Detection of protein–DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy

Kobayashi, Tamiyo; Okamoto, Naoaki; Sawasaki, Tatsuya; Endo, Yaeta

doi:10.1016/j.ab.2004.05.053

Cited by 18 publications

(26 citation statements)

References 26 publications

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“…[18][19][20][21] We found a unique compound, 4,6-dichloro-N-phenyl-1,3,5-triazin-2-amine, named NI241, that inhibits the DNA binding of NFjB ( Fig. 1).…”

Section: The Inhibitory Effect Of Ni241 On the Dna Binding Of Nf-jbmentioning

confidence: 99%

“…We used a single-molecule fluorescence detection system, MF20, for our detailed FCS evaluation of the inhibitory effect of NI241. 20,21 This system allows us to simultaneously measure and distinguish the individual signals of both fluorescence-free and fluorescence-bound molecules in the same solution. The data measured by this system are shown as diffusion times of fluorescence molecules.…”

Section: The Inhibitory Effect Of Ni241 On the Dna Binding Of Nf-jbmentioning

confidence: 99%

“…This method analyzes the difference in each molecular mass and discriminates the protein-bound DNA from the free DNA. 20 The fraction (%) in Figure 4 indicates the amount of Alexa-647-labeled NF-jB probe that is bound to GSThp50 in the solution. tively.…”

Section: The Comparison Of the Inhibitory Effects Among Ni241 Analoguesmentioning

confidence: 99%

“…The 30 ll of the mixture was loaded onto a 384-well, glass-bottomed microplate, and FCS measurement was performed by the single-molecule fluorescence detection system, MF20 (Olympus). 20,21 All experiments were performed under identical conditions with a data acquisition time of 30 s per measurement, and measurements were repeated five times per sample.…”

Section: Fluorescence Correlation Spectroscopy (Fcs) Analysismentioning

confidence: 99%

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A new small molecule that directly inhibits the DNA binding of NF-κB

Kobayashi

Yoshimori

Kino

et al. 2009

Bioorganic & Medicinal Chemistry

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“…[18][19][20][21] We found a unique compound, 4,6-dichloro-N-phenyl-1,3,5-triazin-2-amine, named NI241, that inhibits the DNA binding of NFjB ( Fig. 1).…”

Section: The Inhibitory Effect Of Ni241 On the Dna Binding Of Nf-jbmentioning

confidence: 99%

Section: The Inhibitory Effect Of Ni241 On the Dna Binding Of Nf-jbmentioning

confidence: 99%

Section: The Comparison Of the Inhibitory Effects Among Ni241 Analoguesmentioning

confidence: 99%

Section: Fluorescence Correlation Spectroscopy (Fcs) Analysismentioning

confidence: 99%

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A new small molecule that directly inhibits the DNA binding of NF-κB

Kobayashi

Yoshimori

Kino

et al. 2009

Bioorganic & Medicinal Chemistry

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“…For example, in the former, the binding of two representative transcription factors, AP-1 and NFkB, in nuclear extracts of HeLa cells with each sequence-specific DNA was evaluated quantitatively using the autocorrelation function [28]. The binding rates of these specific interactions were markedly augmented when cells were treated with tumor necrosis factor alpha, which is known to activate both AP-1 and NFkB.…”

Section: Gene Expression Revealed From Protein Dynamicsmentioning

confidence: 99%

Direct Quantification of Gene Expression Using Fluorescence Correlation Spectroscopy

Nomura

Nakamura²,

Feng³

et al. 2007

CPB

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Among the methods for single molecule detection in the field of medicinal chemistry, the importance of fluorescence correlation spectroscopy (FCS) is growing. FCS has the advantage of permitting us to determine the number of fluorescent molecules and the diffusion constant dependent on the molecular weight without any physical separation process such as gel electrophoresis. Thus this method is appropriate for studies on the hybridization of fluorescence-labeled oligonucleotides with RNA or DNA as well as gene expression through translation of a target protein linked with green fluorescent protein. Indeed, several groups have employed FCS for evaluation of gene expression in different ways. Many investigators are particularly interested in using FCS to quantitatively analyze mRNA just after transcription in the living cell. Technical advances in FCS have broadened the research spectrum in medicinal chemistry since it can also be used to study SNPs and molecular interactions between transcription factors and promoter sequences, as well as gene expression in living cells.

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