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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093099-03
Application #
8536840
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Preusch, Peter C
Project Start
2011-09-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$275,942
Indirect Cost
$92,592
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Zeng, Tao; Fleming, Aaron M; Ding, Yun et al. (2017) Interrogation of Base Pairing of the Spiroiminodihydantoin Diastereomers Using the ?-Hemolysin Latch. Biochemistry 56:1596-1603
Fleming, Aaron M; Ding, Yun; Burrows, Cynthia J (2017) Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine. Methods Enzymol 591:187-210
Johnson, Robert P; Fleming, Aaron M; Perera, Rukshan T et al. (2017) Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine. J Am Chem Soc 139:2750-2756
Alenko, Anton; Fleming, Aaron M; Burrows, Cynthia J (2017) Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers. Biochemistry 56:5053-5064
Perera, Rukshan T; Fleming, Aaron M; Peterson, Amberlyn M et al. (2016) Unzipping of A-Form DNA-RNA, A-Form DNA-PNA, and B-Form DNA-DNA in the ?-Hemolysin Nanopore. Biophys J 110:306-14
Riedl, Jan; Fleming, Aaron M; Burrows, Cynthia J (2016) Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps. J Am Chem Soc 138:491-4
Ding, Yun; Fleming, Aaron M; Burrows, Cynthia J (2016) ?-Hemolysin nanopore studies reveal strong interactions between biogenic polyamines and DNA hairpins. Mikrochim Acta 183:973-979
Tan, Cherie S; Riedl, Jan; Fleming, Aaron M et al. (2016) Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the ?-Hemolysin Nanopore. ACS Nano 10:11127-11135
Johnson, Robert P; Fleming, Aaron M; Beuth, Laura R et al. (2016) Base Flipping within the ?-Hemolysin Latch Allows Single-Molecule Identification of Mismatches in DNA. J Am Chem Soc 138:594-603
An, Na; Fleming, Aaron M; Burrows, Cynthia J (2016) Human Telomere G-Quadruplexes with Five Repeats Accommodate 8-Oxo-7,8-dihydroguanine by Looping out the DNA Damage. ACS Chem Biol 11:500-7

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