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A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB.

Ma DL, Xu T, Chan DS, Man BY, Fong WF, Leung CH - Nucleic Acids Res. (2011)

Bottom Line: The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit.In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed.The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. edmondma@hkbu.edu.hk

ABSTRACT
Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.

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The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing the double-stranded DNA substrate with two NF-κB binding sites (0.02 µM) and 40 U of ExoIII as a function of the concentration of BSA (0, 0.08, 0.20 and 0.40 µM).
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Figure 6: The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing the double-stranded DNA substrate with two NF-κB binding sites (0.02 µM) and 40 U of ExoIII as a function of the concentration of BSA (0, 0.08, 0.20 and 0.40 µM).

Mentions: This label-free assay is based on the inhibition of ExoIII catalyzed digestion of the oligonucleotide by the binding of the p50 subunit. To validate the mechanism of this method, we replaced the p50 subunit with the non-DNA binding protein bovine serum albumin (BSA). The luminescence response of the ruthenium complex in the presence of the oligonucleotide containing the double p50 binding site and BSA after ExoIII digestion is shown in Figure 6.Figure 6.


A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB.

Ma DL, Xu T, Chan DS, Man BY, Fong WF, Leung CH - Nucleic Acids Res. (2011)

The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing the double-stranded DNA substrate with two NF-κB binding sites (0.02 µM) and 40 U of ExoIII as a function of the concentration of BSA (0, 0.08, 0.20 and 0.40 µM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105395&req=5

Figure 6: The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing the double-stranded DNA substrate with two NF-κB binding sites (0.02 µM) and 40 U of ExoIII as a function of the concentration of BSA (0, 0.08, 0.20 and 0.40 µM).
Mentions: This label-free assay is based on the inhibition of ExoIII catalyzed digestion of the oligonucleotide by the binding of the p50 subunit. To validate the mechanism of this method, we replaced the p50 subunit with the non-DNA binding protein bovine serum albumin (BSA). The luminescence response of the ruthenium complex in the presence of the oligonucleotide containing the double p50 binding site and BSA after ExoIII digestion is shown in Figure 6.Figure 6.

Bottom Line: The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit.In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed.The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. edmondma@hkbu.edu.hk

ABSTRACT
Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.

Show MeSH
Related in: MedlinePlus