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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)