Spin Labeling and Computer Modeling of DNA

1
Spin Labeling and Computer Modeling of DNA

• Anne Szklarski Xi Jun Chen
• Dr. Donald Hirsh
• The College of New Jersey
• Summer Research Program

2
Introduction

• Many enzymes have reaction centers that consist
  of an organic radical, or spin, and a
  paramagnetic metal center, or enhancer.
• DNA works as a scaffolding to hold the spin
  and the enhancer at a fixed distance apart. Our
  strand is 19 base pairs in length.
• Studying the strength and nature of the
  interactions can be useful for determining
  reaction pathways.

EDTA
T
A
Fe
G
C
O?
3
Labeling Reaction

• A19mer 5NOGCATAGATACATAGATACG 3
• T19mer 3 CGTATCTATGTATCTATGC 5

Amino-modified Guanine
NHS Ester of a Nitroxide Radical
Spin Labeled DNA
.
.
4
Size Exclusion Chromatography

• Larger molecules travel more quickly than smaller
  ones
• A larger DNA strand gives better separation from
  the excess spin label

5
Basic Theory of HPLC

• HPLC separates the components of a sample based
  on their hydrophobicities. When a sample is
  injected into the column, its components bind to
  the column. Solvent being pump into the column
  helps the components move through the column. The
  gradient of the solvent changes from mostly
  aqueous to organic, hence components with
  different polarities in the sample will elute at
  different times through out the gradient. The
  data are saved as a chromatogram, with the UV
  absorbance plotted against time.

6
HPLC Conditions

• Buffers
• A 5 acetonitrile 95 0.1M Triethyl amine
  acetate (TEAA), pH 7
• B 50 acetonitrile 50 0.1M TEAA, pH 7
• Temperature 30?C
• Gradient

7
HPLC Analysis of DNA
244nm
Nitroxide Radical
Labeled DNA
Unlabeled DNA
258nm
280nm

• Figure 1 A chromatogram of the unpurified
  nitroxide-labeling reaction. The nitroxide label
  has an absorption maximum at 244nm. The DNA has
  an absorption maximum at 258nm. The
  N-hydroxysuccinimide had an absorption maximum at
  280nm.

8
HPLC Analysis of Spin-Labeled DNA
Fraction 6
Labeled DNA
Unlabeled DNA
Fraction 7
Fraction 8
Fraction 9

• Figure 2 Fractions 6 to 9 of the first nitroxide
  labeling reaction. The earlier peak is the
  unlabeled DNA and the later one is the labeled
  DNA. 50 of A-19mers are labeled.

9
HPLC Analysis of Spin-Labeled DNA
Fraction 6
Labeled DNA
Fraction 7
Unlabeled DNA
Fraction 8
Fraction 9

• Figure 4 Fraction 6 to 9 of the second nitroxide
  labeling reaction. 80 A-19mers are labeled.

10
HPLC Analysis of DNA Duplex

• Purpose To determine the stability of the
  modified DNA duplex Do the EDTA-labeled T-19mer
  and the nitroxide-labeled A-19mer still possess
  the labels after certain periods.
• Goal Want four resolved peaks
• EDTA-labeled T-19mer
• Unlabeled T-19mer
• Nitroxide-labeled A-19mer
• Unlabeled A-19mer

11
HPLC Analysis of DNA Duplex
Original Buffers
Buffers w/ EDTA

• Figure 5. DNA Duplex under original buffer (top)
  Showed one broad peak, provides no useful
  information. Add EDTA to buffers (bottom) Peaks
  got resolved a bit however, they are still too
  close to provide any useful information.

12
HPLC Analysis of DNA Duplex Under Increased
Temperature
50C with Vydac E970520-9-7
70C with ThermaSep
T-19mer
Labeled A-19mer

• Figure 6 . 50?C UV-melting temperature is
  around 52?C. The one big peak resolved into two
  peaks, however, the peaks are still close. 70?C
  With new column made specifically to withstand
  high temperature Excellent resolution.
  Determined the peak at 19min is the T-19mer and
  the one at 26min is the spin-labeled A-19mer.

13

Problems With The Analysis of DNA Duplex
Unlabeled T-19mer
Unlabeled A-19mer
Unlabeled T-19mer A-19mer

• Figure 8 Retention times for the T-19mer and the
  unlabeled A-19mer are too close. Can not
  accurately determine the ratio between these two
  strands.

14

Trying To Solve The Problem
Unlabeled T-19mer A-19mer

• Figure 9 T-19mer and A-19mer peaks under
  different gradients.

15

Problem with The Analysis of DNA Duplex
EDTA T-19mer w/ Unlabeled T-19mer
Buffer w/o EDTA
Buffer w/o EDTA
Unlabeled T-19mer
Buffer w/ EDTA
EDTA T-19mer

• Figure 10 EDTA- Labeled and Unlabeled T-19mer
  peaks under different gradients.

16

DNA Duplex Under Adjusted Condition

• Condition
• Temperature 80C
• Gradient

Spin-labeled A-19mer
EDTA-labeled T-19mer
Unlabeled T-19mer A-19mer

• Figure 11 DNA Duplex tested under modified
  gradient based on previous experiments.

17
Molecular Modeling

• Possible hydrogen bonding of iron to N6 or N7 of
  adenine through water molecule
• Bond was created in HyperChem and the geometry
  was optimized using the Amber force field
• Distance from N to O of water was restricted to
  2.88 ?1.

N6
N7
Average Range 2.7? to 3.1?
18
Molecular Modeling

• EDTA and Tether Energy 233 300 kcal/mol
• For N6 bonding
• System Energy 428 kcal/mol
• H-N Bond Energy 172 kcal/mol
• Difference 256 kcal/mol

N6

• For N7 bonding
• System Energy 482 kcal/mol
• H-N Bond Energy 141 kcal/mol
• Difference 341 kcal/mol

N7
19
Molecular Modeling
N6 Hydrogen Bonded
N7 Hydrogen Bonded
No Hydrogen Bonding
19.3?
22.5?
19.0?
- Distance also depends on the motion of the
nitroxide radical, which is being studied through
Electron Paramagnetic Resonance Spectrometry
(EPR).
20
Duplex Rendering
21
Future Projects

• Continue to study the motion of the nitroxide
  radical attached to the duplex DNA through EPR
  experiments
• Cool the samples to 0?C to compare the motion of
  single strand and duplex spin-labeled DNA
• Create procedure to examine if hydrogen bonding
  actually occurs between EDTA and adenine base

22
Acknowledgements and References

• Dr. Donald Hirsh
• TCNJ Chemistry Department
• Chimera Technical Support
• Chris Tuohy

• Joseph Schramm
• Heather Skiff
• Nick Vacirca
• Joyce Gaiser
• Alyza Szajna

• Stryer, Lubert. Biochemistry. 3rd ed. New York
  W.H. Freeman and Company, 1975.
• Tissue, Brian. Size Exclusion Chromatography. 18
  Jun. 1996. SCIMEDIA. 30 Jun. 2005
  lthttp//elchem.kaist.ac.kr/vt/chem-ed/sep/lc/size-
  exc.htmgt.
• Basic Theory of HPLC, lthttp//www.med4you.at/la
  borbefunde/techniken/chromatographie/lbef_chromato
  graphie_hplc_gc.htmgt