Development of a Molecular Assay for Rapid Screening of Chemopreventive Compounds Targeting Nrf2

Zhaohui Wang, Vinay Gidwani, Zheng Sun, Donna Zhang, Pak Kin Wong

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection of endogenous Nrf2 was demonstrated using whole-cell lysates treated with sulforaphane. The assay can easily be incorporated into an automated platform for high-throughput screening of chemopreventive compounds targeting Nrf2.

Original languageEnglish (US)
Pages (from-to)243-248
Number of pages6
JournalJALA - Journal of the Association for Laboratory Automation
Volume13
Issue number4
DOIs
StatePublished - Aug 2008

Fingerprint

Assays
NF-E2 Transcription Factor
Screening
DNA
Fluorophores
Antioxidants
High-Throughput Screening Assays
Chemical elements
Single-Stranded DNA
DNA Probes
Chemoprevention
Biosensing Techniques
Fluorescent Dyes
Throughput
Proteins
Transcription factors
Fluorescence
Biosensors
Neoplasms
sulforafan

Keywords

  • biosensor
  • chemoprevention
  • chemotherapy
  • high-throughput screening
  • Nrf2

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biotechnology

Cite this

Development of a Molecular Assay for Rapid Screening of Chemopreventive Compounds Targeting Nrf2. / Wang, Zhaohui; Gidwani, Vinay; Sun, Zheng; Zhang, Donna; Wong, Pak Kin.

In: JALA - Journal of the Association for Laboratory Automation, Vol. 13, No. 4, 08.2008, p. 243-248.

Research output: Contribution to journalArticle

@article{f20e498441ce47b5b26be0b163e78c25,
title = "Development of a Molecular Assay for Rapid Screening of Chemopreventive Compounds Targeting Nrf2",
abstract = "Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection of endogenous Nrf2 was demonstrated using whole-cell lysates treated with sulforaphane. The assay can easily be incorporated into an automated platform for high-throughput screening of chemopreventive compounds targeting Nrf2.",
keywords = "biosensor, chemoprevention, chemotherapy, high-throughput screening, Nrf2",
author = "Zhaohui Wang and Vinay Gidwani and Zheng Sun and Donna Zhang and Wong, {Pak Kin}",
year = "2008",
month = "8",
doi = "10.1016/j.jala.2008.03.007",
language = "English (US)",
volume = "13",
pages = "243--248",
journal = "JALA - Journal of the Association for Laboratory Automation",
issn = "2211-0682",
publisher = "SAGE Publications Inc.",
number = "4",

}

TY - JOUR

T1 - Development of a Molecular Assay for Rapid Screening of Chemopreventive Compounds Targeting Nrf2

AU - Wang, Zhaohui

AU - Gidwani, Vinay

AU - Sun, Zheng

AU - Zhang, Donna

AU - Wong, Pak Kin

PY - 2008/8

Y1 - 2008/8

N2 - Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection of endogenous Nrf2 was demonstrated using whole-cell lysates treated with sulforaphane. The assay can easily be incorporated into an automated platform for high-throughput screening of chemopreventive compounds targeting Nrf2.

AB - Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection of endogenous Nrf2 was demonstrated using whole-cell lysates treated with sulforaphane. The assay can easily be incorporated into an automated platform for high-throughput screening of chemopreventive compounds targeting Nrf2.

KW - biosensor

KW - chemoprevention

KW - chemotherapy

KW - high-throughput screening

KW - Nrf2

UR - http://www.scopus.com/inward/record.url?scp=46449135450&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=46449135450&partnerID=8YFLogxK

U2 - 10.1016/j.jala.2008.03.007

DO - 10.1016/j.jala.2008.03.007

M3 - Article

VL - 13

SP - 243

EP - 248

JO - JALA - Journal of the Association for Laboratory Automation

JF - JALA - Journal of the Association for Laboratory Automation

SN - 2211-0682

IS - 4

ER -