Exo-Dye-based assay for rapid, inexpensive, and sensitive detection of DNA-binding proteins.
We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.
Chen
Zaozao
Z
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Ji
Meiju
M
Hou
Peng
P
Lu
Zuhong
Z
D000595:Amino Acid Sequence; D052160:Benzothiazoles; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D003959:Diamines; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D052160:Benzothiazoles; D004268:DNA-Binding Proteins; D003959:Diamines; D005456:Fluorescent Dyes; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I