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

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

• Emily Cavaliere
• Professor Shing Hos Lab

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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
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Our Plan to Study DNA

• Crystallize different DNA structures
• Compare and contrast DNA structures

http//www.albany.edu/achm110/abzdna.html
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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.htm
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DNA Crystals
Pictures courtesy of Frank Hays
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Introduction to Atomic Force Microscopy
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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 29175-187,2002
http//www.wfmu.org/MACrec/MT.html
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http//www.physics.ucsb.edu/7Ehhansma/afm-acs_new
s.htm
http//www.veeco.com/
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An example of what we can see with AFM
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Smith et al, Biophys. J. 721425-1433, 1997
This image represents a growing calcium carbonate
crystal
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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)

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Ng et al, NAR 252582, 1997
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Summer Project
Growth of Crystal
Buffers and DNA
Artificial Mother Liquor
Atomic Force Microscope imaging
Affixing Crystals
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Tested a variety of buffers to determine which
would yield most stable crystals
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Affixing the Crystals to the Surface
Glass and Plastic Cover Slips
Tiny Clamp
Using agarose as a glue
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Crystal Imaged in Air
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Height v.s. Amplitude
This is a DNA crystal imaged in air, by tapping
mode. Hillocks!
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TCGGTACCGA
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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!

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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.

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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)

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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 252582, 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.

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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.

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Wheres the DNA? Benefit of AFM

• Cannot find the DNA relative to the other
  molecules in the crystal with x-ray diffraction
• Compare the orientation of different types of DNA

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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).

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AFM Image of DNA crystal
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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).

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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
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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.htm
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