Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins

Abstract

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-κB, SP1 and p50, and the target protein of NF-κB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies

“…FRET has outstanding sensitivity of detection, which can be realized at the level of single biomolecules [48]. A number of recent efforts have utilized FRET to quantify binding of protein to DNA [49][50][51]. In one scheme, two duplex DNA "half" protein-binding sites are each separately labeled with a donor and acceptor fluorophores [50].…”

“…This procedure, known as the molecular beacon binding assay, allows real-time optical reporting of the binding event. Another approach exploits the use of dual fluorophore labeled duplex DNA with protein binding detected by exonuclease III protection [51]. A linear duplex DNA is designed with two binding sites occupying each end of the dual-labeled probe.…”

Characterization of the AP Endonucleases fromThermoplasma volcaniumandLactobacillus plantarum: Contributions of Two Important Tryptophan Residues to AP Site Recognition

“…The 3 0 -5 0 exonuclease activity of E. coli ExoIII is often used in molecular biological experiments, such as DNA-protein interaction analysis. 27,28) Since thermophilic TvoExo has not only AP endonuclease activity but also 3 0 -5 0 exonuclease activity, this may expand the molecular biological coverage of the ExoIII family AP endonuclease, especially for high-temperature DNA footprinting. Site-directed mutagenesis experiments were performed on the tryptophan residues of TvoExo and LplExo, corresponding to Trp-212 of ExoIII and Trp-280 of APE1 (Fig.…”

Sensitive detection of adenosine triphosphate by exonuclease III-assisted cyclic amplification coupled with surface plasmon resonance enhanced fluorescence based on nanopore

“…The fluorescence intensity was greatly enhanced by AuNPs by coupling to a plasmonic hotspot of zeptoliter volume [27]. It should be noted that for the S 2 -S 3 hybrid, there was still a 13-nt single-stranded sequence in 3 terminus, resisting the cleavage by Exo-III [28]. Fig.…”