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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 (a) the hairpin DNA (0.02 µM); and (b) the hairpin DNA (0.02 µM) and ExoIII (40 U).
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Figure 1: The luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of (a) the hairpin DNA (0.02 µM); and (b) the hairpin DNA (0.02 µM) and ExoIII (40 U).

Mentions: To validate our label-free detection assay for transcription factors, we designed a hairpin oligonucleotide that contained the NF-κB binding site [-GGGACTTTC-] (56). The hairpin substrate was incubated at 95°C for 5 min, followed by gradual cooling to room temperature to ensure the formation of the double-stranded structure. The luminescence response of the ruthenium complex in the presence of the hairpin substrate was enhanced by 4.6-fold due to intercalation of the ruthenium complex into the DNA (Figure 1). The addition of ExoIII leads to the digestion of the oligonucleotide, converting the hairpin structure into short single-stranded DNA fragments. Due to the weak binding of the ruthenium complex with the single-stranded DNA, emission intensity was decreased by 3.0-fold. Potassium ferrocyanide K3[Fe(CN)6] was used to quench the background emission of the ruthenium complex in aqueous solution or when bound to single-stranded DNA. A ferrocyanide concentration of 600 μM was found to give the highest degree of discrimination between double-stranded and single-stranded DNA.Figure 1.


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 (a) the hairpin DNA (0.02 µM); and (b) the hairpin DNA (0.02 µM) and ExoIII (40 U).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: The luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of (a) the hairpin DNA (0.02 µM); and (b) the hairpin DNA (0.02 µM) and ExoIII (40 U).
Mentions: To validate our label-free detection assay for transcription factors, we designed a hairpin oligonucleotide that contained the NF-κB binding site [-GGGACTTTC-] (56). The hairpin substrate was incubated at 95°C for 5 min, followed by gradual cooling to room temperature to ensure the formation of the double-stranded structure. The luminescence response of the ruthenium complex in the presence of the hairpin substrate was enhanced by 4.6-fold due to intercalation of the ruthenium complex into the DNA (Figure 1). The addition of ExoIII leads to the digestion of the oligonucleotide, converting the hairpin structure into short single-stranded DNA fragments. Due to the weak binding of the ruthenium complex with the single-stranded DNA, emission intensity was decreased by 3.0-fold. Potassium ferrocyanide K3[Fe(CN)6] was used to quench the background emission of the ruthenium complex in aqueous solution or when bound to single-stranded DNA. A ferrocyanide concentration of 600 μM was found to give the highest degree of discrimination between double-stranded and single-stranded DNA.Figure 1.

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