Characterization of DNA at Cryogenic Temperatures Using CD Spectroscopy

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Characterization of DNA at Cryogenic Temperatures
Using CD Spectroscopy

• Heather Skiff
• Dr. Donald J. Hirsh
• Summer Research Program
• The College of New Jersey

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Introduction

• The properties of DNA make it an ideal scaffold
  to which paramagnetic groups can be attached.
  The helical, stable base packing of DNA allows
  for the attachment of molecules at various
  positions along the phosphate backbone and
  therefore at various, known distances from one
  another. However, since the spin-labeled DNA
  will be analyzed at very low temperatures, it is
  imperative to classify the DNA and ensure that it
  will maintain its native B-form conformation,
  even at varying temperatures. Therefore, the
  focus here is on the classification of DNA at 77
  K using CD Spectroscopy to identify the
  molecules conformation.

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Major Groove
Minor Groove
B-Form DNA
A-Form DNA
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Schematic CD Spectra of the Different Forms of DNA
(1)
(1)
nm
nm
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• The cryostat was obtained from the Physics
  Department at The College of New Jersey.
• It is small enough to fit in the sample chamber
  of the CD Spectropolarimeter.
• It has a sleeve and a vacuum valve and therefore
  can be evacuated, thus providing thermal
  stability within the sample space.
• The copper plate allows for good thermal contact,
  given the right adhesive.
• The windows of the cryostat were analyzed for
  transparency and were found to be transparent
  even in the UV-region.

cryogen exhaust
cryogen inlet
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Testing for Artifacts in CD Spectra of the
Cryostat Windows
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Freezing the Cryoprotectant

• It was observed that cryoprotectant is white and
  opaque after freezing slowly.
• The quick freezing method was implemented. This
  presented several new obstacles and challenges.

Can the sample be properly mounted? Can a good
vacuum be obtained after the sample has been
frozen? Can moisture be removed from the windows
of the cryostat and the sample itself?
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Mounting the Sample

• Mounted with Arctic Silver Paste and a rubber
  band
• Breaking the Cuvette led to the use of Quartz
  plates
• The plates demanded several changes to the
  method
• Decrease in DNA Volume / Increase in DNA
  Concentration
• Improved thermal contact
• Screws and rubber tubing for support
• Phenomena such as Linear Dichroism (LD) and
  Linear Birefringence (LB) can have an apparent
  impact on CD Spectra (2)

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The Observed Thin-Film Interference

• What is thin film interference?
• Possible sources
• Quartz Plates
• Thin Layer of Cryoprotectant
• Cryostat Windows

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A CD Spectrum of the DNA Duplex
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References and Acknowledgements

• www.brown.edu/Courses/ BI0020_Miller/dh/guide.html
  Miller, K. A Readers Guide to the Double
  Helix. 2003.
• http//www.jasco-europe.com/Jasco/html/trep/03720
  Artifacts20from20LD20and20LB.pdf Technical
  Report No. 37. Artifacts From LD and LB. April
  2001

• Dr. Donald J. Hirsh
• Dr. Romulo Ochoa
• Dr. John Allison
• TCNJ Chemistry Department
• Nick Vacirca
• Xi-Jun Chen
• Anne Szklarski
• Alyza Szajna
• Joyce Gaiser