title: Fok I cleavage-inhibition strategy for the specific and accurate detection of transcription factors.
abstract: Specific and accurate detection of transcription factors is critical for disease diagnosis and drug development. Here, we developed a novel and versatile fluorescent sensing strategy for specific and accurate detection of transcription factors based on the inhibition of endonuclease Fok I-catalyzed DNA cleavage reaction. A FAM-labeled double-stranded DNA probe (dsDNA probe) with transcription factor binding site and Fok I recognition site was designed for target recognition and signal transduction. With the binding of transcription factors, the dsDNA probes are protected from cleavage by Fok I. These protected dsDNA probes then cannot hybridize with the added BHQ-DNA, keeping the fluorescence of FAM in an on state. However, in the absence of targets, the dsDNA probes were cleaved to release the FAM-DNA, which subsequently hybridized with BHQ-DNA, resulting in the fluorescence of the FAM being quenched. With the Fok I-DNA interaction that can specifically recognize the transcription factor-DNA binding and accurately convert the detection of transcription factors to the detection of DNA, the proposed strategy realized the reliable detection of model target NF-κB p50 with a nanomolar detection limit. This strategy was also employed to detect the inhibition effect of oridonin, a known inhibitor of NF-κB. Furthermore, perfect recoveries were obtained when detecting the targets in HeLa cells lysate, demonstrating the feasibility of this strategy for transcription factor detection in biological samples.
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Gao
authors.affiliation: School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
authors.forename: Desong
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China. Electronic address: wjiang@sdu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D053837:DNA Cleavage / Q000187:drug effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors* / Q000378:metabolism; D045786:Diterpenes, Kaurane / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005240:Feasibility Studies; D006367:HeLa Cells; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000032:analysis*; D013050:Spectrometry, Fluorescence
chemical_list: D045786:Diterpenes, Kaurane; D004791:Enzyme Inhibitors; D052002:NF-kappa B p50 Subunit; C011959:oridonin; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: DNA cleavage; Fluorescence method; Fok I restriction endonuclease; Site-specific; Transcription factor detection