving down

1. Rupture force

Sample moving down

2. Rupture force

Integrin-ligand binding forcesAFM images

of integrin

664 nm & 221 nm scan size

Integrin size: 25 nm x 5 nm

AFM tip was functionalized with RGD-like sequence

Force spectroscopy on densely coated surface

Radius of curvature of AFM tip ~ tens of nm

Rupture Force (pN)

0 60 120 180 240 300 360 420 480

% E

ve

nts

0

3

6

9

12

15

18Loading rate 14,000 pN/secA.

Rupture Force (pN)

Rupture Force (pN)

0 60 120 180 240 300 360 420 480

% E

ve

nts

0

3

6

9

12

15

18

21 42,000 pN/secB.

log (pulling rate, pN/s)10000 100000

Ru

ptu

re F

orc

e (p

N)

75

80

85

90

Rupture Force (pN)

0 60 120 180 240 300 360 420 480

% E

ve

nts

0

3

6

9

12

15

18

21 70,000 pN/secC.

Rupture Force (pN)

0 60 120 180 240 300 360 420 480

% E

ve

nts

0

10

20

30

40

Conclusions from Integrin study

• DFS performed at three different pulling rates (14000, 42000, and 70000) pN/s) yielded rupture forces of 77, 86 and 88 pN;

• Bell model analysis yielded a dissociation constant, koff ~ 0.03 sec-1 and rupture distance x-1~ 0.6 nm.

• Excess cHArGD in solution dramatically reduced the rupture forces, confirming specificity.

• Data are consistent with surface plasmon resonance experiments (Hantgan)

Figure 4: integrin-ligand complex (1TY6.pdb)

Side view of set-up: Top view of set-up:

Linit

Fibrin fiber

AFM tip

Ridge

Ridge

Instrumentation set-up:

Objective lens

AFM tip

substrate

Fibrin fiber

12 m

8 m6 m

x-y-z translatorMicroscope

x-y stage

L’

L’’

A B C

Experimental set-up

Advantages:

• Obtain images & movies of manipulation

• Easy manipulation (nanoManipulator)

• Obtain stress-strain curves of fiber deformation

• Can apply larger force regime than in normal force measurement

• Well-defined geometry

Blood clots ‘perform’ the mechanical task of stemming the flow of

blood.

Our ultimate goal is to build a realistic model of a blood clot, based

on the physical parameters of the fibers.

How does the clot perform, depending on numerous variables

(mutations, environment, crosslinking, diseases, etc).

The properties of any network generally depend on three parameters:

- The properties of the individual fibers

- The properties of the branching points

- The architecture of the network

The major structural component of a blood clot is a network of fibrin fibers*

Image: Yuri Veklich & John Weisel

*ignoring platelets for the time being

Fstress

A

Lstrain :

L

Stress-strain curves of single fibrin fibers

Maximum

extension

Breaking strength

Extensibility: rupture strain = strain at which fiber ruptures.

Energy loss

Elastic limit: Greatest strain a material can withstand without any measurable permanent strain remaining on the complete release of the load.

For elastic deformations: Y… Young’s modulus Y

For viscous fluids: … viscosity t

Polymers usually show viscous and elastic properties

Linit

Extensibility L/Linit

100% 200% 300% 400%

Thr + X

Bat +X

Thr - X

Bat -X

Original length 332 ± 71

226 ± 52

226 ± 72

265 ± 83

0%

H

0

2

4

6

8

10

12

Strain 50 10

015

020

025

030

035

040

045

050

0

Bat-XTr-XBat+XTr+X

G

Cou

nt

= 0

Fibrin fiber

A

= 256%

D

= 183%

C

20 m

E

= 278%

Uncrosslinked

batroxobin

50 m

F

AFM tip

B

= 70%

Ridge

Groove

A

ED

B C

F

G

20µm

Permanent length increase

50% 100% 150%

0

100%

80%

60%

40%

20%

0 strain200%

H

Crosslinked

thrombin

W. Liu et al. Science 313, 634 (2006)

Incremental Stress-strain curves1

1 Silver, F. H., et al. (2000). Biomacromolecules 1(2): 180-185.

Strain (%)0 100 200 300

Str

ess

(n

N)

20

40

60

0 0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300

Total stress

Elastic stressT

ota

l str

ess

(a.

u.) E

lastic stre

ss (a. u

.)

Strain

C

Str

ain

(%

)

100

200

000.5

1

1.5

2

2.5

0 50 100 150 200 250 300 350

Stra

in

A

Time (s)

0 100 200 300

Time (s)

0 100 200 300

Str

ess

(n

N)

20

40

60

00

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350

stre

ss (

a.u)

time (s)

B

Uncrosslinked fibers:

Elastic modulus: ~ 4 MPa

Total modulus: ~ 7 MPa

Incremental Strain Data: Relaxation Rates

2121

tt

ececStress

τ1 = 1.75

τ2 = 30.11

• We measure two relaxation rates

• Relaxation rates are ~ independent of strain

• Relaxation rates are ~ independent of cross-linking

Strain hardening

Strain ()0 100 200

Tot

al m

odul

us (a.

u.)

