Done by: Samuel Lim (4S414) Koh Shun Xiang (4I112) Wee Ting Yit (4S429)

1. Investigation of Interaction Between Estrogen Receptors & Estrogen Response Elements Using Plasmon Rulers Done by: Samuel Lim (4S414) Koh Shun Xiang (4I112) Wee Ting Yit (4S429)

2. Introduction Background Information

3. Plasmon Rulers Used to monitor the spacing between two nanoparticleslinked by DNA Based on plasmon coupling of single gold nanoparticles Plasmon Rulers Wavelength is sensitive to the proximity to other particles

4. Plasmon Rulers Fig 1 & 2: Principle behind plasmon coupling –Wavelengths of light scattered are proportionately related to inter-particle distance

5. Digoxigenin Streptavidin Au Au Anti-Digoxigenin Biotin Plasmon Rulers • Gold nanoparticles and oligonucleotide conjugate  forms dimers, trimers, polymers Nucleotide chain Fig 3: Diagram of a dimer formed by gold nanoparticles and DNA

6. Factors Affecting Dimer Production Assists in DNA Hybridization Ionic Strength Temperature of Heat-shock Factors affecting dimer production Affects DNA’s binding to gold Gold NP to DNA ConcentrationRatios

7. Estrogen Receptors • Contain a specifics site to which only estrogens (or closely related molecules) can bind ER Binds to Estrogen when estrogen molecule enters a cell, thus changing its own shape Activates nearby genes, producing mRNA that guide the synthesis of specific proteins Estrogen-receptor complex binds to specific DNA sites called Estrogen Response Elements (ERE)

8. Estrogen Stimulates female characteristics and sexual reproduction Binds to estrogen receptors, allowing specific genes to be expressed Estrogen Affects only cells that contain ER

9. Beneficial & Harmful Effects of Estrogen

10. Harmful Effects of Estrogen Fig. 4 & 5: Estrogen Receptors lead to proliferation of mutant cells

11. Project Design The details

12. Method Outline Stage 1: Optimizing dimer production Stage 2: Study ER interaction with DNA

13. Key Materials & Apparatuses • Spectrophotometer • Phase contrast optical microscope • Darkfield condenser • Immersion oil • Heat block • Glass slides • Double sided tape • Micropipettes • Microfuge tubes

14. Key Materials & Apparatuses • Deionised H2O • Tris buffer • 40nm gold nanoparticles (coated with Streptavidin) • 40nm gold nanoparticles (coated with anti-Digoxigenin) • Biotin-ended DNA • Digoxigenin-ended DNA • Biotinlyted Bovine Serum Albumin • Estrogen Receptor α • VIT DNA

15. Stage 1: Optimising Dimer Production Procedure & Method

16. Objectives • To develop an efficient method of producing dimers from gold nanoparticles and olignucleotides

17. Hypotheses • Dimers will form in greatest concentrations at lowest ratios of DNA to gold • Dimers will form in greatest concentrations at high heat-shocking temperatures • Dimers will form in greatest concentrations in buffers with high ionic strength

18. Variables

19. Various Methods for Producing Dimers Au Au Au Au Au Au Au Au Au Au Au Au Au Au Au Au Au Au Au

20. Variables • 16 samples are prepared with varying ratios and heat-shock temperatures to test the hypotheses

21. Au Au Au Digoxigenin Biotin Au Au Streptavidin Procedures Dilute DNA and gold nanoparticles to specified concentrations Form monomers Heat shock to promote hybridisation  dimers Target monomer Probe monomer Dimers Fig 6: Dimer production

22. Au Au Au Au Au Au Procedures Immobilize dimers onto slide with biotin Observe under darkfield microscope Biotin Glass slide Fig 7: Immobilizing dimers onto glass slide

24. Results Analysis Dimers will turn up as yellow or orange. Fig 8: Plasmon coupling Monomers or unreacted gold nanoparticles will show up as green Plasmon coupling

25. Monomers Dimers Results Analysis Fig 10: Sample A4 after washing – difference between dimers and monomers under DF microscope Fig 9: Sample A4 (1:20, 70°C) before washing

26. Results Analysis 3 photos were taken as triplicate data ImageJ R channel shows dimers only, as they are yellow or orange Images were split into Red (R) and RedGreen (RG) channels RG channel shows both dimers and monomers

27. Results Analysis Fig 12a: Sample A1 Red channel Fig 12b: Sample A1 RedGreen channel Fig 11: Original image of Sample A1 (1:20, 25°C)

28. Total number of dimers observed X 100% % yield = Total particles observed Number of dots in R channel X 100% = Number of dots in RG channel Results Analysis • Dimer yield (%) was obtained as follows:

29. Fig. 13a: Effect of Differing Ratios of AuNP to DNA on Dimer Yield 40 35 30 25 20 Dimer Yield/% 15 10 5 0 1:20 1:50 1:100 1:200 Ratios of AuNP to DNA Results • More DNA binded to gold  blocks out streptavidin on gold • Prevents gold from being secured to glass slide • Differing Ratios • Affected yield as predicted in our hypothesis • Yield was highest at 1:20 • Therefore • Optimum ratio for dimer synthesis  1:20 Fig 13a

