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Martin Guthold Department of Physics Oct. 16, 2009

Applications of combined atomic force/fluorescence microscopy to medical research and drug discovery. Martin Guthold Department of Physics Oct. 16, 2009. Outline: What is AFM – an imaging and manipulation tool Imaging applications Force Spectroscopy Fiber Manipulations Drug Discovery.

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Martin Guthold Department of Physics Oct. 16, 2009

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  1. Applications of combined atomic force/fluorescence microscopy to medical research and drug discovery Martin Guthold Department of Physics Oct. 16, 2009 • Outline: • What is AFM – an imaging and manipulation tool • Imaging applications • Force Spectroscopy • Fiber Manipulations • Drug Discovery

  2. Potential Establishment of a Nanoimaging NCRR: • Leveraging current instrumentation I.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. • 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

  3. Photodetector Laser Piezo-electric transducer Cantilever Sample Substrate Force-controlled by feedback 20mm 30 nm Schematic of an AFM Gold atoms

  4. Imaging applications • Protein-DNA complexes • Two DNA-drug interactions • Acramtu (Bierbach) • Cisplatin (Scarpinato) • DNA- coated carbon-nanotubes for radiation heat therapy (Gmeiner)

  5. Transcription complexesE. coli RNA polymerase bound to one and two lPR promoters Veeco Nanoscope IIIa  “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

  6. Loop formation in transcription regulation (activation and repression) Veeco Nanoscope IIIa 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

  7. Some AFM imaging studies • DNA, proteins, and Protein-DNA complexes • (Determining the binding constant) • 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

  8. 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

  9. Figure 2: Comparison of unmodified DNA (left) to Acramtu-drugged DNA. Figure 3: Histograms of contour length data 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 repair Acramtu lengthened the DNA by ~23%, at a dosage of 1 Acramtu per 5 base pairs.

  10. Investigating Mismatch Repair Protein-DNA complexes(with Profs. Scarpinato & Salsbury) Veeco Nanoscope IIIa • 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.

  11. Smooth DNA molecule Contour length End-to-end distance Kinked DNA molecule Contour length b End-to-end distance A B C D 80 nm 500 nm 80 nm 500 nm 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).

  12. Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes (Gmeiner) • 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. • 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

  13. 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

  14. 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

  15. G-quadruplexes and Resolvase(with Profs. Vaughn & Akman) Veeco Nanoscope IIIa 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

  16. Atomic Force Microscopy (Force Spectroscopy)  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.

  17. Connection between rupture force and off-rate k-1 Bell model: an applied force lowers the activation energy. Assume a two-state model for the reaction. Dissociation rate without an applied force: Dissociation rate with applied force: G. Bell (1978) Science200, 616-627; E. Evans & K Ritchie (1997) Biophys. J.72, 1541-55

  18. Connection between rupture force and off-rate, k-1 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. The rupture force is related to the off-rate F … rupture force T … temperature k-1 … off-rate x-1 … width of potential kB …Boltzmann constant Data from F. Schwesinger et al. (2000) PNAS97, 9972-77, First done by Rief at al. Science (1997) 276, 1109-12

  19. Integrin-ligand binding forces Force, F (pN) cantilever deflection T-B photodiode signal ligand Sample moving up Sample moving up Sample moving down protein 2. Rupture force 1. Rupture force Sample moving down Sample moving down Tip travel, z (nm)

  20. Integrin-ligand binding forces AFM 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

  21. 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)

  22. AFM tip Fibrin fiber 6 mm 8 mm substrate 12 mm x-y-z translator Microscope x-y stage Objective lens Experimental set-up Instrumentation set-up: Side view of set-up: Top view of set-up: A B C Fibrin fiber Ridge AFM tip Linit L’ L’’ Ridge • 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

  23. The major structural component of a blood clot is a network of fibrin fibers* • 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). Image: Yuri Veklich & John Weisel 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 *ignoring platelets for the time being

  24. Breaking strength Maximum extension Stress-strain curves of single fibrin fibers Energy loss Elastic limit: Greatest strain a material can withstand without any measurable permanent strain remaining on the complete release of the load. Extensibility: rupture strain = strain at which fiber ruptures. For elastic deformations: Y… Young’s modulus For viscous fluids: h … viscosity Polymers usually show viscous and elastic properties

  25. A B Ridge Fibrin fiber AFM tip Groove e = 70% e = 0 C E D 20 mm e = 256% e = 183% e = 278% H Original length 332 ± 71 Thr + X 265 ± 83 Bat +X 226 ± 52 Thr - X 226 ± 72 Bat -X Linit 0% 100% 200% 300% 400% Extensibility DL/Linit Uncrosslinked batroxobin F 50 mm G Count

  26. A B C Crosslinked thrombin e = 80% D E F 20µm e = 230% Permanent length increase G H 100% 80% 60% 40% 20% 0 0 50% 100% 150% 200% strain W. Liu et al. Science 313, 634 (2006)

  27. A 200 Strain (%) 100 0 0 100 200 300 Time (s) C 60 B 60 40 Stress (nN) 40 Stress (nN) 20 20 0 0 100 200 300 0 Strain (%) 0 100 200 300 Time (s) Incremental Stress-strain curves1 Uncrosslinked fibers: Elastic modulus: ~ 4 MPa Total modulus: ~ 7 MPa 1 Silver, F. H., et al. (2000). Biomacromolecules1(2): 180-185.

  28. Incremental Strain Data: Relaxation Rates • We measure two relaxation rates • Relaxation rates are ~ independent of strain • Relaxation rates are ~ independent of cross-linking

  29. Strain hardening Sigmoidal (two-step) strain-hardening

  30. 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)

  31. Energy loss curve Sigmoidal (two-step) energy loss curve

  32. 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

  33. 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).

  34. Conclusions-2 • We observed two relaxation rates t ~ 3 s and t ~ 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.

  35. Molecular Mechanisms of Extensions1 2 3 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

  36. 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 Sci936, 11-30.

  37. Single molecule technique • One or a few cycles New aptamer discovery methodology 1 3a 2a Library of Aptamers Atomic force microscopy 2b 3b AFM image Bind Target Molecules on Substrate & Wash Specific binding Oligo Construct: Fluorescence Fluorophore Fluorescence microscopy 4 bead 6 Primer Random Primer 5 Align and overlay AFM & FRET Isolate Extract Amplify & Characterize

  38. A B fluorescence fluorescence AFM AFM C M 1 2 3 4 5 A N 53 bp 44 bp aptamer D 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.

  39. 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 Collaborators (Cancer Center): Scarpinato lab Gmeiner lab Vaughn/Akman lab Bierbach lab Torti lab Carroll lab (Nanotech center) People: Lolo Jawerth, Harvard Prof. Susan Lord, UNC Prof. Richard Superfine, UNC Prof. Mike Falvo, UNC

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