Sensing of a nucleic acid binding protein via a label-free perylene probe fluorescence recovery assay.
A novel label-free fluorescence recovery assay for the sensing of a DNA binding protein has been developed. A transcription factor c-Jun protein, and a 21 base pair duplex DNA containing the c-Jun protein binding site (J-DNA) were selected. J-DNA was mixed with a cationic fluorescent perylene probe (compound 1), and induced aggregation of the probe. Quenching of the probe's fluorescence was observed. However, when c-Jun protein was mixed with the J-DNA, c-Jun bound to the duplex DNA, which reduced the degree of the induced perylene probe aggregation, and a turn on fluorescence signal was observed. The recovered fluorescence intensity was directly related to the amount of c-Jun added. The method is highly selective, six non-DNA binding proteins and one randomly selected 21 base pair duplex DNA (con-1) were tested. No noticeable compound 1 fluorescence recovery was observed. Mutations were also introduced to the c-Jun recognition sequence and much reduced fluorescence recovery was observed. Our assay is label-free, convenient, inexpensive, and fast. It can be used in biomedical research such as high throughput screening of drugs targeted at DNA-binding proteins.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Dongli
D
Liao
Wenying
W
Li
Jian
J
Chen
Huping
H
Jiao
Huipeng
H
Zhou
Bin
B
Wang
Cong
C
Yu
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D010569:Perylene / Q000737:chemistry*; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D016755:Proto-Oncogene Proteins c-jun; D010569:Perylene; D004247:DNA
Fluorescence; Oligonucleotide; Perylene probe; Protein; Sensing