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

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.

Our Plan to Study DNA Crystallize different DNA structures Crystallize different DNA structures Compare and contrast DNA structures Compare and contrast DNA structures

DNA Crystals DNA crystals allow us to determine the structure of DNA. 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. 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. The MPD draws the water from the aqueous solution in the well. DNA concentration increases: the solubility decreases and DNA falls out of solution DNA concentration increases: the solubility decreases and DNA falls out of solution A DNA crystal is formed. A DNA crystal is formed.

DNA Crystals Pictures courtesy of Frank Hays

Introduction to Atomic Force Microscopy

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: ,2002

nsma/afm-acs_news.htm

An example of what we can see with AFM This image represents a growing calcium carbonate crystal Smith et al, Biophys. J. 72: ,

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

Ng et al, NAR 25:2582, 1997

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

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

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

Crystal Imaged in Air

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

TCGGTACCGA

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

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

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

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

X-Ray Diffraction Currently the only way of finding the atomic structure of DNA crystals. 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). 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. 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. Disadvantage: the phase problem; gaps in the patterns of the crystal.

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

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

AFM Image of DNA crystal

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

Our Plan to Study DNA Crystallize different DNA structures, such as B-DNA and Holliday junctions. Crystallize different DNA structures, such as B-DNA and Holliday junctions. Compare the two types of DNA structures. Compare the two types of DNA structures. Attempt to determine the differences in the two structures at high resolution. Attempt to determine the differences in the two structures at high resolution.

DNA Crystals DNA crystals allow us to determine the structure of DNA. 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. 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. 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. DNA concentration increases: the solubility decreases and DNA falls out of solution, nucleation point. A DNA crystal is formed. A DNA crystal is formed.