This study uses Fenton chemistry to simulate oxidative damage in vitro and examine its effects on nucleic acids like DNA and RNA. The researchers are applying the Fenton reaction, which uses iron and hydrogen peroxide to generate reactive oxygen radicals, to DNA to observe structural changes indicating oxidative damage. They can determine if the DNA is supercoiled, nicked, or linear using gel electrophoresis. By adding tRNA as a competitor, they can compare the relative reactivity of RNA and DNA. The researchers are also analyzing modified RNA structures using PyMOL to predict sites that may be vulnerable to radicals generated by the Fenton reaction. The long-term goal is to study RNA structural changes as indicators of oxidative damage.

1. The use of Fenton chemistry to identify oxidative damage sites in RNA
Sherrell Haney*, Evan Jones, and Dr. Christine Chow
In organisms, cells are constantly undergoing oxidative damage. Much research is being done to understand
the effects of oxidative damage to nucleic acids. Determining if specific nucleic acids are more susceptible to
oxidative damage may lead to a better understanding of its downstream effects. In order to simulate oxidative
damage in vivo, we are using an in vitro system that applies Fenton chemistry to generate oxygen radicals.
Our goal is to generate the radicals and use structural changes of DNA as an indicator of oxidative damage.
By using agarose gel electrophoresis, we can study the DNA structure and identify whether it is supercoiled,
nicked, or linear. This observation serves as an indicator for oxidative damage of the nucleotide. By using
tRNA as a competitor, we can determine the relative reactivity of RNA versus DNA. Furthermore, we are
analyzing modified RNAs using computational approaches to determine sites most likely to be damaged by
hydroxyl radicals generated in the Fenton reaction. The main tool being used is the PyMOL program.
This work was supported in part by NIGMS/NIH grant R25 GM058905-22.
• By varying the reagents of the Fenton reaction, we were able to observe different levels of
DNA damage.
• By using the Fenton reaction and examining the impact on DNA structure, we can also
explore RNA damage through competition.
• Using lower levels of the Fenton mix lowered the pUC19 damage in the presence of tRNA.
• It is important to first optimize the Fenton reaction conditions with DNA, then carry out
oxidative damage of RNA.
• Using PyMOL, U2 snRNA structures were examined for differences in positions of protons
available for abstraction by the hydroxyl radicals.
• The long-term goal is to use oxidative Fenton chemistry and study structural changes of
RNA as an indicator of oxidative damage.
ascorbate
Bacterial cell
Supercoiled
DNA
Nicked DNA
Linear DNA
DNA
isolation
0.1 mM Fe
0.14 mM EDTA
6.94 mM Na
ascorbate
2. FENTON REACTION
1. ABSTRACT
ACKNOWLEDGEMENTS
6. CONCLUSIONS and FUTURE DIRECTIONS
5. DNA DAMAGE WITH ADDITION OF tRNA
3. MATERIALS AND METHODS
+/- modified
tRNA
Nicked
Linear
Super-
coiled
Supercoiled DNA
Nicked
+ + + pUC19
- + - Fenton
- + - Fe
+ + + pUC19
- + - Fenton
- + - ascorbate
Linear
Super-
coiled
4. OBSERVING OXIDATIVE DAMAGE ON DNA
+ + + + + pUC19
- + + + + Fenton
- - 35 350 3500 tRNA
(ng)
Supercoiled DNA
Fe(II)–EDTA + H2O2 ---> Fe(III)–EDTA + ŸOH + OH–
O
H
O
HO Base
P
O
OR
O-
-
OH
O
O
HO Base
P
O
OR
O-
"Fenton
Mix"
A B
C
D
Nicked
Linear
Super-
coiled
PyMOL Images
G1 of U2 snRNA
H5'/H5''
U7
H5'/H5''
Gel quantification
+ + + + + pUC19
- + + + + Fenton
- - 35 350 3500 tRNA
(ng)
E F