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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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A schematic representation of the digestion products for oligonucleotides containing one (a) and two (b) NF-κB subunit binding sites.
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Figure 5: A schematic representation of the digestion products for oligonucleotides containing one (a) and two (b) NF-κB subunit binding sites.

Mentions: We next investigated the effect of introducing two binding sites on the double-stranded oligonucleotide substrate. The luminescence spectrum of the ruthenium complex in the presence of the double-stranded substrate containing two binding sites after digestion is shown in Figure 4. When this oligonucleotide was incubated in the presence of the p50 subunit and subjected to Exo III digestion, a maximal 8-fold increase in the luminescence response was observed, compared to only 4.5-fold for the oligonucleotide containing one binding site. We postulate that in the case of the double-stranded substrate containing one binding site, complete digestion by Exo III from the 3′ would be expected to generate long 5′-overhangs with limited duplex regions (Figure 5a). Thus, even though digestion was inhibited relative to the control, the luminescence response of the ruthenium complex would still be reduced. However, when two p50 subunit binding sites are present, the complete digestion of the double-stranded substrate from the two 3′-termini does not occur (Figure 5b), preserving the duplex structure of the substrate and allowing the intercalation of the ruthenium complex. Using the double-stranded substrate with two p50 subunit binding sites, we observed a linear luminescence response (up to 8-fold intensity enhancement) to changes in the concentration of the p50 subunit in the concentration range of 30–220 nM with a detection limit of 30 nM.Figure 4.


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)

A schematic representation of the digestion products for oligonucleotides containing one (a) and two (b) NF-κB subunit binding sites.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: A schematic representation of the digestion products for oligonucleotides containing one (a) and two (b) NF-κB subunit binding sites.
Mentions: We next investigated the effect of introducing two binding sites on the double-stranded oligonucleotide substrate. The luminescence spectrum of the ruthenium complex in the presence of the double-stranded substrate containing two binding sites after digestion is shown in Figure 4. When this oligonucleotide was incubated in the presence of the p50 subunit and subjected to Exo III digestion, a maximal 8-fold increase in the luminescence response was observed, compared to only 4.5-fold for the oligonucleotide containing one binding site. We postulate that in the case of the double-stranded substrate containing one binding site, complete digestion by Exo III from the 3′ would be expected to generate long 5′-overhangs with limited duplex regions (Figure 5a). Thus, even though digestion was inhibited relative to the control, the luminescence response of the ruthenium complex would still be reduced. However, when two p50 subunit binding sites are present, the complete digestion of the double-stranded substrate from the two 3′-termini does not occur (Figure 5b), preserving the duplex structure of the substrate and allowing the intercalation of the ruthenium complex. Using the double-stranded substrate with two p50 subunit binding sites, we observed a linear luminescence response (up to 8-fold intensity enhancement) to changes in the concentration of the p50 subunit in the concentration range of 30–220 nM with a detection limit of 30 nM.Figure 4.

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