Sensitive detection of transcription factors by isothermal exponential amplification-based colorimetric assay.
Transcription factors regulate gene expression by binding to specific DNA sequences within the regulatory regions of genes and have become potential targets in clinical diagnosis and drug development. However, traditional approaches for the detection of transcription factors are usually laborious and time-consuming with a low sensitivity. Here, we develop an isothermal exponential amplification reaction (EXPAR)-based colorimetric assay for simple and sensitive detection of transcription factor NF-κB p50. In this assay, the presence of NF-κB p50 is converted to the reporter oligonucleotides through protein-DNA interaction, exonuclease III digestion, and isothermal exponential amplification. The subsequent sandwich hybridization of the reporter oligonucleotides with the gold nanoparticle (AuNP)-labeled DNA probes generates a red-to-purple color change, allowing the visual detection of NF-κB p50 with the naked eye. Notably, this method converts the detection of transcription factors to the detection of DNA without the requirement of DNA marker-linked antibodies in the case of immuno-PCR and can sensitively measure NF-κB p50 with a detection limit of 3.8 pM, which has improved by as much as 4 orders of magnitude as compared with the conventional AuNP-based colorimetric assay and the label-free luminescence assay and up to 4 orders of magnitude as compared with fluorescence resonance energy transfer (FRET)-based assay as well. Importantly, this method can be used to measure TNF-α-induced endogenous NF-κB p50 in HeLa cell nuclear extracts and might be further applied for the detection of various DNA-binding proteins and aptamer-binding molecules.
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China.
Yan
Y
Zhang
Juan
J
Hu
Chun-Yang
CY
Zhang
D001483:Base Sequence; D003124:Colorimetry / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry; D052002:NF-kappa B p50 Subunit / Q000737:chemistry / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D052002:NF-kappa B p50 Subunit; D009841:Oligonucleotides; D006046:Gold; D004247:DNA