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Electrochemical based DNA sensors

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

WU, MARY ANN

项目受资助机构

CALIFORNIA INSTITUTE OF TECHNOLOGY

项目编号

5R01GM061077-16

立项年度

2017

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

367607.00美元

学科

Bioengineering; Biotechnology; Cancer; Genetics; Prevention;

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

BARTON, JACQUELINE K

参与机构

NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

项目标书摘要：DESCRIPTION (provided by applicant): 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.

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  • 1.Sensing DNA through DNA Charge Transport

    • 关键词：
    • MEDIATED ELECTRON-TRANSFER; IRON-SULFUR CLUSTER; HUMAN CANCER-CELLS;Z-FORM DNA; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; ENDONUCLEASE-III;REPAIR PROTEINS; ELECTROCHEMICAL DETECTION; 2-ELECTRODE PLATFORM

    DNA charge transport chemistry involves the migration of charge over long molecular distances through the aromatic base pair stack within the DNA helix. This migration depends upon the intimate coupling of bases stacked one with another, and hence any perturbation in that stacking, through base modifications or protein binding, can be sensed electrically. In this review, we describe the many ways DNA charge transport chemistry has been utilized to sense changes in DNA, including the presence of lesions, mismatches, DNA-binding proteins, protein activity, and even reactions under weak magnetic fields. Charge transport chemistry is remarkable in its ability to sense the integrity of DNA.

    ...

  • 2.DNA Charge Transport: from Chemical Principles to the Cell

    • 关键词：
    • MEDIATED ELECTRON-TRANSFER; RANGE OXIDATIVE DAMAGE;BASE-EXCISION-REPAIR; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE;ENDONUCLEASE-III; BINDING PROTEIN; ELECTROCHEMICAL DETECTION; PROTECTIVEANTIGEN; HYDROGEN-PEROXIDE

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair pi-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology.

    ...

  • 3.Redox Signaling through DNA

    • 关键词：
    • bioinorganic chemistry; DNA; electron transport; metalloproteins; redoxchemistry;MEDIATED ELECTRON-TRANSFER; IRON-SULFUR PROTEINS; CYTOCHROME BC(1)COMPLEX; ENZYME ENDONUCLEASE-III; CYTOSOLIC FE/S PROTEINS;BASE-EXCISION-REPAIR; CHARGE-TRANSPORT; ESCHERICHIA-COLI;CRYSTAL-STRUCTURE; OXIDATIVE STRESS

    Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through its -stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S] clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell.

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