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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 luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture with the double-stranded DNA substrate (0.02 µM) containing two NF-κB binding sites incubated with p50 (0.12 µM) with or without oridonin (20 µM).
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Figure 8: The luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture with the double-stranded DNA substrate (0.02 µM) containing two NF-κB binding sites incubated with p50 (0.12 µM) with or without oridonin (20 µM).

Mentions: To provide additional evidence that the selective binding of the p50 subunit is responsible for the inhibition of ExoIII catalyzed digestion of the double-stranded substrate, we repeated the luminescence measurements in the presence of oridonin, a known inhibitor of NF-κB DNA binding activity (58). In the presence of the NF-κB inhibitor (Figure 8, orange), a significant reduction in the luminescence response of the ruthenium complex was observed compared to the sample that does not contain the inhibitor (Figure 8, purple). Oridonin is presumed to inhibit binding of p50 to the double-stranded substrate and ExoIII is able to digest the DNA into short single-stranded fragments resulting in a reduced luminescence response. Taken together, these series of negative control experiments demonstrate that the luminescence enhancement observed in the presence of the p50 subunit is probably due to the binding of the transcription factor to the oligonucleotide, inhibiting the ExoIII catalyzed digestion of the double-stranded substrate.Figure 8.


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 luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture with the double-stranded DNA substrate (0.02 µM) containing two NF-κB binding sites incubated with p50 (0.12 µM) with or without oridonin (20 µM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: The luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture with the double-stranded DNA substrate (0.02 µM) containing two NF-κB binding sites incubated with p50 (0.12 µM) with or without oridonin (20 µM).
Mentions: To provide additional evidence that the selective binding of the p50 subunit is responsible for the inhibition of ExoIII catalyzed digestion of the double-stranded substrate, we repeated the luminescence measurements in the presence of oridonin, a known inhibitor of NF-κB DNA binding activity (58). In the presence of the NF-κB inhibitor (Figure 8, orange), a significant reduction in the luminescence response of the ruthenium complex was observed compared to the sample that does not contain the inhibitor (Figure 8, purple). Oridonin is presumed to inhibit binding of p50 to the double-stranded substrate and ExoIII is able to digest the DNA into short single-stranded fragments resulting in a reduced luminescence response. Taken together, these series of negative control experiments demonstrate that the luminescence enhancement observed in the presence of the p50 subunit is probably due to the binding of the transcription factor to the oligonucleotide, inhibiting the ExoIII catalyzed digestion of the double-stranded substrate.Figure 8.

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