title: Studying forkhead box protein A1-DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy.
abstract: Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods-electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)-as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Khin Moh Moh
authors.initials: KM
authors.lastname: Aung
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Siu Yee
authors.initials: SY
authors.lastname: New
authors.affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Hong
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Laura
authors.initials: L
authors.lastname: Sutarlie
authors.affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Michelle Gek Liang
authors.initials: MG
authors.lastname: Lim
authors.affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A(*)STAR), Singapore.
authors.forename: Si Kee
authors.initials: SK
authors.lastname: Tan
authors.affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A(*)STAR), Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore. Electronic address: cheungcwe@gis.a-star.edu.sg.
authors.forename: Edwin
authors.initials: E
authors.lastname: Cheung
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A(*)STAR), Singapore. Electronic address: xd-su@imre.a-star.edu.sg.
authors.forename: Xiaodi
authors.initials: X
authors.lastname: Su
mesh_terms: D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D006046:Gold / Q000737:chemistry*; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism
chemical_list: D015342:DNA Probes; C496499:FOXA1 protein, human; D051541:Hepatocyte Nuclear Factor 3-alpha; D008024:Ligands; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D006046:Gold
keywords: EMSA; Fluorescent anisotropic; Forkhead box protein; Gold nanoparticles; Protein–DNA interactions