1. Magnetic Tweezers for Accurate Single-Molecule Force Measurements Noah Johnson BRITE 2009

2. Aims Design and calibration Eliminate non-specific interactions Study protein-ligand model systems

3. What are magnetic tweezers? Tiny forces: 0.01-10000pN Single-molecule measurements Optical Tweezers AFM

4. Advantages of magnetic tweezers Record single molecule events in parallel Don’t interfere with biological interactions Requires low energy Maintains constant force

5. Design and calibration F M = F d + F g F d =6π ηr v FgFg FMFM FdFd N CCD 1mm square glass capillary with bead suspension 10x objective 12V electromagnet CCD camera S

6. Gravitational force FgFg The beads are polystyrene with a broken magnetite core ρ Magnetite = 3.3 g/cm 3 ρ Polystyrene = 1.05 g/cm 3 ρ Beads = 1.9 g/cm 3 F g = mg = ρ beads V fluid g = 0.3pN

7. Bead clumping in water

8. Single beads with TWEEN 1.33mM TWEEN 20 in 10X PBS

10. Eliminating non-specific interactions distance glass 11 nm ~ 3 PEG molecules / nm 2 Need at least 20nm separation! Wan, Thomas, Guthrie &Vullev,Annals of Biomed Eng, 2009, 6, 1190-1205.

11. Surface derivatization glass silanization deprotection reductive amination amide coupling protein immobilization glass NH protein glass Wan, Thomas, Guthrie &Vullev,Annals of Biomed Eng, 2009, 6, 1190-1205.

12. Contact angles Validate surface chemistry PEG 3000-72.4 ± 0.48° Clean glass-26.7 ± 0.24° http://en.wikipedia.org/wiki/Contact_angle

13. Bead Derivatization DIC 1,3-Diisopropylcarbodiimide HOBt 1-Hydroxybenzotriazole 13

14. Eliminating non-specific interactions PDMS well filled with magnetic bead suspension molecular layer glass slide NSNS CCD 25x objective mirror

15. Expected results ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° time F beads settle magnetic wash <1 pN NSNS

16. Our results  PEGylated surface  PEGylated beads

17. Our results  PEGylated surface  PEGylated beads

18. Our results 91.3%73.3% 47.2%26.6% Substrate Beads PEG No PEG  Percentage of beads removed by a 0.357pN magnetic wash

19. Progress  Calibration and surface chemistry  Non-specific interactions experiments  Protein-ligand dissociation kinetics and trajectories

20. Future Work Add proteins/ligands to PEG Study dissociation kinetics and trajectories glass bead substrate

21. Acknowledgements Gokul Upadhyayula Dr. Sharad Gupta Stephen Bishop Sean Guthrie Dr. Valentine Vullev Jun Wang

22. Carbonic anhydrase Test magnetic tweezers Well characterized system Determine k d and compare Magnet Stage Objective 10x MirrorCCD