We propose the development of a DNA-based electrochemical device using a new two-electrode strategy for DNA array patterning and detection. This renewal proposal is based on DNA-mediated electrochemistry and should allow the detection of nucleic acid and DNA-binding protein biomarkers with high sensitivity, suitable for quantitative diagnostics and research. Our 2-electrode platform provides a means to fabricate a DNA array on a single electrode, along with quantitative, multiplexed electrocatalytic sensing. We propose first to optimize the platform, including the incorporation of pin- based patterning. Click chemistry with copper activation will be the primary means for potential- dependent array formation. With respect to detection, we will optimize electrocatalysis partners and we will assess limits of detection (attomoles) through analysis of TATA-binding protein binding to DNA on the patterned platform. Microfluidics will be incorporated into the device. Once optimized, we propose developing the platform first for nucleic acid detection, specifically for two target microRNA sequences, miR-200c and let-7a. MicroRNAs are differentially expressed in healthy and cancerous tissues, which make them ideal targets for early cancer detection and profiling. We will monitor differences in expression levels using cultured colorectal cell lines with and without cancerous transformation. We also propose to test this sensor in detecting the human methylase DNMT1. DNA methylation modulates gene regulation and transcription, and both hyper and hypomethylation are associated with disease. We will take advantage of our turn-on methylase/restriction assay. We will quantify DNMT1 from cell lysates differing in expression of DNMT1, followed by measurements of tissue samples. Correlations will be drawn between different cancers and levels of methylase activity in order to establish a new early diagnostic based upon aberrant methylation. We will develop the platform also to screen potential therapeutics that inhibit methylation. Next we will move to simultaneous detection of disease-related miRNA expression and DNMT1 levels. Given the high sensitivity and reproducibility in detection with this device, we will also explore single cell detection of ou biomarkers. We will explore our miRNA and methylase targets to compare results between the bulk average and distribution among single cells. Combining assays for protein binding, RNA and DNA analysis already developed in our laboratory with new array fabrication methods and a two-electrode detection scheme, we propose an innovative approach to multiple biomarker detection through a robust sensor suitable for both basic research in systems biology as well as multiplexed applications for diagnosis and screening.

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 #
5R01GM061077-16
Application #
9220832
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2000-03-01
Project End
2018-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
16
Fiscal Year
2017
Total Cost
$330,845
Indirect Cost
$121,707
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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
Zwang, Theodore J; Tse, Edmund C M; Barton, Jacqueline K (2018) Sensing DNA through DNA Charge Transport. ACS Chem Biol 13:1799-1809
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
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:
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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