Genomic and mitochondrial DNA bases undergo continuous modifications as a result of both natural processes that introduce epigenetic markers as well as exposure to DNA damaging agents through oxidation and alkylation reactions from endogenous sources or toxicants. DNA sequencing techniques do not directly detect DNA damage because the sequencing takes place on PCR-amplified strands that perforce contain only the 4 canonical bases A, C, T, and G. Mutations can be detected by sequencing, and many of these are the ultimate outcome of DNA damage. However, mutations themselves do not provide much information about the chemical identity of the original damage. This project will examine an approach to detection of DNA base modification (e.g. oxidation, alkylation, or excision) by application of chemical and enzymatic methods to convert the modified base to an adduct that yields a detectable signal when individual DNA strands translocate through a membrane-embedded ion channel. This method will provide a direct read-out of DNA damage on single molecules The long-term goal is to develop methodology compatible with microfluidics to analyze very small samples of DNA from cellular sources.
The specific aims of this project are to (1) optimize the conversion of specific DNA lesions to adducts detectable by the nanopore ion channel method by a combination of organic and enzymatic chemistries, (2) optimize the ion channel measurements to detect and quantify single-site DNA damage and demonstrate that DNA strand carrying adducts are translocated through the pore, (3) validate the methods using large DNA targets such as the plasmid M13mp18 after chemical damage, and (4) develop a method to PCR amplify DNA damage by generation of a specific 5th dNTP for enzymatic demarcation of damage sites. Realization of the long-term goals of this project will impact research in human health in 3 areas: (1) personalized drug therapy, (2) early detection of disease, and (3) epigenetics.

Public Health Relevance

The ability to monitor specific DNA base damage sites in tissue samples would have a significant impact on human health by rapid evaluation of genetic damage caused by cancer treatments or environmental toxins. In addition, the ability to detect DNA damage occurring in specific genes may guide physicians to preventative care. The methods developed in this research may also provide epigenetic tools for linking DNA modification to disease states.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Preusch, Peter C
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Utah
Schools of Arts and Sciences
Salt Lake City
United States
Zip Code
Ding, Yun; Fleming, Aaron M; White, Henry S et al. (2014) Internal vs fishhook hairpin DNA: unzipping locations and mechanisms in the ?-hemolysin nanopore. J Phys Chem B 118:12873-82
Johnson, Robert P; Fleming, Aaron M; Jin, Qian et al. (2014) Temperature and electrolyte optimization of the ?-hemolysin latch sensing zone for detection of base modification in double-stranded DNA. Biophys J 107:924-31
Wolna, Anna H; Fleming, Aaron M; Burrows, Cynthia J (2014) Single-molecule detection of a guanine(C8) - thymine(N3) cross-link using ion channel recording. J Phys Org Chem 27:247-251
Johnson, Robert P; Fleming, Aaron M; Burrows, Cynthia J et al. (2014) Effect of an Electrolyte Cation on Detecting DNA Damage with the Latch Constriction of ?-Hemolysin. J Phys Chem Lett 5:3781-3786
An, Na; Fleming, Aaron M; Middleton, Eric G et al. (2014) Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity. Proc Natl Acad Sci U S A 111:14325-31
An, Na; Fleming, Aaron M; Burrows, Cynthia J (2013) Interactions of the human telomere sequence with the nanocavity of the ýý-hemolysin ion channel reveal structure-dependent electrical signatures for hybrid folds. J Am Chem Soc 135:8562-70
Wolna, Anna H; Fleming, Aaron M; An, Na et al. (2013) Electrical Current Signatures of DNA Base Modifications in Single Molecules Immobilized in the *-Hemolysin Ion Channel. Isr J Chem 53:417-430
Jin, Qian; Fleming, Aaron M; Ding, Yun et al. (2013) Structural destabilization of DNA duplexes containing single-base lesions investigated by nanopore measurements. Biochemistry 52:7870-7
Schibel, Anna E P; Fleming, Aaron M; Jin, Qian et al. (2011) Sequence-specific single-molecule analysis of 8-oxo-7,8-dihydroguanine lesions in DNA based on unzipping kinetics of complementary probes in ion channel recordings. J Am Chem Soc 133:14778-84