title: A force-based, parallel assay for the quantification of protein-DNA interactions.
abstract: Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-University, Munich, Germany ; Munich Center for Integrated Protein Science (CIPSM), Munich, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Limmer
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Diana A
authors.initials: DA
authors.lastname: Pippig
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Aschenbrenner
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Hermann E
authors.initials: HE
authors.lastname: Gaub
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA