The overall objective of this renewal application is the development of DNA-mediated electrochemistry as a new technology for sensing DNA-binding proteins and nucleic acids. DNA-mediated electrochemistry is remarkably sensitive in detecting perturbations in the DNA base pairs and hence can be used as a sensor for changes in base stacking that arise with base mismatches or protein binding. Electrical sensors for mutation detection and for base flipping proteins that bind to DNA have been developed using this chemistry. In the program proposed, the scope of this chemistry will be expanded to develop new tools to monitor proteins that bind to DNA. Specifically, DNA-modified gold and graphite electrodes will be used to probe interactions and reactions of proteins on the duplex. DNA-bound redox potentials of redox-active transcriptional activators will be determined. For proteins that lack redox cofactors, covalent redox probes on the DNA duplex will be used as the reporter, and proteins with different DNA binding motifs will be examined. We can also monitor reactions of photolyase and PvuII on DNA to probe the DNA conformational changes that occur during reaction. Thus DNA electrochemistry offers a new tool to probe how proteins bind and react on DNA. DNA-mediated electrochemistry also offers high sensitivity and the ability to multiplex. By systematically reducing the DNA-modified electrode size down to 100 nm, and utilizing electrocatalysis for signal amplification, we will construct sensitive electrical sensors for proteins that bind to DNA and activate transcription. Microelectrodes offer faster kinetics, as well as sensitivity in monitoring protein binding. Multiplexed arrays of DNA-modified electrodes will be fabricated first on the macroscale for optimization. Initial designs will be used to test for methylases that bind different target sequences. A multiplexed assay to test for DNA-binding proteins will then be constructed based upon a competition array with the methylases. Assays for sequence-specific inhibitors of protein binding will also be developed. Using similar approaches, we will examine the scope and limits of RNA detection electrochemically, how DNA-mediated detection can be made robust and quantitative in detecting mRNAs at low concentrations. We will also combine these assays to develop arrays where mRNAs and the transcription factors that activate their synthesis can be detected simultaneously. These sensors offer the potential for a completely new technology to monitor DNA-binding proteins and mRNAs in a label-free format. These tools could provide a sensitive new diagnostic to detect both protein and RNA markers for cancer and other disease states. PUBLIC HEALTHE

Public Health Relevance

This project involves the construction of new electrical sensors for RNA and proteins that bind to DNA. These sensors will serve as completely new diagnostic tools to detect the proteins and RNAs from cells and tissues that are associated with different disease states. These sensors could represent a new technology for the early diagnosis of cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061077-11
Application #
7822767
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2000-03-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
11
Fiscal Year
2010
Total Cost
$352,745
Indirect Cost
Name
California Institute of Technology
Department
Chemistry
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Zwang, Theodore J; Tse, Edmund C M; Barton, Jacqueline K (2018) Sensing DNA through DNA Charge Transport. ACS Chem Biol 13:1799-1809
Zwang, Theodore J; Tse, Edmund C M; Zhong, Dongping et al. (2018) A Compass at Weak Magnetic Fields Using Thymine Dimer Repair. ACS Cent Sci 4:405-412
Tse, Edmund C M; Zwang, Theodore J; Barton, Jacqueline K (2017) The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins. J Am Chem Soc 139:12784-12792
Barton, Jacqueline K; Bartels, Phillip L; Deng, Yingxin et al. (2017) Electrical Probes of DNA-Binding Proteins. Methods Enzymol 591:355-414
O'Brien, Elizabeth; Holt, Marilyn E; Thompson, Matthew K et al. (2017) The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport. Science 355:
Bartels, Phillip L; Zhou, Andy; Arnold, Anna R et al. (2017) Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA. Langmuir 33:2523-2530
Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K (2016) Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin. J Am Chem Soc 138:11290-8
Arnold, Anna R; Grodick, Michael A; Barton, Jacqueline K (2016) DNA Charge Transport: from Chemical Principles to the Cell. Cell Chem Biol 23:183-197
O'Brien, Elizabeth; Silva, Rebekah M B; Barton, Jacqueline K (2016) Redox Signaling through DNA. Isr J Chem 56:705-723
Zwang, Theodore J; Hürlimann, Sylvia; Hill, Michael G et al. (2016) Helix-Dependent Spin Filtering through the DNA Duplex. J Am Chem Soc 138:15551-15554

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