Utilizing A Novel Technique for Analyzing the Atomic Lattice of DNA Crystals

1. Utilizing A Novel Technique for Analyzing the Atomic Lattice of DNA Crystals Emily Cavaliere Professor Shing Ho’s Lab

2. Why study DNA structure? Better understanding of: • The effects of DNA structure on biological processes at the molecular level (e.g. replication). • How small molecules, such as drugs or carcinogens, affect the structure of DNA. http://www.accessexcellence.org/AE/AEPC/NIH/gene02.html

3. Our Plan to Study DNA • Crystallize different DNA structures • Compare and contrast DNA structures http://www.albany.edu/~achm110/abzdna.html

4. DNA Crystals • DNA crystals allow us to determine the structure of DNA. • A solution containing many components is mixed with DNA in one of the 9 wells, with 2-methyl-2,4-pentanediol (MPD) in the reservoir beneath. • The MPD draws the water from the aqueous solution in the well. • DNA concentration increases: the solubility decreases and DNA falls out of solution • A DNA crystal is formed. http://www.hamptonresearch.com/hrproducts/3136.html

5. DNA Crystals Pictures courtesy of Frank Hays

6. Introduction to Atomic Force Microscopy

7. How Atomic Force Microscopy Works • small record player • A very sharp, tiny (5-20nm) probe moves along the surface of the specimen. • This probe is attached to a cantilever, which acts like a spring, which scans the surface of the specimen. Yang, et al, Methods 29:175-187,2002 http://www.wfmu.org/MACrec/MT.html

8. http://www.physics.ucsb.edu/%7Ehhansma/afm-acs_news.htm http://www.veeco.com/

9. An example of what we can see with AFM 1 2 3 4 Smith et al, Biophys. J. 72:1425-1433, 1997 This image represents a growing calcium carbonate crystal

10. Advantages of AFM • Can determine the lattice structure of the specimen. • Less need of higher quality crystals. • Operates under ambient conditions: • In air • In liquid: atomic lattice resolution (possibly)

11. Ng et al, NAR 25:2582, 1997

12. Growth of Crystal Summer Project Buffers and DNA Artificial Mother Liquor Atomic Force Microscope imaging Affixing Crystals

13. Tested a variety of buffers to determine which would yield most stable crystals

14. Affixing the Crystals to the Surface Glass and Plastic Cover Slips Tiny Clamp Using agarose as a “glue”

15. Crystal Imaged in Air

17. Height v.s. Amplitude This is a DNA crystal imaged in air, by tapping mode. Hillocks!

19. TCGGTACCGA

20. We have tried: • Different buffers, and have finally determined, relatively so, what works • Slight increases in the concentrations of the constituents of the mother liquor. • Attaching crystals to the surface: • the crystals: • In agar • In agarose • Under carbon fibers • Growing them on plastic disks • All in order to make them stay put and not dissolve!

21. The Future of this Project • Found an artificial mother liquor to stabilize the DNA crystals • The possible use of agarose or adhesives to hold the crystal for imaging. • Possibly with reliable conditions and environment this project will give lattice resolution of the DNA crystal.

22. Acknowledgments • Professor Shing Ho • Bettye Smith, my mentor • Trish Khuu, Frank Hays, Jeff Watson, and Andrea Voth • Kevin Ahern • Howard Hughes Medical Institute Program • Funding: • Shing Ho Lab (NIH)

23. References • McPherson, A., Kuznetsov, Y., Malkin, A., Plomp, M. 2003. Macromolecular crystal growth as revealed by atomic force microscopy. J. of Struct. Biol. 142, 32-46. • Ng et al, NAR 25:2582, 1997 • Principles of Physical Biochemistry. Johnson, W. C., Shing Ho, P., van Holde, K. E. Prentice Hall. New Jersey. 1998. • Yang, Y., Wang, H., Erie, D. A., 2002. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy. Methods. 29, 175-187. • Smith, B. L., Paloczi, G. T., Walters, D. A., Belcher, A. M., Stucky, G. D., Morse, D. E., Hansma, P. K. 1997. Modification of Calcite Crystal Growth by Abalone Shell Proteins: An Atomic Force Microscope Study. Biophysical Journal. 72, 1425-1433.

24. X-Ray Diffraction • Currently the only way of finding the atomic structure of DNA crystals. • X-ray diffraction: patterns of x-ray diffraction depends on the type and distribution of atoms in the diffracting substance (DNA crystals). • The way a solid deflects x-rays, through complex computations, reveals the size, shape and arrangement of molecules in the specimen. • Disadvantage: the phase problem; gaps in the patterns of the crystal.

25. Cannot find the DNA relative to the other molecules in the crystal with x-ray diffraction • Compare the orientation of different types of DNA Where’s the DNA? Benefit of AFM

26. Need an Artificial Mother Liquor for imaging in fluids • We want atomic lattice resolution • Need to image in liquid • Need liquid, only have 10 µL in the drop • Need AML: • DNA crystals are very sensitive • Need to be stabilized for many hours • Need fluid to be on the tip to prevent breakage (surface tension).

29. AFM Image of DNA crystal

30. Summer Project • DNA crystals have never before been imaged on AFM. • Goals: • Determine stability of DNA crystals under ambient conditions and conducive for AFM imaging. • Be able to attach attained crystals to surface for AFM. • Attain lattice resolution and find the unit cell. • Compare different types of DNA structures (e.g. B-DNA and Holliday Junctions).

31. Our Plan to Study DNA • Crystallize different DNA structures, such as B-DNA and Holliday junctions. • Compare the two types of DNA structures. • Attempt to determine the differences in the two structures at high resolution. http://www.albany.edu/~achm110/abzdna.html

32. DNA Crystals • DNA crystals allow us to determine the structure of DNA. • Recipe For Crystals: salt, buffer, spermine, water and DNA in a 10 µL well, and 2-methyl-2,4-pentanediol (MPD) in the reservoir beneath. • The MPD draws the water from the aqueous solution in the well. • DNA concentration increases: the solubility decreases and DNA falls out of solution, nucleation point. • A DNA crystal is formed. http://www.hamptonresearch.com/hrproducts/3136.html