1

2

3

4

0

B

0

100

200

300

400

500

600

0 50 100 150 200 250

Fo

rce

(nN

)

Strain (%)Strain ()0 100 200

200

400

0

For

ce (nN

)

A

Sigmoidal (two-step) strain-hardening

Repeated stress-strain curves – recovery and energy loss

• Little energy loss below 60% strain

• Up to 70% energy loss increases with increasing strain

• But, fibers still return to original length (for less than 120% strain)

Energy loss curve

Strain ()0 40 12080

0

Stres

s (M

Pa)

15

10

5

A

Strain ()0 100 200

0

Ene

rgy

Loss

(%

)

100

80

60

40

20

B

Sigmoidal (two-step) energy loss curve

Hysteresis and Recovery

Repeated strain:

• Fibrin fibers return to original length upon 120% strain

• Less force required to stretch out second time

• Same force required to stretch third time

Conclusions-1

• Fibrin fiber extensibility is largest of all protein fibers (Mechanism??).

• Cross-linked fibers can be extended to over 4 times their length and

uncrosslinked fibers to over 3 times their length.

• Crosslinked fibers can be extended further than uncrosslinked fibers.

– Crosslinking is directional

• Despite nearly crystalline structure of fibrin fiber, fibers have large elasticities

and extensibilities.

– Monomer must be able to extend while keeping interactions intact

• Crosslinked fibrin fibers can recover elastically from 180% strain.

• Single fibers can be extended further than whole clot (100 – 200%).

– Junctions may break first in clot (more in a bit).

• We observed two relaxation rates ~ 3 s and ~ 50s, for both, crosslinked and uncrosslinked.

• Initial elastic modulus 4 MPa uncrosslinked fibers and 6 MPa for uncrosslinked fibers.

• Total modulus 7 MPa for uncrosslinked and 10 MPa for crosslinked fibers.

• Strain hardening (two stiffnesses) to ~ 3 times stiffer at 100% strain.

• Fibrin fibers show viscoelastic behavior.

• Fibrin fibers show little energy loss up to 60% strain.

• Fibrin fibers show up to 70% energy loss at larger stains.

Conclusions-2

~ 100% strain

100 % = strain = 320 %

A:a interactions

-helix to -strand conversion

Unfolding of -domain

A

B

C

D

E

~ 10% strain

straightening

Molecular Mechanisms of Extensions1

1 Guthold et al (2007) Cell Biochemistry & Biophysics (2007) 49, 165-181

2 Brown et al. (2007) Biophysical Journal 92, L30

3 Averett et al (2008) Langmuir 24, 4979-4988

2

3

Mechanical strength of fiber branching points

• The Y-shaped branching points between fibers have a special, triangular architecture that prevents unzipping, thus making joints also very stable (Perhaps originating from ‘trimolecular junctions’1.

• Joints did not break until the fibers comprising the joint were stretched to over 2.5 times their length.

• Still, rupture at the fiber branching points was twice as likely as rupture of the fibrin fibers. Thus, the branching points are the weakest point in a fibrin clot.

Mosesson et al. (2001) Ann NY Acad Sci 936, 11-30.

& Wash

Bind

Target Molecules on Substrate

Library of Aptamers

1

AFM image

3a

Fluorescence

Specific binding

3b

Align and overlay AFM & FRET

4

Extract

5

Isolate

Amplify & Characterize

6

Atomic force microscopy

2a

Fluorescence microscopy

2b

Random Primer

Primer

Fluorophore

Oligo Construct:

bead

New aptamer discovery methodology• Single molecule technique

• One or a few cycles

Proof-of-concept: selectivity

Pool of 1:1 mixture of thrombin-aptamer:nonsense-DNA

Our method is selective:

Picked up 15 beads, of which 8 yielded DNA in the PCR reaction.

All 8 were the aptamer DNA and none were nonsense DNA.

Odds: 28 = 256.

2 3 4 A51M N

53 bp44 bp

C

B

AFM

fluorescenceA

AFM

fluorescence

D

aptamer

Research support: - NSF

- American Heart Association

- National Cancer Institute (NCI)

- American Cancer Society

- Research Corporation

More collborators (Wake Forest):

Wenhua Liu

Eric Sparks

Christine Carlisle

Patrick Nelli

Corentin Coulais (Lyon)

Christelle der Loughian (Lyon)

Bonin lab

Hantgan lab

Carroll lab

Manoj A. G. Namboothiry

Mary Kearns

Joel Berry

Macosko lab

Salsbury lab

People:

Lolo Jawerth, Harvard

Prof. Susan Lord, UNC

Prof. Richard Superfine, UNC

Prof. Mike Falvo, UNC

Collaborators (Cancer Center):

Scarpinato lab

Gmeiner lab

Vaughn/Akman lab

Bierbach lab

Torti lab

Carroll lab (Nanotech center)