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.
http://www.accessexcellence.org/AE/AEPC/NIH/gene02.html
Our Plan to Study DNA


Crystallize
different
DNA
structures
Compare
and contrast
DNA
structures
http://www.albany.edu/~achm110/abzdna.html
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 2methyl-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
DNA Crystals
Pictures courtesy of Frank Hays
Introduction to Atomic Force
Microscopy
How Atomic Force
Microscopy Works



http://www.wfmu.org/MACrec/MT.html
small record player
A very sharp, tiny (520nm) 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.physics.ucsb.edu/%7Ehha
nsma/afm-acs_news.htm
http://www.veeco.com/
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
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)
Ng et al, NAR 25:2582, 1997
Buffers and
DNA
Growth of
Crystal
Artificial
Mother
Liquor
Summer Project
Atomic
Force
Microscope
imaging
Affixing
Crystals
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


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!
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.
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)
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.
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.
Where’s 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
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).

AFM Image of DNA crystal
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).
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
http://www.albany.edu/~achm110/abzdna.html
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-2,4pentanediol (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