title: Sensitive detection of transcription factors in cell nuclear extracts by using a molecular beacons based amplification strategy.
abstract: Monitoring transcription factor (TF) levels provides an important assessment of the state of cell populations. Unfortunately, traditional methods for monitoring TF concentration are generally cumbersome and time-consuming. We developed an ultrasensitive one-pot TF detection method that uses target-molecular beacons-dependent amplification (TMDA) fluorescence strategy to circumvent the aforementioned limitations in TF detection. In this assay, we employed a DNA1/DNA2 duplex as the reporting probe and a stem-loop DNA molecular beacon (MB) as the signaling probe. The integration of protein-DNA1/DNA2 duplex and exonuclease III (Exo III) digestion can convert the detection of transcription factors to the detection of reporter oligonucleotides. The subsequent hybridization of the reporter oligonucleotides with the molecular beacons opens the stem-loop structure. The formation of the DNA complex triggers amplification reaction and the recovery of the fluorescence. This assay exhibits high sensitivity with a detection limit of 2.2 pM and a detection range of 3 orders of magnitude, which is superior to most currently used methods for transcription factor detection. More importantly, this method is suitable for the direct detection of TFs in crude nuclear extracts of cancer cells.
authors.affiliation: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China. Electronic address: zhangkai@jsinm.org.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors.forename: Minhao
authors.initials: M
authors.lastname: Xie
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D002457:Cell Extracts / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015336:Molecular Probe Techniques; D015335:Molecular Probes; D016328:NF-kappa B / Q000032:analysis; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D014157:Transcription Factors / Q000032:analysis*
chemical_list: D002457:Cell Extracts; D015335:Molecular Probes; D016328:NF-kappa B; D014157:Transcription Factors; D004247:DNA
keywords: Cell nuclear extracts; Exonuclease digestion; Inhibition Assay; Nicking endonucleases; One-pot; Transcription factor