30. Fig. 13b: Effect of Differing Heat Shock Temperatures on Dimer Yield 35 30 25 20 Dimer Yield/% 15 10 5 0 25°C 40°C 55°C 70°C Heat Shock Temperatures Results • Heat-shocking • Was effective in promoting hybridization • Increased yield in dimers • However • Decreased yield @ high temp. • Denaturing of DNA or binding molecules • Therefore • Optimum heat shock temp @ 40 – 55 °C Fig 13b

31. Au Au Purifying Dimers Extract dimers via differential centrifugation Repeat previous procedure with conditions that produce largest % of dimers Fig 14: Dimer Fig 15: Centrifuge

32. Optimum sample with highest % ofdimers 50% glycerol 60% glycerol 70% glycerol 80% glycerol Extracting Dimers Fig 16: Obtaining dimers via differential centrifugation

33. Stage 2: Investigating ER Interaction with DNA Procedure

34. Objectives • To make use of dimers as rulers to study the interaction of estrogen receptors with specific group of DNA sequences, the estrogen response elements (EREs)

35. Hypotheses • Estrogen receptors binds to VIT DNA but not random DNA

36. Variables

37. Immobilize dimer with random DNA onto glass plate Au Au Au Au Flow in ER into glass slides Procedure (Stage 2) Immobilize dimer with VIT DNA onto glass plate Fig 18: Dimers on glass slide

38. Procedure (Stage 2) Take a photograph MATLAB Obtain coordinates (x,y position) of dimers Take spectra of degree of colour change with the spectrophotometer Feed coordinates into spectrophotometer

40. Results Analysis • Shift in spectrum indicates a clear change in colour • Shows that ER binds to DNA, affecting the distance between gold nanoparticles • Distance may be closer or further • No shift in spectrum means no change in colour • ER does not bind to DNA, no change in distance between gold nanoparticles

41. Timeline

42. Potential for Research • Optimizing mass production of dimers • More cost-effective & efficient investigation of DNA using plasmon rulers • Understanding the mechanism of ER-ERE binding • Improving estrogen-regulating drugs • Design • Effectiveness

43. References • Agarawal, A., Deo, R., Wang, G. D., Wang, M. D., Nie, S. (2007). Nanometer-Scale Mapping and Single-Molecule Detection with Color-Coded Nanoparticle Probes. PNAS, 105, 3298-3303 • Chen, G., Wang, Y., Tan, L. H., Yang, M., Tan, L. S., Chen, Y., Chen, H. (2009). High-Purity Separation of Gold Nanoparticles Dimers and Trimers. JACS, 131, 4218-4219 • Krotz, D. (2006, October 11). A Ruler Of Gold And DNA. Research News. Retrieved July 1, 2009, from http://www.lbl.gov/Science-Articles/Archive/LSD-molecular-ruler.html

44. References • Reinhard, B. M., Sheikholeslami, S., Mastroianni, A., Alivisatos, A., Liphardt, J. (2006). Use of Plasmon Coupling to Reveal the Dynamics of DNA bending and Clevage by single EcoRV restriction enzymes. PNAS, 104, 2667-2672 • Parak, W. J., Pellegrino, T., Micheel, C. M., Gerion, D., Williams, S. C., Alivisatos, A. P. (2002). Conformation of Oligonucleotides Attached to Gold Nanocrystals Probed by Gel Electrophoresis. Nano Letters, 3, 33-36 • Raschke, G., Kowarik, S., Franzl, T., Soonichsen, C., Klar, T. A., Feldmann, J. (2003).Biomolecular Recognition Based on Single Gold Nanoparticle Light Scattering. Nano Letters, 3, 935-938

45. References • Reinhard, B. M., Siu, M., Agarwal, H., Alivisatos, A. P., & Liphardt, J. (2005). Cailbration of Dynamic Molecular Rulers Based on Plasmon Coupling between Gold Nanoparticles. Nano Letters, 5, 2246-2252 • Sannomiya, T., Hafner, C., Voros, J. (2008) In Situ Sensing of Single Binding Events by Localized Surface Plasmon Resonance. Nano Letters, 8, 3450-3455 • Sonnichsen, C., Reinhard, B. M., Liphardt, J., Alivisatos, A. P. (2005). A Molecular Ruler Based on lasmon Coupling of Single Gold and Silver Nanoparticles. Nature Biotechnology, 23, 741-745

46. Picture Sources • http://www.technoplas.com.au/images/StandardImage/4010_c.gif • http://www.camlab.co.uk/siteimages/glassware/fl00330_fl00365.gif • http://www.micro-scope.de/scope4.jpg • http://www.labessentials.com/C-5_Centrifuge.jpg • http://biology.kenyon.edu/courses/biol63/chime2001/estrogen/FRAMES/text.htm • www.physics.berkeley.edu/research/liphardt/ • http://www.technoplas.com.au/images/StandardImage/4010_c.gif

47. Thank You for your Attention! Q & A