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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...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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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. |
format | Online Article Text |
id | pubmed-6315435 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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