Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

Abstract The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore...

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Autores principales:	Peter D. Leitner, Ilja Vietor, Lukas A. Huber, Taras Valovka
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d7d7351fa7fa4902828aa877384f06ac
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spelling	oai:doaj.org-article:d7d7351fa7fa4902828aa877384f06ac2021-12-02T13:24:07ZFluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA10.1038/s41598-021-81743-12045-2322https://doaj.org/article/d7d7351fa7fa4902828aa877384f06ac2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81743-1https://doaj.org/toc/2045-2322Abstract The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.Peter D. LeitnerIlja VietorLukas A. HuberTaras ValovkaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Peter D. Leitner Ilja Vietor Lukas A. Huber Taras Valovka Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
description	Abstract The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.
format	article
author	Peter D. Leitner Ilja Vietor Lukas A. Huber Taras Valovka
author_facet	Peter D. Leitner Ilja Vietor Lukas A. Huber Taras Valovka
author_sort	Peter D. Leitner
title	Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
title_short	Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
title_full	Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
title_fullStr	Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
title_full_unstemmed	Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
title_sort	fluorescent thermal shift-based method for detection of nf-κb binding to double-stranded dna
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d7d7351fa7fa4902828aa877384f06ac
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Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

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