scholarly journals Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 126
Author(s):  
Zhaohui Wang ◽  
Pak Wong

Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA–protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems.

Nature ◽  
1986 ◽  
Vol 322 (6080) ◽  
pp. 656-659 ◽  
Author(s):  
Ger J. M. Pruijn ◽  
Wim van Driel ◽  
Peter C. van der Vliet

2003 ◽  
Vol 17 (12) ◽  
pp. 825-836 ◽  
Author(s):  
Vineet Pande ◽  
Rakesh K. Sharma ◽  
Jun-Ichiro Inoue ◽  
Masami Otsuka ◽  
Maria J. Ramos

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