title: Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor.
abstract: DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Xirui
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA and Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts 02215, USA. selim@bu.edu.
authors.forename: George G
authors.initials: GG
authors.lastname: Daaboul
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Philipp S
authors.initials: PS
authors.lastname: Spuhler
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Peter
authors.initials: P
authors.lastname: Dröge
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA and Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts 02215, USA. selim@bu.edu.
authors.forename: M Selim
authors.initials: MS
authors.lastname: Ünlü
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins; D004867:Equipment Design; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D061925:Immobilized Nucleic Acids; D007368:Interferometry / Q000379:methods*; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D040081:Protein Array Analysis / Q000295:instrumentation; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013499:Surface Properties
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D061925:Immobilized Nucleic Acids; D011506:Proteins; D006859:Hydrogen; D004247:DNA