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Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for i...

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Autores principales: Wang, Zhaohui, Wong, Pak Kin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315435/
https://www.ncbi.nlm.nih.gov/pubmed/30544696
http://dx.doi.org/10.3390/bios8040126
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author Wang, Zhaohui
Wong, Pak Kin
author_facet Wang, Zhaohui
Wong, Pak Kin
author_sort Wang, Zhaohui
collection PubMed
description Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA–protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems.
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spelling pubmed-63154352019-01-09 Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B Wang, Zhaohui Wong, Pak Kin Biosensors (Basel) Article Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA–protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems. MDPI 2018-12-10 /pmc/articles/PMC6315435/ /pubmed/30544696 http://dx.doi.org/10.3390/bios8040126 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wang, Zhaohui
Wong, Pak Kin
Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title_full Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title_fullStr Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title_full_unstemmed Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title_short Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
title_sort development of dna pair biosensor for quantization of nuclear factor kappa b
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315435/
https://www.ncbi.nlm.nih.gov/pubmed/30544696
http://dx.doi.org/10.3390/bios8040126
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