1. Modulating back electron transfer between guanine radicals and 2-aminopurine by variations in separation distance
and local sequence in duplex DNA
Daisy Galindo, Priscilla Garcia, Christina Khorozyan, Melissa Marquez, Zitadel Anne Esguerra, Eric Stemp
Mount Saint Mary’s University, Department of Physical Sciences, 12001 Chalon Road Los Angeles, CA 90049
ABSTRACT
Guanine is the DNA base that is most susceptible to 1-electron oxidation. Once formed
by photooxidation, the guanine radical has several different fates: it can either undergo
back electron transfer with the photooxidant or react further to give permanent
products. Here, we used the fluorescent analogue, 2-aminopurine as the photooxidant,
and examined two different systems for modulating the amount of permanent
products from guanine oxidation. In the first system, we monitored the formation of
DNA-protein crosslinks as the trapping reaction for the guanine radical. 2-aminopurine
was incorporated into a oligonucleotide duplex containing a 5’-G(X)nZ-3’ sequence (n=0
or 4, X = A or T and Z= 2-aminopurine) to determine the relative rates of back electron
transfer and crosslinking to protein. Crosslinking was detected by gel shift assay. Upon
325 nm irradiation, only a small change was seen in the mobility of the n=0 duplex,
whereas the n=4 (Z = T) duplex showed a decrease in bands for free DNA or protein and
the appearance of lower mobility bands. This result suggests that at the longer
separation distances, back electron transfer, does not compete as well with the protein
trapping reaction; analogous experiments with intervening adenines are underway. In
the second system, the distance between the photooxidant and the closest guanine
was held constant, but the sequence was designed to either increase or decrease in
guanine oxidation potential with the distance from the photooxidant, to either localize
or pull away the electron hole, respectively. In this case, gel shift assays and
cytochrome c oxidation assays indicated that an increased lifetime of the guanine
radical, as afforded by the guanine environment, leads to more oxidative reaction with
protein.
PHOTO-OXIDATION OF GUANINE BY THIONINE
2-AMINOPURINE
REACTION SCHEME
To show that the yield of permanent
products formed by guanine oxidation can
be modulated by the rate of back electron
transfer of the guanine radical with the 2-
aminopurine photooxidant.
SYSTEM #2: MODULATING BACK ET BY VARYING GUANINE’S OXIDATION POTENTIAL
CONCLUSIONS
The yield of permanent products formed by guanine oxidation can be
modulated by:
• increasing the distance between 2-AP and G to slow down back ET
• changing the oxidation potential of G to either facilitate or inhibit
charge transport away from the photooxidant.
ACKNOWLEDGMENTS
• Anna Arnold and Jacqueline K. Barton
• NSF (MCB 0345478)
• Denault-Loring Research Fellowship
• Loring-Denault Endowed Chair in Chemistry
S
N
NH₂H₂N
Thionine
in poly d(GC)
~ 2 V (singlet states, vs. NHE)
and E0 for G ~ 1.29V
Kelly et al. J. Am. Chem. Soc. 2002, 124, 5518
Lane 3, 10 µM Th
Lane 4, 100 µM Th, 599 nm for 2h
G
+
A
1 2 3 4
-
+
+
-
+
+
+
+
G
+
A
G
G
C
A
A
A
A
A
A
C
G
G
5'
hn
pi
No 5’-G specific damage by Th*
GG
Hopping
Trapping Trapping
kback
if kback > ktrap then no permanent DNA damage is observed
ktrap
Trapping Scheme
time (µsec.)
+ GMP
+ poly d(GC)
0
0.005
0.01
0.015
0.02
0 100 200 300 400 500
Evidence of ET Chemistry
Dohno, Stemp and Barton J. Am. Chem. Soc. 2003, 125, 9586
kforward = 4.0x1012 s-1
kback = 1.3 x 1012 s-1
DNA-Protein
Crosslink
AP , G
AP, G
*AP, G
hn
back et
forward et
(quenching)
trapping
2-aminopurine is a fluorescent analog of
Guanine
Competition between back ET and other
reactions determines the fate of the
Guanine radical
Redox calculations with 2-aminopurine
*Ap + e- → Ap•- E° = + 1.5 V
G → G•+ + e- E° = - 1.29 V
*Ap+ G → Ap- + G•+ ΔE° = + 0.21 V
ΔG° = -(96.5 kJ/V∙mol)(0.21 V)
ΔG° = -20.3 kJ/mol
Forward electron transfer:
Back electron transfer:
Ap•- → Ap + e- E°= + 2.24 V
G•+ + e- → G E°= + 1.29 V
Ap•- + G•+ → Ap + G ΔE°= + 3.53 V
ΔG° = -341 kJ/mol
Back ET slow because of Inverted Region
Quenching Titration
0
1
2
3
4
5
6
7
8
300 350 400 450 500
EmissionIntensity
Wavelength (nm)
Emission Quenching of *AP by GMP
SYSTEM #1: MODULATING BACK ET BY DISTANCE
Goal
5’ … Ap(T)nG … 3’ n = 0, 2, or 4
AAT AXG TAT TAT TGC GTG GAT GGT TCT C
AAT AXT TTT GTT TGC GTG GAT GGT TCT C
G0
G4
X = 2-aminopurine
Labeling Method
Cross-linked
material
Increased Crosslinking
Free DNA
Detecting Crosslinks by Gel Shift Assay
AP-G4AP-G0
time time
Conditions: 2% agarose, TBE buffer, 40 min at 80 V. Samples contained 10 uM Alexafluor546-DNA duplex, 400 ug/mL histone irradiated
for 0 to 32 min with Hg-Xe lamp at 320 nm.
The more T’s (i.e. longer distance) between the photooxidant and G,
the better the yield of DNA-protein crosslink.
Gel Shift Assay Results
Sequence Design
Sequence Design
The ionization potential for guanine follows this trend:
GT = 7.69 eV, GC = 7.68 eV, GA = 7.51 eV, GG = 7.28 eV, GGG = 7.07 eV
DP: 5’ TTA XAA GGG CGG CGT GCG CGC ATA CGT TGT T 3’
A2: 5’ TTA XAA GTG CGA GGA GGA GGG AGG GAG GGT T 3’
Oxidation of cyt c by A2 DNA Oxidation of cyt c: A2 vs DP DNA Gel Shift Assay with Histone
Saito, I.; Takayama, M.; Sugiyama, H.; Nakatani, K. J. Am. Chem. Soc. 1995, 117, 6406-6407.
Gel conditions: 10% polyacrylamide, pH 7.7 MOPS-Tris buffer, 200 V for 25 min. Samples contained 10 uM DNA duplex, 100 ug/mL
histone 2A in 10 mM sodium phosphate, 20 mM NaCl, pH 7 irradiated for 0 to 16 min with the 325 nm output (~4 mW) of a HeCd
laser (Kimmon)
time time